Identification and Validation of Operational Pain Indicators in Anurans

Abstract

Amphibian welfare, particularly pain assessment in anurans, remains understudied despite their ecological and biomedical significance. This study aimed to identify and validate operational pain indicators for adult anurans under professional care. A four-phase approach was used: a systematic literature review, expert validation with risk analysis, field validation in a zoological facility, and development of a preliminary pain index. From 158 publications, 16 potential indicators were identified, encompassing behavioural, clinical, and physiological signs. Expert evaluation by 28 professionals from 12 institutions refined this to seven indicators, achieving over 60% consensus: feeding behaviour changes, abnormal behaviour, impaired locomotion, oedema, reduced movement, retained skin post-moulting, and altered respiration. Field validation in 53 anurans confirmed high observability and ease of measurement, with feeding behaviour changes and oedema scoring highest for practicality (93.5% and 93.0%, respectively). These validated indicators provide a science-based foundation for routine welfare monitoring, enabling timely interventions. Their integration into husbandry protocols can enhance ethical standards, improve conservation outcomes, and increase public confidence in amphibian care, paving the way for a standardised anuran pain index.

1. Introduction

Amphibians and, in particular, adult anurans such as frogs and toads, constitute one of the most evolutionary ancient and ecologically indispensable groups of vertebrates [1]. Having occupied both aquatic and terrestrial environments for over 370 million years, their evolutionary resilience is paralleled by their vital roles in contemporary ecosystems [2]. These include regulating insect populations, facilitating nutrient cycling, and serving as bioindicators of environmental health [3]. Furthermore, amphibians have become an increasingly important focus in biomedical research due to their physiological plasticity, regenerative capabilities, and the presence of pharmacologically significant compounds within their skin secretions [4,5,6]. However, anurans are among the most threatened vertebrates globally, with approximately 41% of species categorised under some level of conservation concern by the International Union for Conservation of Nature [5,7,8]. The current trajectory of amphibian population decline is frequently cited as indicative of a sixth mass extinction, driven by multifactorial pressures including habitat destruction, climate change, disease, and anthropogenic exploitation [9,10]. The precipitous loss of wild amphibian populations has intensified the role of ex situ conservation strategies, which include professional management of amphibians in zoological parks, rehabilitation centres, research institutions, and controlled breeding facilities [11,12,13]. These environments are now not only conservation tools but also ethical spaces that must adhere to evolving standards of animal welfare [14,15,16,17]. Within this framework, pain management has emerged as a critical, albeit historically overlooked, aspect of amphibian welfare [18,19,20]. In mammals and birds, pain is now widely accepted as a core dimension of welfare, and a range of validated tools are routinely employed to detect, measure, and mitigate it [21,22,23]. In contrast, amphibians have long been excluded from such considerations, in part due to persistent misconceptions regarding their neurobiological capacity to experience pain [24], as well as practical challenges associated with their behavioural assessment [25,26]. Over the last two decades; however, scientific understanding of amphibian nociception has advanced significantly. It is now well-established that anurans possess nociceptors capable of detecting thermal, mechanical, and chemical stimuli, and that they exhibit both spinal and supraspinal processing of such signals [19,20,25,27,28,29]. Amphibians show protective behavioural responses to noxious stimuli such as limb withdrawal, rubbing, decreased movement, or altered postures that are reduced when analgesic substances like opioids are administered, thereby satisfying criteria commonly used to infer pain in other non-human animals [19,30,31]. While this body of evidence supports the presence of pain perception in anurans, there remains a stark paucity of validated, operational indicators of pain that can be implemented in the routine care of these animals. Unlike mammals, for whom facial expressions, vocalisations, and detailed ethograms provide a basis for pain evaluation [21,32,33,34], anurans lack facial mobility and exhibit a relatively cryptic behavioural repertoire, particularly under observation [20,35]. These characteristics demand the development of tailored approaches for pain detection that are grounded in species-specific knowledge and adapted to the practical constraints of zoological and laboratory environments as in fish species [36].

