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1 Applied Animal Behaviour Science xxx (2005) xxx–xxx

www.elsevier.com/locate/applanim

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3 Training in behaviour-based post-operative pain
4 scoring in rats—An evaluation based on improved
recognition of analgesic requirements

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John V. Roughan *, Paul A. Flecknell
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7 Comparative Biology Centre, University of Newcastle Upon Tyne, Framlington Place,
8 Newcastle Upon Tyne NE2 4HH, UK

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9 Accepted 24 June 2005
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12

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Abstract
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The objective of the study was to evaluate whether an objective technique based on quantitative
15
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16 behaviour analysis represents a methodological improvement upon a simple subjective approach for
17 assessing post-operative pain in rats. Data were collected during three Phases from 332 treatment-
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18 blinded volunteer observers with varying prior experience of animal husbandry and care. Aside from
19 Phase 3, initially, they each used the subjective approach (visual analogue scale, VAS) to estimate
20 pain severity after observing four randomly presented 5 min videotaped clips of the behaviour of rats.
21 The rats had each undergone surgery (lower abdominal laparotomy) for an unrelated study, and the
22 four clips corresponded to individual rats given saline or one of three dosages of meloxicam (0.5, 1, or
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23 2 mg/kg) 1 h prior to surgery. Following VAS assessment, the volunteers received approximately
24 10 min of training in behaviour scoring. They were then asked to re-observe the four clips and score
25 the occurrence of behaviour with the clips presented in another random sequence. The data were
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26 collated for each Phase of the exercise and a measure of success was calculated for scores obtained
27 with each assessment method (VAS and behaviour). Success or failure relied upon each volunteer’s
28 ability to differentiate between the rats according to the treatment received. Success rates with VAS
29 scoring were considerably more varied than with behaviour scoring, and although some groups were
30 quite accomplished with VAS scoring, success rates with behaviour scoring were even higher in these
31 groups. Irrespective or prior experience of rat or other laboratory animal husbandry, behaviour
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32 scoring was overall superior for differentiating between the effects of analgesic treatment. Overall
33 success rate for differentiating between treatments by subjective (VAS) assessment was 54%,

* Corresponding author. Tel.: +44 191 2226715; fax: +44 191 2228688.
E-mail address: J.V.Roughan@newcastle.ac.uk (J.V. Roughan).
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0168-1591/$ – see front matter # 2005 Published by Elsevier B.V.

doi:10.1016/j.applanim.2005.06.012

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34 compared with 75% with behaviour scoring. We concluded that this technique of behaviour scoring
35 represents a significant methodological improvement that can be rapidly learned by inexperienced
36 staff and used to recognise and assess pain following laparotomy in rats.
37 # 2005 Published by Elsevier B.V.

38 Keywords: Rat; Pain; Behaviour; Training; Surgery

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1. Introduction
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42 It is generally accepted that surgical procedures that would be expected to cause pain in

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43 people are likely to cause pain in animals. Despite this, in a recent survey of post-operative
44 analgesic use in laboratory animals (Richardson and Flecknell, 2005) some respondents
45 commented that analgesics were unnecessary, in some case even after major surgery,
46 because the animals showed no signs of pain. From comments made in a number of
47 individual published articles (Grau and Steiniger, 2003; Labat et al., 2002; Lawson et al.,
2001), we suggest that poor ability to recognise pain and the uncertainties that then arise

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49 concerning the need for pain relief are main reasons that analgesics are often withheld from
50 laboratory rodents. This problem is not unique to laboratory animals and a similar lack of
51 analgesic treatment and lack of pain assessment techniques has been noted in farm and
52 companion animals (Capner and Lascelles, 1999; Hugonnard et al., 2004; Lascelles et al.,
53 1999). In addition to the need to recognise the presence of pain reliably, assessing the
54 intensity of that pain is essential for evaluating the efficacy of any intervention therapy.
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55 Although, few practically useful pain assessment techniques are currently available for any
56 species, studies in companion animals (Al-Gizawiy and Rude, 2004; Firth and Haldane,
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57 1999; Hardie et al., 1997), farm animals (Kent et al., 2004; Molony et al., 2002) and
58 laboratory rats (Affaitati et al., 2002; Giamberardino et al., 2000; Gonzalez et al., 2000;
59 Roughan and Flecknell, 2001, 2003, 2004) have indicated that objective pain scoring
60 systems can be developed and can be successfully applied.
61 A potential problem in building acceptance of newer behaviour-based techniques is
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62 the widespread assumption that those working with animals are already able to recognise
63 the presence of pain and to assess its severity. A survey of current practice in recognising
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64 pain, suffering and distress noted that the majority of research establishments used
65 subjective assessment methods or scoring systems that have not been validated
66 (Hawkins, 2002). The inadequacies of subjective pain scoring techniques have been
67 noted in several independent studies (Conzemius et al., 1997; Holton et al., 1998; Reese
68 et al., 2000), yet most of the interviewees in the Hawkins survey were confident that
69 pain, suffering and distress were being detected effectively within their own research
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70 establishment.
71 We have developed an objective (behaviour-based) system for scoring pain after
72 laparotomy in rats (Roughan and Flecknell, 2001) and shown that scoring can be carried
73 out rapidly (Roughan and Flecknell, 2003) and can be used to assess the duration of pain
74 and the relative efficacy of different analgesic regimens (Roughan and Flecknell, 2004). To
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75 evaluate the practicability of this scoring technique, we investigated whether individuals

76 could be taught to score behaviour in a short space of time. We tested the hypothesis that

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77 this approach could increase the proportion of animal carers that are able to identify
78 animals that may require analgesics, or that need additional treatments, than could be
79 achieved with a simple subjective assessment.

