Abstract
The objective of this study was to investigate the effect of ground transportation on guineapigs. Physiological parameters, i.e. heart rate (HR), body temperature (BT) and activity (ACT), were measured before and after transport, using previously implanted radiotelemetry transmitters. Body weight was measured before and after transport. After a postsurgical recovery period and data recording at the breeder's facility, the animals were transported for 2.25 h (Group 1) and for 7.5 h (Group 2) to a different animal facility. Data collection started immediately after arrival at the second animal facility. All parameters measured changed significantly after transport. These results suggest that a 10- to 12-day period is required for guineapigs to return to pre-transport levels of HR, BT and ACT.
Keywords
Significant gains and improvements have been made in recent decades not only to reduce, refine and replace animals in experiments, but also to provide for improved housing conditions, exercise and environmental enrichment. One area of animal welfare that has not received sufficient attention in the scientific literature is the transportation of laboratory animals from breeding facilities to research institutions. Animal welfare regulations have stimulated consolidation within the research animal breeding industry, leading to the elimination of many smaller regional facilities and breeding activities within research institutes. As a result, a greater percentage and growing number of animals (mostly rodents) are bred in fewer licensed facilities and transported over longer distances to research locations. Animals received by research facilities are usually immediately quarantined (generally for a minimum of 7 to 10 days), as protection against the introduction of infectious agents and also to permit the animals to recover from any stress associated with transportation.
The published guideline on the transport of laboratory animals states that ‘change is stressful to animals, and transport is an especially powerful stressor that should be regarded as a major life event and not undertaken unless absolutely necessary’. 1
As summarized in this guideline, there are many aspects that have a direct impact on animal welfare. After transport the animals have to get used to a different facility, different caretakers and a different environment (light, water, noise, etc.), including food and different bedding. The packing and unpacking of the animals before and after transportation also cause stress. In general, animals subjected to the environmental changes that occur during and after transportation react with changes in physiological parameters such as body weight (BW), plasma hormonal levels, heart rate (HR) and blood pressure (BP). 1 Animals can be stressed by either psychological stresses (restraint, handling or novelty) or physical stresses (hunger, thirst, fatigue, injury or thermal extremes) that can cause variability in the condition of individual animals and thus lead to unreliable research results. 2 To foster good scientific practice, animals should be used in experimental procedures only after complete adaptation to their new situation and the stabilization of their physiological parameters.
Therefore, after each transport, an acclimatization period is necessary before laboratory animals are used in scientific research projects. In general – this is almost a gold standard – a seven- to 10-day acclimatization period is advised after transport. However, in practice, acclimatization varies from two days to two weeks. When measurements of physiological parameters are performed using conventional techniques, the results must be interpreted with caution, as these techniques also affect the animals. Radiotelemetry provides an alternative means of obtaining physiological measurements from awake and freely moving laboratory animals, without introducing stress artefacts. 3 It is being increasingly used as a procedure for stress-free measurement of physiological parameters in small laboratory animals, and in a wide range of physiological, pharmacological and/or behavioural-based research. Totally implantable miniaturized radiotelemetry systems are a valuable tool for animal welfare research studies because these systems can eliminate handling and other factors that may influence results. 3 Moreover, HR and body temperature (BT) can be used as indicators of animal stress and a measurement of adaptation to the new environment. 3
Few studies have been carried out to assess the impact of transport on stress and the time required for values to return to baseline levels. 1 Radiotelemetry has also so far not been reported as a method for measuring animal stress and adaptation after transport. Recently, our group 4 demonstrated – using radiotelemetry – that a three-day period was required for rats to return to pre-transport levels of HR, BT, activity (ACT) and BW.
This study was designed to investigate the extent of physiological stress responses (HR, BT, ACT and BW) in guineapigs after van transport. After a recovery period from surgery (7 days) and data recording (Group 1: 15 days; Group 2: 22 days) at the breeder's unit, the animals were transported for 2.25 h (Group 1) and for 7.5 h (Group 2) to a different animal facility. Data collection (Group 1: 21 days; Group 2: 14 days) started immediately after arrival. HR, BT and ACT were measured in guineapigs before and after transport using previously implanted radiotelemetry transmitters. BW was also measured before and after transport.
