Orbital sinus blood sampling in rats: effects upon selected behavioural variables H. van Herck1, V. Baumans2, H. A. G. Boere1, A. P. M. Hespl, H. A. van lith2 & A. C. Beynen2 I Central Laboratory Animal Institute (GOL), Utrecht University and 20epartment of Laboratory Animal Science, Faculty of Veterinary Medicine, Utrecht University, The Netherlands
Summary The question addressed was whether the behaviour of rats is changed after orbital sinus blood sampling while they are under diethyl-ether anaesthesia. Twelve rats were subjected to sham anaesthesia, diethyl-ether anaesthesia and anaesthesia plus orbital puncture according to a Latin square. After each treatment, the behaviour of the rats was automatically monitored using the so-called LABORAS method, which discriminates between grooming, locomotion and inactivity. Treatment ended, and behaviour monitoring began, when the light period changed over to the dark period. The various behaviours were quantified as relative duration and frequency. Anaesthesia versus sham anaesthesia reduced the relative duration of grooming during the first 5 h after treatment. Anaesthesia plus orbital puncture versus anaesthesia alone did not significantly influence grooming, but orbital puncture did reduce the relative duration and the frequency of locomotion during the entire 20 h period, which was mainly due to a decrease in the dark period. After orbital puncture, the animals were also less frequently inactive. It is concluded that orbital puncture has an effect on behaviour superimposed on that of diethyl-ether anaesthesia. This observation may contribute new arguments to the debate on the acceptability of the orbital puncture technique. Keywords Rat; discomfort; orbital sinus blood sampling; orbital puncture; behaviour; inactivity; grooming; locomotion; LABORAS
Orbital sinus puncture is frequently used to obtain blood samples from rats. However, the technique is controversial because it possibly causes pain and distress and because of aversion (van Herck et a1. 1992a). The BVA/FRAME/RSPCA/UFAW joint working group on refinement has produced a report entitled 'Removal of blood from laboratory mammals and birds' in which the group states that orbital puncture is acceptable only as a terminal procedure while the animal is under anaesthesia (Morton et a1. 1993). Correspondence to: H van Herck, PO Box 80.190, NL 3508 TD Utrecht, The Netherlands. B-mail:
[email protected] Accepted 5 July 1999
Orbital sinus blood sampling causes tissue damage (Beynen et al. 1988a, van Herck et a1. 1992b), the severity of which can be limited, depending on the technique and skill of the person performing the puncture (van Herck et a1. 1998). In addition to tissue damage, various aspects of behaviour may serve as indicators of discomfort. Barclay et a1. (1988) suggested that in rats, being a prey species, responses of injury are likely to include a reduced level of activity or even immobility. They developed a 'Disturbance Index' to assess the severity of procedures on rodents. The Disturbance Index uses the difference between the totalled number of movements © Laboratory Animals Ltd. Laboratory Animals (2000) 34. 10-19
Orbital sinus blood sampling in rats: effects on behaviour
11
of test and control animals in a given time period as an index of the degree of disturbance of the animals caused by the tested procedure. Also Flecknell and Liles (1992)use a reduction in locomotor activity as an index of post-surgical stress and/or pain. Orbital sinus blood sampling, while the rats were under diethyl-ether anaesthesia, did not influence telemetrically determined body temperature, heart rate rhythm, locomotor and eating activity when compared with diethyl-ether anaesthesia alone [van Herck et aI. 1997).However, it could be argued that these measurements were not sufficiently sensitive and precise to detect any effects of orbital puncture. In addition, behavioural variables such as grooming and inactivity were not determined. This prompted us to study the effect of orbital puncture in rats on the behavioural variables grooming, locomotion and inactivity, using an automated system for recording and analyses of behaviour. The automated system can be considered sensitive as the i.p. injection of 1ml of sterile saline in mice has been shown to produce less locomotion but more grooming and inactivity (Baumans et al. 1997). Furthermore, it seems worthwhile to measure these behavioural variables in ratsi for instance Vermeulen et al. (1997)described that in rats, fasting caused an increase in locomotion and grooming. In the present study the same variables were measured in rats after they had undergone orbital sinus blood sampling while under diethyl-ether anaesthesia. For comparison we also studied the influence of diethyl-ether anaesthesia alone and that of sham anaesthesia.
in Macrolon cages type III, provided with a layer of sawdust (~iBMI, Helmond, The Netherlands). The animals had free access to tap water and pelleted food (RMH-B;Hope Farms BV,Woerden, The Netherlands). The cages were placed in a room with relative humidity of 50-60%, temperature of 2024°C and ventilation rate of 15-20 air changes per hour. The lighting regime was adapted so that the light period was between 23:1511:15h with a light intensity in the cage of 60-80 lux. The change from light to dark was gradual, between 11:15-11:45h, and vice versa between 23:15-23:45h. Daily care for the animals was provided between 09:00 and 11:00h.