2. Materials and methods

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81 The exercise used video-recordings of the post-operative behaviour of young Fischer 344
82 rats (50 g) obtained during a previous investigation (Roughan and Flecknell, 2003). This
83 original study did not involve additional animal use, as the rats underwent surgery for an
84 unrelated tumour growth study. Details of the husbandry, anaesthesia, and surgery are given

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85 elsewhere (Roughan and Flecknell, 2003). Briefly, each rat received a subcutaneous injection
86 0.2 mls/100 g of saline (Sal) or meloxicam at 0.5, 1 or 2 mg/kg (M0.5, M1, M2) 1 h before
87 surgery (a 1 cm laparotomy followed by injection of 0.2 mls of a tumour cell suspension into
88 the bladder wall). It should be noted that use of saline controls raised ethical concerns;
89 however, these were necessary to determine whether drug treatment would influence tumour
growth (the main aim of the primary study). No such effects were observed. The rats were

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91 allowed to fully recover from isoflurane anaesthesia (20–30 min) in an incubator; until
92 capable of exploratory behaviour without any signs of ataxia. Ten minutes later, each rat was
93 placed into a separate clear polycarbonate cage and filmed with a digital video camera for
94 10 min. In the original study, this was repeated three times at 2 h intervals.
95 Following behaviour analysis by the behaviour score system developer (JVR), four 5 min
sequences (clips) of behaviour from the immediate post-operative period were selected for
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97 observation by groups of volunteers. Each clip corresponded to one rat that had received one
98 of the four treatments (saline or meloxicam). Clip selection was random, except that
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99 recordings when behaviours were obscured or the camera poorly focused were excluded.
100 TheprojecthadthreePhases.Phase1wasanexercisetoexplorewhethervolunteerobservers
101 at the host institution (CBC) could be successfully instructed in behaviour-based pain scoring.
102 DuringPhase 2, volunteers were a diverse mix of technicians, veterinary surgeons and research
103 workerswhoobservedtheclipsaspartofaseriesofworkshopsonpainassessmentundertakenin
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104 the UK and elsewhere. Workshop locations included conferences (2), pharmaceutical
105 companies (4), and universities (2). Phase 3 also included technicians, researchers and
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106 veterinary surgeons, but the participating groups (2) had extensive prior experience in rat
107 husbandry and career emphasis on improving the welfare of laboratory animals.
108 For Phases 1 and 2, the procedure for testing the volunteers was virtually the same. The
109 VAS and behaviour data were collected in the following sequence; obtain VAS scores for
110 each clip, and following a short period of instruction on recognising the relevant pain-
related behaviours, re-test for ability to recognise behavioural differences between the rats
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112 shown in each of the four clips.

2.1. Phase 1 (pilot study)

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114 Eighteen volunteers from CBC staff were divided into three categories to reflect prior
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115 experience of rat husbandry, or more general experience of caring for rats or other
laboratory animals: Group 1 were animal technicians (n = 11), Group 2 were research

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117 workers (n = 4), and Group 3 were clerical/administrative staff (n = 3). All of the
118 technicians had several years of experience with assessing the welfare implications of
119 surgical procedures involving rats. The research workers had less experience, but each had
120 been involved with projects requiring basic rat husbandry. The administrative staff had no
121 experience of rat or other laboratory animal husbandry.
122 Each volunteer observed the four 5 min sequences of behaviour alone and undisturbed.

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123 They were aware that the rats had undergone surgery, but not the treatment that each rat had
124 received. The four clips were presented in random order and the category of observer called
125 to observe was also random, but only insofar as was permitted by the daily work schedule.
126 Following observation of each clip, each volunteer used a visual analogue scale to
127 estimate the extent of any pain they thought the rat shown might be experiencing. This was

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128 by making a mark on a 10 cm VAS line corresponding to the relevant clip (Fig. 1a). They
129 were also required to comment upon the factors that influenced their assessment in a
130 separate box (not shown).
131 Following observation of the four clips, the volunteers were individually given
132 approximately 10 min of instruction on recognising and scoring the occurrence of back-
arching, writhing, stagger/fall and twitching, the key behaviours shown to be useful for

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134 scoring pain after this surgical procedure. They then used behaviour scoring software (The
135 Observer, Version 3.0, Noldus IT, Wageningen, the Netherlands) to assess the four clips a
136 second time presented in another random sequence. Data input consisted of different key
137 presses upon occurrence of the key behaviours.

2.2. Phase 2
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139 The procedure for observation of the four clips of behaviour was essentially the same as
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140 for Phase 1, except that after assessing the video-recordings with the VAS the observers
141 simultaneously received training in scoring behaviour in groups of from 8 to 44 individuals.
142 Training in behaviour scoring in Phase 2 was also aided by use of material on a Compact
143 Disk (Pain Assessment in the Rat, Version 5.0, available from http://www.digires.co.uk/
144 dpain.html).
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145 The Phase 2 exercise was part of a broader lecture on assessing post-operative pain in rats.
146 The order of presentation of the clips was again randomised between groups and within each
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147 observation session (VAS or behaviour). As in Phase 1, the VAS stage was completed with the
148 sheet shown in Fig. 1a. For behaviour scoring, each observer was given a second scoring sheet
149 (Fig. 1b) and asked to score the four clips a second time, presented in another random
150 sequence (without computer aid). For reasons of confidentiality, it was not possible to identify
151 the individual institutions at which Phase 2 testing/training was conducted. Due to the limited
time available, and because the larger number of volunteers were unknown to the session(s)
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153 organiser, it was also not possible to estimate prior experience as a possible factor in the
154 outcomes of the pre-training (VAS) stage of the exercise.

2.3. Phase 3
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156 Phase 3 volunteers were two groups of highly experienced researchers and senior animal
technicians attending an advanced training program. For the present purposes, these have

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Fig. 1. The VAS (a) and behaviour (b) score sheets. Treatment-blinded participants were asked to view four clips
of rat behaviour and follow the instructions provided. Phase 1 (VAS) also required that observers commented on
any factors influencing their assessment in a separate box (not shown). The score sheets have been truncated.

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been classified as experts (Group 1, n = 81; Group 2, n = 54). Due to the short time available,
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159 data collection was restricted to a post-training exercise and only two of the behaviour clips
160 were assessed. The clips that were shown corresponded to the rat given saline and the rat
161 given M2. Pre-training evaluation involved only a ‘show-of-hands’ as to which of the two
162 clips the participants thought corresponded to each treatment, i.e. which rat they thought may
163 or may not have received an analgesic. No data on this were collected other than a rough
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164 estimate as to whether any majority of opinion was evident. As in Phase 2, training and
165 behaviour scoring was subsequently conducted using the sheet shown in Fig. 1b.