Materials and methods
Animals and housing
Eighteen female guineapigs (Group 1: HsdPoc:DH, BW 251 ± 5 g; Harlan NL, Horst, The Netherlands) and 22 female guineapigs (Group 2: HsdPoc:DH, BW 343 ± 27 g; Harlan NL) were housed in a specific pathogen free (SPF) barrier (following Federation of European Laboratory Animal Science Associations' (FELASA) recommendations) in a light- and temperature-controlled room at the Harlan facility. During the experiment, a 14 h:10 h light/dark cycle (lights on at 04.30, lights off at 18.30 [from 04:30 h to 06:00 h and from 18.00 h to 18.30 h there was twilight]), room temperature (17–20°C), and relative humidity (52–75%) were maintained. There were 12 100% fresh air changes per hour.
The animals were housed in Makrolon type 4 cages with Tierwohl Classic® bedding (Rettenmaier & Söhne, Rosenberg, Germany). Guineapigs had free access to a standard irradiated diet (Harlan Teklad Global Guinea Pig Diet, 2040) and processed (acidified [pH 5.8–6.4]), chlorinated (6–8 ppm), softened and filtered (0.02 micron) water
During transport three animals were transported in one box (55 × 35 × 20 cm, Williton Box Co, Taunton, UK) with standard food pellets (Harlan Teklad Global Guinea Pig Diet, 2040) and Hydrogel™ (Harlan, IN, USA) as a water source. After transport the animals were housed in Makrolon type 4 cages with Aspen bedding in a light- and temperature-controlled room (lights on at 07:00 h, lights off at 19:00 h; room temperature 20–22°C; relative humidity 45–55%) at the receiving facility. The guineapigs had free access to a standard diet (Harlan Teklad Global Guinea Pig Diet, 2040) and normal tap water
Transmitter implantation
Two days before surgery, the animals received Vitamin C in their drinking water (100 mg/100 mL). For analgesia, the guineapigs were given a subcutaneous injection (s.c.) of Tramal 100® (Tramadolhydrochloride) (Group 1: 0.05 mL; Group 2: 0.1 mL; ALTANA Pharma BV, Hoofddorp, The Netherlands) 2 h before surgery. Just prior to surgery, the animals also received 0.2 mL Baytril® (enrofloxacin) 2.5% s.c. (Bayer BV, Mijdrecht, The Netherlands).
Six guineapigs (Group 1) and seven guineapigs (Group 2) were implanted with a radiotelemetry transmitter (TA11CTA-F40, Data Sciences International, St Paul, MN, USA). One animal in Group 2 died of unknown causes during surgery, but this guineapig was replaced by another one. The animals were anaesthetized with an intraperitoneal injection (i.p.) with Narketan® (ketamine) 10 (Group 1: 0.15 mL; Group 2: 0.2 mL; Vetoquinol BV, Den Bosch, The Netherlands), followed by an intramuscular injection (i.m.) of Rompun® (xylazine) (Group 1: 0.05 mL; Group 2: 0.08 mL; Bayer BV, Mijdrecht, The Netherlands). During surgery, local anaesthesia was administered (Lidocaine®, Eurovet Animal Health, Cuijck, The Netherlands) and the eyes were protected with Duodrops® (Produlab Pharma, Raamsdonkveer, The Netherlands).
The implantation procedure was carried out under strict aseptic conditions as described by Kramer
Experimental design
At the Harlan facility, pre-weaned guineapigs were normally housed in plastic tubs before being transferred to stainless steel cages at the time of weaning. The stainless steel cages could not be used in this study due to interference with the radiotelemetry signal system. Therefore, one week before the start of the study the animals in both groups were placed in Makrolon type 4 cages, in order to become accustomed to the cages.
Two days before the start of the experiment and five days after surgery, the animals were provided with Vitamin C through their drinking water. This supplement was given prophylactically to compensate for possible lower postoperative food consumption. Surgery took place behind the barrier of the breeder (Harlan NL), thus ensuring that no animals were transported prior to the study. Six animals in Group 1 and seven in Group 2 were prepared for surgery by animal caretakers and surgery was performed by an experienced surgical technician and a veterinarian. As soon as each animal regained consciousness after surgery, it was group-housed with two non-implanted guineapigs.
