Journal of the American Association for Laboratory Animal Science Copyright 2014 by the American Association for Laboratory Animal Science
Vol 53, No 2 March 2014 Pages 161–166
The Social Buffering Effect of Playful Handling on Responses to Repeated Intraperitoneal Injections in Laboratory Rats Sylvie Cloutier,1,* Kim Wahl,1 Chelsea Baker,1 and Ruth C Newberry1,2 Handling small animals for veterinary and experimental procedures can negatively affect animal wellbeing. We hypothesized that playful handling (tickling) would decrease stress associated with repeated injections in adult laboratory rats, especially those with prior tickling experience. We compared responses of 4 groups of male Sprague–Dawley rats to intraperitoneal injection of saline daily for 10 d. Rats either tickled or not tickled as juveniles (2 min/d for 21 d) were exposed as adults to either a passive hand or tickling for 2 min immediately before and after injections. Rates of vocalization (22- and 50-kHz ultrasonic vocalizations (USV), indicative of negative and positive affective states, respectively, and audible calls indicative of pain and discomfort) were quantified before, during, and after injection. Tickling before and after injection, especially when combined with juvenile tickling experience (ending 40 to 50 d earlier), increased 50-kHz USV rates before and after injection, reduced audible call rate during injection, and decreased the duration of the injection procedure. The treatments did not affect indicators of physiologic stress (body weight change; fecal corticosteroid levels). We conclude that playful handling performed in association with a mildly aversive procedure serves as a useful refinement by inducing a positive affective state that mitigates the aversiveness of the procedure and makes rats easier to handle, especially when they have been accustomed to tickling as juveniles. Abbreviation: M, exposure to minimal handling; P, exposure to a passive hand; T, exposure to tickling; USV, ultrasonic vocalization.
Small animals can be stressed by handling for veterinary and experimental medical procedures such as injection, blood sampling, and ear notching, and the stress is magnified if an aversive procedure has to be repeated for several days.2,19 Handling also can result in injuries to both humans and animals when fearful animals struggle to escape and scratch or bite the handler. To avoid adverse effects on overall animal welfare and experimental outcomes, habituation of animals to handling at times when they are not exposed to stressful procedures has been recommended so that routine handling does not become associated with occasional aversive experiences.27 Although repeated brief exposures to a passive hand or hand restraint have some habituating effects on juvenile laboratory rats, the use of a playful handling technique is more effective in overcoming fear of humans.16 Friendly social contact between animals can serve to buffer them against aversive events.1,25 Considering that laboratory rats respond similarly to humans as to conspecifics when engaged in positive interaction,31,38,43 humans as well as conspecifics could perhaps serve as social buffers. Therefore, handling rats in a manner that simulates the bodily contact that occurs during social play could have effects similar to social play between rats in dampening negative emotional responses33 and attenuating the effects of unpleasant medical procedures. Humans can mimic the playful rough-and-tumble behavior of rats by alternately tickling the rat’s nape (dorsal contact)
Received: 06 Jun 2013. Revision requested: 12 Jul 2013. Accepted: 30 Aug 2013. 1Center for the Study of Animal Well-being, Department of Integrative Physiology and Neuroscience, Pullman, Washington, DC; 2Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, Ås, Norway. *Corresponding author. Email: [email protected]
and ventral surface (pinning) by using vigorous, rapid finger movements. This form of interaction (tickling) has been shown to induce a positive motivational state in rats and to be actively solicited by the animals.8,16,31 Tickling also shows some of the attributes of a social buffer as it involves positive physical contacts and the release of dopamine and opioids, all of which are important in the transmission of social buffering effects.6,10,11,25 Therefore, tickling could be a useful and practical refinement by attenuating the aversiveness of veterinary and experimental medical procedures. In a previous study in which intraperitoneal injections were given on a daily basis, we found no significant benefit of tickling adult male rats immediately after each injection compared with the provision of a food reward, stroking, or control treatments.14 Therefore, repeatedly experiencing a positive stimulus immediately after injections was insufficient to overcome the aversiveness of future injections; providing a positive stimulus both before and after an aversive procedure may have a stronger, more lasting effect. In addition, in another study we found that rats that had been tickled by their caretakers during late adolescence spent more time rearing near an experimenter (interpreted as nonfearful monitoring) after a single intraperitoneal injection compared to minimally handled rats.16 Therefore, experiencing tickling as juveniles, when rats are most playful29,32 and correspondingly most receptive to tickling,30 could facilitate adaptation to stressful procedures later in life. We hypothesized that tickling decreases stress associated with repeated exposure to aversive medical procedures in adult laboratory rats, especially when accompanied by prior tickling experience during the juvenile period. We investigated this hypothesis by using rats from a prior study13 in which they either received or did not receive tickling experience as juveniles (25 to 161
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45 d of age). In adulthood (85 to 94 d of age), we exposed these rats to either a passive hand or tickling for 2 min immediately before and after the administration of a daily intraperitoneal injection for 10 d. To assess treatment outcomes, we measured the production of ultrasonic and audible vocalizations. Rat 50-kHz ultrasonic vocalizations (USV) have been validated as indicators of positive affective states,4,7,44 whereas 22-kHz USVs provide information about fear and anxiety,4,7 and audible vocalizations serve as indicators of physical pain and discomfort.4 We expected that tickling immediately before and after injection would increase the production of 50-kHz USV and reduce the production of 22-kHz USV and audible calls before, during, and after injections, compared with exposure to a passive hand. Because tickling reduces fear of humans,16 and hence struggling and escape attempts when handled, we also expected the adult tickling treatment would reduce the duration of the injection procedure as well as physiologic stress associated with repeated injections. We predicted that juvenile tickling experience would enhance the effectiveness of the adult tickling treatment in reducing the aversiveness of repeated injections.