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2.4. Data analysis
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167 For each clip, the mean VAS and post-instruction cumulative behaviour score (the sum
168 of the scores for the occurrence of back arch, stagger/fall, writhe and twitch) was calculated
169 for each group of observers. For Phase 1, further calculations were made within each
170 observer category (level of prior experience).

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171 The main aim of the study was to compare the effectiveness of behaviour scoring with
172 VAS scoring for rating pain severity. The major difficulty, however, was that in order to
173 determine which approach was the most effective for assessing pain; it would be necessary
174 to make some sort of comparison between the two techniques. Although, desirable,
175 statistically it was impossible to make direct comparisons between the raw data obtained

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176 for each technique. The analysis was designed to partially circumvent this problem, by
177 comparing measures of success with each technique rather than absolute scores. The
178 rationale was that this comparison would allow us to establish which was the most
179 consistently effective technique for differentiating between an untreated rat, and rats given
180 different dosages of an analgesic.

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2.5. Statistical analysis
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182 The study had a repeated measures design, with the within subjects factor being
183 behaviour clip (as the same individuals rated each clip consecutively). It was assumed that
184 the rats were experiencing pain differing in intensity dependent upon the treatment
received (untreated or different analgesic dose rate). The behaviour scores were therefore
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186 treated as scale data. Due to current uncertainties as to the numerical properties of the VAS
187 scale, it was inappropriate to make direct comparisons between the techniques.
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188 The data from the three Phases of the study were assessed separately. This was because,
189 as described above, following Phase 1, the participants were not classed according to prior
190 experience, and because computerised data entry was not possible. Also, the VAS scoring
191 procedure was omitted from Phase 3, when only two behaviour clips were assessed by pre-
192 supposed ‘experts’. The instruction on behaviour recognition given to the groups during
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193 Phase 2 also did not adhere to a strict format (other than being of approximately 10 min
194 duration).
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195 Due to the low number of volunteers in the categories ‘research’ and ‘laypersons’ of in
196 Phase 1, it was not meaningful to apply formal statistical analysis. Table 1 therefore only
197 shows the results of analysis of the technicians’ scores and the research and laypersons data
198 are only described qualitatively in the text.
199 The Phases 2 and 3 data were tested for normality and homogeneity of variance. A
modification of the Kolmogorov–Smirnov test was used (Shapiro–Wilk) when sample
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200
201 sizes were less than 50. The homogeneity test was the Levene Statistic. The VAS data from
202 each workshop were, with only a few exceptions, normally distributed and homogeneous.
203 However, the behaviour data showed several violations depending on the group and the
204 variable tested (score for each clip). Data transformations failed to achieve homoscedasity
205 so all further comparisons between the VAS and behaviour success rates were made with a
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206 non-parametric equivalent of the paired samples t-test; the Wilcoxon signed ranks test
207 (two-tailed).

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Table 1
Phase 1 (P1, pilot study, row 1, technicians only) and Phases 2 and 3 (P2, P3) median and lower and upper quartiles for VAS and cumulative behaviour scores
Venue Treatment VAS (quartiles) Behaviour (quartiles) Wilcoxon test Success (%)
(paired comparisons, p value)

J.V. Roughan, P.A. Flecknell / Applied Animal Behaviour Science xxx (2005) xxx–xxx
25th Median 75th 25th Median 75th VAS Behaviour VAS Behaviour
P1, CBC (n = 26) 1. Saline 56 80 86 18 23 38 1 vs. 2 = ns, 1 vs. 2 = ns, 1 < 2 = 27, 1 < 2 = 54,
2. M0.5 27 61 82 17 30 41 1 vs. 4 = ns, 1 vs. 4 = 0.02, 1 > 4 = 73, 1 > 4 = 91,
3. M1 63 76 83 6 16 29 3 vs. 4 = ns 3 vs. 4 = ns 3 > 4 = 73 3 > 4 = 64
4. M2 31 60 86 8 15 24

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P2, conference 1 (n = 26) 1. Saline 28 48 63 9 17 24 1 vs. 2 = 1.02, 1 vs. 2 < 0.01, 1 < 2 = 54, 1 < 2 = 14,
2. M0.5 40 59 76 6 9 15 1 vs. 4 = 0.56, 1 vs. 4 < 0.01, 1 > 4 = 31, 1 > 4 = 89,
3. M1 22 38 66 5 9 15 3 vs. 4 = 0.04 3 vs. 4 < 0.01 3 > 4 = 31 3 > 4 = 79

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4. M2 47 57 71 3 6 8
P2, conference 2 (n = 10) 1. Saline 50 75 90 17 18 25 1 vs. 2 = 5.5, 1 vs. 2 = 2.2, 1 < 2 = 40, 1 < 2 = 60,
2. M0.5 60 75 91 17 21 26 1 vs. 4 = 0.68, 1 vs. 4 = 0.5, 1 > 4 = 60, 1 > 4 = 80,

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3.
4.
M1
M2
35
38
49
58
78
82
15
7
16
12
21
16
3 vs. 4 = 4.7 3 vs. 4 = 0.28 3 > 4 = 60 3 > 4 = 80

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P2, pharm. 1 (n = 44) 1. Saline 37 50 65 7 9 14 1 vs. 2 = 0.08, 1 vs. 2 < 0.01, 1 < 2 = 75, 1 < 2 = 76,
2. M0.5 43 65 83 12 20 24 1 vs. 4 = 0.09, 1 vs. 4 < 0.01, 1 > 4 = 56, 1 > 4 = 78,
3. M1 24 37 58 8 11 15 3 vs. 4 = 2.79 3 vs. 4 < 0.01 3 > 4 = 57 3 > 4 = 91

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4. M2 23 35 50 3 5 7
P2, pharm. 2 (n = 33) 1. Saline 27 38 54 20 25 37 1 vs. 2 = 1.9, 1 vs. 2 = 4.2, 1 < 2 = 58, 1 < 2 = 49,