Current welfare assessments for anurans in captivity tend to rely on indirect or nonspecific measures such as survival rates, reproductive output, general activity levels, and indices of physical condition [29,37]. Although such parameters are valuable in a general health context, they lack the sensitivity and specificity required to identify and quantify the experience of pain. Moreover, physiological indicators such as elevated glucocorticoid levels, while indicative of stress, cannot distinguish between pain-induced and environmentally induced distress [38]. This ambiguity in interpretation limits their utility as standalone tools for pain assessment [39]. In practice, caregivers and veterinarians are often left to rely on anecdotal observations or subjective judgments when attempting to identify pain in anurans, which not only increases the risk of misinterpretation but also delays or prevents the implementation of appropriate analgesic interventions [40]. The absence of validated pain indicators in amphibians is not merely a scientific gap but also an ethical failure. The lack of such tools compromises the ability of institutions to comply with internationally accepted standards of animal care and impairs efforts to safeguard individual welfare [41,42]. Any efforts to define operational indicators of pain must consider the unique morphology and sensory ecology of anurans, including their semi-permeable skin, reliance on environmental temperature regulation, and variable activity patterns across circadian cycles [43]. These biological attributes not only shape the ways in which pain might manifest but also influence the feasibility of observation and measurement. For instance, behavioural signs such as refusal to feed, impaired locomotion, abnormal posture, or disrupted shedding cycles may serve as reliable proxies for underlying discomfort, provided they are standardised and contextualised within normal behavioural baselines [44]. Also, the establishment of reliable pain indicators in anurans has broader implications for public perception. As public concern for animal welfare continues to rise, zoos and aquaria are increasingly expected to provide transparent evidence of humane treatment, particularly for species that have traditionally received less attention [45,46,47]. The demonstration of systematic pain assessment protocols for amphibians could help reshape societal narratives about these animals, highlighting their capacity for suffering and reinforcing their moral relevance. In doing so, such efforts could foster deeper public engagement with amphibian conservation and contribute to a cultural shift in the way these species are valued and protected [48]. From a scientific perspective, the creation of a pain index or validated indicator suite would also lay the groundwork for future comparative studies across taxa, environments, and developmental stages. It would facilitate longitudinal monitoring of welfare trajectories and enable the evaluation of interventions aimed at improving husbandry practices. Moreover, it would generate a valuable body of baseline data that could inform policy development at institutional, national, and international levels. This is particularly relevant considering emerging legislative frameworks that increasingly recognise the sentience of animals across a wider range of taxa [49,50].

The objective of this study was to identify pain indicators both bibliographically and directly from people that work with anurans routinely (zoo staff, research institutions, universities worldwide) and then validate those which are truly applicable and operational through both experts and stakeholders’ surveys and practicability.

2. Materials and Methods

The methodological design consisted of four sequential phases: (i) A systematic literature review of potential pain indicators in anurans, (ii) expert validation of these indicators combined with a risk analysis, (iii) field validation under professional care conditions, and (iv) the development of a pain index.

The statistical approach employed in this study was designed to balance methodological rigour with the practical constraints inherent to welfare research involving a relatively small number of expert respondents and field observations. Given the exploratory nature of the investigation, and the fact that the primary objective was to reach a consensus on the most reliable and observable indicators of pain in adult anurans, the analysis emphasised descriptive and consensus-based methods rather than inferential statistical modelling.

Literature Review of Pain Indicators in Anurans: The initial phase comprised the identification of potential operational indicators of pain in adult anurans through a targeted literature review. Bibliographic research was carried out using Scopus and Web of Science (Core Collection) due to their comprehensive coverage of peer-reviewed scientific literature in the fields of animal welfare, zoology, and veterinary sciences. The search strategy used combinations of the following keywords: “pain”, “frog”, “indicators”, and “welfare”. Each term was used individually and in various Boolean combinations to maximise retrieval sensitivity. Retrieved records were screened initially at the title and abstract level to exclude irrelevant publications, non-peer-reviewed sources, or studies not addressing welfare or pain in amphibians. Eligible articles were subjected to full-text review, during which any described behavioural, physiological, or clinical variables with potential utility as operational pain indicators in adult anurans were extracted (Supplementary Materials). We followed the same procedure for a systematic review, using non-standard keywords to amplify the results from searches. This may produce bias, but we believe we have increased the chances of finding more results. This process yielded a list of 16 candidate indicators for subsequent validation.

Expert Validation and Risk Analysis: Following literature recollection, the identified candidate indicators were submitted for expert validation. A structured questionnaire was developed in Google Forms and translated into three languages (Spanish, English, and Portuguese, see Supplementary Materials) to increase accessibility for a wider pool of respondents. The survey consisted of four sections: (i) Informed Consent: participants were required to accept or decline consent before continuing with the questionnaire; (ii) Participant Identification: non-sensitive demographic and professional background information was collected; (iii) Indicator Evaluation: each potential indicator identified in the previous phase was assessed for perceived risk, magnitude, and field applicability using a 5-point Likert scale (1 = 0%, 5 = 100%). Closed-ended questions were supplemented with an open-ended question inviting respondents to propose additional indicators not included in the list, and (iv) Conclusion and Satisfaction: final comments and self-reported satisfaction with the survey process were collected.

