# Development and validation of a pain scale in guinea pig
(Cavia porcellus)

Floriane Benedetti, Charly Pignon, Patricia Muffat-Es-Jacques, Caroline

Gilbert, Loïc Desquilbet

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Floriane Benedetti, Charly Pignon, Patricia Muffat-Es-Jacques, Caroline Gilbert, Loïc Desquilbet. Development and validation of a pain scale in guinea pig (Cavia porcellus). Journal of Exotic Pet Medicine, 2024, 50, pp.36-41. 10.1053/j.jepm.2024.06.002. hal-04972382

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Research Article

# Development and validation of a pain scale in guinea pig (*Cavia porcellus*)

Floriane Benedetti<sup>a,*</sup>, Charly Pignon<sup>a</sup>, Patricia Muffat-es-Jacques<sup>a</sup>, Caroline Gilbert<sup>b,c</sup>, Loïc Desquilbet<sup>d,e</sup>

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ARTICLE INFO

Keywords:

Cavia porcellus

Guinea pigs

Pain

Evaluation

Assessment

Scale

Validation

ABSTRACT

**Background:** This study aimed to develop and validate a pain assessment scale for guinea pigs to be performed in clinics by veterinarians. The scale was established using pre-existing pain scales from other species and was based on signs expressed by guinea pigs in pain already identified in the literature. The scale consists of 14 descriptors grouped into 4 categories: appearance, functional, physiological, and behavioral. Each item on the scale was assigned a score based on the observation and handling of the animals. The total score obtained ranged from 0 to 26, with more intense pain scoring higher.

**Methods:** Fifty-eight adult guinea pigs presenting at the authors' Teaching Hospital between March 2021 and November 2022 were assessed. Guinea pigs were filmed during their consultation and an information sheet was completed. The information sheet provided details on the animals' appetite, fecal production, and behavior at home. The videos and corresponding information sheets were then reviewed to attribute a pain score to each animal. The pain scores were measured twice by a same observer 1 month apart to assess repeatability and by a second observer to assess reproducibility. The validity was assessed by correlating the pain scores given by the novel scale with scores using a numerical rating scale (NRS) as a gold standard method.

**Results:** The initial Cronbach's alpha obtained was 0.77, which was satisfactory. However, it highlighted that items concerning pruritus and spasmodic movements should be deleted to strengthen internal consistency. Then, Lin's coefficient and Bland and Altman's graphical method highlighted that the scale had an almost strong repeatability and intra-operator reproducibility. Moreover, the correlation between pain intensities obtained with the novel scale and the scores given using NRS was correct (Kappa = 0.72). The specificity was 87.5%, but the sensitivity of the scale was 52.0%. Each item's repeatability and reproducibility were assessed to identify items that would benefit from added clarification.

**Conclusions and clinical relevance:** Our work is the first step in establishing a pain scale in guinea pigs. However, it should not yet be used in a clinical context. Indeed, the scale was not good at detecting pain when a guinea pig was actually painful. Further studies, including improvement of certain items and testing a larger sample of animals, are ongoing.

Introduction

Pain is an unpleasant sensory and emotional experience associated with or resembling that associated with actual or potential tissue damage, according to the 2020 IASP definition [1]. Assessing and managing pain is an integral part of clinical veterinary practice and an ethical duty for veterinarians [2]. During the last twenty years, the recognition and management of pain in veterinary medicine has progressively improved following the development of pain scales in many domestic species [3–7]. Composite pain scales already exist for cats and dogs. Such scales require an assessment of specific criteria that enable pain measurement

and are structured as a questionnaire based on behavioral and physiological changes occurring with pain [8]. Although guinea pigs are one of the most common species used in laboratories and a popular pet in Europe, little information is available regarding pain in guinea pigs [9]. Postoperative ethograms (highlighting subtle body movements such as twitching, back arching and weight shifting) have been described, but no pain assessment methods have yet been validated in this species [10]. As a prey species, guinea pigs usually hide signs of pain, which makes its observation challenging [10]. Guinea pigs represent a small percentage of patient for small animal veterinarians, raising the difficulty of detecting specific pain behaviors for this species [11]. Keown et