Experimental design From the first day of the acclimatization period, which lasted 8 days, the animals received a clean cage daily. The experiment had a balanced Latin square design (Table 1). There were three treatments and three periods. The Latin square was extended in such a way that each sequence of treatments occurs twice. With this extended Latin square design, from the treatment effects, time influences and carry-over effects will be eliminated. The three treatments were sham anaesthesia, diethyl-ether anaesthesia and diethyl-ether anaesthesia plus orbital puncture. Treatments were performed at 48 h intervals, namely on Monday (Day I), Wednesday (Day 3) and Friday (Day 5) between 10:45 and 11:15h. In previous experiments, the effect of diethyl-ether anaesthesia on behavioural variables did not exceed 24 h [Beynen et al. 1988b, van Herck et al. 1997), so that carry-over effects in the present Animals, materials and methods experiment were considered to be absent. The animals were transported one by one for Th~ research project was approved by the treatment, while in their home cage, to an Animal Ethics Committee of the Faculty of adjacent room. Before an animal was placed Veterinary Medicine and the protocol of the in the Perspex anaesthesia box (25x 15x experiment by the Animal Ethics Committee 15cm, I x w X hI, a paper tissue was put on of the Department of Laboratory Animal the bottom and the box was flushed for 1min Science, Utrecht University. with either room air (sham anaesthesia) or with room air that had been led through a Animals, housing and diet bottle containing diethyl-ether mixed with Twelve male, virus-antibody-free U:WU(Cpb) water. Between treatments, the box was rats, aged 8 weeks, were housed individually cleaned with warm water and dried with a Laboratory Animals (2000) 34
van Herck et a/.
12
paper tissue. The rat was exposed to either sham anaesthesia for 1min or to diethylether anaesthesia until the palpebral reflex had disappeared. The rat was then removed from the Perspex anaesthesia box, including the paper tissue it was lying on. For orbital sinus blood sampling, an experienced animal technician (van Herck et al. 1998; 'technician B'l penetrated the conjunctiva near the inner canthus of the right eye with an intact, heparinized Pasteurs' pipette (QTE 250, length l50mm; Bilbate, Daventry, UK). The pipette was gently rotated as it was advanced through the conjunctiva alongside the eyeball. As soon as blood appeared in the pipette, it was held still. One millilitre of blood was collected. After removal of the pipette, furOther bleeding was prevented by pressing a tissue on the inner canthus of the punctured eye. Immediately after treatment, the animal was put back in its home cage and trans-
Table 1 Treatments per animal. Overview anaesthesia (SA), diethyl-ether anaesthesia
ported to the test cage, which was placed on a balance platform.
Observations during treatment Treatment was defined to start when the animal was put in the Perspex anaesthesia box and to end when it was taken out again (sham anaesthesia) or as soon as the righting reflex was positive (anaesthesia with or without orbital sinus blood sampling). During treatment, the following variables were scored if appropriate: time needed to induce anaesthesia (palpebral reflex absent) and needed for recovery (righting reflex present). While in the anaesthesia box, animals were scored for signs of excitation (O=no excitation, l=slightly excited, 2=clearly excited). During treatment the amount of faeces (no. of faecal boli and faecal weight) and urine produced (weight) were determined. Urine
of periods and treatments per animal: sham (A) and anaesthesia plus orbital puncture (AP) Sensing platform'
Week
Day
Period
0 1
Sunday Monday Tuesday Wednesday Thursday Friday Saturday
Day prior to first treatment Treatment Day after treatment Treatment Day after treatment Treatment
Sunday Monday Tuesday Wednesday Thursday Friday Saturday
Day prior to first treatment Treatment Day after treatment Treatment
Sunday Monday Tuesday Wednesday Thursday Friday
Day prior to fi rst treatment Treatment Day after treatment Treatment Day after treatment Treatment
Saturday
Day after treatment
2
3
A
B
C
D
Rat 1
Rat 2
Rat 3
Rat 4
AP
A
SA
SA
A
AP
A
AP
SA
SA
AP
A
Rat 5
Rat 6
Rat 7
Rat 8
A
AP
AP
A
SA
SA
A
AP
AP
A
SA
SA
Rat 9
Rat 10
Rat 11
Rat 12
SA
SA
A
AP
A
AP
SA
SA
AP
A
AP
A
Day after treatment
Day after treatment Treatment Day after treatment
*See 'Behavioural observations after treatment', under Animals, Materials and Methods section Laboratory Animals (2000) 34
Orbital sinus blood sampling in rats: effects on behaviour
13
weight was determined as weight increase of the paper tissue.