These three guineapigs remained together during the entire study. During the first week after surgery, the animals were weighed and checked daily. After one week of recovery HR, BT and ACT were measured every 3 min for 10 s, 24 h per day. The guineapigs of Group 1 were transported three weeks after surgery (1 week of recovery and 2 weeks of collecting control data at the breeder) and the guineapigs of Group 2 were transported four weeks after surgery (1 week of recovery and 3 weeks of collecting control data at the breeder). The animals were prepared for transport in the same room in which they were housed. The guineapigs were moved from their cage, weighed and allocated with their social pairs in solid floor plastic transport boxes with filters (55 × 35 × 20 cm, Williton Box Co). Wood bedding were placed in the boxes (Tierwohl, Classic® bedding), diet pellets (Harlan Teklad Global Guinea Pig Diet, 2040), irradiated hay and Hydrogel™ as a water source. The lids of the boxes were closed and taped to prevent escape.
The animals in Group 1 were packed in the transport boxes at 13:00 h and, after 2.75 h, the boxes were taken to the holding area, where the lights are on from 08:00 h to 17:00 h. The temperature in the holding area was approximately 18°C. The next day, at 06.00 h, the animals were loaded onto a van and transported to the second animal facility. The journey lasted 2.25 h. As soon as the animals arrived at the receiving facility, they were placed on the telemetry receivers for one hour before they were unpacked and weighed.
The guineapigs in Group 2 were packed in the transport boxes at 07:20 h, and at 12:55 h, the boxes were taken to the holding area. The next day at 07:00 h, the animals were loaded on the van and transported to the second animal facility. The journey lasted 7.5 h. On the way to the receiving facility, the van stopped at five other animal facilities to unload animals. As soon as the guineapigs arrived at the receiving facility, they were placed on the telemetry receivers for one hour before they were unpacked and weighed. At the receiving facility, data recording resumed every 3 min for 10 s, 24 h a day for three weeks (Group 1) and for two weeks (Group 2).
Statistical analysis
For consistent and reliable analysis, the individual mean hour HRs, BTs and ACTs were first calculated (i.e. means over all data from the whole hour until one minute before the next hour). These individual mean values were analysed using maximum likelihood analysis. If only one measurement was included to calculate the individual mean, this mean was not used in the analysis.
First the factors ‘hour’, ‘day’ and ‘subject’ were included as categorical variables to see whether the values increased continually day by day or not. The complete study period was thereafter divided into four (Group 1) or three (Group 2) different periods to distinguish between pre- and post-transport and between periods with rising, falling or stable levels.
The second maximum likelihood analysis was performed on the same data with the categorical factors ‘hour’, ‘period’ and ‘subject’ and the continuous factor ‘day’. The value of hour is a representation of the number of hours after the start of the light period in the dark/light cycle. By including the factor ‘hour’, the overall circadian rhythm effect could be excluded from any other estimation. By including the ‘subject’ as a random factor, individual subject variations were excluded from the analysis.
The overall means per period and daily increase or decrease per period were obtained from the statistical analysis. Period means are a representation of the mean value in the middle of the respective period. Differences between periods in overall means and daily increase/decrease were calculated using linear contrasts.
The estimated means based on the first maximum likelihood analysis with ‘day’ as a categorical factor are presented in the tables below. Based on these estimates, the periods per group are divided as follows: Group 1: day −15 to day −11 (HR levels remain relatively stable); day −10 to day −1 (HR levels decrease before transport); day 0 to day 9 (HR levels decrease after transport); day 10 to day 21 (HR levels remain relatively stable). BT and ACT do not show a distinct pattern but to be consistent: the same division was used as for HR. Group 2: day −22 to day −17 (HR levels are relatively high); day −16 to day −1 (HR levels and other variables are relatively stable); day 0 to day 14 (levels of all variables are relatively stable).
Results
Both groups
Data obtained from six guineapigs (Group 1) and seven guineapigs (Group 2) were included in the analysis. One of the animals from Group 1 was euthanized after 17 days because it developed an inflammation at the surgical site.
Group 1
Comparison of heart rate, body temperature and activity data pre- and post-transport
Statistically significant differences were detected between hours, periods and the increase or decrease in daily levels within periods.
The mean HR declined throughout the study (Table 1). This was mainly due to a fall in the periods from day 10 before transport until 10 days after transport. The mean for the post-transport period (day 0 to day 9) was significantly lower than that for the pre-transport period (day −10 to day −1). However, the steepness of the fall in HR during the two periods was not significantly different. This indicates that the decline in HR that started in the pretransport period (day −10 to day −1) continued at the same rate during the post-transport period (day 0 to day 9).
