Table 3.

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

Voluntary interaction	First scruff		Third scruff		24 h after 3rd scruff
(a) Before scruff restraint
Handling method	F2,56 = 220.1	P < 0.0001	F2,56 = 175.9	P < 0.0001	F2,55 = 247.02	P < 0.0001
Prior hold duration	F3,56 = 1.28	P = 0.29	F3,56 = 2.63	P = 0.06	F3,55 = 2.35	P = 0.082
Sex	F1,56 = 0.70	P = 0.41	F1,56 = 2.05	P = 0.16	F1,55 = 1.21	P = 0.28
Method × duration	F6,56 = 0.44	P = 0.85	F6,56 = 1.95	P = 0.09	F6,55 = 1.50	P = 0.20
Method × sex	F2,56 = 0.13	P = 0.88	F2,56 = 0.52	P = 0.60	F2,55 = 0.63	P = 0.54
Duration × sex	F3,56 = 0.46	P = 0.72	F3,56 = 0.05	P = 0.98	F3,55 = 0.46	P = 0.71
3 way interaction	F6,56 = 0.68	P = 0.67	F6,56 = 0.19	P = 0.98	F6,55 = 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	F2,56 = 117.1	P < 0.0001	F2,56 = 122.3	P < 0.0001	F2,55 = 208.84	P < 0.0001
Prior hold duration	F3,56 = 2.04	P = 0.12	F3,56 = 2.54	P = 0.07	F3,55 = 4.90	P = 0.004
Sex	F1,56 = 4.16	P = 0.046	F1,56 = 1.82	P = 0.18	F1,55 = 0.08	P = 0.78
Method × duration	F6,56 = 1.42	P = 0.22	F6,56 = 1.58	P = 0.17	F6,55 = 2.69	P = 0.023†
Method × sex	F2,56 = 0.54	P = 0.59	F2,56 = 0.28	P = 0.76	F2,55 = 0.05	P = 0.95
Duration × sex	F3,56 = 0.25	P = 0.86	F3,56 = 0.65	P = 0.59	F3,55 = 0.91	P = 0.44
3 way interaction	F6,56 = 0.25	P = 0.96	F6,56 = 1.55	P = 0.18	F6,55 = 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	F1,56 = 2.97	P = 0.09	F1,56 = 2.22	P = 0.14	F1,55 = 21.02	P < 0.0001
Method × before/after	F2,56 = 7.25	P = 0.002	F2,56 = 4.49	P = 0.016	F2,55 = 2.79	P = 0.07
Tail	F1,8 = 5.43	P = 0.048	F1,8 = 1.62	P = 0.24	F1,8 = 1.93	P = 0.20
Tunnel	F1,24 = 9.64	P = 0.005	F1,24 = 2.67	P = 0.11	F1,24 = 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	F1,12 = 0.70	P = 0.42
Cup: females	F1,12 = 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.