Schematic representations of the parental care test in a mated pair isolated in an old (b–d) or new (n–p) cage. After cohabiting with their pups as a family unit (a), the parents were separated from the pups for 10 min (b–e) and then reunited with five pups (f–i). Subsequent pup retrieval behaviour was then observed (j–m), and the number of family members (new sire or dam) showing retrieval was scored and expressed as a percentage in (z) (one trial per mouse). In (a–m), the pups were placed in a new holding box (e, yellow), and the parents remained in the old box (b–d, grey) during the separation period (n=35 families each). The number on the right indicates the sires or dams with retrieval out of subjects tested. In (a,n–y), the pups remained in the old cage (q, grey), while the parents (separately or together) were placed in new isolation boxes (n–p, yellow; n=40 families each). The number on the right indicates sires or dams with retrieval out of subjects tested. (z) Percentages of males (M) and females (F) showing retrieval behaviour upon reuniting after separation alone (MA or FA) or with the mate (MW or FW). Left, results of the experiments in sires, dams, and sires and dams that were separated for 10 min in old cages. Right, results of the experiments on animals separated into new cages. Fisher’s test, two-tailed, *, **P<0.01, 0.001, respectively).

(a) Duration of various maternal behaviours performed by the sires in their home cages for 30 min before and after 10-min separation from pups in a new cage together with their mates. Mean±s.e.m. (n=5 sires; Student’s t-test, *P<0.05). (b,c) Effects of cohabitation duration on retrieval behaviour in parental males (b) and females (c). Parents were isolated alone (squares) or together with their mates (circles) in a new cage for 3, 5, 10 or 30 min. Subsequently, the animals showing retrieval upon reunion in the old cages were counted. In two different separation groups, 24 families were examined at each time point. Experiments were grouped for statistical analysis, and one-way ANOVA followed by Bonferroni’s post hoc test was performed (F_4,15=3.56, *P<0.05 from 0 min, **P<0.01 from 5-min cohabitation). Student’s t-test for two different separation groups at 10 and 30 min, ^#P<0.01.

(a–d) Schematic views of the social interaction chamber. New sires were separated from pups either alone (a, experiment I) or with their mates (c, experiment II) for 3, 5 or 10 min in a central compartment in the Perspex box. Then, five pups were placed in the left compartment (filled box); the opposite compartment was empty (open box), and the sire was then placed alone in the centre compartment. The movements of the sire were recorded online using tracking software. (e,f) Two representative traces of the sires’ movements during social exploration with pups in the left compartment and in the empty cylinder on the right, after separation for 10 min. Left, experiment I (b); and right, experiment II (d). (g,h) By analysing the time spent in three chambers as a function of the isolation period, social preference of the sires was quantified when they were isolated alone (g; n=20), or isolated with their mates (h; n=20). Filled, grey and white bars represent time spent in left, centre and right chambers, respectively (mean±s.e.m.). One-way ANOVA followed by Bonferroni’s post hoc test, the letters (a-c) above the bars indicate significant difference from duration in the central room at P<0.001, P<0.01 and P<0.05, respectively, and (d) denotes significant difference from the left zone at P<0.05.

(a–h) A schematic representation of the care behaviour test in a mouse pair housed in the same cage (c,f) and isolated from pups in (b) and away from the home nursing cages (a). During the 10-min separation, the parental female was housed either in an open-top box (c) or in a close box (f). The male was then reunited with the pups (d,g), and his subsequent retrieval behaviour (one trial per male) was observed (e,h). (i) Percentage of sires with retrieval responses. The first two columns show responses when the dam was placed in the open-top (c) or sealed box (f). Columns 3–5 show the retrieval behaviour by sires with deafness (with wax ear plugs), anosmia (with 5% ZnSO₄ solution poured into both nostrils) or both. N=12 sires each, Fisher’s test, two-tailed, *P<0.05. (j) Acoustic and olfactory cues independently induced paternal parental behaviour. USVs were recorded for 10 min from a dam in a new cage with a sire after his removal from a home cage or from virgin females; USVs from the maternal mate (columns 1 and 3) were then played back in a soundproof chamber to the father of the same couple in a new cage (column 1) or in the cage that had previously housed the dam (excreta; column 3). Sires were exposed either to USVs from virgins in a new cage (column 2) or only to the virgins’ excreta (column 5), as above. The sires were then scored for retrieval behaviour when they were returned to their old home cages with pups. N=9–15 subjects, Fisher’s test, two-tailed, *P<0.05.

USVs emitted by a dam co-housed with a sire during a separation from their pups (a–c) or by a virgin female to another virgin female (d–f). Representative sonograms (a,d) show the acoustic power (sound pressure level, SPL) of the vocalizations as a function of time and frequency (b,c and e,f). Note the clusters of very brief USVs of ~38 kHz in b and c and the continuous sounds of two distinct USVs at ~47 and 68 kHz in e and f. r.m.s., root mean squared.