Improving the practicality of using non-aversive handling methods to reduce background stress and anxiety in laboratory mice

Abstract

Handling can stimulate stress and anxiety in laboratory animals that negatively impacts welfare and introduces a confounding factor in many areas of research. Picking up mice by the tail is a major source of handling stress that results in strong aversion to the handler, while mice familiarised with being picked up in a tunnel or cupped on the open hand show low stress and anxiety, and actively seek interaction with their handlers. Here we investigate the duration and frequency of handling required for effective familiarisation with these non-aversive handling methods, and test whether this is sufficient to prevent aversion and anxiety when animals then experience immobilisation and a mild procedure (subcutaneous injection). Very brief handling (2 s) was sufficient to familiarise mice with tunnel handling, even when experienced only during cage cleaning. Brief but more frequent handling was needed for familiarisation with cup handling, while pick up by tail induced strong aversion even when handling was brief and infrequent. Experience of repeated immobilisation and subcutaneous injection did not reverse the positive effects of tunnel handling. Our findings demonstrate that replacing tail with tunnel handling during routine cage cleaning and procedures provides a major refinement with little if any cost for familiarisation.

Figure 1

Effect of handling method and hold duration on voluntary interaction and anxiety (mean ± sem). Duration of voluntary interaction with the handling device (hand for tail and cup, or hand holding tunnel) (A) after the first and fifth daily handling session according to hold duration and method, and (B) immediately before (b) and after (a) handling in the first and fifth sessions pooled across hold durations. (C,D,E) Measures of anxiety in an open field test conducted two days after the fifth handling session (m: males, f: females). P values from univariate (A) or repeated measures ANOVAs (B,C) shown in Tables 1 and 2. Asterisks indicate significance of planned comparisons between tunnel or cup versus tail (*P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001). Data for tunnel and cup handled mice pooled across groups experiencing same or tail handling at cage cleaning prior to the experiment. Number of mice/cages: Tail = 16/8 m + 16/8 f (8/4 mf per duration), Tunnel = 32/16 m + 32/16 f (16/8 mf per duration), Cup = 32/16 m + 32/16 f (16/8 mf per duration).

Figure 2

Effect of handling method and restraint by scruff on voluntary interaction and anxiety (mean ± sem). (A) Duration of voluntary interaction with handling device (hand for tail and cup, or hand holding tunnel) assessed immediately before (b) and after (a) scruff restraint among mice familiarised for one week with tail, tunnel or cup methods in first and third daily restraint sessions, and when picked up but not restrained 24 h later. (B) Measures of anxiety in an elevated plus maze test conducted two days after experience of 3^rd scruff restraint among males (m) and females (f) familiarised with tail, tunnel or cup handling. Data for mice held for different durations during handling familiarisation pooled. P values from repeated measures ANOVAs shown in Tables 3 and 4. Asterisks indicate significance of planned comparisons between tunnel or cup versus tail (*P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001). Data for tunnel and cup handled mice pooled across groups experiencing same or tail handling at cage cleaning prior to the experiment (N sizes as for Fig. 1).

Figure 3

Effect of handling method and frequency on willingness to interact with handler and anxiety. Mice were handled briefly only at cleaning for the first 4 fortnightly cage cleans. Between 4^th and 5^th cage clean, mice received supplementary brief (2 s) daily handling for nine sessions (d) or continued only to be handled at cage cleaning (c). (A) Duration of voluntary interaction with the handling device (hand for tail and cup, or hand holding tunnel) assessed immediately after cage cleaning (mean ± sem). (B) Percentage of mice reluctant to be handled when approached by the handler, measured immediately following post-handling voluntary interaction test. (C) Measures of anxiety in an elevated plus maze test conducted two days following the fifth cage clean (m: males, f: females). P values from univariate (A, Table 5) or repeated measures (C, Table 6) ANOVAs, or contingency chi-squared tests (B). Asterisks indicate significance of planned comparisons between tunnel or cup versus tail (*P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001). N sizes for mice/cages: Tail = 16/8 m + 16/8 f, Tunnel = 16/8 m + 16/8 f, Cup = 16/8 m + 16/8 f (half handled daily (d) and half at caged clean only (c) following 4^th cage clean).

Figure 4

Response of mice familiarised with tunnel or tail handling to subcutaneous injection. Mice were familiarised with brief (2 s) pick up by tunnel or tail in 10 daily sessions. They were then either picked up, restrained by scruff and given a subcutaneous injection (i) or just picked up briefly without restraint or injection (c). (A) Duration of voluntary interaction with the handling device (hand for tail and cup, or hand holding tunnel) assessed immediately after 10^th handling familiarisation, 1^st injection or control pick up, and after 5^th injection or control pick up (mean ± sem). Pre-inject: for the 10^th handling familiarisation, mice assigned to the injection treatment are compared to those in the control pick up treatment, although neither group had experienced injection at this point. (B) Percentage of mice reluctant to be handled when approached by the handler. (C) Exploratory (positive score) versus anxious behaviour (negative score) in a modified open field test conducted after 5^th injection or control pick up (mean ± sem). P values from Mann-Whitney (A, Table 7), repeated measures ANOVA (C, Table 8), or contingency chi-squared tests (B). N = 20 females/10 cages per treatment group.