study, vibration patterns other than the validated ones were assigned to the category {undefined' (results not shown).
Behavioural observations after treatment Registrations were performed from 11:30h on Day 0 (Sunday) until 07:30 on Day 6 (Saturday). The test cages were located in the home room and were similar to the home cages, including bedding, food and water. They were placed on one of four sensing platforms of the LABORASsystem (Laboratory Animal Behaviour Observation, Registration and Analysis System; Metris System Engineering, Hoofddorp, The Netherlands). LABORASis a fully automated device for the recording and analysis of the behaviour of individually-housed mice or rats. The system consists of a triangular-shaped sensing platform, which is positioned on two orthogonally placed sensors and a third fixed point. A Macrolon type II cage (375cm2) or type III cage (840cm2) is placed on the sensing platform. Each sensor transforms the mechanical vibrations caused by the movements of the animal in the macrolon cage into electrical signals, which are amplified and filtered to eliminate noise and then stored on a computer. The software (Metris System Engineering, Hoofddorp, The Netherlandsl contains an analysis and classification module, which processes the stored data and compares the signals with the predetermined characteristic vibration patterns and thus classifies the data into behavioural categories. LABORASfollows the movements continuously, but classifies these signals into behavioural categories each 10s. Details of LABORASare described by Bulthuis et a1. (1997). Van de Weerd et al. (submitted) have validated the LABORASregistrations by comparing them with data from observations of videotapes by human observers. Therefore, LABORASis considered a reliable system for the automated registration of the behaviours 'grooming' (shaking, scratching, wiping or licking fur, snout, ear or genitals), 'locomotion' (walking, running, jumping), and 'inactivity' (no movements while the animal is in a lying or sitting position; short movements, e.g. turning over while sleeping, are not considered as an interruption) in rats. In this
Data processing and statistical analyses Figure 1 shows the average (n = 12)patterns of grooming, locomotion and inactivity on the day prior to the first treatment (Day 0). For 65% of 20 h observation period the rats were inactive. They spent 9% of the time on grooming and 3% on locomotion. Twentythree per cent of the 20 h period was spent on other behaviours{ not recognized ('undefined') by LABORAS.The locomotion pattern compares well with our earlier observations using another method (van Herck et al. 1997). For 15min intervals the recorded behaviours were quantified as relative duration (s/h) and frequency (number/h). In the light period, rats were inactive most of the time. Grooming and locomotion showed activity peaks at the beginning and end of the dark period. During the light period these behaviours were negligible. Based on this activity profile, we arbitrarily identified four time intervals during which animals showed distinct activity patterns, namely 11:3016:30h, 16:30-21:30h{ 21:30-01:00h and 01:00-07:30 h. We anticipated that distinguishing between the four time intervals would increase the chance of detecting treatment effects on behaviour. The behaviours were averaged per rat per treatment for either the whole 20 h period of observation (11:30-07:30 h), the dark period (11:30-23:15 hI, part of the light period (23:1S-07:30h) or for each of the four arbitrarily chosen intervals. Results in the tables are presented as means ± SD. For the behavioural measurements, treatment comparisons were done only within [but not between) the predetermined observational periods. The data for each measure, except for the excitation scores and the number of faecal boli, were subjected to MANOVA (repeated measurements) to identify treatment effects. For excitation score and number of faecal boli, the significance of the differences between treatments was calculated by Friedman two-way ANOVA, whereas the Student's one-sample t-test for paired data Laboratory Animals (2000) 34
van Herck et al.