<footnote><sup>a</sup> Exotics Medicine Service, Ecole Nationale Vétérinaire d'Alfort, Maisons-Alfort, France</footnote>

<footnote><sup>b</sup> Behavior Medicine Service, Ecole Nationale Vétérinaire d'Alfort, Maisons-Alfort, France</footnote>

<footnote><sup>c</sup> Laboratoire MECADEV, UMR 7179, CNRS/MNHN, Brunoy, France</footnote>

<footnote><sup>d</sup> Biostatistics and Clinical Epidemiology Service, Ecole Nationale Vétérinaire d'Alfort, Maisons-Alfort, France</footnote>

<footnote><sup>e</sup> INSERM, IMRB, Maisons-Alfort, France</footnote>

<footnote>* Corresponding author.
E-mail address: dr.florianebenedetti@gmail.com (F. Benedetti).</footnote>
al. (2011), who reviewed analgesia in rabbits and guinea pigs, highlighted that many general practitioners felt inadequate in recognizing and treating pain in these species in contrast to dogs and cats whose pain signs are better documented [11].

The goal of this study was to develop and validate a pain assessment scale for guinea pigs that could be performed in clinics by veterinarians. Pre-existing pain scales from other species, such as composite pain scales for rabbits, were used [3,12,13]. Criteria linked to the presence of pain in guinea pigs, which have been described in the literature, were also included [14,15]. The scale was tested for repeatable and reproducible pain scores between 2 operators. The validity of the scale was then evaluated. This was performed by comparing the pain scores obtained with this novel scale to the scores obtained using a NRS (numerical rating scale) which was performed by a third naive observer [13,16].

## Materials and methods

### Pain scale

The guinea pig pain scale is a multidimensional scale composed of fourteen descriptors grouped into 4 categories (Fig. 1). The first category is based on appearance with descriptors that include posture, appearance of the fur, eyes and nostrils, presence of spasmodic body movements and facial expression, such as closed eyelids or vibrissae clumping together. The second category is a functional part with descriptors that include appetite and fecal production. The third category relates to physiological criteria and focuses on the respiratory rate and breathing pattern. The fourth category is a behavioral assessment, with descriptors that include activity of the animal, interactions with the environment, owner or veterinarian, and reaction of the animal to handling or

palpation. Depending on their intensity, a score ranging from 0 to 3 was assigned to each item. The maximum total score for all items was 26. Intervals were assigned based on total score to further classify the pain intensity observed. Five intervals were established and were divided as: no pain (for scores from 0 to 4), mild pain (for scores from 5 to 9), moderate pain (for scores from 10 to 14), severe pain (for scores from 15 to 20) and intolerable pain (for scores from 21 to 26). The intervals were arbitrarily chosen to divide the pain scale into 5 equal sections of the total score obtained.

### Protocol

The study obtained the approval of the Clinical Research Ethics Committee of the institution where the research project was conducted (number 2019-04-12). It involved guinea pigs over four months old at the authors' Teaching Hospital between March 2021 and November 2022.

### Population

Selected animals were healthy guinea pigs presented for health checks or elective surgeries such as neutering and guinea pigs presented with painful conditions such as dental diseases, urinary tract diseases, gastric dilation, otitis, or ovarian cysts. Guinea pigs under 4 months were not included. No restriction on breed, sex, or weight of the guinea pigs was performed. However, as the animals' coat quality was evaluated, and the presence of alopecia was associated with pain, guinea pigs that presented with skin disease were excluded. Owners' consent was obtained before including their animal in this study.

