Table 1 Effects of handling method and hold duration on voluntary interaction with the handler.

Voluntary interaction	First handling session		Fifth handling session
(a) Response after handling
Handling method	F_2,52 = 138.4	P < 0.0001	F_2,56 = 331.8	P < 0.0001
Hold duration	F_3,52 = 0.69	P = 0.56	F_3,56 = 2.38	P = 0.08
Sex	F_1,52 = 0.24	P = 0.63	F_1,56 = 2.59	P = 0.11
Method × duration	F_6,52 = 0.59	P = 0.74	F_6,56 = 0.77	P = 0.60
Method × sex	F_2,52 = 1.78	P = 0.18	F_2,56 = 1.82	P = 0.17
Duration × sex	F_3,52 = 0.21	P = 0.89	F_3,56 = 0.72	P = 0.55
3 way interaction	F_6,52 = 0.40	P = 0.88	F_6,56 = 0.61	P = 0.72
Planned contrasts:
tail vs tunnel		P < 0.0001		P < 0.0001
tail vs cup		P = 0.07		P < 0.0001
(b) Immediately before vs after handling
Handling method	F_2,52 = 159.0	P < 0.0001	F_2,56 = 402.0	P < 0.0001
Hold duration	F_3,52 = 0.47	P = 0.71	F_3,56 = 1.62	P = 0.19
Sex	F_1,52 = 0.01	P = 0.98	F_1,56 = 2.51	P = 0.12
Before vs after handling	F_1,52 = 0.42	P = 0.52	F_1,56 = 16.85	P = 0.0001
Method × duration	F_6,52 = 0.23	P = 0.96	F_6,56 = 0.84	P = 0.54
Method × before/after	F_2,52 = 3.25	P = 0.047	F_2,56 = 2.05	P = 0.14
Duration × before/after	F_3,52 = 0.81	P = 0.50	F_3,56 = 1.79	P = 0.16
Sex × before after	F_1,52 = 1.06	P = 0.31	F_1,56 = 0.08	P = 0.78
Before vs after handling:
tail	F_1,8 = 19.41	P = 0.002	F_1,8 = 1.34	P = 0.34
tunnel	F_1,24 = 0.69	P = 0.42	F_1,24 = 7.04	P = 0.014
cup	F_1,24 = 0.74	P = 0.40	F_1,24 = 20.24	P = 0.0001
Planned contrasts:
tail vs tunnel		P < 0.0001		P < 0.0001
tail vs cup		P = 0.39		P = 0.001

Mice were picked up by their assigned method (tail, tunnel or cup) and held for 2, 10, 30 or 60 s per day each day for five days. Voluntary interaction was measured before and after handling on days 1 and 5, averaged for both mice tested together in the same cage (data shown in Fig. 1A,B and Supplementary File 1). Data were log transformed to meet assumptions for parametric analysis. (a) Univariate ANOVA with planned contrasts between tail and non-aversive methods. (b) Repeated measures ANOVA comparing response before and after handling (higher order interactions not shown, but all were non-significant). Significant effects (P < 0.05) are in bold. Sample size for the first handling session was reduced because of technical problems with 4 video files, though all animals underwent their assigned handling.

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