Materials and Methods
Animals and husbandry. Male Sprague–Dawley rats (n = 48; Rattus norvegicus; Sim:[SD] fBR Albino, Simonsen Laboratories, Gilroy, CA) were retained from a previous study in which they had been housed singly, in pairs, or in triplets from 21 to 52 d of age and singly after 52 d.13 For the 10 d of the current study, they remained singly housed in standard clear plastic cages (46 cm × 24 cm × 20 cm) with wood-fiber bedding (Biofresh Comfort Bedding Natural Soft Cellulose, Absorption, Ferndale, WA), a paper hut (Shepherd Specialty Paper, Chicago, IL), and a metal lid. Rodent chow (2018 Teklad F6, Harlan, Indianapolis, IN) and water were provided ad libitum. Room temperature (mean ± SE) was 21 ± 2 °C, and humidity averaged 37% ± 15% across the period from 21 to 94 d of age. Rats were exposed to a 12:12-h light dark photoperiod (lights on, 1500). Each rat was identified by lines drawn on the tail by using a nontoxic marker. The animals were monitored daily and routine cage cleaning was performed weekly. Sentinel rats exposed to dirty bedding from the cages housing study animals were found to be negative for rat coronaviruses, Sendai virus, rodent pneumovirus, parvoviruses (rat parvovirus, Kilham rat virus, Toolan H1 virus) and parvoviral protein (rNS1), rat minute virus, Mycoplasma pulmonis, Theiler murine encephalomyelitis virus (mouse poliovirus), reovirus type 3, lymphocytic choriomeningitis, Syphacia spp., and fur mites. The experiment was performed in accordance with the Guide for the Care and Use of Laboratory Animals26 at an AAALACaccredited facility and was approved by the Washington State University IACUC (protocol no. 3382). Experimental treatments. From 25 to 45 d of age during the juvenile period, half of the rats (randomly assigned within group) received only the routine, minimal handling (M) once weekly on cage cleaning day, and the remaining rats received an extra 2 min of daily handling involving tickling (T) the nape and ventral surface of the body in a manner intended to mimic a positive, playful social interaction with another rat. Tickling was conducted in repeated 15-s bouts of hand contact alternating with 15-s bouts during which the hand remained motionless. The rats from each juvenile handling treatment were assigned randomly to one of 2 adult handling treatments—exposure to a passive hand (P) or to tickling (as described earlier)—which was applied for 2 min immediately before and for 2 min immediately after a daily intraperitoneal injection given on 10 consecutive days from 85 to 94 d of age. Thus there were 4 handling
treatment combinations—MP, TP, MT, and TT—with 12 rats per treatment condition. No significant interactions between handling treatments and juvenile group-size treatments were detected, and so they are not considered further. To control for potential effects of tier level and locus of human activity in the room,15,22 cages and treatments were distributed across 3 tier levels on 2 standard rodent racks. To apply adult handling treatments, an experimenter carried the cage to the workbench in the animal room, provided the assigned handling treatment, and then returned the cage to its normal location on the rack. Adult rats were injected intraperitoneally with 1 mL/kg physiologic sterile saline (0.9% NaCl) based on body weight on days −1 and 5 of injection by using a short (15.2 mm), fine (27-gauge) needle and a 1-mL syringe. Using the left hand, the experimenter cradled the rat’s back and thorax, with the forefinger and middle finger placed on either side of the animals’ body at shoulder level and the thumb over the right hindleg, preventing it from moving. To avoid major blood vessels and injury to internal organs, the rat’s head was tilted lower than the body, and the needle was gently inserted into the lower right quadrant of the abdomen. Treatments were administered to rats under red lighting during the dark phase of the light cycle in a predetermined random order, which was different each day. Experimenters were blind to the juvenile handling treatment experienced by the rats but not to the adult handling treatment. To ensure consistency, 2 experimenters were trained in the tickling, restraint, and injection techniques prior to the experiment, and each handled half of the rats per treatment. Similar methods were used by each of 2 caretakers when applying tickling (without injection) during the juvenile period.16 Measurements. Audible calls and USV were recorded on days 1 through 10 of injection by using a 10- to 120- kHz range microphone (P48 Electret Ultrasound Microphone; Avisoft Bioacoustics, Berlin, Germany) suspended 12 cm from the cage side and connected to a computer via an audio–MIDI interface (model 0404 USB2, E-MU Systems, Scotts Valley, CA). To minimize aliasing artifacts, sounds were sampled at a rate of 192 kHz and a bit depth of 24 bits during capture on a PC laptop. A real-time high frequency USV recording system (SEA Pro Ultra, Nauta, Milan, Italy) was used for sound capture and visualization of calls by trained observers. Audible, 50-kHz, and 22-kHz USV rates were determined separately for preand postinjection handling (2 min each) and during injection. Interrater concordance, assessed prior to data collection, was greater than 90% for the different vocalizations. The time taken to make each injection, from the moment the hand entered the cage until the rat was returned to the cage after injection, was recorded by a second experimenter, blind to the treatments, who was present in the room. To compare chronic physiologic stress between handling treatments across the 10 d of injections, body weight was measured on days −1 and 11 by using an electronic scale (CS2000, Ohaus, Pinebrook, NJ). Fecal samples for the determination of corticosterone metabolite levels also were obtained on days −1 (baseline) and 11. To increase reliability by reducing sample variability, a minimum of 2 fresh (soft) fecal pellets per rat were collected from the litter surface of the home cage, with minimal disturbance to the occupant. All fecal samples were collected around 1500 to minimize the effect of circadian variation in corticosterone levels41 and were stored at −20 °C until processed. Fecal corticosteroid levels were quantified by radioimmunoassay as previously,13,15 by using the ImmuChem double antibody 125I RIA kit for rats and mice (MP Biochemicals,
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Orangeburg, NY) according to the manufacturer’s instructions. Samples were freeze dried, and ground into a fine powder. Aliquots (0.2 g) were extracted with 100% ethanol, vortexed for 30 min, and centrifuged at approximately 655 × g for 20 min. The supernatant was removed and dried by using a water bath (37 °C), and the residue was reconstituted with 20 μL 100% ethanol and 280 μL of diluent (provided in the kit). All samples were assayed in duplicate. Concentrations are expressed as ng/g of fecal dry matter. The assays had a sensitivity of 7.7 ng/g, the interassay coefficient of variation was 3.3%, and the intraassay coefficient of variation was 2.6%. Statistical analysis. The rat (n = 48) was the unit of analysis. Vocalization data were averaged over days 1 through 10 of injection prior to analysis, and all statistical tests were conducted by using SAS statistical software.36 Normality of residuals was assessed by using the Shapiro–Wilk test in the SAS univariate procedure. A general linear model ANOVA was used to assess the effects of handling treatment on 50-kHz USV before and after injection; square-root–transformed 50-kHz USV during injection, log-transformed audible calls during injection, duration of injection, and the difference (that is, after minus before the 10 d of injection) in body weight and fecal corticosteroid levels. A Kruskal–Wallis test was used for 22-kHz USV before, during, and after injection and for audible calls before and after injection, because residuals were not normally distributed even after transformation. Student t tests were used to assess the change in body weight and fecal corticosteroid levels. Means comparisons were made based on differences in least-squares means, with P values adjusted for multiple comparisons by using the Tukey option after ANOVA and by using the Mann–Whitney U test, with P values adjusted for multiple comparisons by using the sequential Bonferroni adjustment35 after Kruskal–Wallis tests. Results are presented as mean ± SE for normally distributed variables, and as median and interquartile range for nonnormally distributed variables. For all tests, the level of statistical significance was set at a P value of less than 0.05.