<table><thead><tr><th colspan="2">APPEARANCE</th></tr></thead><tbody><tr><td colspan="2">Posture</td></tr><tr><td colspan="2">Position:</td></tr><tr><td>Relaxed, vigilant, resting position and abdomen touching the ground</td><td>0</td></tr><tr><td>Grouped or curled up position, prostrated animal</td><td>1</td></tr><tr><td colspan="2">Stance: Abdomen raised from the cage floor and/or weight shifting (transfer of body weight by shifting of limbs in the absence of locomotion) and/or back arching (even temporary)</td></tr><tr><td>Not present</td><td>0</td></tr><tr><td>Present</td><td>1</td></tr><tr><td colspan="2">Appearance of hair, eyes, and nostrils</td></tr><tr><td>Shiny coat, clean eyes, and nostrils</td><td>0</td></tr><tr><td>Dull but clean coat, clean eyes, and nostrils</td><td>1</td></tr><tr><td>Slightly stitched/sticky fur and/or moderate presence of nasal or ocular secretions</td><td>2</td></tr><tr><td>Very soiled fur, piloerection and/or large amounts of nasal and ocular secretions</td><td>3</td></tr><tr><td colspan="2">Pruritus / alopecia area</td></tr><tr><td>Not present</td><td>0</td></tr><tr><td>Present</td><td>1</td></tr><tr><td colspan="2">Facial expression</td></tr><tr><td colspan="2">Evelid closure: (narrowing of orbital area, a wrinkle may be visible around the eye)</td></tr><tr><td>Not present</td><td>0</td></tr><tr><td>Moderately present (half-closed eyes, even temporary)</td><td>1</td></tr><tr><td>Obviously present (closed eyes or half-closed eyes permanently)</td><td>2</td></tr><tr><td colspan="2">Vibrissae orientation: (clump together, lose their natural "downward" curve)</td></tr><tr><td>Not present</td><td>0</td></tr><tr><td>Moderately present</td><td>1</td></tr><tr><td>Obviously present</td><td>2</td></tr><tr><td colspan="2">Presence of subtle body movements, trembling (spasmodic movements of the fur, skin, and muscles over the dorsum and flank)</td></tr><tr><td>Not present</td><td>0</td></tr><tr><td>Rarely present and/or localized</td><td>1</td></tr><tr><td>Permanent and/or generalized</td><td>2</td></tr></tbody></table>

<table><caption>Fig. 1. The guinea pig pain scale: The guinea pig pain scale is a multidimensional scale composed of fourteen descriptors grouped into four categories. Depending on their intensity, a score ranging from 0 to 3 is attributed to each item. The maximum total score is 26.</caption><thead><tr><th colspan="2">FONCTION</th></tr></thead><tbody><tr><td colspan="2">Appetite</td></tr><tr><td>Goes to the food and eats instantly, eats its entire ration in 24 hours</td><td>0</td></tr><tr><td>Feeds less (2/3 of its ration in 24h)</td><td>1</td></tr><tr><td>Feeds very little (1/3 of its ration in 24h), needs to be stimulated to eat</td><td>2</td></tr><tr><td>Not self-feeding or eat only with force-feeding for 24 hours or more</td><td>3</td></tr><tr><td colspan="2">Stool production</td></tr><tr><td>Normal appearance and quantity</td><td>0</td></tr><tr><td>Drier or softer stools or smaller stools or decreased quantity (less than 130 over 24 hours) (1 item present)</td><td>1</td></tr><tr><td>Dry or loose stools and/or smaller and/or decreased quantity (at least 2 items present)</td><td>2</td></tr><tr><td>Absence of stool</td><td>3</td></tr><tr><td colspan="2">PHYSIOLOGY</td></tr><tr><td colspan="2">Respiratory pattern</td></tr><tr><td>Normal</td><td>0</td></tr><tr><td>Dyspnea, discordance</td><td>1</td></tr><tr><td colspan="2">Respiratory rate</td></tr><tr><td>Normal</td><td>0</td></tr><tr><td>Increased (&gt;100 mpm)</td><td>1</td></tr><tr><td colspan="2">BEHAVIOUR</td></tr><tr><td colspan="2">Activity / vigilance</td></tr><tr><td>Moves, goes to food, explores, grooms (licks, chews and rubs coat with paws without insisting on an area), is attentive to its environment</td><td>0</td></tr><tr><td>Less activity, little movement and/or lameness, and/or decreased alertness</td><td>1</td></tr><tr><td>Lack of movement, lethargy, total disinterest in the environment</td><td>2</td></tr><tr><td colspan="2">Interactive behavior</td></tr><tr><td>Comes spontaneously or lets the hand approach, lets himself be petted</td><td>0</td></tr><tr><td>Lets the hand approach only with food, does not let itself be petted</td><td>1</td></tr><tr><td>Goes away even when food is offered, is aggressive or unresponsive</td><td>2</td></tr><tr><td colspan="2">Handling, palpation of the painful area</td></tr><tr><td>Remains relaxed</td><td>0</td></tr><tr><td>Becomes aggressive, avoidance reaction</td><td>1</td></tr><tr><td>No reaction, remains silent and lethargic</td><td>2</td></tr><tr><td colspan="2">Total score</td></tr></tbody></table>
## Videos