The questionnaire was sent via email to professional networks, including targeted outreach to members of the Latin American Association of Zoos and Aquariums (ALPZA), the World Association of Zoos and Aquariums (WAZA), and other regional or national equivalents. Institutions contacted included the National Zoo of the Metropolitan Park of Santiago, the Wildlife Rehabilitation Centre of Universidad Andrés Bello (UFAS), Universidad de Concepción, and the Corral La Mañosa breeding facility. International contacts encompassed the Zoological Society of London, Stanford University, the Zoológico de Cali, and the Pontificia Universidad Católica del Ecuador. ALPZA further supported dissemination by circulating the survey link among its expert networks. The number of questionnaires sent was 38. The response window remained open for three weeks.

The use of Likert-type scales to quantify expert judgements on risk, magnitude, and field applicability allowed for the capture of nuanced opinions across a continuum, rather than restricting responses to binary options. Likert scales are widely accepted in animal welfare assessment studies as they accommodate subjective judgements while enabling quantitative aggregation of data [51,52]. The transformation of Likert scores into percentages facilitated standardisation across the three dimensions being evaluated, enabling direct comparability and the computation of composite scores [53].

The analysis followed the European Food Safety Authority (EFSA) approach for expert knowledge elicitation [54,55], averaging the Likert scores for risk and magnitude for each indicator, and subsequently averaging these with the applicability score. Only indicators achieving ≥60% overall weighting proceeded to the next phase of validation. Averaging scores across respondents was selected as the primary method for combining expert inputs. This decision was supported by its common use in Delphi-method style consensus-building exercises, where the aim is to reduce inter-individual variability by identifying central tendencies in the group’s opinions [56]. While median values are sometimes preferred in ordinal data analysis, the mean was chosen here to preserve the sensitivity of the percentage scale and allow for fine-grained ranking of indicators, which is critical when small differences in score may influence indicator inclusion or exclusion [57]. The threshold of 60% for indicator retention was determined a priori, aligning with thresholds used in similar welfare tool development studies [55]. This cut-off represents a pragmatic balance between ensuring sufficient consensus and avoiding excessive exclusion of potentially valuable indicators at an early stage. Retaining only those indicators with ≥60% agreement across both expert opinion and field applicability phases helped ensure that the final pain index is both scientifically defensible and practical for use under professional care conditions.

Field Validation of Pain Indicators: Field validation was conducted at the National Zoo of the Metropolitan Park of Santiago, selected for its large and diverse collection of anurans in both exhibition and rehabilitation contexts. Limiting the study to a single facility minimised cross-institutional pathogen transmission risk (e.g., ranavirus, chytridiomycosis). The biosecurity protocols included the use of disposable protective equipment (hair covers, masks, shoe covers, aprons) and active footbaths at each entry point to amphibian enclosures. The facility houses approximately 49 aquaria containing various anuran species with different group sizes. For inclusion, at least 10% of aquaria had to contain individuals presenting visible pathology or physical abnormalities (e.g., oedema, swelling, trauma, haemorrhage, fractures, or multiple injuries). Healthy individuals without overt abnormalities were excluded. Hands-off observations were conducted in dedicated anuran conservation areas and during the frog exposition named “Expoanfibios” over one month. For each selected aquarium or terrarium, all indicators selected previously from the expert validation phase were assessed for both observability and ease of measurement in the field, using the same 5-point Likert scale (1 = strongly disagree/very difficult to measure, 5 = strongly agree/very easy to measure). Assessments were performed twice daily (morning and afternoon) to increase the chance to capture the indicators due to potential temporal variation in behaviour or clinical presentation. In the field validation phase, the same scoring approach was maintained to preserve methodological continuity and to facilitate the integration of expert-derived and field-derived data into a single composite measure. This approach is consistent with triangulation in research methodology, whereby multiple data sources are systematically combined to enhance the validity and robustness of findings [58].

Data were recorded manually on paper using pre-prepared tables listing each indicator with check boxes for the Likert score with contextual variables such as number of individuals, enclosure environmental conditions (ambient temperature, visual barriers) and enrichment, if present. All records were subsequently transcribed into Google Forms to facilitate data collated in Microsoft Excel for percentage and mean score calculations for each indicator. Given the modest sample size of expert participants and the targeted nature of field validation (conducted in a single zoological institution), the use of more complex inferential statistics (e.g., regression analysis, factor analysis) was not warranted at this stage. Such techniques require larger and more diverse datasets to yield reliable parameter estimates [59]. Instead, the focus remained on descriptive aggregation, which is appropriate for initial validation of indicators prior to wider multi-institutional testing.