Results
Production of 50-kHz USV. The number of 50-kHz USV produced during the 2 min period before (F3, 44 = 9.59, P < 0.0001; Figure 1 A) and after (F3, 44 = 8.74, P = 0.0001; Figure 1 B) injection was affected by treatment. Both before and after injection, rats with both juvenile and immediate tickling experience (TT) uttered more (P < 0.05) 50-kHz USV than did rats exposed to a passive hand (MP and TP). Rats with immediate tickling experience only (MT) uttered an intermediate number of calls. During the injection procedure, the rate (mean ± SE) of 50-kHz USV produced (15 ± 1.4 USV/min) was not affected by treatment. Production of 22-kHz USV. The production of 22-kHz USV during the 2 min before (H = 8.53, df = 3, P = 0.04) and during (H = 7.83, df = 3, P = 0.0497) injection was marginally affected by treatment, but post hoc comparisons of the medians did not reveal any significant differences (Table 1). In the 2 min after injection, the production of 22-kHz USVs was robustly affected by treatment (H = 18.05, df = 3, P = 0.0004; Table 1). Rats with immediate tickling experience only (MT) produced more (P ≤ 0.008) 22-kHz USV than did rats exposed to a passive hand (MP and TP). Rats with both juvenile and immediate tickling experience (TT) uttered an intermediate number of calls. Production of audible calls. The production of audible calls during the 2 min before (0.007 ± 0.0052 calls/min) and after (0.01 ± 0.004 calls/min) injection was not affected by treatment (P > 0.05), but the rate of calling during injection differed with
Figure 1. Effect of an intraperitoneal injection procedure on the rate (mean ± SE; n = 48) of 50-kHz USV (A) before and (B) after the procedure by adult male rats exposed to minimal handling (M) or tickling (T) as juveniles and a passive hand (P) or tickling immediately before and after injection, yielding 4 treatment combinations (MP, TP, MT, TT). Bars with different letters (a, b, c) indicate differences in pairwise comparisons (Tukey–Kramer, P < 0.05). Table 1. Effect of an intraperitoneal injection procedure on production (median, interquartile range; n = 48) of 22-kHz USV (no./min) before, during, and after the procedure Handling Time
MP
TP
MT
TT
Before
0, 0–0
0, 0–0
0, 0–0.45
0, 0–0
During
0, 0–0
0, 0–0
0, 0–0.65
0, 0–0
After
0, 0–0
0, 0–0
0.9, 0.08–1.53
0, 0–0.35
Adult male rats were exposed to minimal handling (M) or tickling (T) as juveniles and a passive hand (P) or tickling immediately before and after injection, yielding 4 treatment combinations (MP, TP, MT, TT).
treatment (F3, 44 = 5.31, P = 0.003; Figure 2). Rats with no tickling experience (MP) produced audible calls at a higher (P < 0.05) rate during injection than did rats with immediate tickling experience (MT, TT), whereas rats with juvenile tickling experience only (TP) called at an intermediate rate. Duration of injection procedure. Duration of injection was affected by treatment (F3, 44 = 2.89, P = 0.046; Figure 3). Rats with no tickling experience (MP) took more (P < 0.05) time to inject than did rats with immediate tickling experience (MT), whereas rats with juvenile tickling experience (TP and TT) had intermediate durations. Body weight change and fecal corticosteroid levels. The change in body weight from day −1 (345 ± 4 g) to day 11 (364 ± 4 g) was not affected by handling treatment but was influenced by time (t = 15.98, P < 0.0001; difference, 19 ± 1 g). Similarly, the 163
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Figure 2. Effect of an intraperitoneal injection procedure on the rate (mean ± SE; n = 48) of audible calls given during the procedure by adult male rats exposed to minimal handling (M) or tickling (T) as juveniles and a passive hand (P) or tickling immediately before and after injection, yielding 4 treatment combinations (MP, TP, MT, TT). Bars with different letters (a, b, c) indicate differences in pairwise comparisons (Tukey–Kramer, P < 0.05).
Figure 3. Duration (mean ± SE; n = 48) of an intraperitoneal injection procedure administered to adult male rats exposed to minimal handling (M) or tickling (T) as juveniles and a passive hand (P) or tickling immediately before and after injection, yielding 4 treatment combinations (MP, TP, MT, TT). Bars with different letters (a, b, c) indicate differences in pairwise comparisons (Tukey–Kramer, P < 0.05).
change in fecal corticosterone levels from before (506 ± 42 ng/g) to after (406 ± 29 ng/g) the 10 daily injections was unaffected by handling treatment but was influenced by time (t = −2.77, P = 0.008; difference, −106 ± 38 ng/g).
Discussion
The results provide support for the hypothesis that tickling, when provided both before and after injection, is effective in mitigating the aversiveness of an intraperitoneal injection procedure in rats. Tickling increased the production of 50-kHz USV before and after injection, decreased the rate of audible calls produced during injection, and decreased the duration of the procedure. This outcome contrasts with those of a previous study,14 which found no beneficial effect of tickling when provided only immediately after injections. From the treatments we tested in the current study, we are unable to say whether tickling only before an aversive event would be effective or whether the positive result was due to the combined effect of tickling both before and after the injections. We interpret the greater number of 50-kHz USVs uttered by tickled compared with nonhandled rats as indicating that tickling induced a positive affective state.7,9 Fewer 50-kHz USV were produced during the injections when no tickling
was performed, but they increased to similar levels during the 2 min after as in the 2 min before injection, suggesting that the injection procedure did not prevent rats from having a positive emotional response to tickling immediately after receiving an intraperitoneal injection. These results are consistent with those of other colleagues,15 who reported that tickling by caretakers induced a more positive affective state than did the mere presence of a passive human hand. Because 22-kHz USV occur mainly in association with—or anticipation of—aversive events, they are interpreted to reflect negative affective states.4,7 In the current study, the lack of elevated 22-kHz USV rates prior to and during injection suggests that repeated injections did not induce a state of anxiety in anticipation of, or during, injection. Contrary to prediction, rats tickled only as adults (MT) produced more 22-kHz USV after injection than did rats exposed to a passive hand (MP and TP). This result is in agreement with a previous report in which rats with little or no prior handling experience were more likely to produce 22-kHz USV when handled.5 The relative novelty of tickling combined with the injection procedure likely contributed to the higher 22-kHz USV rates in the MT group. In addition, rats experiencing tickling only in adulthood might perceive tickling as mildly alarming at first, because adult rats are less playful than juveniles, and play becomes more dominance-related as rats, especially males, mature.30,34 The tickling-inexperienced rats might, therefore, initially have been more resistant to the loss of control associated with being subjected to handling mimicking rough-and-tumble play. Nonetheless, these effects appear to be transient given that, overall, few 22-kHz USV were produced by MT rats, and their production of 50-kHz USV was similar to that of rats with both previous and immediate tickling experience (TT). In addition, the duration of injection was shorter for MT rats than for minimally handled rats (MP), indicating that, even with only a few tickling experiences provided in adulthood, rats struggled less and were easier to handle. Audible vocalizations have previously been associated with agonistic encounters,23 defensive situations,3 and pain and discomfort.4 Rats exposed to a passive hand and tickled rats produced similar, low numbers of audible calls before and after the injection, suggesting that human contact during tickling did not cause pain or discomfort. The increased production of audible calls by rats in all treatment groups when given injections confirms that intraperitoneal injection is a stressful procedure, albeit a relatively mild one, as suggested by generally low levels of 22-kHz calls, rapid recovery of 50-kHz calling immediately afterward, and lack of effects on body weight or fecal corticosteroids. Tickling both before and after injection, rather than afterward only,14 lowered the rate of audible calls produced during injection, suggesting that tickling mitigated the discomfort associated with this procedure. We previously reported that tickling has long-lasting effects in reducing rat fear of humans.13 In the current study, rats with juvenile tickling experience but without exposure to tickling at the time of injection (TP group), showed intermediate responses in production of 50-kHz calls before and after injection, audible calls during injection, and duration of the procedure, compared with rats with immediate (average of MT and TT) or no tickling (MP) experience. Furthermore, there were some additive effects of combining juvenile and immediate tickling experiences, in that TT rats produced the most 50-kHz USV when tickled and the fewest audible calls during injection. These findings confirm that tickling of juvenile rats has long-lasting effects, which in this case were still evident 40 to 50 d after discontinuation of tickling during the juvenile period.