Group 1 (
Confidence interval (CI) stands for the reliability of the values. Slope means the increase or decrease per day
*
The mean BT was stable in the pre-transport period (day −15 to day −1), but significantly lower in the post-transport period (day 0 to day 21) (Table 2). During the pre-transport (day −10 to day −1) and post-transport period (day 0 to day 21), BT remained stable (a drop of 0.001 and 0.002°C/day, respectively) indicating that the decrease in temperature was caused by a sudden change between both these periods. This could be due to the new environment after transport.
Group 1 (
Due to rounding the difference between periods can be slightly different than expected based on the presented values. Confidence interval (CI) stands for the reliability of the values. Slope means the increase or decrease per day
*
The mean ACT score was lower in the period before transport (day −10 to day −1) and in the second period (day 11 to day 21) after transport (Table 3). In the post-transport phase (day 0 to day 10), the mean ACT score started high and dropped considerably (–0.263/day) during the next period (day 11 to day 21). From these results, it can be concluded that activity levels were higher after transport, but fell as the animals got used to their new environment.
Group 1 (
Due to rounding the difference between periods can be slightly different than expected based on the presented values. Confidence Interval (CI) stands for the reliability of the values. Slope means the increase or decrease per day
*
Comparison of body weight data pre- and post-transport
The total time the animals spent in the boxes was 20.25 h. During this time, the animals lost 7.4 ± 3.1% of their BW despite having enough food, hydrogel and hay. After transport it took 17 days before all the animals regained their pre-transport BW levels.
Group 2
Comparison of heart rate, body temperature and activity data pre- and post-transport
With regard to HR and ACT, statistically significant differences were detected between hours, periods and between increase or decrease in daily levels per period.
For BT, there were statistically significant differences between periods and between the increase or decrease in daily levels within periods.
A drop in HR from period 2 (day −17 to day −1) onwards is shown in Table 4. The means of the three periods also differed significantly. During period 2 (day −17 to day −1) and period 3 (day 0 to day 14), there was a significant decline in HR. In addition, the fall in HR in period 3 (day 0 to day 14) was significantly higher than in period 2 (day −17 to day −1).
Group 2 (
Due to rounding the difference between periods can be slightly different than expected based on the presented values. Confidence interval (CI) stands for the reliability of the values. Slope means the increase or decrease per day
*
No overall significant differences in BT on an hourly basis could be observed throughout the study, indicating that no clear overall circadian rhythm was distinguished. Table 5 shows that the mean BT was significantly lower in period 3 (day 0 to day 14) than in period 2 (day −17 to day −1) and there appeared to be a sharper fall in BT during period 3. This could be explained by a few unexpected lower measurements on day 12.
Group 2 (
Due to rounding the difference between periods can be slightly different then expected based on the presented values. Confidence interval (CI) stands for the reliability of the values. Slope means the increase or decrease per day
*
As can be seen from Table 6, the mean ACT score in period 3 (day 0 to day 14) was significantly lower than in period 1 (day −22 to day −18) but not significantly different from period 2 (day −17 to day −1). The reduction in ACT was significant both in period 2 (day −17 to day −1) and in period 3 (day 0 to day 14). However, the decline in period 3 was significantly less than in period 2.
Group 2 (
Due to rounding the difference between periods can be slightly different then expected based on the presented values. Confidence interval (CI) stands for the reliability of the values. Slope means the increase or decrease per day
*
Comparison of body weight data pre- and post-transport
The total time the guineapigs spent in the boxes was 32 h, 10 min. During this time, the guineapigs lost 7.7 ± 2.2% of their BW despite being provided with enough food, hydrogel and hay. After transport it took 14 days for all the guineapigs to regain their pre-transport BW.
Discussion
Our results suggest that a 10- to 12-day recovery period is required for guineapigs to return to pre-transport levels of HR, BT and ACT. It took 14 (Group 1) to 17 (Group 2) days before all the guineapigs regained their pre-transport BW.
This recovery period is longer than the recovery period that rats are reported to require. Recently, it was reported that after a 5 h transport to a different animal facility a three-day recovery period was required for rats to return to pre-transport levels of BW, HR, BT and ACT. 4
This adaptation period is similar to results published by Van Ruiven
Mice required an adaptation period from several hours to four days after transport.