Animals were filmed with their owners for a total of 3 minutes during their first consultation or before and after surgery for animals admitted for elective surgery. The animals were initially filmed at close range for 60 seconds. Right and left lateral views, as well as dorsal and frontal views of the animal, were obtained. The animals were then placed on the floor and filmed for 120 seconds, during which they could explore the room. The videos provided information regarding the appearance, physiological processes and behavior of each animal. The owner completed an information sheet to record information regarding their animals' appetite, fecal production, and behavior at home. Once recordings for all animals were performed, the videos and associated information sheet (appetite, fecal production, behavior at home) were transmitted to the observers for pain scoring. The observers had different levels of experience with guinea pigs. The first observer was a final-year veterinary student, the second observer was a first-year European College of Zoological Medicine small mammal resident and the third was an Exotics Medicine intern. The observers were blinded to the condition of the animal and whether it had received any analgesia. Animals undergoing elective procedures received analgesia prior to their surgery. For animals presenting for clinical examination, analgesia was provided after the videos were taken.

## Statistical analyses

Firstly, the Cronbach's alpha coefficient was calculated to assess the internal consistency of the scale [17]. This test highlighted items that should be removed from the scale to improve internal consistency. Secondly, the validity of the scale was evaluated by assessing the repeatability, reproducibility, and congruence between the pain score obtained using this novel scale and score obtained when using a NRS scale [18].

To assess repeatability, evaluations were performed one month apart (t1 and t2) by the first observer and compared. The videos from all guinea pigs, including those which received analgesia, in different orders, were assessed twice by the same observer.

To assess reproducibility, the evaluations performed by the first 2 observers were compared. The videos from all guinea pigs, including those which received analgesia, were assessed. These comparisons were performed based on Lin's coefficient and Bland and Altman's graphical method using an Excel spreadsheet that summarized the recorded results and automatically made calculations and graphs [18,19]. Interpreting Lin's concordance correlation coefficient value was made according to Landis and Koch's classification (1977) [20].

When performing the statistical analysis for repeatability and reproducibility, information from the owner (appetite, stool production, behavior at home) was excluded to ensure the score would not vary between observers. We arbitrarily considered an item to be repeatable or reproducible if the difference in absolute value between the 2 series was less than 4, meaning within 15% of the maximal score when using Bland and Altman's graphical method.

To assess the ability of the pain scale to detect pain in guinea pigs, the correlation between the pain scores obtained with the novel scale and a numerical rating scale (NRS), used as a gold standard, was assessed. The NRS is based on a score given out of 10 (with 0 given when no pain is observed and 10 represented the most intolerable pain). To limit bias, the NRS pain scoring was performed by a third observer blinded to the study. The correlation was assessed by calculating the Spearman correlation coefficient and the Kappa coefficient [21,22]. To reduce penalization for small differences in agreement, quadratic weighting was used rather than linear weighting for the Kappa coefficient calculation [22]. The Kappa coefficient's value was interpreted according to Landis and Koch's classification (1977) [20].

Repeatability and reproducibility of specific items from the pain scale were then estimated by calculating the Kappa coefficient. This could be performed as each item was an ordinal qualitative character.

This calculation, however, was not performed for items present on the information sheet (appetite, fecal production, behavior at home) as their interpretation and value would not vary from one observer to the other. SAS software (SAS Studio 3.8, SAS Institute Inc., Cary, NC, USA) was used to calculate the Cronbach's alpha coefficient by using a SAS macro describe in [23].