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We found that injections took less time in tickled rats than in nonhandled rats. Provision of tickling does require that handlers spend extra time with the rats and thus increases the total handling time associated with the medical procedure. However, on the basis of our results, we assert that this time investment results in reduced fear of humans, attenuation of discomfort associated with injections, increased positive affect in the time immediately after the aversive procedure, and reduced time spent attempting to restrain naïve, fearful animals for injection. When rats are less fearful of humans and more accepting of the procedure, rats struggle less, thus reducing the threat of injury to themselves and handlers. Currently, depending on facilities, rat intraperitoneal injections are performed by using either a one- or a 2-person technique. The 2-person technique, in which one person restrains the rat and the other performs the injection, is often preferred because the rat is controlled more effectively, thereby reducing the risk of injury. Although our study did not compare both procedures, our results show that applying tickling in association with giving intraperitoneal injections makes it easy for a single person to handle the rat in a manner safe for both rat and handler. Therefore, using a one-person rather than a 2-person technique could partially compensate for the extra time invested in tickling. As reviewed previously,37 several studies have shown that standard handling (that is, the minimal amount of handling required for husbandry and experimental procedures) can be associated with poor repeatability of studies. Our results show that when handling is playful and mimics the rats’ own social play, it avoids the problem of animals developing an anticipatory stress response to aversive procedures.27 Therefore, integrating tickling into laboratory procedures could improve both rat welfare and the quality and validity of data collected from these animals, consequently potentially reducing the number of animals used and the need to repeat experiments. We had expected that exposure to the procedure for 10 consecutive days would lead to increases in corticosteroid levels and decreases in body weight suggestive of chronic stress. Changes in body weight (a measure used to gauge stress28) and corticosteroid levels in feces collected from the home environment were not affected by treatment, providing evidence that repeated, daily injections did not disrupt the rats sufficiently to cause chronic stress. Body weights continued to increase over time and were within the normal range for the strain.20 Our findings are consistent with previous studies assessing the effect of repeated injection on body weight42 and plasma corticosterone levels.24 Changes in plasma corticosterone levels have been reported after 30-min to 3-h periods of restraint in samples collected 3 h or less after the stressor.39,40 Considering that the restraint period of our procedure was less than 40 s and that the postinjection samples were collected more than 24 h after the last test, it is likely that the duration of the procedure was too short to induce long-term detectable changes in corticosteroid levels or to affect the release of corticotropin-releasing factor in quantities sufficient to affect body weight gain.21 Furthermore, rats may have habituated to the intraperitoneal procedure and interaction with humans, because corticosteroid levels decreased over time and were comparable to the prestressor levels reported previously.12 We conclude that tickling is a positive handling technique that can mitigate the aversiveness of intraperitoneal injections and facilitate handling in male rats, especially those with prior tickling experience as juveniles, consequently improving laboratory rat welfare. We expect our results also apply to female rats, because they respond to tickling similarly to male rats.30 However, sex-associated differences in response to painful,
stressful stimuli17,18 could affect results. If tickling during the juvenile period is not practically feasible, tickling in adulthood has benefits. A social buffering effect of playful handling may be applicable to other species that display play behavior that can safely be mimicked through human interaction and to other mildly aversive medical procedures performed for veterinary or experimental reasons. In this way, playful handling could serve as a valuable refinement, especially when it is necessary to perform procedures multiple times.
Acknowledgments
We thank the staff of the EALB Vivarium for animal care and Mr MD McNabb for assisting with preparation of the manuscript. This material is based on work supported by a Washington State University Veterinary Summer Research Fellowship Program (to KW) and an Animal Welfare Enhancement Award from the Animal Welfare Institute (to SC).
References
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17. Craft RM. 2007. Modulation of pain by estrogen. Pain 132:S3–S12. 18. Faraday MM. 2002. Rat sex and strain differences in responses to stress. Physiol Behav 75:507–522. 19. Gärtner K, Büttner D, Döhler K, Friedel R, Lindena J, Trautschold I. 1980. Stress response of rats to handling and experimental procedures. Lab Anim 14:267–274. 20. Harlan Laboratories. [internet] 2008. Combined data from multiple barriers. [Cited 3 June 2013] Available at: www.harlan. com/products_and_services/research_models_and_services/ research_models/sprague_dawley_outbred_rat.hl. 21. Harris RBS, Mitchell TD, Simpson J, Redmann SM Jr, Youngblood BD, Ryan DH. 2002. Weight loss in rats exposed to repeated acute restraint stress is independent of energy or leptin status. Am J Physiol Regul Integr Comp Physiol 282:R77–R88. 22. Izidio GS, Lopes DM, Spricigo L Jr, Ramos A. 2005. Common variations in the pretest environment influence genotypic comparisons in models of anxiety. Genes Brain Behav 4:412–419. 23. Kaltwasser MT. 1990. Acoustic signalling in the black rat (Rattus rattus). J Comp Psychol 104:227–232. 24. Kant GJ, Bunnell BN, Mougey EH, Pennington LL, Meyerhoff JL. 1983. Effects of repeated stress on pituitary cyclic AMP, and plasma prolactin, corticosterone, and growth hormone in male rats. Pharmacol Biochem Behav 18:967–971. 25. Kikusui T, Winslow JT, Mori Y. 2006. Social buffering: relief from stress and anxiety. Philos Trans R Soc Lond B Biol Sci 361:2215–2228. 26. Institute for Laboratory Animal Research. 2011. Guide for the care and use of laboratory animals, 8th ed. Washington (DC): National Academies Press. 27. New South Wales Animal Research Review Panel. 2004. ARRP Guideline 20: guidelines for the housing of rats in scientific institutions. Orange (Australia): Animal Welfare Branch, NSW Department of Primary Industries. 28. Olsson IAS, Dahlborn K. 2002. Improving housing conditions for laboratory mice: a review of environmental enrichment. Lab Anim 36:243–270. 29. Panksepp J. 1981. The ontogeny of play in rats. Dev Psychobiol 14:327–332. 30. Panksepp J. 1998. Affective neuroscience. The foundations of human and animal emotions. New York (NY): Oxford University Press.
