The authors have declared that no competing interests exist. The commercial organisation Harlan UK donated the animals used in the study. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: MCL AAT PAF. Performed the experiments: MCL AAT MCL. Analyzed the data: MCL SCJK. Wrote, read, edited and approved the final manuscript: SCJK AAT PAF MCL.
Ear tattooing is a routine procedure performed on laboratory, commercial and companion rabbits for the purpose of identification. Although this procedure is potentially painful, it is usually performed without the provision of analgesia, so compromising animal welfare. Furthermore, current means to assess pain in rabbits are poor and more reliable methods are required. The objectives of this study were to assess the physiological and behavioural effects of ear tattooing on rabbits, evaluate the analgesic efficacy of topical local anaesthetic cream application prior to this procedure, and to develop a scale to assess pain in rabbits based on changes in facial expression.
In a crossover study, eight New Zealand White rabbits each underwent four different treatments of actual or sham ear tattooing, with and without prior application of a topical local anaesthetic (lidocaine/prilocaine). Changes in immediate behaviour, heart rate, arterial blood pressure, serum corticosterone concentrations, facial expression and home pen behaviours were assessed. Changes in facial expression were examined to develop the Rabbit Grimace Scale in order to assess acute pain. Tattooing without EMLA cream resulted in significantly greater struggling behaviour and vocalisation, greater facial expression scores of pain, higher peak heart rate, as well as higher systolic and mean arterial blood pressure compared to all other treatments. Physiological and behavioural changes following tattooing with EMLA cream were similar to those in animals receiving sham tattoos with or without EMLA cream. Behavioural changes 1 hour post-treatment were minimal with no pain behaviours identifiable in any group. Serum corticosterone responses did not differ between sham and tattoo treatments.
Ear tattooing causes transient and potentially severe pain in rabbits, which is almost completely prevented by prior application of local anaesthetic cream. The Rabbit Grimace Scale developed appears to be a reliable and accurate way to assess acute pain in rabbits.
The marking of animals for identification is one of the most common procedures carried out on laboratory, farm and some pet animal species. Although a wide range of methods are available and routinely used we know little about the effect of these on the animals being marked. Being able to clearly identify individuals is a fundamental and often a legal requirement for many species, particularly for those undergoing treatments or procedures, breeding animals, and those being transported, sold or shown. While a number of identification methods are available for use in rabbits
EMLA (Eutectic Mixture of Local Anesthetics) cream is a eutectic mixture of 2.5% prilocaine and 2.5% lidocaine that can easily be applied to provide topical anaesthesia. The use of EMLA cream has been used in a range of species to reduce or prevent pain associated with intravenous and arterial catheter placement, vaccination, biopsies, skin graft removal, and even minor surgical procedures with minimal side effects
In order to evaluate both the potential negative effects associated with tattooing and the positive influence of EMLA administration, we must be able to recognise pain effectively and assess its severity. Unfortunately, validated methods of assessing the pain associated with tattooing and other routine husbandry and veterinary procedures are lacking for many species including rabbits and our ability to recognise pain is often poor
The inherent limitations of conventional pain assessment methods have prompted the search for reliable cage-side indicators of pain. Changes in facial expression have recently been evaluated in the mouse and rat and have proven to be an accurate, repeatable and valid way to identify pain in these species
The objectives of the current study were to assess the physiologic and behavioural impact of ear tattooing on rabbits, evaluate the analgesic efficacy of EMLA cream during this procedure and to develop a pain scale based on facial expressions in rabbits.
Treatment and time were the only factors to significantly influence the parameters measured. Treatment order had no significant influence on any measure.
No vocalisation or struggling was observed when the animals underwent the sham tattooing with or without prior application of EMLA. One rabbit in the group that was tattooed after application of EMLA exhibited vocalisation but no struggling was observed in any animals in this group. Considerable and significantly greater struggling and vocalisation was observed in animals tattooed without prior application of EMLA (P = 0.000), with all animals in this group vocalising and struggling violently to escape restraint for up to 10 seconds during the tattoo procedure.