14
Frequency (number/h)
Relative duration (s/h)
~ 01
c: E 0 0
•.. C)
450
0
00
EO)
c: 0 ~ 0 E
3X)
0 u
0 ....J
o
0
36:XJ
~ ':;
••
15
u ca
c:
o
0
Dark 11 30
1630
Dark
...... Light 21:30
02:>0
0730
time (h)
11:30
LiQ.ht
1630
0230
time (h)
Fig 1 Behaviour patterns prior to the first treatment. Patterns of grooming, locomotion and inactivity on the day prior to the first treatment (Day 0). The behaviours are quantified for 15 min intervals during the period 11:30-07:30 h and represent the means for 12 rats. The relative duration and frequency of each behaviour are expressed per hour. In the animal room, lights were on from 23:15-11:15h
was used for the parameters 'time needed to induce anaesthesia' and 'time needed for recovery'. The pre-set P value was < 0.05. Day of observation, observation period and treatment were used as main within-subject factors. If treatment and/or day of observation after MANOVA (main effect and/or interaction) were found to have a significant effect (p < 0.05) on the behavioural measurements, the Student's one sample t-test for paired data was used to compare the three Laboratory Animals (2000) 34
treatments and/or the days per treatment. To take into account the increased risk of a type I error due to multiple comparisons, the preset P value was reduced to < 0.05/number of meaningful comparisons (Bonferroni correction) i.e. < 0.05/3 = 0.0167 for the 20 h period, to < 0.05/4 = 0.0125 for the dark/light periods and to < 0.05/6 = 0.0083 for the activity blocks. Pairwise comparisons of means for excitation score and number of faecal boli were performed with the Wilcoxon matched-
Orbital sinus blood sampling in rats: effects on behaviour
1S
pairs signed rank test if treatment after Friedman two-way ANOVA was found to have a significant effect on these parameters. The level of significance was then pre-set according to Bonferroni's adaptation at P < 0.025.If treatment after MANOVA was found to have a significant effect on faeces and urine production (both expressed in g)the Student's one-sample t-test for paired data (P < 0.025,Bonferroni correction) was used to compare the three treatments. All statistical analyses were carried out according to Steel and Torrie (19811,using a SPSS/PC+ computer program (SPSSInc. 19901.
Behavioural observations after treatment (Tables 3, 4 and 5) The type of treatment significantly influenced grooming (Table 3), locomotion (Table 4) and inactivity (Table 5). On the day after treatment no significant differences were found between the treatment groups. Anaesthesia reduced the relative duration of grooming during the first part of the dark period (11:30-16:30h) compared with sham anaesthesia. Anaesthesia plus orbital puncture did not significantly influence grooming, compared with anaesthesia alone. However, when compared with anaesthesia alone, anaesthesia plus orbital puncture significantly reduced the frequency and the relative duration of locomotion during the 20 h period (11:30-07:30 h). This was mainly due to a decrease in the dark period (11:30-23: 15h). Also the frequency of inactivity was decreased significantly during the largest part of the dark period (11:30-21 :30h) after orbital puncture under diethyl-ether anaesthesia compared to diethyl-ether anaesthesia alone. Compared to sham anaesthesia, anaesthesia plus orbital puncture caused a decrease in the variables locomotion (frequency: 20 h period, 11:30-07:30h, and relative duration: 20h period, 11:30-07:30hj qark period, 11:3023:15hj first activity block, 11:30-16:30h) and inactivity (frequency: largest part of the dark period, 11:30-16:30h).
Results Observations during treatment (Table 2) Anaesthesia with or without orbital sinus blood sampling had similar duration for induction of anaesthesia and recovery. On average, it took 18s (SD= 4 s, n = 12)to perform blood sampling. During sham anaesthesia, hardly any excitation was observed. There was a significant treatment effect on excitation and production of faeces and urine. The differences between pairs of treatment groups did not reach statistical significance in the paired Student's t-test (faeces and urine production, g) or in the Wilcoxon test for two related samples (excitation score and no. of faecal boli I.
Table 2 Results of observations anaesthesia (A) and anaesthesia
during treatment for sham anaesthesia plus orbital puncture (AP)
(SA), diethyl-ether
Treatment Measure
Significant
A
AP
98 ±21 39 ± 15
97 ±23 43 ± 16
0.17 ± 0.39
1.25 ± 0.75
1.17 ± 0.72
T
0.83 ± 1.27 0.33 ± 0.46 0.16 ± 0.34
2.58 ± 1.08 0.90 ± 0.39 0.53 ±0.27
1.75 ± 1.22 0.65 ±0.47 0.72±0.58
T
SA
Anaesthesia Induction (s) Recovery (s)
difference·
Excitation (score 0,1,2) Faeces production (no. of boli) (g) Urine production
(9)
T T
• Significant (P < 0.05) effects of treatment according to MANOVA (faeces production (g) and urine production (g)) or Friedman two-way ANOVA (excitation score and faeces production. no. of boli) are indicated by T Results are expressed as means ± SD (n = 12) Laboratory Animals (2000) 34
16
van Herck et a/.