31. Panksepp J, Burgdorf J. 2000. 50-kHz chirping (laughter?) in response to conditioned and unconditioned tickle-induced reward in rats: effects of social housing and genetic variables. Behav Brain Res 115:25–38. 32. Pellis S, Pellis V. 1997. The prejuvenile onset of play fighting in laboratory rats (Rattus norvegicus). Dev Psychobiol 31:193–205. 33. Pellis S, Pellis V. 2009. The playful brain—venturing to the limits of neuroscience. Oxford (UK): Oneworld Publications. 34. Pellis SM, Field EF, Smith LK, Pellis VC. 1997. Multiple differences in the play fighting of male and female rats. Implications for the causes and functions of play. Neurosci Biobehav Rev 21:105–120. 35. Rice WR. 1989. Analyzing tables of statistical tests. Evolution 43:223–225. 36. SAS Institute. 1999. SAS/STAT user’s guide, version 8. Cary (NC): SAS Institute. 37. Sherwin CM. 2004. The influences of standard laboratory cages on rodents and the validity of research data. Anim Welf 13:S9–S15. 38. Sloan LR, Latané B. 1974. Social deprivation and stimulus satiation in the albino rat. J Comp Physiol Psychol 87:1148–1156. 39. Song X, Tian H, Bressler J, Pruett S, Pope C. 2002. Acute and repeated restraint stress have little effect on pyridostigmine toxicity or brain regional cholinesterase inhibition in rats. Toxicol Sci 69:157–164. 40. Strausbaugh HJ, Dallman MF, Levine JD. 1999. Repeated, but not acute, stress suppresses inflammatory plasma extravasation. Proc Natl Acad Sci USA 96:14629–14634. 41. Touma C, Sachser N, Mostl E, Palme R. 2003. Effects of sex and time of day on metabolism and excretion of corticosterone in urine and feces of mice. Gen Comp Endocrinol 130:267–278. 42. Wang Y, Huang C, Cao Y, Han J-S. 2000. Repeated administration of low-dose ketamine for the treatment of monoarthritic pain in the rat. Life Sci 67:261–267. 43. Werner C, Latané B. 1974. Interaction motivates attraction: rats are fond of fondling. J Pers Soc Psychol 29:328–334. 44. Wöhr M, Houx B, Schwarting RK, Spruijt B. 2008. Effects of experience and context on 50-kHz vocalizations in rats. Physiol Behav 93:766–776.
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The Social Buffering Effect of Playful Handling on Responses to Repeated Intraperitoneal Injections in Laboratory Rats Sylvie Cloutier,1,* Kim Wahl,1 Chelsea Baker,1 and Ruth C Newberry1,2 Handling small animals for veterinary and experimental procedures can negatively affect animal wellbeing. We hypothesized that playful handling (tickling) would decrease stress associated with repeated injections in adult laboratory rats, especially those with prior tickling experience. We compared responses of 4 groups of male Sprague–Dawley rats to intraperitoneal injection of saline daily for 10 d. Rats either tickled or not tickled as juveniles (2 min/d for 21 d) were exposed as adults to either a passive hand or tickling for 2 min immediately before and after injections. Rates of vocalization (22- and 50-kHz ultrasonic vocalizations (USV), indicative of negative and positive affective states, respectively, and audible calls indicative of pain and discomfort) were quantified before, during, and after injection. Tickling before and after injection, especially when combined with juvenile tickling experience (ending 40 to 50 d earlier), increased 50-kHz USV rates before and after injection, reduced audible call rate during injection, and decreased the duration of the injection procedure. The treatments did not affect indicators of physiologic stress (body weight change; fecal corticosteroid levels). We conclude that playful handling performed in association with a mildly aversive procedure serves as a useful refinement by inducing a positive affective state that mitigates the aversiveness of the procedure and makes rats easier to handle, especially when they have been accustomed to tickling as juveniles. Abbreviation: M, exposure to minimal handling; P, exposure to a passive hand; T, exposure to tickling; USV, ultrasonic vocalization.
Small animals can be stressed by handling for veterinary and experimental medical procedures such as injection, blood sampling, and ear notching, and the stress is magnified if an aversive procedure has to be repeated for several days.2,19 Handling also can result in injuries to both humans and animals when fearful animals struggle to escape and scratch or bite the handler. To avoid adverse effects on overall animal welfare and experimental outcomes, habituation of animals to handling at times when they are not exposed to stressful procedures has been recommended so that routine handling does not become associated with occasional aversive experiences.27 Although repeated brief exposures to a passive hand or hand restraint have some habituating effects on juvenile laboratory rats, the use of a playful handling technique is more effective in overcoming fear of humans.16 Friendly social contact between animals can serve to buffer them against aversive events.1,25 Considering that laboratory rats respond similarly to humans as to conspecifics when engaged in positive interaction,31,38,43 humans as well as conspecifics could perhaps serve as social buffers. Therefore, handling rats in a manner that simulates the bodily contact that occurs during social play could have effects similar to social play between rats in dampening negative emotional responses33 and attenuating the effects of unpleasant medical procedures. Humans can mimic the playful rough-and-tumble behavior of rats by alternately tickling the rat’s nape (dorsal contact)
Received: 06 Jun 2013. Revision requested: 12 Jul 2013. Accepted: 30 Aug 2013. 1Center for the Study of Animal Well-being, Department of Integrative Physiology and Neuroscience, Pullman, Washington, DC; 2Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, Ås, Norway. *Corresponding author. Email: [email protected]
and ventral surface (pinning) by using vigorous, rapid finger movements. This form of interaction (tickling) has been shown to induce a positive motivational state in rats and to be actively solicited by the animals.8,16,31 Tickling also shows some of the attributes of a social buffer as it involves positive physical contacts and the release of dopamine and opioids, all of which are important in the transmission of social buffering effects.6,10,11,25 Therefore, tickling could be a useful and practical refinement by attenuating the aversiveness of veterinary and experimental medical procedures. In a previous study in which intraperitoneal injections were given on a daily basis, we found no significant benefit of tickling adult male rats immediately after each injection compared with the provision of a food reward, stroking, or control treatments.14 Therefore, repeatedly experiencing a positive stimulus immediately after injections was insufficient to overcome the aversiveness of future injections; providing a positive stimulus both before and after an aversive procedure may have a stronger, more lasting effect. In addition, in another study we found that rats that had been tickled by their caretakers during late adolescence spent more time rearing near an experimenter (interpreted as nonfearful monitoring) after a single intraperitoneal injection compared to minimally handled rats.16 Therefore, experiencing tickling as juveniles, when rats are most playful29,32 and correspondingly most receptive to tickling,30 could facilitate adaptation to stressful procedures later in life. We hypothesized that tickling decreases stress associated with repeated exposure to aversive medical procedures in adult laboratory rats, especially when accompanied by prior tickling experience during the juvenile period. We investigated this hypothesis by using rats from a prior study13 in which they either received or did not receive tickling experience as juveniles (25 to 161
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45 d of age). In adulthood (85 to 94 d of age), we exposed these rats to either a passive hand or tickling for 2 min immediately before and after the administration of a daily intraperitoneal injection for 10 d. To assess treatment outcomes, we measured the production of ultrasonic and audible vocalizations. Rat 50-kHz ultrasonic vocalizations (USV) have been validated as indicators of positive affective states,4,7,44 whereas 22-kHz USVs provide information about fear and anxiety,4,7 and audible vocalizations serve as indicators of physical pain and discomfort.4 We expected that tickling immediately before and after injection would increase the production of 50-kHz USV and reduce the production of 22-kHz USV and audible calls before, during, and after injections, compared with exposure to a passive hand. Because tickling reduces fear of humans,16 and hence struggling and escape attempts when handled, we also expected the adult tickling treatment would reduce the duration of the injection procedure as well as physiologic stress associated with repeated injections. We predicted that juvenile tickling experience would enhance the effectiveness of the adult tickling treatment in reducing the aversiveness of repeated injections.