Treatment had a significant effect on the pre to post procedure change in peak heart rate (P = 0.002). A significantly greater increase from pre to post procedure was observed with tattooing without prior application of EMLA compared to tattooing with prior application of EMLA and sham tattooing with and without prior application of EMLA (P = 0.022, P = 0.031, P = 0.004 respectively), with no further differences between the treatments (see
Change in mean heart rate (beats per minute ±1 SE) from pre to post-procedure observed in rabbits (y-axis) when undergoing the various treatments (x-axis); sham tattooing with and without prior application of EMLA and tattooing with and without prior application of EMLA (n = 8 per treatment) (★P = 0.022, ★★P = 0.031, ★★★P = 0.004).
Treatment had a significant effect on the pre to post procedure change in peak SAP (P = 0.001). A significantly greater increase from pre to post procedure was observed with tattooing without prior application of EMLA compared to tattooing with prior application of EMLA and sham tattooing with and without prior application of EMLA (P = 0.024, P = 0.028, P = 0.003 respectively), with no further significant differences between the treatments (see
Change in mean systolic arterial pressure (mmHg ±1 SE) from pre to post-procedure observed in rabbits (y-axis) when undergoing the various treatments (x-axis); sham tattooing with and without prior application of EMLA and tattooing with and without prior application of EMLA (n = 8 per treatment) (★P = 0.024, ★★P = 0.028, ★★★P = 0.003).
Treatment had a significant effect on the pre to post procedure change in peak MAP (P = 0.004). A significantly greater increase from pre to post procedure was observed with tattooing without prior application of EMLA compared to tattooing with prior application of EMLA and sham tattooing with and without prior application of EMLA (P = 0.05, P = 0.035, P = 0.01 respectively), with no further significant differences between the treatments (see
Change in mean arterial pressure (mmHg ±1SE) from pre to post-procedure observed in rabbits (y-axis) when undergoing the various treatments (x-axis); sham tattooing with and without prior application of EMLA and tattooing with and without prior application of EMLA (n = 8 per treatment) (★P = 0.05, ★★P = 0.035, ★★★P = 0.01).
Treatment did not have a significant effect on the pre to post procedure change in peak, lowest or area-under-the-curve for DAP.
Time and a time*treatment interaction had significant effects on corticosterone concentration (P<0.001, P<0.001 respectively). Treatment alone did not significantly influence corticosterone concentration. The corticosterone concentration was significantly higher compared to baseline at 15 and 30 minutes post-procedure for sham tattooing without prior application of EMLA (P = 0.002, P = 0.02 respectively), tattooing without prior application of EMLA (P = 0.001, P = 0.02 respectively) and tattooing with prior application of EMLA (P = 0.000, P = 0.02 respectively). For sham tattooing with prior application of EMLA corticosterone concentration was significantly higher compared to baseline at 15 minutes post-procedure (P = 0.01) but not at 30 minutes post procedure. Corticosterone concentration was not significantly different to baseline at 1 hour, 2 hours or 3 hours post-procedure for any of the treatment groups (see
Mean corticosterone concentration (ng/ml ±1SE) observed in rabbits (y-axis) at baseline and various post-procedure time points (x-axis) when undergoing the various treatments; sham tattooing with and without prior application of EMLA and tattooing with and without prior application of EMLA (n = 8 per treatment) (Comparison to baseline: ★P<0.05, ★★P<0.01, ★★★P<0.001, Comparison between treatments: +P = 0.05, ++P = 0.01).
The change in corticosterone concentration from baseline was significantly influenced by treatment at 15 minutes post-procedure (P = 0.05), but not at 30 minutes, 1 hour, 2 hours or 3 hours post-procedure. At 15 minutes post-procedure, the change in corticosterone concentration from baseline was significantly greater for tattooing with and without prior application of EMLA compared to sham tattoo with prior application of EMLA (P = 0.05, P = 0.01 respectively), with no significant differences between the remaining treatments. For area-under-curve (AUC) there was no significant effect of treatment (P = 0.81).
