Male and female Slc:ICR, C57BL/6, and BALB/c mice were obtained from Japan SLC, Inc. (Hamamatsu, Japan) via a local distributor (Sankyo Laboratory Service Corporation, Toyama, Japan). The ICR mice were originally obtained from Charles River Laboratories in 1965 and since then bred in Japan with the alternative name Swiss CD1. The offspring of these mice were born in our laboratory colony, weaned at 21–28 days of age, and housed in same-sex groups of 3–5 animals until pairing (Liu et al., 2013). The animals were paired and kept in our laboratory under standard conditions (24°C; 12-h light/dark cycle, lights on at 08:00) with food and water ad libitum. The mice were housed together continuously in standard mouse maternity cages. The experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Kanazawa University.

Virgin males and females were paired at 45–55 d. A single male and a single female were continuously housed together in a standard mouse maternity cage from the mating period until the delivery of pups. In some experiments, the males were separated in new cages 1 day before parturition to prevent formation of family relationships and kept in the new cages for 3 days. Then, the males were allowed to meet their pups with or without pairmates from day 3 to day 5. All family units composed of a new sire (first-time father), dam, and their first litter were experimentally naïve.

One male parent was placed for 10 min in the original cage or new cage alone or with his pairmate (separation environment). Five pups were randomly selected from the litter and placed individually at a site remote from the nest in the original cage. The sires were returned to the original home cage or a new cage in the presence of their five biological or foster pups to assess parental behavior. Parental retrieval behavior (percentage of sires exhibiting retrieval) was examined for 10 min following reunion. The behavioral tests were performed in a randomly mixed sequence of experimental groups. Experiments were usually performed at 10:00–15:00. We defined retrieval as positive if the sires carried all 5 pups to the original nesting place or within two thirds of the distance between the nest and the place at which the pups had been placed (Liu et al., 2013). We also observed other parental behaviors (grooming, crouching, and huddling) as defined by Gubernick and Alberts (1987, 1989). The animals in this and subsequent experiments were tested only once.

Experiments were carried out in a soundproof chamber measuring 600 × 500 × 500 mm (model MC-050/VA; Muromachi Kikai, Tokyo, Japan). USVs were detected with a condenser microphone (Type 7016; Aco, Tokyo, Japan) and a preamplifier (type 4116; Aco) designed for sound pressure level (SPL) measurements between 20 Hz and 90 kHz. A 4-kHz band-pass filter was used to minimize background noise during recordings; however, most WAV files still contained a considerable amount of “non-USV” signal. Extraneous noise was identified and removed from the sonograms as far as possible. When a rater found an ultrasound signal that was difficult to interpret, the call was evaluated by a minimum of one additional trained observer and identification required a consensus by all raters. Each sonogram was then evaluated with a series of automated parameters. The microphone was placed 50 cm above the cage in a soundproof chamber and connected to an amplifier (model UMA-2; Muromachi Kikai). Acoustic signals were transmitted to a vocalization analyzer system (model MK-1500; Muromachi Kikai) with functions such as an analog-to-digital converter (192 kHz), frequency filters, a digital fast-Fourier-transform analyzer, and signal input—output terminals. Input signals were visualized on SpectraLAB (Sound Technology Inc., State College, PA) in the analyzer system on a personal computer. USVs were recorded as WAVE files and analyzed; the number of calls, frequency, and wave width (>40 ms) were measured using a USV monitor (Muromachi Kikai).

The data were calculated as the means or the means ± s.e.m. Two-tailed Fisher's exact probability test was used for single comparisons of retrieval behaviors. The remaining data were analyzed by two-tailed Student t-test.

The experimental paradigms for each experiment are shown schematically in each figure. In Figure 1, we first reproduced our previous results (Liu et al., 2013). Male and female ICR strain mice were paired and housed together continuously in a standard mouse maternity cage (Figure 1A). The mice were left undisturbed during the first 3 days after the birth of their pups (Figure 1B), during which they displayed distinct paternal and maternal behaviors as described previously (Liu et al., 2013). The sire and dam nursed the pups. This involved nest-building, pup retrieval, licking, and huddling over the pups and lactating. However, as described in the Methods section, we mainly analyzed the male's retrieval behavior, as a parental role, in the following experiments.