Materials and Methods
Animals and husbandry. Male Sprague–Dawley rats (n = 48; Rattus norvegicus; Sim:[SD] fBR Albino, Simonsen Laboratories, Gilroy, CA) were retained from a previous study in which they had been housed singly, in pairs, or in triplets from 21 to 52 d of age and singly after 52 d.13 For the 10 d of the current study, they remained singly housed in standard clear plastic cages (46 cm × 24 cm × 20 cm) with wood-fiber bedding (Biofresh Comfort Bedding Natural Soft Cellulose, Absorption, Ferndale, WA), a paper hut (Shepherd Specialty Paper, Chicago, IL), and a metal lid. Rodent chow (2018 Teklad F6, Harlan, Indianapolis, IN) and water were provided ad libitum. Room temperature (mean ± SE) was 21 ± 2 °C, and humidity averaged 37% ± 15% across the period from 21 to 94 d of age. Rats were exposed to a 12:12-h light dark photoperiod (lights on, 1500). Each rat was identified by lines drawn on the tail by using a nontoxic marker. The animals were monitored daily and routine cage cleaning was performed weekly. Sentinel rats exposed to dirty bedding from the cages housing study animals were found to be negative for rat coronaviruses, Sendai virus, rodent pneumovirus, parvoviruses (rat parvovirus, Kilham rat virus, Toolan H1 virus) and parvoviral protein (rNS1), rat minute virus, Mycoplasma pulmonis, Theiler murine encephalomyelitis virus (mouse poliovirus), reovirus type 3, lymphocytic choriomeningitis, Syphacia spp., and fur mites. The experiment was performed in accordance with the Guide for the Care and Use of Laboratory Animals26 at an AAALACaccredited facility and was approved by the Washington State University IACUC (protocol no. 3382). Experimental treatments. From 25 to 45 d of age during the juvenile period, half of the rats (randomly assigned within group) received only the routine, minimal handling (M) once weekly on cage cleaning day, and the remaining rats received an extra 2 min of daily handling involving tickling (T) the nape and ventral surface of the body in a manner intended to mimic a positive, playful social interaction with another rat. Tickling was conducted in repeated 15-s bouts of hand contact alternating with 15-s bouts during which the hand remained motionless. The rats from each juvenile handling treatment were assigned randomly to one of 2 adult handling treatments—exposure to a passive hand (P) or to tickling (as described earlier)—which was applied for 2 min immediately before and for 2 min immediately after a daily intraperitoneal injection given on 10 consecutive days from 85 to 94 d of age. Thus there were 4 handling
treatment combinations—MP, TP, MT, and TT—with 12 rats per treatment condition. No significant interactions between handling treatments and juvenile group-size treatments were detected, and so they are not considered further. To control for potential effects of tier level and locus of human activity in the room,15,22 cages and treatments were distributed across 3 tier levels on 2 standard rodent racks. To apply adult handling treatments, an experimenter carried the cage to the workbench in the animal room, provided the assigned handling treatment, and then returned the cage to its normal location on the rack. Adult rats were injected intraperitoneally with 1 mL/kg physiologic sterile saline (0.9% NaCl) based on body weight on days −1 and 5 of injection by using a short (15.2 mm), fine (27-gauge) needle and a 1-mL syringe. Using the left hand, the experimenter cradled the rat’s back and thorax, with the forefinger and middle finger placed on either side of the animals’ body at shoulder level and the thumb over the right hindleg, preventing it from moving. To avoid major blood vessels and injury to internal organs, the rat’s head was tilted lower than the body, and the needle was gently inserted into the lower right quadrant of the abdomen. Treatments were administered to rats under red lighting during the dark phase of the light cycle in a predetermined random order, which was different each day. Experimenters were blind to the juvenile handling treatment experienced by the rats but not to the adult handling treatment. To ensure consistency, 2 experimenters were trained in the tickling, restraint, and injection techniques prior to the experiment, and each handled half of the rats per treatment. Similar methods were used by each of 2 caretakers when applying tickling (without injection) during the juvenile period.16 Measurements. Audible calls and USV were recorded on days 1 through 10 of injection by using a 10- to 120- kHz range microphone (P48 Electret Ultrasound Microphone; Avisoft Bioacoustics, Berlin, Germany) suspended 12 cm from the cage side and connected to a computer via an audio–MIDI interface (model 0404 USB2, E-MU Systems, Scotts Valley, CA). To minimize aliasing artifacts, sounds were sampled at a rate of 192 kHz and a bit depth of 24 bits during capture on a PC laptop. A real-time high frequency USV recording system (SEA Pro Ultra, Nauta, Milan, Italy) was used for sound capture and visualization of calls by trained observers. Audible, 50-kHz, and 22-kHz USV rates were determined separately for preand postinjection handling (2 min each) and during injection. Interrater concordance, assessed prior to data collection, was greater than 90% for the different vocalizations. The time taken to make each injection, from the moment the hand entered the cage until the rat was returned to the cage after injection, was recorded by a second experimenter, blind to the treatments, who was present in the room. To compare chronic physiologic stress between handling treatments across the 10 d of injections, body weight was measured on days −1 and 11 by using an electronic scale (CS2000, Ohaus, Pinebrook, NJ). Fecal samples for the determination of corticosterone metabolite levels also were obtained on days −1 (baseline) and 11. To increase reliability by reducing sample variability, a minimum of 2 fresh (soft) fecal pellets per rat were collected from the litter surface of the home cage, with minimal disturbance to the occupant. All fecal samples were collected around 1500 to minimize the effect of circadian variation in corticosterone levels41 and were stored at −20 °C until processed. Fecal corticosteroid levels were quantified by radioimmunoassay as previously,13,15 by using the ImmuChem double antibody 125I RIA kit for rats and mice (MP Biochemicals,
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Orangeburg, NY) according to the manufacturer’s instructions. Samples were freeze dried, and ground into a fine powder. Aliquots (0.2 g) were extracted with 100% ethanol, vortexed for 30 min, and centrifuged at approximately 655 × g for 20 min. The supernatant was removed and dried by using a water bath (37 °C), and the residue was reconstituted with 20 μL 100% ethanol and 280 μL of diluent (provided in the kit). All samples were assayed in duplicate. Concentrations are expressed as ng/g of fecal dry matter. The assays had a sensitivity of 7.7 ng/g, the interassay coefficient of variation was 3.3%, and the intraassay coefficient of variation was 2.6%. Statistical analysis. The rat (n = 48) was the unit of analysis. Vocalization data were averaged over days 1 through 10 of injection prior to analysis, and all statistical tests were conducted by using SAS statistical software.36 Normality of residuals was assessed by using the Shapiro–Wilk test in the SAS univariate procedure. A general linear model ANOVA was used to assess the effects of handling treatment on 50-kHz USV before and after injection; square-root–transformed 50-kHz USV during injection, log-transformed audible calls during injection, duration of injection, and the difference (that is, after minus before the 10 d of injection) in body weight and fecal corticosteroid levels. A Kruskal–Wallis test was used for 22-kHz USV before, during, and after injection and for audible calls before and after injection, because residuals were not normally distributed even after transformation. Student t tests were used to assess the change in body weight and fecal corticosteroid levels. Means comparisons were made based on differences in least-squares means, with P values adjusted for multiple comparisons by using the Tukey option after ANOVA and by using the Mann–Whitney U test, with P values adjusted for multiple comparisons by using the sequential Bonferroni adjustment35 after Kruskal–Wallis tests. Results are presented as mean ± SE for normally distributed variables, and as median and interquartile range for nonnormally distributed variables. For all tests, the level of statistical significance was set at a P value of less than 0.05.