Treatment and a time*treatment interaction had significant effects on RbtGS score (P = 0.004, P = 0.001, respectively). Time alone did not significantly influence RbtGS score. There was no significant difference between the treatments in the pre-procedure period. Intra-procedure, RbtGS score was significantly higher when animals underwent tattooing without prior application of EMLA compared to tattooing with prior application of EMLA and sham tattooing with and without prior application of EMLA (P = 0.001, P = 0.018, P = 0.002 respectively), with no other significant differences between treatments (see
Mean RbtGS scores (±1SE) observed in rabbits (y-axis) at baseline and intra-procedure (x-axis) when undergoing the various treatments; sham tattooing with and without prior application of EMLA and tattooing with and without prior application of EMLA (n = 8 per treatment) (★P = 0.018, ★★P = 0.002, ★★★P = 0.001, ★★★★P = 0.000).
Example images and associated RbtGS scores of a rabbit in the pre-tattoo (a) and intra-tattoo (b) periods without prior application of EMLA and of a rabbit in the pre-sham tattoo (c) and intra-sham tattoo (d) periods without prior application of EMLA.
The average accuracy of global pain assessment was 83.6%, with misses being slightly more prevalent (10.6%) than false positives (5.8%). Individual accuracy varied from 62.5% to 86.5%. The Rabbit Grimace Scale also demonstrated high inter-rater reliability with an overall intraclass correlation coefficient (ICC) value of 0.91. All of the comprising facial action units also showed high ICC values; 0.94 for orbital tightening, 0.86 for cheek flattening and 0.84 for pointed nose.
Treatment had a significant effect on the duration of grooming (P = 0.034) and movement around the home pen (P = 0.026), and the frequency of rearing (P = 0.015). Treatment showed no significant influence on any of the other behaviours scored including previously validated pain behaviours (flinch, twitch, etc.
At 1-hour post-treatment, the duration of grooming the head and ears was significantly longer than baseline following the sham tattooing with and without prior application of EMLA (P = 0.03 respectively), and tattooing without prior application of EMLA (P = 0.05), but not with the tattooing with prior application of EMLA. There were no significant differences between the treatment groups at baseline or 1 h post-treatment (see
Behaviour | Baseline | Sham tattoo withoutEMLA | Sham tattoo with EMLA | Tattoo without EMLA | Tattoo with EMLA |
Groom | 50.0±29.7 | 220.5±40.8 | 179.1±51.7 | 242.6±70.9 | 136.3±63.6 |
Movement | 219.5±62.0 | 43.4±12.5 | 55.8±26.8 | 29.6±3.8 | 58.3±40.9 |
Rear | 8.1±2.4 | 1.0±0.4 | 1.8±0.7 | 0.5±0.1 | 0.6±0.2 |
At 1-hour post-treatment, the duration of movements were significantly shorter than baseline following the sham tattooing with and without prior application of EMLA (P = 0.01, P = 0.038 respectively), and tattooing with and without prior application of EMLA (P = 0.013, P = 0.05 respectively) (see
At 1-hour post-treatment, the frequency of rearing was significantly lower than baseline following sham tattooing with and without prior application of EMLA (P = 0.03 respectively), and tattooing with and without prior application of EMLA (P = 0.013, P = 0.012 respectively) (see
Application of a needle tattoo device without prior application of EMLA cream caused clear physiologic and behavioural changes during and immediately following the procedure, which were absent following sham tattoo application. Consistent with previous findings, acute increases in heart rate and blood pressure occurred indicating a short period of intense pain
Beyond the immediate post-procedure period, home pen behaviours and corticosterone responses were similar irrespective of whether the animal underwent a tattoo or sham procedure with or without EMLA cream. Pain may be expressed through general behaviours, such as decreases in activity
Changes in serum corticosterone also provide evidence of the transient nature of the pain and stress response. While concentrations were initially higher than baseline at 15 to 30 minutes post-procedure, corticosterone concentrations declined rapidly to return to baseline between 30 minutes to 1 hour post-procedure with all treatments. EMLA cream application had minimal effect on corticosterone concentrations suggesting that tattooing does not induce greater changes than those caused by handling and restraint alone, which are integral to both the tattoo and sham tattoo procedures. Although, application of a needle tattoo was associated with greater increase in corticosterone serum concentrations initially (15 minutes post-procedure) compared to the sham procedure, this effect did not persistent beyond 30 minutes post-procedure. These changes are not unexpected as changes in stress hormones are routinely observed in animals undergoing other potentially stressful procedures such as handling and introduction to novel environments
