Optimization of overhead enclosure monitoring in juvenile male Dunkin Hartley guinea pigs (Cavia porcellus)

Abstract

Overhead enclosure monitoring provides objective quantitative mobility measurements for animals undergoing open-field testing. Notably, protocols for testing optimization have been minimally established for the guinea pig. It is unknown whether (a) repeated exposure, (b) time-of-day, or (c) length of testing duration influence outcome parameters. We hypothesized that guinea pigs would display decreased activity following repeated exposure to the open field; heightened activity during the earliest testing period; and that 10 min would be adequate for data collection. The study was conducted in two separate phases to distinguish between enclosure habituation and time-of-day effects, respectively. Two cohorts of male Dunkin Hartley guinea pigs were allowed voluntary movement in an open-field enclosure for 14 min to quantify mobility outcomes, including total distance traveled, total time mobile, average speed while mobile, and total time spent in the shelter. For both phases, testing occurred at four different times of day, and overhead monitoring software was programmed to divide the total testing duration into 2-min bins. Habituation phase results showed time mobile and distance traveled were influenced significantly by repeat exposure, as animals were most active during the first testing event. Time-of-day phase animals spent significantly more time mobile during the earliest testing period. Interestingly, significant differences were observed across 2-min bins for the time-of-day phase but not during the habituation phase. Specifically, progressively decreased ambulatory activity was observed as testing duration increased. Thus, habituation and time-of-day should be accounted for when possible. Finally, a trial period greater than 10 min may not yield additional data.

Keywords: Guinea pig behavior; animal welfare; habituation; open field testing; overhead enclosure monitoring.

Figure 1.

Guinea pig activity is highest during the first testing event, as evidenced by increased time mobile and distance traveled, with shorter durations spent in the red hut shelter. (a) Total time mobile in seconds (left y axis) and as a percentage of total testing duration (right y axis) in 10, five-month-old, intact male guinea pigs. Significant differences were found between testing orders (P = 0.0013), with animals demonstrating the greatest mobility during the first (0530–0700) testing period. (b) Total distance traveled in meters also differed significantly (P = 0.0047) among testing bouts, with animals traveling further in the first testing period. (c) Average speed while mobile in cm/s did not vary significantly between testing periods. (d) Total time in red hut was not significant (P = 0.0769); however, animals spent less time in the shelter during preliminary periods; dashed line represents maximum testing duration (840 s). Representative guinea pig shows increased mobility during the first testing bout by track plot (e) and heat map spectrum (f) of the animal’s head position for total distance traveled during the test duration, bar scale 0 s (left) to 15 s (right). Statistical analyses were performed using RM one-way ANOVA. Data presented as median (IQR). *P ≤ 0.05.

Figure 2.

Mobility outcomes in the time-of-day phase revealed guinea pig activity was highest at the earliest (0530–0700) timepoint. (a) Total time mobile in seconds (left y axis) and as a percentage of total testing duration (right y axis) in 24, two-month-old guinea pigs. Total time mobile differed significantly between times of day (P = 0.0489). (b) Mean differences in total distance traveled approached significance (P = 0.0553) with animals traveling further in the 0530–0700 period. (c) Average speed while mobile in cm/s and (d) total time in red hut did not vary significantly between time-of-day. However, animals did spend the least time in red hut during the 0530–0700 timepoint; dashed line represents maximum testing duration (840 s). Statistical analyses were performed using RM one-way ANOVA. Data presented as median (IQR). *P ≤ 0.05.

Figure 3.

Open-field enclosure monitoring software, programmed to create distinct 2-min bins (x axis) within 14-min testing period, identified diminishing returns of data collection beyond 10 min in the habituation phase. Mean values across 2-min intervals were not statistically significant for (a) time mobile, (b) total distance traveled in meters or (c) average speed while mobile in cm/s (median with IQR). As per data provided in Figure 1, animals demonstrated increased activity during the first exposure to the enclosure (P = 0.0013). (d) Total time in red hut in seconds (median with IQR) showed no significant differences overall; however, animals spent extensive time in the red hut during the third and fourth tests. (e) Median cumulative percent distance traveled approached 90% in all timepoints within the first 10-min of the 14-min testing interval.

Figure 4.

Open-field enclosure monitoring software, programmed to create distinct 2-min bins (x axis) revealed decreasing mobility outcomes and increasing time spent in the red hut shelter as testing duration progressed in the time-of-day phase. Mean values across 2-min intervals were statistically significant (P < 0.0001) for all outcome measures: (a) time mobile, (b) total distance traveled in meters, (c) average speed while mobile in cm/s and (d) total time in red hut in seconds, all median with IQR. (e) Median cumulative percent distance traveled was over 85% in all timepoints within the first 10 min of the 14-min testing interval. Differences in testing periods and bins were analyzed using RM two-way ANOVA with Geisser-Greenhouse correction.