Results
Production of 50-kHz USV. The number of 50-kHz USV produced during the 2 min period before (F3, 44 = 9.59, P < 0.0001; Figure 1 A) and after (F3, 44 = 8.74, P = 0.0001; Figure 1 B) injection was affected by treatment. Both before and after injection, rats with both juvenile and immediate tickling experience (TT) uttered more (P < 0.05) 50-kHz USV than did rats exposed to a passive hand (MP and TP). Rats with immediate tickling experience only (MT) uttered an intermediate number of calls. During the injection procedure, the rate (mean ± SE) of 50-kHz USV produced (15 ± 1.4 USV/min) was not affected by treatment. Production of 22-kHz USV. The production of 22-kHz USV during the 2 min before (H = 8.53, df = 3, P = 0.04) and during (H = 7.83, df = 3, P = 0.0497) injection was marginally affected by treatment, but post hoc comparisons of the medians did not reveal any significant differences (Table 1). In the 2 min after injection, the production of 22-kHz USVs was robustly affected by treatment (H = 18.05, df = 3, P = 0.0004; Table 1). Rats with immediate tickling experience only (MT) produced more (P ≤ 0.008) 22-kHz USV than did rats exposed to a passive hand (MP and TP). Rats with both juvenile and immediate tickling experience (TT) uttered an intermediate number of calls. Production of audible calls. The production of audible calls during the 2 min before (0.007 ± 0.0052 calls/min) and after (0.01 ± 0.004 calls/min) injection was not affected by treatment (P > 0.05), but the rate of calling during injection differed with
Figure 1. Effect of an intraperitoneal injection procedure on the rate (mean ± SE; n = 48) of 50-kHz USV (A) before and (B) after the procedure by adult male rats exposed to minimal handling (M) or tickling (T) as juveniles and a passive hand (P) or tickling immediately before and after injection, yielding 4 treatment combinations (MP, TP, MT, TT). Bars with different letters (a, b, c) indicate differences in pairwise comparisons (Tukey–Kramer, P < 0.05). Table 1. Effect of an intraperitoneal injection procedure on production (median, interquartile range; n = 48) of 22-kHz USV (no./min) before, during, and after the procedure Handling Time
MP
TP
MT
TT
Before
0, 0–0
0, 0–0
0, 0–0.45
0, 0–0
During
0, 0–0
0, 0–0
0, 0–0.65
0, 0–0
After
0, 0–0
0, 0–0
0.9, 0.08–1.53
0, 0–0.35
Adult male rats were exposed to minimal handling (M) or tickling (T) as juveniles and a passive hand (P) or tickling immediately before and after injection, yielding 4 treatment combinations (MP, TP, MT, TT).
treatment (F3, 44 = 5.31, P = 0.003; Figure 2). Rats with no tickling experience (MP) produced audible calls at a higher (P < 0.05) rate during injection than did rats with immediate tickling experience (MT, TT), whereas rats with juvenile tickling experience only (TP) called at an intermediate rate. Duration of injection procedure. Duration of injection was affected by treatment (F3, 44 = 2.89, P = 0.046; Figure 3). Rats with no tickling experience (MP) took more (P < 0.05) time to inject than did rats with immediate tickling experience (MT), whereas rats with juvenile tickling experience (TP and TT) had intermediate durations. Body weight change and fecal corticosteroid levels. The change in body weight from day −1 (345 ± 4 g) to day 11 (364 ± 4 g) was not affected by handling treatment but was influenced by time (t = 15.98, P < 0.0001; difference, 19 ± 1 g). Similarly, the 163
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Figure 2. Effect of an intraperitoneal injection procedure on the rate (mean ± SE; n = 48) of audible calls given during the procedure by adult male rats exposed to minimal handling (M) or tickling (T) as juveniles and a passive hand (P) or tickling immediately before and after injection, yielding 4 treatment combinations (MP, TP, MT, TT). Bars with different letters (a, b, c) indicate differences in pairwise comparisons (Tukey–Kramer, P < 0.05).
Figure 3. Duration (mean ± SE; n = 48) of an intraperitoneal injection procedure administered to adult male rats exposed to minimal handling (M) or tickling (T) as juveniles and a passive hand (P) or tickling immediately before and after injection, yielding 4 treatment combinations (MP, TP, MT, TT). Bars with different letters (a, b, c) indicate differences in pairwise comparisons (Tukey–Kramer, P < 0.05).
change in fecal corticosterone levels from before (506 ± 42 ng/g) to after (406 ± 29 ng/g) the 10 daily injections was unaffected by handling treatment but was influenced by time (t = −2.77, P = 0.008; difference, −106 ± 38 ng/g).
