Table 6 Effects of handling method and frequency on anxiety in an elevated plus maze test.

	Protected stretch attend		Entry to open arms (n)		Time on open arms (s)		Entry to closed arms (n)		Time in central hub (s)
Handling method	F_2,31 = 11.93	P = 0.0001	F_2,31 = 8.30	P = 0.001	F_2,31 = 0.74	P = 0.49	F_2,31 = 7.04	P = 0.003	F_2,31 = 3.86	P = 0.03
Frequency	F_1,31 = 0.01	P = 0.95	F_1,31 = 0.46	P = 0.50	F_1,31 = 1.73	P = 0.20	F_1,31 = 2.57	P = 0.12	F_1,31 = 1.62	P = 0.21
Sex	F_1,31 = 5.22	P = 0.03	F_1,31 = 17.91	P = 0.0002	F_1,31 = 0.95	P = 0.34	F_1,31 = 10.21	P = 0.003	F_1,31 = 3.74	P = 0.06
Method × frequency	F_2,31 = 0.26	P = 0.77	F_2,31 = 0.25	P = 0.78	F_2,31 = 0.52	P = 0.60	F_2,31 = 0.12	P = 0.89	F_2,31 = 0.55	P = 0.58
Method × sex	F_2,31 = 3.46	P = 0.04	F_2,31 = 0.14	P = 0.87	F_2,31 = 3.12	P = 0.058	F_2,31 = 0.65	P = 0.53	F_2,31 = 2.65	P = 0.09
Frequency × sex	F_1,31 = 0.75	P = 0.39	F_1,31 = 0.12	P = 0.73	F_1,31 = 0.21	P = 0.65	F_1,31 = 0.20	P = 0.66	F_1,31 = 0.74	P = 0.40
3 way interaction	F_2,31 = 0.54	P = 0.59	F_2,31 = 0.02	P = 0.98	F_2,31 = 2.41	P = 0.11	F_2,31 = 0.16	P = 0.85	F_2,31 = 1.68	P = 0.20
Planned contrasts:
tail vs tunnel		P < 0.0001		P = 0.002		P = 0.23		P = 0.013		P = 0.024
tail vs cup		P = 0.001		P = 0.001		P = 0.54		P = 0.001		P = 0.025
Handling method
Males only	F_2,16 = 2.22	P = 0.14			F_2,16 = 0.41	P = 0.67
tail vs tunnel		P = 0.07				P = 0.42
tail vs cup		P = 0.13				P = 0.49
Females only	F_2,15 = 9.62	P = 0.002			F_2,15 = 3.66	P = 0.05
tail vs tunnel		P = 0.001				P = 0.02
tail vs cup		P = 0.004				P = 0.10

Mice were handled by their assigned method (tail, tunnel or cup) to transfer them to a clean cage at five fortnightly cage cleans. Half also received brief daily handling between the 4^th and 5^th cage cleans. Mice were delivered to the elevated plus maze test by their assigned method on the day after their 5^th cage clean (data for measures of anxiety (protected stretch attend, entry and time on open arms) shown in Fig. 3C). Repeated measures ANOVAs took into account two mice per cage as a within-subjects factor, with planned contrasts between tail and tunnel or cup methods. Significant effects (P = < 0.05) are in bold. The effect of handling method was examined for each sex separately where there was a significant or almost significant interaction between handling method and sex.

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