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2. M0.5 31 45 63 21 25 29 1 vs. 4 = 0.83, 1 vs. 4 < 0.01, 1 > 4 = 63, 1 > 4 = 87,
3. M1 20 29 49 16 20 26 3 vs. 4 = 5.4 3 vs. 4 < 0.01 3 > 4 = 62 3 > 4 = 94
4. M2 20 35 50 9 11 15
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P2, pharm. 3 (n = 8) 1. Saline 31 64 79 16 18 37 1 vs. 2 = 0.64, 1 vs. 2 = 2.9, 1 < 2 = 37, 1 < 2 = 50,
2. M0.5 29 45 51 24 27 35 1 vs. 4 = 0.85, 1 vs. 4 = 0.1, 1 > 4 = 62, 1 > 4 = 87,
3. M1 41 58 60 11 18 30 3 vs. 4 = 2.9 3 vs. 4 = 0.07 3 > 4 = 63 3 > 4 = 100

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4. M2 26 43 58 6 11 17

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Table 1 (Continued )
Venue Treatment VAS (quartiles) Behaviour (quartiles) Wilcoxon test Success (%)
(paired comparisons, p value)
25th Median 75th 25th Median 75th VAS Behaviour VAS Behaviour

J.V. Roughan, P.A. Flecknell / Applied Animal Behaviour Science xxx (2005) xxx–xxx
P2, pharm. 4 (n = 24) 1. Saline 32 52 62 24 31 37 1 vs. 2 = 2.7, 1 vs. 2 = 0.12, 1 < 2 = 61, 1 < 2 = 63,
2. M0.5 30 46 70 30 37 40 1 vs. 4 = 2.1, 1 vs. 4 = 0.01, 1 > 4 = 56, 1 > 4 = 75,
3. M1 20 38 58 19 25 33 3 vs. 4 = 2.5 3 vs. 4 = 0.05 3 > 4 = 48 3 > 4 = 75
4. M2 26 44 56 15 18 25
P2, uni. 1 (n = 24) 1. Saline 36 48 59 18 26 38 1 vs. 2 = 2.6, 1 vs. 2 = 0.24, 1 < 2 = 58, 1 < 2 = 63,
2. M0.5 28 60 70 26 32 38 1 vs. 4 = 3.6, 1 vs. 4 < 0.01, 1 > 4 = 50, 1 > 4 = 96,

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3. M1 26 35 50 17 23 32 3 vs. 4 = 1.8 3 vs. 4 < 0.01 3 > 4 = 45 3 > 4 = 100
4. M2 23 45 65 10 14 20
P2, uni. 2 (n = 29) 1. Saline 24 35 49 17 26 35 1 vs. 2 = 2.0, 1 vs. 2 = 2.8, 1 < 2 = 38, 1 < 2 = 40,

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3.
4.
M0.5
M1
M2
21
31
28
27
54
41
39
72
51
21
22
12
25
23
15
30
23
17
1 vs. 4 = 1.9,
3 vs. 4 = 0.16
1 vs. 4 = 0.02,
3 vs. 4 < 0.01
1 > 4 = 45,
3 > 4 = 76
1 > 4 = 77,
3 > 4 = 87

P3, experts 1 (n = 80)

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2.
Saline
n/c
n/c
n/c
n/c
n/c
n/c
n/c
22
n/c
28
n/c
38
n/c
n/c 1 vs. 4 < 0.01 n/c 1 > 4 = 94

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3. n/c n/c n/c n/c n/c n/c n/c
4. M2 n/c n/c n/c 9 13 20

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P3, experts 2 (n = 54) 1. Saline n/c n/c n/c 20 26 32 n/c 1 vs. 4 < 0.01 n/c 1 > 4 = 100
2. n/c n/c n/c n/c n/c n/c n/c
3. n/c n/c n/c n/c n/c n/c n/c

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4. M2 n/c n/c n/c 6 11 16
The results of Wilcoxon signed ranks tests for three within-groups comparisons are shown: 1 vs. 2 (saline vs. M0.5), 1 vs. 4 (saline vs. M2), and 3 vs. 4 (M1 vs. M2). In Phase
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2, groups of observers from conferences (2), pharmaceutical companies (4), and universities (2) attending a workshop on pain assessment. P3 participants observed clips

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saline and M2 only. P2 individual probability values were adjusted (6) to account for multiple comparisons. Success rates were based on the percentage of ranked scores
for the VAS or behaviour that were in agreement with hypothetical pain severity, whereby treatment 1 (saline) VAS or behaviour scores should be greater than treatment 4
(M2) (total positive ranks/n (100)). The two other hypothesis tests were 1 < 2 (negative ranks/n (100)) and 2 > 4 (positive ranks).

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Fig. 2. Cumulative behaviour scores (back-arch, stagger/fall, writhe, and twitch) for each of the four behaviour
clips as assessed by the score system developer (JVR). Each clip corresponds to the score for one rat given saline,

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or meloxicam at 0.5, 1 or 2 mg/kg.

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208 No absolute values for the VAS were possible, so our assumptions concerning the ‘true’
209 level of pain that each rat was experiencing were based on the data in Fig. 2. This shows the
score for the four randomly selected clips that had previously been made by the score
210
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211 system developer (JVR). As shown, pain score was high in the rat given saline and least
212 following M2. The highest score occurred in the rat given M0.5 and the rat given the
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213 highest dose of meloxicam (M2) showed less pain behaviour than the rat given M1. The
214 null hypothesis was that there would be no difference in success rate between VAS and
215 behaviour-based assessments. With four treatments, there were a maximum of 12 pair-wise
216 comparisons for each workshop dataset. To minimise the Type I error rate, this was reduced
217 to six (three VAS and three behaviour comparisons) to test the following alternative
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218 hypothesis for the pattern of pain severity between rats given each treatment:
219 saline < M0.5; saline > M2 and M1 > M2 (see Fig. 2).
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220 These comparisons were selected as the most rigorous test possible of the extent to
221 which the participants could use each scoring technique to differentiate between the four
222 treatments. To further account for the number of comparisons made, a post-hoc probability
223 correction was also used (Bonferroni) whereby the observed p-values were multiplied by 6.
224 This was not necessary for the Phase 3 data, as there were only two levels of the within-
subjects factor (treatment), saline, and M2, and no VAS data.
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225
226 To provide a measure of observer success, each workshop participant’s VAS and
227 behaviour scores were tested for agreement with the hypothesised pattern of pain severity.
228 Success was then calculated as the proportion of rank scores that conformed to the pattern
229 (positive ranks, M0.5 saline; negative ranks, M2 saline and M2 M1). The mean
230 VAS and behaviour success rates (saline versus M0.5 + saline versus M2 + M1 versus M2/
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231 3) were then compared (Wilcoxon) to determine whether one technique was superior to the
232 other, in other words, which resulted in overall greater success in differentiating between

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233 treatments. All VAS and behaviour data are expressed as median and interquartile range,
234 and success rates as percentages (mean  1 S.D.).