7–10
Tuli
HR has been used as an indicator for measuring animal welfare and stress.
12,13
Directly after transport and for a period of 10 days thereafter we found a reduction in HR (bradycardia) in both groups. We also observed, after an initial increase, a fall in HR after surgery. From five to six days after surgery, we observed a single case of bradycardia that stabilized after another six to seven days (3 to 4 days before transport). We do not have a full explanation for the bradycardia seen several days after surgery. Harper
It is well documented that severe stress situations such as during transportation may cause bradycardia. Recently, bradycardia was also reported in rats after transport. 4 A lower HR has also been described in mice by Chin, 16 when the mice were exposed to stress by being put in a room with bright fluorescent light and noise. Several other publications have reported a reduction in HR in mice and rats placed in restrainers, which was also considered to be stressful. 17–19
Meerlo
In our study, BT fell significantly after transport. In both groups, post-transport BT was significantly lower than pretransport BT (
The significant increase we found in ACT after transport is in accordance with other transport studies. 4,9,26 We also share their conclusion that the increase in ACT is explained by increased exploratory behaviour after transportation.
In both groups we found a reduction in BW of almost 8% after transportation. However, recently we have found (data not shown) that during a pre-transport period (23 h and 40 min), when the animals were placed in transport boxes and remained in the barrier, guineapigs lost 5.4% of their BW. Another group of guineapigs, which were also packed in transport boxes and then remained in the barrier for 5 h and 35 min, followed by hand transportation (transport time: 2 min) to the holding area where they remained for another 18 h and 5 min, lost 7.3% of their BW. Although these preliminary results indicate that most of the loss of BW is caused by the stress of packing and transportation to the holding area, and not by the transport itself, further studies need to be performed to examine the effect of holding time on the BW of animals before, during and after transportation. It is also suggested that, if a light–dark shift occurs during transport, the period of adaptation is likely to be extended significantly.
4,26
In our study, the light cycle was not modified, because light cycles could not be programmed in the transport van. The animals therefore experienced a light to dark change during the time that they were transported (Group 1: 2.25 h; Group 2: 7.5 h), and this took place in total darkness. Hasegawa
Moreover, in this study there was a difference in light and dark cycles between the breeder's and the receiving facility, i.e. going from 14 h of light at the breeder's facility to 12 h of light at the receiving facility. Although we realized at the beginning of this study that an acute change in light and dark cycles can have an impact on the physiological state of the animals and a potential impact on animal welfare, we decided not to synchronize the light and dark cycles between both facilities. From our own experience we know that there will usually be a difference in light and dark cycles between breeders and the receiving companies. But, in this first transport study, we were only interested in the possible changes in the physiological parameters under standard housing and transport conditions.
In future transport studies, we will focus our research on more details, i.e. synchronizing the light and dark cycles between the breeder's and the receiving facility; synchronizing the light and dark cycles during the whole transport period (including the holding area at the breeder and the transport van), measuring food and fluid intake during transport, measuring stress hormones, recording vocalization, aligning the conditions for the animals between the breeder's and the receiving facility (e.g. the same food, water quality, bedding, room temperature, humidity and type of cages).
Information on HR, BT, ACT and BW can be used to assess the acclimatization time after transportation for guineapigs moved from breeders to other facilities. This study suggests that the acclimatization period for guineapigs transported for several hours is 10–12 days. However, the parameters measured in this first pilot study provide only a few reference points, and many other parameters, such as levels of circulating corticosterone and glucose, adrenal gland mass, behaviour, and food and water intake, could also be modified by transportation of various durations. With more knowledge of the effects of transport stress on the physiology of small laboratory animals, we will be able to obtain objective information regarding acclimatization, which can be used to define more accurate acclimatization periods and provide information for local, national and international guidelines and standard operation procedures for the transport of small laboratory animals.
Footnotes
ACKNOWLEDGEMENTS
This study was financially supported by Harlan Netherlands and the authors would especially thank General Manager Mr Jan Bartels as well as the animal caretakers who work in Barrier 3 at Harlan Netherlands. Dr Robert J Russell from Harlan USA, Dr Markus Josten from Harlan/RCC and the anonymous referees are acknowledged for their helpful comments on this manuscript.