Table 3 Effects of scruff restraint and prior handling on voluntary interaction with the handler.

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

Voluntary interaction	First scruff		Third scruff		24 h after 3^rd scruff
(a) Before scruff restraint
Handling method	F_2,56 = 220.1	P < 0.0001	F_2,56 = 175.9	P < 0.0001	F_2,55 = 247.02	P < 0.0001
Prior hold duration	F_3,56 = 1.28	P = 0.29	F_3,56 = 2.63	P = 0.06	F_3,55 = 2.35	P = 0.082
Sex	F_1,56 = 0.70	P = 0.41	F_1,56 = 2.05	P = 0.16	F_1,55 = 1.21	P = 0.28
Method × duration	F_6,56 = 0.44	P = 0.85	F_6,56 = 1.95	P = 0.09	F_6,55 = 1.50	P = 0.20
Method × sex	F_2,56 = 0.13	P = 0.88	F_2,56 = 0.52	P = 0.60	F_2,55 = 0.63	P = 0.54
Duration × sex	F_3,56 = 0.46	P = 0.72	F_3,56 = 0.05	P = 0.98	F_3,55 = 0.46	P = 0.71
3 way interaction	F_6,56 = 0.68	P = 0.67	F_6,56 = 0.19	P = 0.98	F_6,55 = 2.00	P = 0.08
Planned contrasts:
tail vs tunnel		P < 0.0001		P < 0.0001		P < 0.0001
tail vs cup		P = 0.017		P < 0.0001		P < 0.0001
(b) After scruff restraint
Handling method	F_2,56 = 117.1	P < 0.0001	F_2,56 = 122.3	P < 0.0001	F_2,55 = 208.84	P < 0.0001
Prior hold duration	F_3,56 = 2.04	P = 0.12	F_3,56 = 2.54	P = 0.07	F_3,55 = 4.90	P = 0.004
Sex	F_1,56 = 4.16	P = 0.046	F_1,56 = 1.82	P = 0.18	F_1,55 = 0.08	P = 0.78
Method × duration	F_6,56 = 1.42	P = 0.22	F_6,56 = 1.58	P = 0.17	F_6,55 = 2.69	P = 0.023^†
Method × sex	F_2,56 = 0.54	P = 0.59	F_2,56 = 0.28	P = 0.76	F_2,55 = 0.05	P = 0.95
Duration × sex	F_3,56 = 0.25	P = 0.86	F_3,56 = 0.65	P = 0.59	F_3,55 = 0.91	P = 0.44
3 way interaction	F_6,56 = 0.25	P = 0.96	F_6,56 = 1.55	P = 0.18	F_6,55 = 1.73	P = 0.13
Planned contrasts:
tail vs tunnel		P < 0.0005		P < 0.0005		P < 0.0001
tail vs cup		P < 0.0005		P < 0.0005		P < 0.0001
(c) Before vs after restraint
Before vs after	F_1,56 = 2.97	P = 0.09	F_1,56 = 2.22	P = 0.14	F_1,55 = 21.02	P < 0.0001
Method × before/after	F_2,56 = 7.25	P = 0.002	F_2,56 = 4.49	P = 0.016	F_2,55 = 2.79	P = 0.07
Tail	F_1,8 =5.43	P = 0.048	F_1,8 =1.62	P = 0.24	F_1,8 =1.93	P = 0.20
Tunnel	F_1,24 = 9.64	P = 0.005	F_1,24 = 2.67	P = 0.11	F_1,24 = 10.90	P = 0.003
Cup	Interaction with sex		F1,24 = 5.76	P = 0.025	F1,24 = 15.56	P = 0.001
Cup: males	F_1,12 = 0.70	P = 0.42
Cup: females	F_1,12 = 16.97	P = 0.001

During the week prior to testing, mice had been picked by their assigned method (tail, tunnel, cup) and held for 2, 10, 30 or 60 s per day (prior hold duration). Mice were then picked up by their assigned method prior to 10 s scruff restraint on three successive days. Voluntary interaction, averaged for both mice tested together in the same cage, was assessed immediately before and after scruff restraint on the first and third day, and 24 h later, before and after pick up by the assigned method for 2 s without scruff restraint (data shown in Fig. 2A). Data were log transformed to meet assumptions of parametric analysis. (a,b) Univariate and (c) repeated measures ANOVAs with planned contrasts between tail and non-aversive methods. Significant effects (P < 0.05) are in bold. Repeated measures ANOVA (c) included prior hold duration and sex but these were not significant. Response to first scruff among those handled by cupping depended on sex, with females showing significantly greater interaction after scruffing than before. ^†Significant interaction between handling method and prior hold duration 24 h after 3^rd scruff was because cupped mice showed lowest interaction when previously held for 2 s and highest interaction when held for 60 s per day (Bonferoni post-hoc comparison, P = 0.002) while prior hold duration did not influence those handled by tail or tunnel.

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Table 3 Effects of scruff restraint and prior handling on voluntary interaction with the handler.

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