3. Results
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236 Data were obtained from a total of 332 volunteer observers.

3.1. Phase 1
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238 3.1.1. CBC volunteers

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239 The Phase 1 VAS and behaviour scores of the technicians are shown in Table 1. The
240 Wilcoxon signed ranks test was used to make three comparisons between the technicians
241 VAS scores (saline versus M0.5, saline versus M2, and M1 versus M2). Similar
242 comparisons were then made for the post-training behaviour scores.
243 Using the VAS scale, eight out of 11 technicians (73%) scored the saline treated rat as
experiencing more pain than the rat given M2. The same number thought the rat given M2

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245 experienced less pain that the rat given dose M1, however, neither result was significant
246 after probability correction. There was also no significant difference in the technicians’
247 VAS scores for rats saline and M0.5, when only 27% of technicians (3 out of 11) assigned a
248 larger pain score to the drug treated rat. Of the remainder of the CBC volunteers, only one
249 of the four research workers and two of the three laypersons gave higher scores for the
saline treated rat than the rat given the highest dosage, M2. Two research staff and two
250
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251 laypersons used the VAS correctly to differentiate between rats M0.5 and Sal, and two in
252 each category also correctly identified differences between rats M1 and M2.
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253 Comments on the factors that influenced the VAS score centred on the perceived gross
254 level of mobility, and the condition of the coat and eyes. On the whole, more active
255 descriptions were used to justify scores for the saline treated rat. The rat made several
256 exploratory ventures before settling in one corner of the cage. The rat that received
257 meloxicam was described as less active (‘sitting in the corner’, ‘wound licking’) but the
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258 coat condition, and appearance of the rats’ eyes was thought better, ‘coat normal’ and ‘eyes
259 bright’ (or similar) were frequent descriptors. Several technicians thought the saline treated
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260 rat was recovering from anaesthesia, and made comments, such as ‘sleepy’ and
261 ‘uncoordinated’ or noted that the eyes were closed. However, descriptions of the saline
262 treated rat also included ‘active’ and ‘alert’. There was a similar disparity of opinion for the
263 rats given meloxicam, and qualitatively little agreement as to the treatment received
264 irrespective of the observer groups’ prior experience.
During behaviour scoring, 10 out of 11 technicians returned lower cumulative scores for
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266 rat M2 compared to Sal (91%, p = 0.02). All four researchers also successfully differentiated
267 between these, but only one of the three laypersons. One layperson gave a behavioural pain
268 score that was higher for the rat given M2 than the saline treated rat (one result tied). Three
269 researchers and two laypersons correctly distinguished between Sal and rat M0.5. Six
270 technicians (54%) were also successful but the magnitude of the individual differences was
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271 not significant. Seven of the technicians (64%) and three researchers assigned higher
272 behaviour scores to rat M1 than M2, but none of the laypersons recognised a difference.

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3.2. Phase 2
273
274 As in the analysis of the Phase 1 technician’s scores, for each Phase 2 workshop, the
275 Wilcoxon signed ranks test was used for comparisons between the assessors’ initial VAS
276 and subsequent behaviour success rates. The results of this are shown are shown in Table 1.

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277 3.2.1. Conferences
278 Analysis of the VAS data from conferences provided only one significant result. Overall,
279 conference 1 participants attributed higher VAS scores significantly more often (i.e. more
280 pain) to the rat given M2 than the rat M1, M1 ( p = 0.04). This conflicted with the
281 hypothetical score (i.e. M2 < M1). The success rates for the three VAS comparisons were

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282 generally low and ranged from 31 to 51%.
283 Following training the vast majority of conference 1 participants (89%) successfully
284 scored behaviour, and significantly more frequently attributed higher pain scores to rat Sal
285 compared with the rat given M2 ( p < 0.01). Although 80% successfully discriminated
286 between rats M1 and M2 ( p < 0.01), only 14% of conference 1 delegates successfully
differentiated between the behaviour of rats Sal and M0.5.

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287
288 conference 2 delegates were unsuccessful in using the VAS and apparently also had
289 difficulties in learning and applying behaviour-based scoring. Although success rates of up
290 to 60% (VAS) and 80% (behaviour) were obtained, these were based on relatively few
291 participant’s scores and the magnitude of the differences between the scores for each rat
292 were comparatively small.
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293 3.2.2. Pharmaceutical companies
294 Using VAS scoring, although there were occasions when up to 63% of individuals
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295 successfully identified differences between treatments, the individual group comparisons
296 were non-significant due to large within group variability. Using the behavioural technique,
297 a much greater majority of observers correctly differentiated between the treatments,
298 irrespective of the establishment visited. Difficulties were encountered, however, in
299 recognising differences in behaviour between the rat given saline or M0.5 (Pharm. 2 and
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300 Pharm. 4). The mean success rates obtained at Pharm. 3 suggested the participants were
301 unable to score behaviour as effectively as some other groups, although again, the group
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302 was relatively small (n = 8). Nevertheless, all participants correctly differentiated between
303 the medium and high dosages of meloxicam and more frequently assigned lower pain
304 scores to rat M2. The result, however, was not significant after probability correction. This
305 was due to large variation in this group’s scores for the two treatments. Most groups,
306 especially Pharm. 1, detected significant differences between treatments in line with the
hypothetical results ( p < 0.01), and in these, success rates in recognising more severe pain
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307
308 in the control (saline treated rat) compared to the rat that received the highest dose of
309 meloxicam ranged from 75% (Pharm. 4) to 87% (Pharm. 2).

