Table 5.

Effects of handling method and frequency on voluntary interaction with the handler.

Voluntary interaction	1st cage clean		4th cage clean		5th cage clean
Response after handling
Handling method	F2,36 = 54.72	P < 0.0001	F2,36 = 92.76	P < 0.0001	F2,36 = 65.33	P < 0.0001
Frequency	F1,36 = 0.05	P = 0.83	F1,36 = 0.10	P = 0.75	F1,36 = 34.25	P < 0.0001
Sex	F1,36 = 0.24	P = 0.63	F1,36 = 0.04	P = 0.85	F1,36 = 0.14	P = 0.71
Method × frequency	F2,36 = 0.04	P = 0.97	F2,36 = 0.34	P = 0.71	F2,36 = 5.73	P = 0.007
Method × sex	F2,36 = 0.18	P = 0.84	F2,36 = 0.28	P = 0.76	F2,36 = 4.03	P = 0.026
Frequency × sex	F1,36 = 1.25	P = 0.27	F1,36 = 0.04	P = 0.84	F1,36 = 0.08	P = 0.78
3 way interaction	F2,36 = 0.57	P = 0.57	F2,36 = 0.03	P = 0.97	F2,36 = 1.78	P = 0.18
Planned contrasts:
tail vs tunnel		P < 0.0001		P < 0.0001		P < 0.0001
tail vs cup		P = 0.93		P = 0.34		P < 0.0001
Daily vs cage clean only†
tail					F1,12 = 1.30	P = 0.28
tunnel					F1,12 = 12.12	P = 0.005
cup					F1,12 = 24.47	P = 0.0003
Male response after handling‡
Handling method					F2,18 = 47.76	P < 0.0001
Frequency					F1,18 = 16.27	P = 0.001
Method × frequency					F2,18 = 2.29	P = 0.13
Planned contrasts:
tail vs tunnel						P < 0.0001
tail vs cup						P = 0.010
Female response after handling‡
Handling method					F2,18 = 22.79	P < 0.0001
Frequency					F1,18 = 17.99	P < 0.0001
Method × frequency					F2,18 = 5.08	P = 0.018
Planned contrasts:
tail vs tunnel						P < 0.0001
tail vs cup						P = 0.001

Mice were picked up briefly by their assigned method (tail, tunnel or cup) to transfer them between cages at four fortnightly cage cleans; between the 4^th and 5^th cage clean, half were assigned to brief daily handling (approx. 2 s) while the other half were only handled at cage cleaning. Voluntary interaction, averaged for both mice tested together in the same cage, tested immediately after mice were transferred to a clean cage (data shown in Fig. 3A). Univariate ANOVAs after 1^st, 4^th and 5^th cage clean with planned contrasts between tail and non-aversive tunnel or cup methods. Significant effects (P < 0.05) are in bold. ^†Daily vs fortnightly handling was assessed separately for each handling method due to a significant interaction between handling method and frequency at 5^th cage clean. ^‡Male and female response assessed separately due to a significant interaction between handling method and sex at 5^th cage clean. A significant interaction between handling method and frequency among females was due to much greater voluntary interaction among females handled daily by cupping between 4^th and 5^th cage clean compared to those handled only at cage cleaning.