The sire in the first family was left alone in the vacated cage during the period of separation (Figure 1C), whereas the pups and dam were removed and placed in a new cage (Figure 1D) separated from the family cage. After 10 min, the five selected pups of the sire (biological offspring) were returned to the nursing cage in a remote area away from the nest, where the sire was present (Figure 1E). The sire retrieved the offspring over 10 min (86% of the sires, n = 15; Figures 1F,S).

If the non-biological (foster) pups (Figure 1N) of the third family (Figures 1M,N) were introduced into the vacated home cage with the second sire (Figures 1I,K) in the second family (Figures 1G,H), instead of the biological pups (Figures 1H,J), 33% of the 15 sires displayed pup retrieval (Figures 1L,S; two-tailed Fisher's exact probability test between sires toward biological (F) and non-biological (L) pups, P < 0.01).

When a sire from the third family (Figure 1N) was placed and isolated for 10 min in the home cage of the second family (Figure 1O), the third sire did not retrieve any of the foster (second family's) pups (Figures 1R,S; n = 15, two-tailed Fisher's exact probability test between unrelated sires (R) and sires with non-biological (L) or biological (F) pups, P < 0.05 and P < 0.0001, respectively). These results suggested that paternal pup retrieval behavior in the home cage is maintained by biological family cues of their mate dams and remaining pups.

Male parental care in Figure 1 may have been induced by the fact that the males were left in the nursing environment during parent–pup separation. To select out pup information during isolation, we used the co-housing paradigm presented in Figure 2. We examined whether sires developed paternal behavior following time spent with the family. Pup retrieval increased on a daily basis after parturition, while dams displayed a higher retrieval ratio from the first day of parturition than the sires (Table 2).

The sires alone (Figure 2D) or together with the mate dams (Figure 2H) were placed in a new cage for 10 min, whereas the pups alone (Figure 2G) or together with dams (Figure 2C) were left in the home cage. Then, the sires were returned to the home cages in which five pups remained (Figures 2E,I). The male's retrieval behavior was undiminished when housed with the pairmate (66%, n = 30; Figures 2J,U) but was strongly reduced when housed alone (24%, n = 41; Figures 2F,U). As expected, a high level of sire care was displayed after isolation in the new environment together with mate dams and pups (as the whole family (Figures 2K,L) (66%, n = 15; Figure 2U): two-tailed Fisher's exact probability test between sires separated alone (F) and together (J) or as a whole family (N), P < 0.001, equally.

The latter was specifically associated with co-habitation with the pairmate dam during the separation period (Figure 2H), because negligible retrieval behavior was apparent if the sire was housed with the dam of another brood (Figures 2O–T; 20%, n = 15); two-tailed Fisher's exact probability test shows no significant difference between sires separated together with unrelated dams (T) and alone (F); and separated together (J), P < 0.01; and separated as a whole family (N), P < 0.05, Figure 2U). Thus, it appears that the mate dam provides some signal(s) during the separation period to induce parental behavior in the sire, in agreement with the results reported previously (Liu et al., 2013). Whereas parental care by the dam is independent of the presence of the male or the housing environment, that by the male is strongly dependent on cues from the pairmate dam and/or home cage.