Table 3 Grooming behaviour in rats after they had been subjected to either sham anaesthesia ether anaesthesia (A) or anaesthesia plus orbital puncture (AP) Relative duration (s/h) Day of treatment Period'
SA
20h (11:30-07:30 h) Mean 322.5 SD 60.6 Dark/light 11:30-23:15 h Mean 464.5 SD 85.3 23:15-07:30 h Mean 109.4 SD 33.5 Activity blocks 11:30-16:30 h Mean 527.7" 5D 84.8 16:30-21 :30 h Mean 369.9 SD 90.5 21:30-01 :00 h Mean 417.9 SD 165.5 01:00-07:30 h Mean 76.7 SD 18.5
(SA), diethyl-
Frequency (number/h) Day after treatment
Day of treatment
Day after treatment
SA
SA
A
AP
A
A
AP
SA
A
AP
AP
298.4 57.7
279.7 103.4
334.9 74.6
312.9 88.3
313.6 97.4
13.5 1.6
12.9 1.5
12.0 2.8
13.2 1.3
13.4 1.4
12.9 1.9
428.4 78.1 103.5 37.5
408.0 156.3 87.2 31.5
491.0 109.9 101.1 39.2
464.5 129.5 85.5 33.5
451.9 137.4 106.3 43.3
17.4 2.6 7.7 1.2
16.7 1.8 7.2 1.4
15.5 3.6 6.7 1.8
17.5 1.7 6.7 1.3
17.8 1.7 6.8 1.6
17.0 2.3 6.6 1.7
447.1" 104.9 355.7 94.3 431.1 1-18.3 68.5 27.3
399.9' 171.7 375.1 183.6 379.0 131.1 60.3 23.3
527.2 156.5 412.8 144.3 428.8 111.8 76.8 39.4
482.5 162.1 409.0 150.2 399.2 81.4 62.2 29.4
537.0' 176.7 340.5 145.0 383.9 154.9 83.4 41.9
20.3 2.1 13.5 3.2 17.3 4.6 6.3 1.2
18.1 2.4 14.2 1.8 16.9 3.5 5.8 1.3
16.7 4.3 12.4 4.4 16.8 3.6 5.4 1.8
18.9 2.0 14.9 2.4 16.6 3.2 5.6 1.4
19.3 2.4 14.9 3.1 17.2 1.6 5.6 1.6
18.5 2.3 14.3 3.8 16.1 3.8 5.8 1.8
'The periods for which the data are presented correspond with the time axis illustrated in Fig 1 Results are given for 12 rats. Within one row of a variable, values with the same let1er are si9nificantly different
Table 4 Locomotion behaviour in rats after they had been subjected to either sham anaesthesia ether anaesthesia (A) or anaesthesia plus orbital puncture (AP) Relative duration (s/h)
Period'
(SA), diethyl-
Frequency (number/h)
Day of Treatment
Day after treatment
Day of treatment
Day after treatment
SA
SA
SA
SA
A
AP
20h (11:30-07:30 h) Mean 80.9" 81.8b 63.2"·b.< SD 19.1 20.5 15.2 Dark/light 11:30-23:15 h Mean 116.5" 116.3b 89.0",b SD 29.6 32.9 28.3 23:15-07:30h Mean 27.5 29.9 24.5 5D 6.1 5.7 9.6 Activity blocks 11:30-16:30 h Mean 151.1" 144.2 100.5" SD 38.6 47.5 34.0 16:30-21 :30 h Mean 75.5 64.6 84.7 SD 33.2 24.6 31.3 21:30-01:00h Mean 114.6 113.0 105.1 SD 39.4 59.9 38.6 01:00-07:30h Mean 12.9 14.7 11.0 SD 5.0 7.8 4.7
A
AP 75.3< 18.4
9.6d 2.2
A
78.8 13.9
79.4 15.2
113.4 24.3 26.9 8.4
113.7 25.7 27.9 8.7
105.6 27.3 30.0 9.9
13.8 3.4 3.3 0.7
13.6 3.6 3.6 0.6
134.4 40.6 80.9 22.4 118.1 41.5 13.4 7.1
132.3 27.9 80.2 31.6 120.4 41.2 16.0 8.6
118.5 30.5 75.2 28.8 122.3 66.8 17.1 10.8
18.0 4.4 9.2 3,6 13.0 4.5 1.6 0.7
16.3 4.7 10.5 3.2 13.3 6.7 1.8 0.6
9.6" 2.2
AP 8.1d," 2.2
A
AP
9.3 1.8
9.4 1.7
9.0 2.5
11.5 3.8 3.0 1.1
13.5 3.2 3.1 1.1
13.4 3.0 3.3 0.8
12.9 3.7 3.3 0.9
12.8 4.0 8.4 4.0 13.5 4.8 1.4 1.0
16.1 5.0 9.8 2,8 13.0 4.1 1.7 1.0
15.7 3.3 9.8 3.8 13.5 4.0 1.9 0.8
14.6 4.0 9.3 3.9 13.7 7.1 2.0 1.1
• See legend to Table 3 Results are given for 12 rats. Within one row of a variable. values with the same let1er are significantly different Laboratory Animals (2000) 34
Orbital sinus blood sampling
in rats: effects on behaviour
17
Table 5 Inactivity behaviour in rats after they had been ether anaesthesia (A) or anaesthesia plus orbital puncture Relative duration
(s/h)
Oay of treatment
Dark/light 11:30-23:15h 23:15-07:30h Activity blocks 11 :30-16:30 h 16:30-21 :30 h 21:30-01:00h 01 :00-07:30 h
to either
sham
Frequency Oay after treatment