Evaluation of facial expression is a relatively recently developed means of assessing pain in animals. The growing popularity of this method is likely due to our previously poor ability to recognise pain in many species
Evaluation of the RbtGS scores assigned by blinded evaluators determined that the overall accuracy is lower than that of the MGS (97%)
EMLA cream is the most widely used topical anaesthetic, with demonstrated analgesic efficacy in superficial procedures, as well more invasive procedures such as the excision of subcutaneous lesions and skin graft removal
The ability of treatment-blinded evaluators to identify pain based on facial expressions in rabbits is in contrast to findings from a previous study using behaviour, which found that evaluators were less successful in correctly identifying pain in rabbits that had undergone ovariohysterectomy the more time they spent looking at the face
The findings from the current study clearly demonstrate the pain and distress caused by ear tattooing without analgesia, and that prior application of EMLA cream is effective in preventing almost all pain associated with this procedure. EMLA cream application can be considered a safe, effective, accessible method of analgesia that eliminates the need for general anaesthesia and can be used in non-veterinary environments. The Rabbit Grimace Scale has also been shown to correctly identify rabbits experiencing acute pain and may be a helpful tool when evaluating other husbandry or experimental procedures.
All procedures were carried out under project and personal licences approved by the Secretary of State for the Home Office, under the United Kingdom’s 1986 Animals (Scientific Procedures) Act and the Local Ethical Review Committee at Newcastle University. This study employed a strict ‘rescue’ analgesia policy. If any animal was deemed to be in greater then mild pain (assessed by an independent veterinarian), then buprenorphine (0.01 mg/kg
Eight barrier-reared New Zealand White Rabbits (4 male and 4 female) weighing 2–3 kg were obtained from a commercial supplier (Harlan, UK Ltd, Bicester, UK). Rabbits were housed singly in floor pens within sight, sound and touch of animals in adjacent pens, with males and females being housed in separate rooms. The rabbits were housed singly throughout the study to enable video footage and still images to be obtained and to prevent changes in behaviour or facial expressions resulting from transient separation from their cage mates. The animal room was maintained at 22±2°C, 50±10% humidity and on 12/12 h light/dark cycle. Food (Rabma pellets, SDS Ltd, Essex, UK) and tap water were provided
A crossover design was employed, with the research team involved in data collection and application of the tattooing device being blinded to the treatments. Each rabbit underwent 4 treatments in semi-randomised order undergoing either an ear tattoo or a sham procedure, with either prior application of aqueous cream or EMLA cream. The tattoo was applied using handheld horizontal tattooing pliers (Haptner-Herberholz, GmBh and Co, Germany) loaded with 4×5 mm numbers, each consisting of 9 sharp pins of approximately 1 mm diameter that penetrated the full thickness of the ear, while the sham procedure consisted of application of the same device with a flat, unloaded plate. The rabbits underwent both tattoo treatments 7 days after the sham procedures were applied. This was to avoid hyperalgesia or allodynia, which could have been caused by tattooing, altering the responses to the sham procedure. Within each treatment block (sham or real tattoo) the sequence of prior application of EMLA or aqueous cream application and specific ear treated were randomized ensuring different ears were used for tattoo application with and without EMLA cream. A minimum ‘washout’ period of 2 days was implemented between treatment applications for each rabbit in each block. The only procedures that differed between the treatments was the application of a tattoo or not and the presence/absence of EMLA cream. All of the other common procedures (handling, restraint, catheterisation etc.) conducted on the rabbits in order to carry out the treatments remained constant.