We recorded USVs (with >40 ms in wave width) to determine their role as one form of critical interactive information in this paradigm. We detected 38-kHz USVs identical to those reported previously (Liu et al., 2013) under isolation conditions in new cages for 10 min between sires and mate dams at a frequency of 25.9 ± 4.8 calls/10 min (n = 8, Table 3; P < 0.01 from other values, two-tailed Student t-test). No identical 38-kHz USVs were recorded between sires and unrelated dams. Instead, 30–80-kHz USVs were recorded infrequently at 40.7 ± 26.7 calls/10 min (n = 11) between unfamiliar couples. These 30–80-kHz USVs were emitted when a sire was co-housed with a virgin female at 313.6 ± 64.9 calls/10 min (n = 11, P < 0.001 from two other values, two-tailed Student t-test). These data clearly support the suggestion that paternal retrieval is essentially triggered by the pairmate's 38-kHz USVs.

The retrieval behavior displayed by males may have been induced by family formation in the nursing cage environment. To assess this possibility, data were obtained from parting males (Figures 3, 4) that remained with the paired pregnant females 1 day before parturition of their first litter and were then separated into a new cage (Figures 3A–C). The males were then isolated alone for 3 days (Figure 3E). When sire paternal retrieval was examined immediately on day 3 in the sire home cage (Figure 3K), 21.4% of sires with no prior contact with their biological pups and pairmate dam, i.e., the paternity unformed state (Figure 3E), displayed retrieval behavior (n = 42; Figure 3N). Next, when the isolated males were relocated in the home cage and stayed with the family (pups and pairmate dam) for 3 days (Figure 3F), the rate of retrieval in their home cage was only 4% (n = 25; Figures 3G–J). Although the sire lived together with the family for 3 days, such treatment made no contribution to the formation of paternity (two values in Figure 3O were equally very low; no significance, two-tailed Fisher's exact probability test).

To further analyze the relevance of family interaction during the stay as a whole family on postnatal days 3–5 (P3–P5) (Figure 3), we used the short-term pup exposure method (twice for 3 h for a total 6 h a day; Figure 4) to acquire or learn the process of paternity for the family. Males were isolated in new cages prior to parturition (Figures 4B,C) and kept in the cages for 2 days (Figures 4D,E). Then, pairmate dams and pups were relocated to the male's cage, and the whole family was kept there for 3 days (Figure 4F). Retrieval behavior was displayed by 8 (62%) of 13 sires (Figures 4G–J). The high level of retrieval appears to have been caused by continuously living in new cages that had been established as the male's territory.

In this suitable condition, we examined whether the presence of the dam was necessary for parental behavior by the isolated males. From P3 to P5, the pups and dam were kept together in their original home cages (Figure 4K), but the pups were temporarily transferred to the sire's cage twice for 3 h (a total of 6 h) per day (Figure 4L), and the males were otherwise alone for the rest of the day (18 h; Figure 4M). These sires showed retrieval behavior at a very high rate (17 (85%) of 20 sires tested; Figures 4O–Q). In both cases, the sires displayed a very high frequency of retrieval after living as the whole family or only with pups shortly in new cages that had, nevertheless, been established as the territory and established nest of the male, although no significant differences were observed between two types of sire (J and Q in Figure 4R; not significant, two-tailed Fisher's exact probability test). Furthermore, these results indicated that direct interaction with the mate dam is not necessary if the home territory is established by the sires.

Finally, we further examined the impact of territorial information on male retrieval behavior. Family cues were learned by individual sires in a manner identical to that shown in Figure 4 (Figures 5A–F) during P3–P5, but in this case, via short exposure by transferring of their biological pups with their dams in new cages to the nursing cage with the sires. Then, retrieval behavior was examined under two housing conditions: in the sire's home cage in which the sire had stayed continuously (Figures 5F,G,I–L), or in a new cage (to the sires) in which the mate dams and pups had been staying (Figures 5G,H,M–P). In the home cages, 10 (50%) of 20 sires showed retrieval (Figure 5L), whereas only 3 (15%) of 20 sires in new cages displayed retrieval behavior (P < 0.05 between testing in old (L) and new (P) cages shown in Figure 5Q, two-tailed Fisher's exact probability test). In both cages, nests were established by the sire and dam. However, the new cages established by the sires' mate dams were quite new to the sires, even if the cages were fully filled with the mate dam's olfactory information.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.