Use of a conditioning technique to reduce stress associated with repeated intra-peritoneal injections in laboratory rats

We hypothesized that using classical conditioning to pair a stressful procedure such as injection with a rewarding experience decreases the stress associated with repeated exposure to this procedure. We investigated the effectiveness of pairing repeated intra-peritoneal injections with a positive stimulus to reduce physiological and behavioural stress responses of laboratory rats by comparing injection with 1 ml/kg of saline followed by one of three different reward types (food treat (F), stroking (S) and tickling (T)), delivered in a treatment box, with two control conditions (handling without injection (CH) and injected (CI)) followed by an equivalent period in the treatment box. Treatments were applied to rats (N = 40; 8 rats/treatment) daily for 10 days. Audible and 50 kHz (laughing) vocalizations were assessed during handling and while in the treatment box. Chromodacryorrhea (an indicator of stress) was assessed daily in the home cage. Body weight, and behaviour in the Human Approach (HA) test, were assessed before (baseline) and after the 10-day treatment period. Behaviour in the Elevated Plus Maze (EPM) test was assessed after the 10-day treatment period. Rats on all treatments produced more chromodacryorrhea during the treatment period compared to baseline, suggesting that all treatments induced stress. Principal component analyses on behaviour in the HA and EPM tests revealed two and three factors that explained 86.8% and 98.7% of total variance, respectively. Multivariate analysis of variance on these factors, along with the other variables, revealed a significant contrast between CH and the other treatments. CH rats performed fewer audible vocalizations when handled compared to rats given injections. We found no clear benefit of providing a food treat, stroking, or tickling to alleviate stress associated with injection under the conditions of this study, possibly due to differences between rats in their perception of the aversiveness of handling restraint and injection, their stress response style (active or passive), and their perception of the pleasantness of the different rewards. Rats on all treatments emitted 50 kHz chirps in the treatment box, suggesting that being placed in the box after handling was rewarding regardless of treatment.