Twenty minutes prior to a treatment, each rabbit was picked up and gently restrained in its home pen and a 1 mm thick layer of topical local anaesthetic cream (EMLA, Astra AstraZeneca, Luton, UK) or non-anaesthetic aqueous cream (E45 cream, Reckitt Benckiser Healthcare UK Ltd, UK) was applied to both inner and outer surfaces of the ear that was to be tattooed or handled (test ear) and covered with an occlusive dressing. EMLA cream was applied to the other ear, to prevent pain during vessel catheterisation. Application of either cream was carried out by a member of the research team who was not involved in any of the animal assessments to ensure that all other procedures were undertaken by treatment-blinded observers. After 15 minutes each animal was transferred to a procedure room, placed onto a non-slip table and gently restrained, the bandages and cream removed from the ears and 22G “over-the-needle” catheters (Abbocath, Abbott, Maidenhead, UK) placed in the ear vein and artery of the non-treated ear for blood sampling and direct measurement of arterial pressure. Adhesive ECG electrodes were attached to the forelimbs and left hindlimb. Rabbits were connected to a multi-parameter monitor (Kontron UK, Chichester, UK) to allow recording of the ECG and arterial blood pressure. Three high definition video cameras (Sony High Definition HandyCam model HDR-XR155, Sony, Japan) were positioned to allow recording of the monitor screen, the rabbit face and head, and the behavioural responses of the rabbit. Five minutes after being brought into the procedure room each rabbit underwent one of the 4 treatments: either ear tattooing or the sham procedure, with prior application of either aqueous cream or EMLA cream. The same operator carried out all treatments in all animals with the tattoo pliers being applied for 3 seconds in all treatments. Following each procedure the rabbit was then returned to its home pen for further data collection. No intra-procedure complications were reported and all rabbits recovered uneventfully.
The immediate physiological response to the treatments was assessed by recording systolic, diastolic and mean blood pressure (mmHg) every 10 seconds for 120 seconds before and after treatment administration using a multi-parameter monitor (Kontron UK, Chichester, UK). The immediate behavioural reaction to the treatments was assessed by recording the frequency of struggling and vocalisation observed during the application of each treatment The assessment was made by a treatment blind observer from the video recordings taken using an ethogram of rabbit behaviour (see
Behaviour | Description |
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|
Struggle | Struggling against gentle restraint during handling and procedures |
Vocalisation | Emitting a audible sound during handling and procedures |
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|
Unaffected ear | The ear that has not undergone a treatment |
Affected ear | The ear that has undergone a treatment |
Head/face | Head or face using front or hind feet |
Body & limbs | Any other part of the body by licking or scratching |
Unknown | Unable to identify where due to rabbits position |
Groom overall | All of the above behaviours |
|
|
Movement | Movement around the pen by hopping, jumping or walking |
Out of sight | Not visible due to hiding behind or under objects in pen |
Head shake | Shaking the head from side-to-side |
Body shake | Shaking of entire body from side-to-side |
Object interact | Interaction with objects in the floor pen |
Digging | Digging in the pen substrate |
Eat/Drink | Eating and/or drinking |
Stretch | Stretching of the body in various ways |
Other | Behaviour not listed in ethogram |
Inactive | Time spent completely motionless and inactive |
|
|
Twitching | Rapid movement of fur on back |
Flinching | Body jerks upwards for no apparent reason. |
Wincing | Rapid movement of the backwards in a rocking motion accompanied by eye closing and swallowing action |
Staggering | Partial loss of balance |
Falling | Complete loss of balance when moving |
Pressing | Abdomen pushed towards floor, usually before walking |
Pain | Undefined behaviour (not observed in any other individual) that is believed to indicate pain |
Arching | Full arching of the back upwards |
Writhing | Contraction of the oblique flank muscles |
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|
Flat | Ears flat on the back. |
Erect | Ears erect and pointing in a specific direction. |
Relaxed | Ears positioned between ‘flat’ and ‘alert’ |
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|
Open | Eyes wide open |
Closed | Eyes tightly closed |
Slit | Eyes semi-closed |
Not visible | Eyes not visible due to rabbits position |
|
|
Crouch/sit | Sitting relaxed with hind limbs tucked under the rump and fore limbs underneath |
Lie down | Lying down either with legs tucked under body or on right/left side of body |
Rear | Rearing up with placement of fore limbs with or without support the wall of the pen or other object |
Stand | Standing up on all four feet with abdomen off the floor |
Plasma corticosterone concentrations were determined from blood samples taken the day prior to the study beginning (baseline) by venepuncture and at 15 minutes, 30 minutes, 1 hour, 2 hours and 3 hours post-treatment from the catheter in the non-treated ear. The venepuncture for baseline blood sampling involved each rabbit being picked up and gently restrained by an assistant. Each sample was taken within 3 minutes of the operators entering the floor pen. The corticosterone concentrations were assessed using a competitive enzyme-immunoassay (EIA) kit (IDS Plc, Boldon, UK).