## Results

### Population

Fifty-eight guinea pigs from 4 months old to 6 years old were included in this study. There were thirty-four males and twenty-four females with a mean age of 1.9 years. Twelve guinea pigs were neutered. Twenty-two were considered healthy and thirty-six had a painful condition. Healthy guinea pigs were presented for health checks or neutering. Guinea pigs with painful conditions were presented for disorders, which included dental diseases, urinary tract diseases, gastric dilation, otitis, or ovarian cysts.

### Internal consistency

The alpha was 0.77, and as it was superior to 0.7, it was deemed satisfactory. However, it was recognized that the internal consistency could be strengthened if items such as pruritus and spasmodic movements were removed from the novel pain scale. The alpha obtained without these values was 0.82. This resulted in the new maximum total score being 23 instead of 26.

### Pain score

Following the deletion of 2 items, the intervals of pain were revised. The 5 intervals were divided as no pain (for scores from 0 to 4), mild pain (for scores from 5 to 9), moderate pain (for scores from 10 to 14), severe pain (for scores from 15 to 19) and intolerable pain (for scores from 20 to 23). The median pain score recorded for all guinea pigs was 3.0, with a range of 0-15. Healthy guinea pigs presented for health checks or elective surgeries obtained a median pain score of 1.0, with a range of 0-10. The median score of guinea pigs with a painful condition who did not receive analgesia was 5.0, with a range of 0-15 (Fig. 2).

### Repeatability

Repeatability was estimated, according to Lin's concordance correlation coefficient, as being almost strong with a coefficient of 0.93 (95% CI [0.88; 0.96]) [19]. The small lack of consistency resulted from a lack of precision (94%) between the sets of measurements rather than from a lack of accuracy (6%). In this study, the bias was 0.4 (95% CI [0; 0.8]), meaning that, on average, the score was overestimated by 0.4 for series 2 (observation at t2) compared to series 1 (observation at t1) (Fig. 3). The overestimation during the second observation was not significant, as the 95% confidence interval had a value of 0. The ordinates of the limit of agreement are -2.58 (95% CI [-3.28; -1.89]) and 3.38 (95% CI [2.68; 4.08]). In this study, 95% of the measurement differences between observations at t1 and at t2 were between -2.58 and 3.38. So 95% of all measurement discrepancies were less than 4, meaning that the scale can be considered repeatable according to Bland and Altman's graphical method [18].

### Reproducibility

Reproducibility was estimated as almost strong according to Lin's concordance correlation coefficient of 0.89 (95% CI [0.82; 0.93]). The lack of consistency between the 2 observers resulted almost exclusively from a lack of precision (98%) and was not due to a lack of accuracy (2%). On the Bland and Altman graph (Fig. 4), the bias was 0.12 (95% CI [-0.4; 0.64]), meaning that, on average, the second observer overestimated the score by
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Pain score repartition: Box plot of pain scores given by the scale for guinea pigs without pain and for guinea pigs with a painful condition, before receiving analgesia. The dotted line represents the median, the upper and lower fence represents the minimum and the maximum values. The dots indicate outsiders values.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Graph for repeatability assessment: In the Bland and Altman graph, the x-coordinate of a point was the mean of the 2 measurements performed on a same individual and the y-ordinate was the difference between the 2 measurements. The red line represents the observed bias, which is the averaged differences between the 2 series of measurements. The 95% limit of agreement is represented by the 2 green lines.</figcaption></figure>

0.12 compared with the first observer. However, this overestimation was not significant as the 95% confidence interval had a value of 0. Furthermore, 95% of the measurement differences between the 2 observers were within the agreement limits, which were -3.75 (95% CI [-4.65; -2.84]) and 3.99 (95% CI [3.08; 4.9]). So 95% of the measurement discrepancies were less than 4, indicating that the scale can be considered reproducible according to Bland and Altman's graphical method [18].

### Repeatability of specific items

The consistency between observations at t1 and t2 was moderate for the items. Position, Eyelid closure, and Vibrissae orientation (Table 1). The agreement was substantial for the items Stance, Appearance of hair, eyes, and nostrils, and respiratory rate. It was almost strong for the items Respiratory pattern, and Activity/vigilance.

