Supplementary Figure S1. Parental behaviour by sires separated alone. The duration spent outside or inside the nest, huddling and licking, retrieval, and burrowing under chips during 30 min preceding the 10-min separation alone in a new cage and after reunion in the old cage is illustrated. Mean ± s.e.m. (n = 6); n.s., not significant.
Supplementary Figure S2. Immobilisation times in the interacting zone of a 3-chamber apparatus. Sires were isolated alone (A) or with mates (B) in new cages for the indicated times (3, 5, and 10 min). The immobilisation time in the left interacting zone with 5 pups (filled bars) or in the right zone without pups (open bars) is illustrated. One-way ANOVA followed by Bonferroni s post hoc test: n = 20, F 2,55 = 4.41, *p < 0.05, F 2,51 = 6.45,**p < 0.01.
Supplementary Figure S3. 38-kHz USVs emitted in the home cage. USVs were recorded from the dam and pups and a sire that had been first isolated in a new cage for 10 min and then returned to its family. USVs were recorded from 10 family units for 10 min each.
Supplementary Figure S4. USVs emitted from virgin females and sires. USVs were recorded from a female co-housed with an unrelated sire in the home cage (A) or a new cage (B). USVs recorded from 5 pairs are illustrated.
Supplementary Figure S5. The acoustic startle responses as a function of increasing sound intensity (100, 110 and 120 db) in mice with intact hearing (blue) and ear plugs (red). An assessment of the acoustic startle response in males in response to randomised 40-ms bursts of 100 db, 110 db and 120 db white noise. The unreadable values in the ear-plug mice were 0.098 ± 0.01, 0.14 ± 0.05, and 0.13 ± 0.04, respectively. In the absence of a weight correction, the acoustic startle response was significantly higher in the control mice than in ones with ear-plugs. (100 db, F = 56.1, p < 0.0001). All values refer to mean values ± SEM (n=5). Oneway ANOVA followed by Bonferroni s post hoc test, F 4,12 = 30.99, *p < 0.002, **p < 0.001.
Supplementary Figure S6. Photomicrographs of sections of mouse olfactory epithelium (OE) stained with haematoxylin (blue; cell nuclei) and eosin (pink; cell cytoplasm) in the nasal cavities of control (A, B, and C) and ZnSO 4 -treated (D, E, and F) male mice. Lamina propria (LP) and bundles of olfactory axons (a) are shown. Note that ZnSO 4 causes severe damage to the OE. The damage generally consists of a complete separation of the OE from the LP, disruption of the OE and loss of columnar organisation. The bundles of olfactory axons are also destroyed. Scale bar = 200 µm in A and D. Scale bar = 50 µm in B, C, E, and F.
Supplementary Table S1. Call number and duration of USVs recorded under different conditions Conditions Cage Number of calls* Call duration (calls/min) (n) (msec) (n) Dam with a sire and pups old 0 (10) with a sire old 0 (10) with pups old 0 (15) with pups then the sire** old 0.06 (10) 55.0 ± 6.9 (6) with a sire and pups new 0 (10) with a sire new 5.1 ± 0.8 (12) 121 ± 26 (200) with pups new 0 (5) with pups then the sire** new 0 (5) Virgin female alone old 0 (5) alone new 0 (5) with a sire*** old 1.5 ± 1.3**** (5) 59.4 ± 2.3 (70) with a sire*** new 2.8± 1.8**** (5) 72.5 ± 2.6 (82) with a virgin female old 0***** (5) with a virgin female new 0***** (8) Sire alone old 0 (10) * USVs with 35-45 khz were analysed for 10 min in (n) observations. **USVs were recorded from an old or new cage after reunion with the sire, in which a dam and pups were placed. ***Non-mating sires. ****Most of USVs were presumably emitted from sires, according to previous reports (18,20). *****USVs with different frequencies were recorded as shown in Fig. 5d-f.
Supplementary Methods Startle response The acoustic startle response was measured using a commercially available behavioural testing system (San Diego Instruments, San Diego, CA, USA). This apparatus consisted of a soundproof box with a 6-cm speaker to deliver the acoustic stimuli, coupled to an accelerometer to monitor the animal's movement. The behavioural testing was performed in a separate room, and the cages were moved into the room at least 2 h before the start of the experiment. Prior to testing, each mouse was placed inside the startle apparatus and allowed to acclimatise for 5 min. To test the acoustic startle response, acoustic stimuli were presented as 40-ms impulses of white noise with three different intensities (100, 110 and 120 db) a total of 20 times, each in a pseudorandom order and spaced at random intervals between 10 and 20 s. Responses were expressed in arbitrary units. Haematoxylin eosin staining A solution of 5% ZnSO4 (0.06 ml) was irrigated into the ICR male s nose. After 2 h, the mice were fixed by transcardial perfusion with ice-cold 1X PBS followed by internal fixation by 4% PFA perfusion. Next, the mouse snouts were cut and put into a 4% PFA solution overnight for external fixation. The snouts were rinsed in 5% formic acid solution for decalcification for 2 days. After neutralisation of the solution with 5% sodium sulfate for 1 day, the snouts were washed with running water, embedded in the OCT compound, and sectioned into 10-µm-thick slices. The samples were dried for 10 min, stained with one drop of haematoxylin for 2-3 minutes, and washed for 1 minute with pure water. One drop of 0.5% Eosin in 80% ethanol was poured to the samples on slides for 2 s, washed in 50%, 80%, 90%, and 95% ethanol for 2 s, followed by 100% ethanol for 5 min. After treatment with xylene for 5 min, the samples were covered with coverglasses in MGK-S solution.