anaesthesia
(SA), diethyl-
(number/h)
Oay of treatment
Oay after treatment SA
SA
A
AP
SA
A
AP
SA
A
AP
Mean SO
2500.5 82.2
2531.7 81.7
2551.5 146.6
2468.3 104.4
2493.1 97.4
2480.9 120.7
16.4 1.9
16.2 1.3
14.7 2.0
16.1 1.4
16.1 1.9
16.0 1.2
Mean 50 Mean SO
2072.4 124.3 3142.6 59.9
2126.2 123.3 3140.0 66.9
2159.0 230.6 3140.4 120.7
1998.1 183.8 3173.0 59.0
2033.5 142.3 3182.1 113.8
2065.8 184.2 3103.3 204.6
19.8 2.7 11.2 1.3
19.6 2.0 10.9 0.8
17.4 3.0 10.8 1.6
19.8 2.' 10.5 1.1
19.6 2.7 10.8 1.6
19.5 1.9 10.7 1.5
Mean SO Mean SO Mean SO Mean SO
1765.6 229.2 2490.0 222.6 2108.1 455.6 3285.0 86.2
1915.7 219.5 2438.2 179.9 2140.8 340.1 3288.1 71.0
2051.1 348.7 2440.9 322.2 2102.4 284.8 3263.5 136.5
1781.3 272.6 2343.0 294.4 2100.8 370.1 3289.8 76.3
1834.9 237.3 2403.7 239.7 2066.3 333.1 3297.3 81.4
1871.0 228.6 2443.0 273.0 2059.9 408.9 3205.4 216.4
22.7" 3.4 16.6 2.9 18.8 4.9 10.1 1.4
21.0b 2.9 18.1" 2.7 18.8 3.7 9.6 1.0
17.4"·b
21.3 2.9 17.7 2.6 18.6 4.2 9.4 0.9
20.7 2.9 18.1 3.1 18.5 4.0 9.7 1.8
20.1 1.6 17.5 3.0 19.6 4.3 9.7 1.9
Period' 20h (11 :30-07:30 h)
subjected (AP)
2.8 15.2" 4.2 19.6 3.4 9.7 1.7
A
AP
, See legend to Table 3 . Results are given for 12 rats. Within one row of a variable. values with the same letter are significantly different
Discussion In this study we assessed the impact on behaviour of orbital sinus blood sampling in rats while they were under diethyl-ether anaesthesia. To monitor behaviour, the socalled LABORAS system (Bulthuis et al. 1997) was used. This system allows automatic behaviour registration without disturbing the animals. The various types of behaviour were quantified in terms of relative duration and frequency for different periods during the dark and light phases. Treatments were performed immediately before the dark period. Registration of behaviour began with the onset of the dark period. In this way the normal behaviour during the light period was minimally disturbed and the effect of treatments was registered from the beginning of the a'ctive period. Grooming, locomotion and inactivity were identified. Treatment had no significant effect on behaviour registered one day later. This indicates that the effects of treatments vanished within a period of 24 h. Likewise, in earlier studies with rats, no long-term
(> 24 h) effects of diethyl-ether anaesthesia and orbital puncture were found, neither on heart rate, body temperature, locomotor and eating activity (van Herck et al. 1997), nor on plasma levels of catecholamines and corticosterone (van Herck et al. 1991). Compared to sham anaesthesia, animals punctured while under diethyl-ether anaesthesia spent significantly less time on locomotion and had a significantly higher frequency of inactivity, mainly during the first activity period after treatment (11:30l6:30h). If changes in locomotion and/or inactivity are associated with discomfort, it can be concluded that orbital bleeding under diethyl-ether anaesthesia elicits discomfort for a period of about 5 h. Diethyl-ether anaesthesia versus sham anaesthesia caused a significant decrease in time spent on grooming, but this was seen only during the first 5 h after treatment. Compared to diethyl-ether anaesthesia alone, orbital sinus blood sampling caused a significant decrease in the frequency and the relative duration of locomotion during the entire 20h period (11:30-07:30 h) and in the Laboratory Animals (2000) 34
van Herck et a/.