310 3.2.3. Universities

311 The results of pain scoring at the two university based workshops were broadly similar
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312 to the conference and pharmaceutical company findings. None of the groups successfully
used VAS scoring to identify significant differences between the treatments. Behaviour

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314 scoring was considerably more successful. As before, there were some individuals who
315 could not differentiate between Sal and M0.5, but differences were recognised between rats
316 Sal and M2 when success ranged from 77% (uni. 2; p = 0.02) to 96% (uni. 1; p < 0.01), and
317 between M1 and M2 ranging from 87% (uni. 2; p < 0.01) to 100% (uni. 1; p < 0.01).

3.3. Phase 3 (experts)

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318
319 Following instruction, the ‘experts’ achieved success rates of between 94% (experts 1;
320 p < 0.01) and 100% (experts 2; p < 0.01) and gave higher pain scores when observing the
321 untreated rat. No VAS data were collected in these groups, but a ‘show of hands’ indicated
322 that, prior to instruction, about half of participants had failed to detect a difference in pain

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323 severity between the untreated rat and the rat given M2.

3.4. Method comparison

324
325 The averaged success rate for differentiating between treatments for all Phase 2
exercises was then compared (paired t-test). This showed that the overall probability of

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326
327 successfully identifying a rat experiencing pain was greater with behaviour scoring than
328 with the VAS approach. The mean success rate (%  1 S.D.) for VAS scoring over all
329 workshop venues was 54  7% while equivalent rate following instruction on using
330 behaviour scoring was 75  8% ( p = 0.012). E
4. Discussion
331
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332 The results of this study showed that behavioural assessment was more successful than
333 VAS scoring in allowing naı̈ve and experienced observers to gauge pain severity following
334 surgery in rats.
335 The VAS provided a convenient means of recording each assessor’s opinion as to the
336 degree of pain experienced by each of the four rats. This approach has been used in other
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337 animals and in human infants (Lascelles et al., 1994, 1995; Nolan and Reid, 1993; Slingsby
338 and Waterman-Pearson, 2001, 1998, 2000; Stephens and Anand, 2000) as a relatively
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339 simple and subjective approach to pain scoring. Its major limitations, however, are a lack of
340 consistency and validity (Conzemius et al., 1997; Holton et al., 1998; Reese et al., 2000;
341 Stephens and Anand, 2000) so researchers continue to seek more objective techniques to
342 improve pain assessment methods for animals and other non-verbal subjects. Developing
343 these techniques has been the focus of the research of the Newcastle Pain Systems group
for the past decade.
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344
345 An essential step in acceptance of our present findings, however, is confidence that the
346 behaviour scores assigned to each of the four rats by the ‘expert’ are truly representative of the
347 pain that each was experiencing. In an ideal world, we would have a score to act as a ‘gold
348 standard’ with which to compare the volunteers’ VAS and behaviour results, but
349 unfortunately this does not exist. In its absence, we have used adopted the ‘nest best’
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350 approach, and used a score founded on an extensive research program into the post-operative
behaviour of rats. The behaviour scoring system was initially developed following a blinded,

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351
352 controlled study of rats undergoing laparotomy (Roughan and Flecknell, 2000). A
353 subsequent more detailed study enabled us to identify behaviours that could be used to
354 differentiate between anaesthetised and drug or saline treated controls, from rats given either
355 an analgesic or saline prior to laparotomy (Roughan and Flecknell, 2001). Subsequently, we
356 evaluated whether shorter observation periods could be used (ca. 5 min) as this would make
357 scoring potentially useful for routine application in research units (Roughan and Flecknell,

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358 2003). Following this, we used behaviour scoring to determine the efficacy of other
359 analgesics (Roughan and Flecknell, 2004) and found our conclusions were supported by
360 similar behavioural studies by other groups working independently (Affaitati et al., 2002;
361 Giamberardino et al., 2000; Gonzalez et al., 2000). As our initial behavioural analyses gave
362 no precedence to any behavioural characteristics as potential indices of pain, we believe we

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363 achieved a high level of objectivity in identifying the key behavioural signs of abdominal pain
364 in rats. We therefore think it reasonable to conclude that the behavioural technique is valid for
365 assessing the degree of pain that rats experience and are confident that the present study
366 demonstrates an improvement of the ability of volunteers to assess pain; albeit limited to the
367 effects of abdominal surgery so far.
Considerable attention was given to the most appropriate method of analysis of the data.

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368
369 A literature survey by (Mantha et al., 1993) found that approximately 50% of studies
370 involving VAS measurements applied parametric statistics, presumably when the
371 assumptions of normality and homogeneity of variance were met. Opinions vary as to
372 the linearity of the VAS scale, that is, whether a score of eight represents twice as much
373 pain as a score of four in the same individual. This has important implications for the type
of statistical tests that can be applied. Although, some reputable texts advocate use of the
374
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375 non-parametric approach (Altman, 1991), other authors contend that the scale is linear (at
376 least for mild to moderate pain in humans), so conventional parametric statistics can be
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377 applied (Myles et al., 1999; Philip, 1990). Our previous work has shown that scores based
378 on the occurrence of the relevant pain-related behaviours are related to the dose of
379 analgesic given (Roughan and Flecknell, 2003), so these data can be treated fundamentally
380 as linear. Although, it might appear that an analysis based on method comparison could be
381 undertaken (Bland and Altman, 1986), the subjective VAS and the behaviour-based pain
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382 system essentially scored different measures of pain, using different scales. As such, it was
383 impossible to make direct comparisons of the results obtained with each technique.
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384 Because of this our approach to the analysis of the data placed emphasis on determining
385 which of the two assessments was most successful in detecting a difference between
386 treatments in four video clips. This seemed to be the key attribute in a pain scoring system
387 intended for use in a research environment: firstly, because it would be used to assess
388 whether an analgesic is needed; secondly, to determine whether additional treatment(s) are
necessary. In cases where surgery of a more severe nature is conducted, it might also be
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389
390 helpful in applying more humane end-points. Furthermore, in obtaining these results, we
391 also demonstrated that the basics of the scoring system could also be taught to naı̈ve
392 observers in a very short time (10 min). Experience within our own research group has
393 shown that reproducibility of scoring and reliability of identification of behaviours
394 increases with experience, but the basic scheme can be learnt rapidly. Although the
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395 behaviour score performed well with most comparisons, several groups failed to
differentiate between the saline treated animal and the animal received a low (ineffective)