Finally, with the results, we created a proposed Protocol for Daily Evaluation, a Companion Guide to the protocol and an Excel datasheet for recording. We also created a prototype of anuran pain index code in HTML. This index assumes equal weighting in all indicators, and the researcher/clinician/manager can move the intensity of each indicator (according to the daily examination of animals) and obtain a total pain score. All these are attached as Supplementary Materials.

3. Results

Literature Review of Potential Pain Indicators in Anurans: A systematic search in Scopus and Web of Science retrieve a total of 158 records (Figure 1). After screening titles and abstracts for relevance to amphibian welfare and pain, 54 manuscripts were selected for full-text review. Only 16 manuscripts met the inclusion criteria and described behavioural, clinical, or physiological variables potentially indicative of pain in adult anurans. The final list of 16 candidate indicators derived from the literature were feeding behaviour changes, abnormal behaviour, impaired locomotion, presence of oedema, reduced movement, retained skin post-moulting, altered respiration, corticosterone, altered heart rate, blood glucose, intraspecific social interaction, skin microbiome, sex, environmental temperature, shelter use, and vocalisation.

Expert Validation and Risk Analysis: The expert survey received 28 responses from professionals in 12 institutions across Latin America, Europe, and North America, including veterinarians, zoologists, herpetologists, and welfare specialists. The response rate was 74%. The 16 candidate indicators were scored for risk, magnitude, and field applicability on a 5-point Likert scale, subsequently converted to percentages and averaged.

Table 1. Mean expert scores (%) for potential operational pain indicators in adult anurans.

Indicator	Risk (%)	Magnitude (%)	Applicability (%)	Composite Score (%)
Feeding behaviour changes	71.0	71.0	66.0	69.0
Abnormal behaviour	78.0	75.0	74.0	75.0
Impaired locomotion	71.0	70.0	77.0	74.0
Presence of oedema	72.0	67.0	65.0	67.0
Reduced movement	78.0	74.0	79.0	78.0
Retained skin post-moulting	61.0	54.0	66.0	62.0
Altered respiration	71.0	71.0	67.0	69.0
All others (<60%)	—	—	—	<60.0

presents the rounded mean percentage scores from expert evaluation. Only seven indicators (43.8%) exceeded the 60% threshold, which were feeding behaviour changes, abnormal behaviour, impaired locomotion, presence of oedema, reduced movement, retained skin post-moulting, and altered respiration.

Field Validation of Indicators: A total of 53 individual anurans were assessed across the retained indicators. Observations were performed twice daily (AM and PM), producing 106 observation sessions and 308 indicator-specific data points. All indicators had 100% score for observability in the field while ease of measurement scores ranged from 74% to 100% (

Table 2. Field validation scores (%) for expert validated indicators.

Indicator	Observability (%)	Ease of Measurement (%)	Mean Field Score (%)
Feeding behaviour changes	100.0	100.0	100.0
Oedema	100.0	75.0	88.0
Abnormal behaviour	100.0	99.0	100.0
Impaired locomotion	100.0	100.0	100.0
Reduced movement	100.0	99.0	100.0
Retained skin post-moulting	100.0	75.0	88.0
Altered respiration	100.0	79.0	90.0

).

Finally, we created a Field Protocol, a Companion Guide for the Field Protocol, and an Excel spreadsheet to easily record the validated anuran pain indicators, in the Supplementary Materials. We have also uploaded a HTML file as Supplementary Materials, with a prototype coding of an online app to measure anuran pain. This code can be seen as a starting point and be modified according to the real conditions of every lab, zoo, etc.