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Graph for reproducibility assessment: In the Bland and Altman graph, the x-coordinate of a point was the mean of the 2 measurements performed on a same individual and the y-ordinate was the difference between the 2 measurements. The red line represents the observed bias, which is the averaged differences between the 2 series of measurements. The 95% limit of agreement is represented by the 2 green lines.</figcaption></figure>
<table><caption>**Table 1**
Repeatability of the descriptors</caption><thead><tr><th>Items</th><th>Kappa coefficient<br>(quadratic weighting)</th><th>P-value</th><th>Interpretation</th></tr></thead><tbody><tr><td>Position</td><td>0.41</td><td>0.006</td><td>Moderate</td></tr><tr><td>Stance</td><td>0.62</td><td>0.001</td><td>Substantial</td></tr><tr><td>Appearance of hair, eyes,<br>and natural orifices</td><td>0.75</td><td>-</td><td>Substantial</td></tr><tr><td>Respiratory pattern</td><td>0.92</td><td>&lt;0.001</td><td>Almost perfect</td></tr><tr><td>Respiratory rate</td><td>0.59</td><td>&lt;0.001</td><td>Substantial</td></tr><tr><td>Activity / vigilance</td><td>0.82</td><td>-</td><td>Almost perfect</td></tr><tr><td>Eyelid closure</td><td>0.58</td><td>-</td><td>Moderate</td></tr><tr><td>Vibrissae orientation</td><td>0.47</td><td>-</td><td>Moderate</td></tr></tbody></table>

The *P*-value was not computable for items with a score >1 (more than 2 ordinal characters).

<table><caption>**Table 2**
Reproducibility of the descriptors</caption><thead><tr><th>Items</th><th>Kappa coefficient<br>(quadratic weighting)</th><th>P-value</th><th>Interpretation</th></tr></thead><tbody><tr><td>Position</td><td>0.41</td><td>0.020</td><td>Moderate</td></tr><tr><td>Stance</td><td>0.24</td><td>0.193</td><td>Fair</td></tr><tr><td>Appearance of hair, eyes,<br>and natural orifices</td><td>0.70</td><td>-</td><td>Substantial</td></tr><tr><td>Respiratory pattern</td><td>0.91</td><td>&lt;0.001</td><td>Almost perfect</td></tr><tr><td>Respiratory rate</td><td>0.39</td><td>0.004</td><td>Fair</td></tr><tr><td>Activity / vigilance</td><td>0.73</td><td>-</td><td>Substantial</td></tr><tr><td>Eyelid closure</td><td>0.59</td><td>-</td><td>Moderate</td></tr><tr><td>Vibrissae orientation</td><td>0.45</td><td>-</td><td>Moderate</td></tr></tbody></table>

The *P*-value was not computable for items with a score >1 (more than 2 ordinal characters).

## Reproducibility specifics of items

The agreement between the 2 observers was fair for the items Stance and Respiratory rate (Table 2). It was average for the items Position, Eyelid closure and Vibrissae orientation. The agreement was substantial for the items Appearance of hair, eyes, and natural nostrils, and Activity/vigilance and almost strong for the item Respiratory pattern.

## Validity

The correlation between the score given by the NRS and the guinea pig pain scale was correct ($\rho_0 = 0.67; P < 0.001$) (Fig. 5). The Kappa coefficient was 0.72. This value corresponds to a good agreement

between the pain intensities obtained by the scale and the gold standard method.

The sensitivity of the scale was 52.0%, and the specificity was 87.5%.

## Discussion

The aim of this study was to develop and validate a pain assessment scale for guinea pigs. The pain scale was developed based on observable pain signs in guinea pigs as well as those previously described in the literature. This scale is intended for use on pet guinea pigs in a clinical setting. Statistical analysis based on evaluating the videos indicated almost strong repeatability and inter-operator reproducibility. Agreement regarding pain intensities obtained when comparing our pain score to the score given by a numerical rating scale was good. However, items as Stance, Respiratory rate and appearance of hair, eyes, and nostrils had poor repeatability and/or reproducibility, highlighting that changes must be made before fully validating this scale.