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397 dose of meloxicam (0.5 mg/kg). This is consistent with the results from the original study
398 which included these video clips, in which the mean scores of the saline and 0.5 mg/kg
399 groups were not significantly different ( p > 0.1, Fig. 2).
400 The results obtained using the VAS indicate that a high percentage of laboratory animal
401 scientists, veterinary surgeons, and animal technicians have a relatively poor ability to
402 recognise this type of pain and the distress it causes in rats. We suspect that more severe

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403 pain would be identified more readily, but that pain of moderate intensity, as used in this
404 exercise, is more difficult to assess. Participants in one pharmaceutical workshop were
405 much more successful in using the VAS, although behavioural scoring was even more
406 successful. This group, however, included technicians, veterinarians and research workers
407 who were amongst the most experienced in day-to-day post-operative care of rodents. Far

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408 from being seen as a negative result, this was reassuring in that at least some groups had
409 developed an ability to recognise pain by evaluating the animal’s overall appearance.
410 Indeed, other studies employing blinded study designs have also demonstrated successful
411 use of the VAS in differentiating pain severity between animals given placebo, or different
412 doses of analgesics (Lascelles et al., 1994, 1995; Nolan and Reid, 1993; Slingsby and
Waterman-Pearson, 1998, 2000).

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413
414 It is possible that direct evaluation of the animal, enabling handling would have
415 improved the accuracy of the VAS. A consideration of the logistics of pain assessment
416 suggests that only a limited time may be available for pain assessment. Descriptions of
417 effective behaviour-based pain assessments can be complex (Holton et al., 2001) and
418 excessive training requirements may preclude their routine application. These difficulties
may explain Hawkins finding (2002) that the simplest schemes are the ones most often
419
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420 implemented. We believe it important that those undertaking pain assessments should
421 adopt the most accurate system that can be applied in a relatively brief observation period.
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422 Based on present results, we believe this scheme, by being developed on sound scientific
423 evidence goes a considerable way towards achieving its objectives of achieving greater
424 usability and accuracy. One limitation, however, is that the scheme is probably only useful
425 for assessing the effects of acute post-operative pain in rats undergoing ventral abdominal
426 procedures. Much effort is needed to develop behavioural schemes affording the possibility
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427 of assessing pain following other common procedures or when more prolonged effects
428 could be anticipated, such as in neuropathic and other chronic pain syndromes.
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429 An obvious source of bias in the design of the study was the consistent collection of VAS
430 data prior to behaviour sampling. This was unavoidable, as clearly, it was not possible to
431 collect unbiased VAS data following training in behaviour scoring. Neither was it possible
432 to refrain from demonstrating both approaches to participants in the workshops used for
433 data collection.
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5. Conclusion
434
435 We have demonstrated that a behaviour-based pain assessment system is superior to one
436 based on general clinical impression, and that the behaviour scoring system can be taught
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437 and applied rapidly. Numerous problems still remain to be overcome before all types of
post-surgical pain can be assessed and alleviated. The scoring system has so far only been

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438
439 applied to surgical procedures involving the abdomen. A different scoring system may be
440 needed for procedures, such as craniotomy. The use of anaesthetic regimens which result in
441 very prolonged recovery may also mask some of the relevant pain-related behaviour.
442 Finally, scoring systems for other laboratory species are required. Ultimately, progress of
443 this type should enable research workers to better identify a need for analgesic therapy, and
444 administer analgesics at dose rates and dose frequencies appropriate to the needs of the

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445 individual animal.

Acknowledgements
446

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447 We wish to thank all of the volunteers who participated in the study. The study was
448 funded by the UK Medical Research Council.

References

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449
450
Affaitati, G., Giamberardino, M.A., Lerza, R., Lapenna, D., De Laurentis, S., Vecchiet, L., 2002. Effects of
451
tramadol on behavioural indicators of colic pain in a rat model of ureteral calculosis. Fundam. Clin.
452 Pharmacol. 16, 23–30.
453
Al-Gizawiy, M.M., Rude, E.P., 2004. Comparison of preoperative carprofen and postoperative butorphanol as
454 postsurgucal analgesics in cats undergoing ovariohysterectomy. Vet. Anaesth. Analg. 31, 164–174.
455 Altman, D.G., 1991. Practical Statistics for Medical Research. Chapman and Hall, London, UK, pp. 172–173.
456
E
Bland, M., Altman, D.G., 1986. Statistical methods for assessing agreement between two methods of clinical
457 measurement. Lancet 1 (8476), 307–310.
458
Capner, C., Lascelles, B.D.X., 1999. Current British veterinary attitudes to perioperative analgesia for dogs. Vet.
CT
459 Rec. 145, 95–99.
460
Conzemius, M.G., Hill, C.M., Sammarco, J.L., Perkowski, S.Z., 1997. Correlation between subjective and
461
objective measures used to determine severity of postoperative pain in dogs. JAVMA 210 (11), 1619–
462 1622.
463
Firth, A.M., Haldane, S.L., 1999. Development of a scale to evaluate postoperative pain in dogs. JAVMA 214 (5),
464 651–659.
E

465
Giamberardino, M.A., Affaitati, G., Lerza, R., Vecchiet, L., 2000. Pre-emptive analgesia in rats with artificial
466
ureteric calculosis. Effects on visceral pain behaviour in the post-operative period. Brain Res. 878, 148–
467 154.
RR

468
Gonzalez, M.I., Field, M.J., Bramwell, S., McCleary, S., Singh, L., 2000. Ovariohysterectomy in the rat: a model
469 of surgical pain for evaluation of pre-emptive analgesia? Pain 88 (1), 79–88.
470 Grau, V., Steiniger, B., 2003. Transplantation of both kidneys from one donor rat. Lab. Anim. 37, 162–165.
471
Hardie, E., Hansen, B., Carroll, G.S., 1997. Behavior after ovariohysterectomy in the dog: what’s normal? Appl.
472 Anim. Behav. Sci. 51, 111–128.
473
Hawkins, P., 2002. Recognizing and assessing pain, suffering and distress in laboratory animals: a survey of
CO