Home cage behaviour was assessed from video recordings taken in the home pen. Fifteen-minute recordings were made on the day prior to treatment (baseline) and then at 1 hour post-treatment using a High definition video camera (Sony High Definition HandyCam model HDR-XR155, Sony, Japan). The camera was placed at a fixed distance from the front of the pen. This arrangement gave the highest probability of capturing the behaviour and face of the rabbit during filming. The behaviour observed in each video sequence (10 min epoch: same start time used for all clips) was scored using Observer XT (Version 9: Noldus Information Technology, Wageningen, Netherlands) according to an ethogram developed for assessing post ovariohysterectomy pain in rabbits
The RbtGS was developed using the methods developed by Langford et al.
The Rabbit Grimace Scale with images and explanations for each of the 5 facial action units (FAU); orbital tightening, cheek flattening, nose shape, whisker position and ear position. Each FAU is scored according to whether it is not present (score of 0), moderately present (score of 1) and obliviously present (score of 2).
In order to explore the effect of time (pre vs. intra procedure) and treatment (sham tattoo vs. real tattoo with and without EMLA), the mean RbtGS scores were calculated for each image across all participants. The Rabbit Grimace Scale score for each image was determined by summing the score provided for each FAU with the exception of whisker position. Whisker position was excluded, as the majority of assessors were unable to score whisker position for many of the images, as they were not of high enough quality for whisker position to be clearly seen.
A total of 10 observers participated in this study and were recruited and tested in 2011 at Newcastle University. The observers were from diverse backgrounds and included veterinary surgeons, veterinary nurses, research scientists, animal technicians, psychology students and non-animal related occupations.
All statistical analyses were conducted using SPSS 18 (SPSS Inc., Chicago, USA). The data were normally distributed with homogeneity of variance, so parametric analyses were carried out. Differences were considered to be statistically significant if P<0.05. A Chi-square analysis was used to compare the number of rabbits struggling and vocalising during the application of the various treatments. Repeated measures General Linear Models (GLM) were used to analyse the rabbit grimace scale and serum corticosterone concentration data with the time points (pre, intra & post-procedure), treatment group and treatment order as the within-subjects factors. Any time*treatment interactions were further investigated using repeated measures analysis of variance. In addition for the rabbit grimace scale, the change from pre to post procedure for each treatment group was calculated and compared to zero using a 1-sample t-test. The reliability of the scale was determined using inter-class correlation coefficient to compare mean scores for each of the facial action units across all of the participants. Accuracy was determined by comparing the global pain or no pain judgement made by the participants with actual pain state of the rabbit in each photograph (e.g. sham or actual tattoo with or without EMLA) as scored by treatment and session blind observer (MCL) with considerable experience in scoring post-procedural pain in rabbits using RbtGS and other measures. Repeated measures ANOVAs were used to analyse the change from pre to post-procedure and the area-under-curve for systolic arterial pressure (SAP), mean arterial pressure (MAP) and diastolic arterial pressure (DAP) and home case behaviour with treatment as the within subjects factor. All post-hoc analysis for the repeated measures ANOVA and GLM was conducted using a comparison of main effects with a Bonferroni interval adjustment.
The authors would like to thank Mr Jon Gledhill for assistance with video recordings and image processing, as well as Ms Caroline Fox, Ms Denise Reed and Ms Emma Gilmore for their technical assistance with treatment application and blood sampling.