Discussion
The results provide support for the hypothesis that tickling, when provided both before and after injection, is effective in mitigating the aversiveness of an intraperitoneal injection procedure in rats. Tickling increased the production of 50-kHz USV before and after injection, decreased the rate of audible calls produced during injection, and decreased the duration of the procedure. This outcome contrasts with those of a previous study,14 which found no beneficial effect of tickling when provided only immediately after injections. From the treatments we tested in the current study, we are unable to say whether tickling only before an aversive event would be effective or whether the positive result was due to the combined effect of tickling both before and after the injections. We interpret the greater number of 50-kHz USVs uttered by tickled compared with nonhandled rats as indicating that tickling induced a positive affective state.7,9 Fewer 50-kHz USV were produced during the injections when no tickling
was performed, but they increased to similar levels during the 2 min after as in the 2 min before injection, suggesting that the injection procedure did not prevent rats from having a positive emotional response to tickling immediately after receiving an intraperitoneal injection. These results are consistent with those of other colleagues,15 who reported that tickling by caretakers induced a more positive affective state than did the mere presence of a passive human hand. Because 22-kHz USV occur mainly in association with—or anticipation of—aversive events, they are interpreted to reflect negative affective states.4,7 In the current study, the lack of elevated 22-kHz USV rates prior to and during injection suggests that repeated injections did not induce a state of anxiety in anticipation of, or during, injection. Contrary to prediction, rats tickled only as adults (MT) produced more 22-kHz USV after injection than did rats exposed to a passive hand (MP and TP). This result is in agreement with a previous report in which rats with little or no prior handling experience were more likely to produce 22-kHz USV when handled.5 The relative novelty of tickling combined with the injection procedure likely contributed to the higher 22-kHz USV rates in the MT group. In addition, rats experiencing tickling only in adulthood might perceive tickling as mildly alarming at first, because adult rats are less playful than juveniles, and play becomes more dominance-related as rats, especially males, mature.30,34 The tickling-inexperienced rats might, therefore, initially have been more resistant to the loss of control associated with being subjected to handling mimicking rough-and-tumble play. Nonetheless, these effects appear to be transient given that, overall, few 22-kHz USV were produced by MT rats, and their production of 50-kHz USV was similar to that of rats with both previous and immediate tickling experience (TT). In addition, the duration of injection was shorter for MT rats than for minimally handled rats (MP), indicating that, even with only a few tickling experiences provided in adulthood, rats struggled less and were easier to handle. Audible vocalizations have previously been associated with agonistic encounters,23 defensive situations,3 and pain and discomfort.4 Rats exposed to a passive hand and tickled rats produced similar, low numbers of audible calls before and after the injection, suggesting that human contact during tickling did not cause pain or discomfort. The increased production of audible calls by rats in all treatment groups when given injections confirms that intraperitoneal injection is a stressful procedure, albeit a relatively mild one, as suggested by generally low levels of 22-kHz calls, rapid recovery of 50-kHz calling immediately afterward, and lack of effects on body weight or fecal corticosteroids. Tickling both before and after injection, rather than afterward only,14 lowered the rate of audible calls produced during injection, suggesting that tickling mitigated the discomfort associated with this procedure. We previously reported that tickling has long-lasting effects in reducing rat fear of humans.13 In the current study, rats with juvenile tickling experience but without exposure to tickling at the time of injection (TP group), showed intermediate responses in production of 50-kHz calls before and after injection, audible calls during injection, and duration of the procedure, compared with rats with immediate (average of MT and TT) or no tickling (MP) experience. Furthermore, there were some additive effects of combining juvenile and immediate tickling experiences, in that TT rats produced the most 50-kHz USV when tickled and the fewest audible calls during injection. These findings confirm that tickling of juvenile rats has long-lasting effects, which in this case were still evident 40 to 50 d after discontinuation of tickling during the juvenile period.
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We found that injections took less time in tickled rats than in nonhandled rats. Provision of tickling does require that handlers spend extra time with the rats and thus increases the total handling time associated with the medical procedure. However, on the basis of our results, we assert that this time investment results in reduced fear of humans, attenuation of discomfort associated with injections, increased positive affect in the time immediately after the aversive procedure, and reduced time spent attempting to restrain naïve, fearful animals for injection. When rats are less fearful of humans and more accepting of the procedure, rats struggle less, thus reducing the threat of injury to themselves and handlers. Currently, depending on facilities, rat intraperitoneal injections are performed by using either a one- or a 2-person technique. The 2-person technique, in which one person restrains the rat and the other performs the injection, is often preferred because the rat is controlled more effectively, thereby reducing the risk of injury. Although our study did not compare both procedures, our results show that applying tickling in association with giving intraperitoneal injections makes it easy for a single person to handle the rat in a manner safe for both rat and handler. Therefore, using a one-person rather than a 2-person technique could partially compensate for the extra time invested in tickling. As reviewed previously,37 several studies have shown that standard handling (that is, the minimal amount of handling required for husbandry and experimental procedures) can be associated with poor repeatability of studies. Our results show that when handling is playful and mimics the rats’ own social play, it avoids the problem of animals developing an anticipatory stress response to aversive procedures.27 Therefore, integrating tickling into laboratory procedures could improve both rat welfare and the quality and validity of data collected from these animals, consequently potentially reducing the number of animals used and the need to repeat experiments. We had expected that exposure to the procedure for 10 consecutive days would lead to increases in corticosteroid levels and decreases in body weight suggestive of chronic stress. Changes in body weight (a measure used to gauge stress28) and corticosteroid levels in feces collected from the home environment were not affected by treatment, providing evidence that repeated, daily injections did not disrupt the rats sufficiently to cause chronic stress. Body weights continued to increase over time and were within the normal range for the strain.20 Our findings are consistent with previous studies assessing the effect of repeated injection on body weight42 and plasma corticosterone levels.24 Changes in plasma corticosterone levels have been reported after 30-min to 3-h periods of restraint in samples collected 3 h or less after the stressor.39,40 Considering that the restraint period of our procedure was less than 40 s and that the postinjection samples were collected more than 24 h after the last test, it is likely that the duration of the procedure was too short to induce long-term detectable changes in corticosteroid levels or to affect the release of corticotropin-releasing factor in quantities sufficient to affect body weight gain.21 Furthermore, rats may have habituated to the intraperitoneal procedure and interaction with humans, because corticosteroid levels decreased over time and were comparable to the prestressor levels reported previously.12 We conclude that tickling is a positive handling technique that can mitigate the aversiveness of intraperitoneal injections and facilitate handling in male rats, especially those with prior tickling experience as juveniles, consequently improving laboratory rat welfare. We expect our results also apply to female rats, because they respond to tickling similarly to male rats.30 However, sex-associated differences in response to painful,
stressful stimuli17,18 could affect results. If tickling during the juvenile period is not practically feasible, tickling in adulthood has benefits. A social buffering effect of playful handling may be applicable to other species that display play behavior that can safely be mimicked through human interaction and to other mildly aversive medical procedures performed for veterinary or experimental reasons. In this way, playful handling could serve as a valuable refinement, especially when it is necessary to perform procedures multiple times.
Acknowledgments
We thank the staff of the EALB Vivarium for animal care and Mr MD McNabb for assisting with preparation of the manuscript. This material is based on work supported by a Washington State University Veterinary Summer Research Fellowship Program (to KW) and an Animal Welfare Enhancement Award from the Animal Welfare Institute (to SC).
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