474 current practice in the UK with recommendations. Lab. Anim. 36 (4), 378–395.
475
Holton, L., Reid, J., Scott, E.M., Pawson, P., Nolan, A., 2001. Development of a behaviour-based scale to measure
476 pain in dogs. Vet. Rec. 148, 525–531.
477
Holton, L.L., Scott, E.M., Nolan, A.M., Reid, J., Welsh, E., Flaherty, D., 1998. Comparison of three methods used
478 for assessment of pain in dogs. JAVMA 212 (1), 61–66.
479
Hugonnard, M., Leblond, A., Keroack, S., Cadore, J.-L., Troncy, E., 2004. Attitudes and concerns of French
480 veterinarians towards pain and analgesia in dogs and cats. Vet. Anaesth. Analg. 31, 154–163.
UN

481
Kent, J.E., Thrushfield, M.V., Molony, V., Hosie, B.D., Sheppard, B.W., 2004. Randomised, controlled field trial of
482 two new techniques for the castration and tail docking of lambs less than 2 days of age. Vet. Rec. 154, 193–200.

APPLAN 2391 1–16

 DTD 5

16 J.V. Roughan, P.A. Flecknell / Applied Animal Behaviour Science xxx (2005) xxx–xxx

482
483 Labat, A., Calise, D., Thiers, J.C., Pieraggi, M.T., Cerene, A., Fournial, G., Thomsen, M., Dambrin, C., 2002.
484 Simultaneous orthotopic transplantation of carotid and aorta in the rat by the sleeve technique. Lab. Anim. 36
485 (4), 426–431.
486 Lascelles, B., Capner, C., Waterman-Pearson, A., 1999. Current British veterinary attitudes to perioperative
487 analgesia for cats and small mammals. Vet. Rec. 145, 601–604.
488 Lascelles, B.D.X., Butterworth, S.J., Waterman, A.E., 1994. Postoperative analgesic and sedative effects of
489 carprofen and pethidine in dogs. Vet. Rec. 134, 187–191.

OF
490 Lascelles, B.D.X., Cripps, P., Mirchandani, S., Waterman, A.E., 1995. Carprofen as an analgesic for postoperative
491 pain in cats: dose titration and assessment of efficacy in comparison to pethidine hydrochloride. J. Small.
492 Anim. Pract. 36, 535–541.
493 Lawson, D.M., Duke, J.L., Zammit, T.G., Collins, H.L., Dicarlo, S.E., 2001. Recovery from carotid artery
494 catheterization performed under various anesthetics in male, Sprague–Dawley rats. Contemp. Top. Lab. Anim.
495 Sci. 40 (4), 18–22.

RO
496 Mantha, S., Thisted, R., Foss, J., Ellis, J.E., Roizen, M.F., 1993. A proposal to use confidence intervals for visual
497 analog scale data for pain measurement to determine clinical significance. Anaesth. Analg. 77 (2), 1041–1047.
498 Molony, V., Kent, J.E., McKendrick, I.J., 2002. Validation of a method for assessment of an acute pain in lambs.
499 Appl. Anim. Behav. Sci. 76 (3), 215–238.
500 Myles, P.S., Troedel, S., Boquest, M., Reeves, M., 1999. The pain visual analog scale: is it linear or nonlinear.
501 Anaesth. Analg. 89 (6), 1517–1522.
502 Nolan, A., Reid, J., 1993. Comparison of the post-operative analgesic and sedative effects of carprofen and

DP
503 papaveretum in the dog. Vet. Rec. 133, 240–242.
504 Philip, B.K., 1990. Parametric statistics for evaluation of the visual analog scale. Anaesth. Analg. 71, 710.
505 Reese, C.J., Trotter, E.J., Short, C.E., Erb, H.N., Barlow, L.L., 2000. Assessing the efficacy of perioperative
506 carprofen administration in dogs undergoing surgical repair of a ruptured cranial cruciate ligament. J. Am.
507 Anim. Hosp. Assoc. 36, 448–455.
508 Richardson, C., Flecknell, P.A., 2005. Anaesthesia and post-operative analgesia following experimental surgery in
509 laboratory rodents—are we making progress? ATLA, in press.
510
E
Roughan, J.V., Flecknell, P.A., 2000. Effects of surgery and analgesic administration on spontaneous behaviour in
511 singly housed rats. Res. Vet. Sci. 69, 283–288.
512 Roughan, J.V., Flecknell, P.A., 2001. Behavioural effects of laparotomy and analgesic effects of ketoprofen and
CT
513 carprofen in rats. Pain 90 (1–2), 65–74.
514 Roughan, J.V., Flecknell, P.A., 2003. Evaluation of a short duration behaviour-based post-operative pain scoring
515 system in rats. Eur. J. Pain 7 (5), 397–406.
516 Roughan, J.V., Flecknell, P.A., 2004. Behaviour-based assessment of the duration of laparotomy-induced
517 abdominal pain and the analgesic effects of carprofen and buprenorphine in rats. Behav. Pharmacol. 15,
518 461–472.
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519 Slingsby, L.S., Waterman-Pearson, A., 2001. Analgesic effects in dogs of carprofen and pethidine together
520 compared with the effects of either drug alone. Vet. Rec. 148, 441–4.
521 Slingsby, L.S., Waterman-Pearson, A.E., 1998. Comparison of pethidine, buprenorphine, and ketoprofen for
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522 postoperative analgesia after ovariohysterectomy in the cat. Vet. Rec. 143, 185–189.
523 Slingsby, L.S., Waterman-Pearson, A.E., 2000. Postoperative analgesia in the cat after ovariohysterectomy by use
524 of carprofen, ketoprofen, meloxicam or tolfenamic acid. J. Small Anim. Pract. 41, 447–450.
525 Stephens, B.J., Anand, K.J.S., 2000. An overview of neonatal pain. In: Anand, K.J.S., Stevens, B.J., McGrath, P.J.
526
526 (Eds.), Pain in Neonates. Elsevier, Amsterdam.
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