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frequency of inactivity during the largest part of the dark period (11:30-21:30). Several automated systems have been used to register behaviour for the purpose of assessing possible discomfort caused by experimental procedures. The Disturbance Index of Barclay et al. (1988) is based on influencing the investigative and scent marking behaviour of rodents in a novel environment by a preceding procedure. The number of movements of the cage of test and control animals is registered with an automated system and totalled per selected time period. Hayes and Flecknell (1999) studied locomotor activity. They divided the cage into 12 areas of 15 x 12cm by using an array of four by three beams and took the number of beam interrupts per one-hour period as a measure. Van Herck et al. (1997) determined locomotor activity telemetrically. Compared to those approaches, LABORASprovided detailed information on a continuous time scale of both the frequency of and the time spent on the behaviours grooming, locomotion and inactivity. The Disturbance Index does not provide information about specified behaviours. The infrared beam array system addresses only the frequency of locomotion. The telemetry system id,entifies both the frequency and the time but also only of locomotor behaviour. The importance of identifying several behavioural categories was shown by Baumans et al. (1997), investigating the effect of an i.p. injection of saline in mice with a balance platform. They found an increase in grooming and resting and a decrease in climbing and locomotor behaviour in the first hour after injection. Such opposite changes may affect the results obtained by the Disturbance Index. Because behaviours occur in distinct phases, all methods mentioned divide the observational period into phases before analysing the results. Assuming that the Disturbance Index gives an indication of locomotor activity, all methods mentioned in general show a decrease of locomotion after experimental procedures. This corroborates the suggestion of Barclay et al. (1988) that rats respond to injury with a reduced level of activity or even immobility. However, the differences both between the registration Laboratory Animals (2000) 34
systems and between the way the studies were performed and data were analysed are too large to compare the results obtained by the methods used in detail. Although injuries to the body surface are often followed by grooming and licking (Spruijt et al. 1992Lwe did not find an increase of grooming after orbital puncture under diethyl-ether anaesthesia. This might indicate that the animals experience the wound caused by orbital puncture to be minor. If self-grooming reflects the process of de-arousal due to the termination of or habituation to a stressful situation (Spruijt etal. 1992),the period of arousal after diethylether anaesthesia with and without orbital puncture was limited to the first activity block 111:30-16:30h). As far as we know, this is the first study showing an effect of orbital sinus blood sampling on behaviour that is superimposed on the effect of diethyl-ether anaesthesia alone. In our earlier studies, orbital puncture versus diethyl-ether anaesthesia alone was found not to affect open-field behaviour of rats (Beynen et aI. 1988b) and not to affect telemetrically determined locomotor activity (van Herck et al. 1997).Thus, we now report that orbital puncture influences locomotion, whereas we previously found (van Herck et aI. 1997) that it did not. This discrepancy possibly lies in the fact that puncture was now performed at the end of the light period, immediately before the first activity peak of the animals during dusk. In conclusion, this study shows, for the first time, that orbital sinus blood sampling depressed locomotion and diminished the frequency of inactivity periods during the dark period. Assuming that these changes are a sign of discomfort, it then follows that orbital puncture per se causes discomfort. This assumption however, may be subject to debate. It could be suggested that the use of appropriate analgesia is required to demonstrate first that the behavioural changes observed in this study are indeed caused by discomfort (Flecknell & Liles 1992). Acknowledgments
The authors thank F. A. Schlingmann for his technical assistance when using LABORAS, Dr S. F. de Boer for his advice on data
Orbital sinus blood sampling in rats: effects on behaviour
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analyses and for critically reading the manuscript and J. D. Tanner for her advice on spelling and grammar.