4. Discussion

This study represents, to our knowledge, the first systematic attempt to identify and validate operational pain indicators for adult anurans maintained under professional care conditions. By integrating evidence from scientific literature with structured expert opinion and empirical field validation, the research advances the capacity for evidence-based welfare assessment in a taxonomic group historically underrepresented in pain research [41]. From the initial 158 papers, a final 16 papers were used to obtain the potential indicators, and only seven indicators were retained following expert consensus and field validation. These indicators (feeding behaviour changes, abnormal behaviour, impaired locomotion, presence of oedema, reduced movement, retained skin post-moulting, and altered respiration) demonstrated both high perceived relevance (risk and magnitude) and practical feasibility of detection under zoo conditions. Feeding behaviour changes emerged as the highest-ranked indicator across all stages, reflecting the well-documented relationship between illness or distress and appetite suppression in amphibians. The strong performance of abnormal behaviour and impaired locomotion is consistent with findings in other vertebrate taxa, where changes in activity patterns, gait, or posture often constitute early warning signs of pain or compromised welfare [60,61]. In reptiles, reduced movement and altered locomotion have been associated with musculoskeletal injuries, systemic infections, and suboptimal environmental conditions [62]. The inclusion of oedema is likewise unsurprising, as it represents a visible and objectively measurable clinical sign linked to a range of pathological states, including renal disease, trauma, and infectious processes in anurans [63]. Retained skin post-moulting and altered respiration ranked lower but still surpassed the threshold for inclusion. Retained skin has been linked to compromised hydration, inadequate environmental humidity, or systemic disease [64], while altered respiration may signal systemic distress, anaemia, pulmonary compromise, or toxin exposure [65]. Although these signs may be more transient or species-specific than the higher-ranking indicators, their consistent recognition by both experts and field observers supports their inclusion in a comprehensive assessment tool.

Pain assessment in amphibians remains a relatively nascent field compared with mammals and birds. Previous attempts to identify amphibian pain indicators have largely been descriptive and anecdotal, often embedded within clinical case reports or husbandry manuals. Also, cortisol levels, although sometimes measured, are ambiguous as standalone indicators due to variability in stress responses, environmental influences, and inter-species differences. These studies show the importance of developing operational, behaviourally, and clinically observable indicators, such as those of this study.

The use of expert opinion is similar as methods used in developing welfare assessment protocols for other taxa, such as reptiles [66], zoo mammals [37], and livestock [55]. However, amphibians present unique challenges due to their permeable skin, ectothermic physiology, and often cryptic behavioural responses to injury or disease. As [41] highlighted, the paucity of species-specific data necessitates an approach that synthesises available knowledge while remaining adaptable to new findings.

Several of the indicators identified here have analogues in mammalian welfare science. For example, reduced feeding, impaired locomotion, and abnormal posture are frequently incorporated into pain scoring systems for laboratory rodents, farm animals, and companion species [67,68]. Their recurrence across diverse vertebrate groups suggests conserved physiological and behavioural responses to noxious stimuli. However, amphibian-specific manifestations such as retained skin or buoyancy abnormalities highlight the need for taxon-tailored indices.

Field validation at a single institution was selected to minimise cross-institutional biosecurity risks, a concern of relevance to amphibians given the global prevalence of chytridiomycosis and ranaviral infections [69,70]. While this necessarily limits generalisability, it allows intensive, standardised observation under controlled conditions.

The consistent high scores for feeding behaviour changes and oedema across both phases suggest these may be among the most robust indicators for routine use. However, the lower and more variable field scores for altered respiration and retained skin indicate that observer training and species-specific knowledge may be necessary to ensure reliable detection.

We also identified several limitations that should be taken into consideration. First, although the expert panel was international, the sample size was modest (n = 28), and participation was weighted towards Latin American institutions. Follow-up studies should consider broader geographic representation that may reveal regional or species-specific differences in indicator relevance. Second, field observations were restricted to a relatively small number of individuals (n = 53) and species (Telmatobius dankoi, Rhinella arunco, and Callyptocephalella gayi), which may not capture the full range of responses across the diverse order Anura, such as the model species, the African clawed toad (Xenopus laevis). Future studies should aim to validate the indicators across multiple institutions, climates, and species to ensure robustness and generalisability. Third, the reliance on visual assessment means that subtle or internal indicators of pain such as physiological parameters were not included, though these may complement behavioural signs in future indices. Also, it is recommended that follow-up studies should include inter-observer assessment to ensure consistent application by different staff members with varying levels of training.

5. Conclusions

This study provides the first validated set of operational pain indicators for adult anurans under professional care, bridging a long-standing gap in amphibian welfare assessment. By combining a targeted literature review, expert consensus, and field validation, we were able to identify seven indicators: feeding behaviour changes, abnormal behaviour, impaired locomotion, presence of oedema, reduced movement, retained skin post-moulting, and altered respiration, which are both biologically relevant and practically feasible to detect and measure. The robustness of these indicators, particularly feeding changes and oedema, revealed their potential for integration into routine husbandry protocols, enabling timely recognition and alleviation of pain. Beyond their immediate application, these findings contribute to a broader ethical imperative such as ensuring amphibians receive humane care. The proposed indicators offer a foundation for future refinement into a standardised anuran pain index, with the potential to influence welfare policy, enhance public trust in zoological institutions, and ultimately improve the quality of life for these ecologically vital yet vulnerable species.