Elements present on the information sheet (appetite, stool production, behavior at home) were commonly completed before the animal received any analgesia. The pre-filled information could have led to inconsistencies with the pain felt by the animal, as videos sometimes took place after analgesia administration. However, these possible inconsistencies were negligible when evaluating the scale's repeatability and reproducibility.

The intensity of pain perceived is patient-dependent and can be highly variable between patients suffering from the same condition. Pain perceived by an individual depends on factors such as the individual's experience with pain, concomitant presence of other conditions, age, and sex [24]. Using a numerical rating scale as a gold standard method allowed to overcome this bias, as results did not depend on the condition of each animal.

As for limitations, a major concern was the lack of established pain behavior and pain scale in guinea pigs. A grimace scale is not yet available in guinea pigs, although it has been adapted for many other species. Cohen *et al.* (2020) even suggested that grimace score is not a good tool to evaluate pain in guinea pigs [25]. However, since NRS is a subjective assessment based on individual's appearance, the reliability of this method as a gold standard may be questioned. A study using precise measurement software would be required, as performed for the development of grimace scales in rats and mice to analyze guinea pigs' specific facial expressions when experiencing pain [26,27]. Another limitation was the small number of guinea pigs recruited, and especially the lack of animals with intolerable pain, preventing all pain intensities from being assessed with our scale. In fact, when a guinea pig seemed truly painful, the clinician's first reflex was to provide analgesia to the animal before filming.

The observers were blinded to the condition of the animal as the videos were presented in a random order, anonymized and analyzed several

<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Scatter chart of the pain score: Scatter chart of the pain scores given by the guinea pig pain scale correlated to the pain scores obtained with the numerical rating scale (NRS). The dotted line represents the linear regression line. This graph shows a positive correlation between the 2 methods.</figcaption></figure>
weeks after the data was collected. But, as the two first observers collected data, they could have remembered some animals.

Moreover, observers with different experiences were selected to ensure that this scale could be performed by veterinarians with different experiences, including specialists, general practitioners and new graduates. However, the difference in experience could have resulted in the lack of reproducibility seen in this study. Some authors argue that good knowledge of the species, especially its appearance and behaviors, is essential for pain assessment [28]. It would be interesting to re-evaluate reproducibility with 2 individuals having the same experience or to add further descriptions for some items to reduce observer subjectivity. Items such as Position and Stance, which had unsatisfactory repeatability and reproducibility could benefit from picture illustrations, as was done by Weaver et al. (2010) to enable people with various levels of experience to have a reference [29]. This would enable the observer to be more objective when assigning scores. Adding pictures could, therefore, let this scale be used by a broader audience, including nurses and non-veterinarian evaluators. Although this scale aimed to be used in a clinical setting, it would be interesting to test its validity with owners to ensure it is understandable by all. Observation by owners using this scale would let them test the repeatability and reproducibility of all items and highlight items requiring longer observation, especially items such as activity, interactions, appetite, and fecal production. To make it available to a broad public, the creation of a mobile phone application would allow easier use and would permit data collection for a standardized experimental study as well as collecting users' opinions.

Although the specificity of this scale was 87.5% and good, its sensitivity was not satisfactory at 52.0%. Thus, the scale was not good at indicating pain when a guinea pig was actually painful, so this scale is not ideal. However, it was good at excluding pain in non-painful animals.

In conclusion, this is the first step towards a standardized assessment of pain in guinea pigs. However, improvements are needed to obtain a scale that can be used in a clinical context to allow better management of pain in this species.

## Financial disclosure

None reported.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## CRediT authorship contribution statement

**Floriane Benedetti:** Writing – review & editing, Writing – original draft, Resources, Methodology, Investigation, Data curation, Conceptualization. **Charly Pignon:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Conceptualization. **Patricia Muffat-es-Jacques:** Writing – review & editing, Writing – original draft, Investigation, Data curation. **Caroline Gilbert:** Writing – original draft, Validation, Supervision. **Loïc Desquilbet:** Writing – review & editing, Software.

## Acknowledgments

The authors would like to thank the students and nursing staff who contributed to this study, in particular Delphine Lehuédé for data collection and her collaboration during the whole study and Camille Lapous for video analyses.

## Formatting of funding sources

This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.

## References

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