Morton DB, Abbot D, Barclay R, et a1.11993) Removal of blood from laboratory mammals and birds. Laboratory Animals 27, 1-22 Spruijt BM, van Hooff JARAM, Gispen WH (1992) Ethology and neurobiology of grooming behaviour. Physiological Reviews 72, 825-51 SPSS Inc (1990) SPSS/PC+® 4.0 Advanced Statistical Manual for the IBM/PC/XT/AT and PS/2V (Release 4.0). SPSS Inc. Chicago, 11,USA Steel RGD, Torrie JH (1981) PrinCiples and Procedures of Statistics. A Biomedical Approach, 2nd edn. Singapore: McGraw-Hill van de Weerd HA, Bulthuis RJA, Bergmann AF, et a1. (submitted) Validation of a new system for the automatic registration of behaviour in mice and rats van Herck H, Baumans V, de Boer SF, van der Gugten J, van Woerkom AB, Beynen AC (1991) Endocrine stress response in rats subjected to singular orbital puncture while under diethyl ether anaesthesia. Laboratory Animals 25, 325-9 van Herck H, Baumans V, Stafleu FR, Beynen AC 11992a) A questionnaire-based inventory of the orbital puncture method in The Netherlands. Scandinavian lournal of Laboratory Animal Science 19, 189-96 van Herck H, Baumans V, van der Craats NR, et a1. (1992b) Histological changes in the orbital region of rats after orbital bleeding. Laboratory Animals 26, 53-8 van Herck H, de Boer SF, Hesp APM, van Lith HA, Baumans V, Beynen AC (1997) Orbital bleeding in rats does not influence telemetrically determined heart rate, body temperature, locomotor and eating activity when compared with anaesthesia alone. Laboratory Animals 31, 271-8 van Herck H, Ballmans V, Brandt CJWM, et a1. (1998) Orbital sinus blood sampling in rats as performed by different animal technicians: the influence of technique and expertise. Laboratory Animals 32, 377-86 Vermeulen JK, de Vries A, Schlingmann F, Remie R 11997) Food deprivation: common sense or nonsense? Animal Technology 48, 43-54
References Barclay RJ, Herbert WJ, Poole TB (1988) The Disturbance Index: a behavioural method of assessing the severity of common laboratory procedures on rodents. In: UFAW Animal Welfare Research Report 2. Potters Bar (UK): Universities Federation for Animal Welfare, pp 1-35 Baumans V, Schlingmann FA, van Herck H, Boere HAG, Tolboom J (19971 Assessment of discomfort in the mouse by means of a balance device. In: Harmonization of Laboratory Animal Husbandry, Proceedings of the 6th FELASA Symposium IO'Donoghue PN, ed). London: Royal Society of Medicine Press, pp 14--16 Beynen AC, Baumans V, Haas JWM, van Hellemond KK, Stafleu FR, van Tintelen G 11988a) Assessment of discomfort induced by orbital puncture in rats. In: New Developments in Biosciences: their Implications for Laboratory Animal Science. Dordrecht: Martinus Nijhoff, pp 431-6 Beynen AC, van Tintelen G, Baumans V 11988b) Orbital puncture may not influence open field behaviour in rats. Zeitschrift fUr Versuchstier· kunde 31, 121-3 Bulthuis RJA, Bergman AF, Nijessen S, et a1. (1997) Automated behaviour classification: the LABORAS project. In: Harmonization of Laboratory Animal Husbandry, Proceedings of the 6th FELASA Symposium (O'Donoghue PN, ed). London: Royal Society of Medicine Press, pp 17-18 Flecknell PA, Liles JH (19921 Evaluation of locomotor activity and food and water consumption as a method of assessing postoperative pain in rodents. In: Animal Pain (Short CE, van Poznak A, edsJ. New York: Churchill Livingstone, pp 482-8 Hayes JH, Flecknell PA (1999) A comparison of preand post-surgical administration of bupivacaine or buprenorphine following laparotomy in the rat. Laboratory Animals 33, 16-23
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