Attraction of female house mice to male ultrasonic courtship vocalizations depends on their social experience and estrous stage

Male house mice (Mus musculus) produce complex ultrasonic vocalizations (USVs), especially during courtship and mating. Playback experiments suggest that female attraction towards recordings of male USVs depends on their social experience, paternal exposure, and estrous stage. We conducted a playback experiment with wild-derived female house mice (M. musculus musculus) and compared their attraction to male USVs versus the same recording without USVs (background noise). We tested whether female attraction to USVs is influenced by the following factors: (1) social housing (two versus one female per cage); (2) neonatal paternal exposure (rearing females with versus without father); and (3) sexual receptivity (pro-estrous and estrous stages versus non-receptive metestrous and diestrous stages). We found that females showed a significant attraction to male USVs but only if females were housed with another female. Individually housed females showed the opposite response. We found no evidence that pre-weaning exposure to a father influenced females’ preferences, whereas sexual receptivity influenced females’ attraction to male USVs: non-receptive females showed preferences towards male USVs but receptive females did not. Finally, we found that individually housed females were more likely to be in sexually receptive estrous stages than those housed socially, and that attraction to male USVs was most pronounced amongst non-receptive females that were socially housed. Our findings indicate that the attraction of female mice to male USVs depends upon their social experience and estrous stage, though not paternal exposure. They contribute to the growing number of studies showing that social housing and estrous stage influence the behavior of house mice and we show how such unreported variables can contribute to the replication crisis.

Introduction 141 (ABEDD, Austria), nesting material (Nestlet, Ehret, Austria), a translucent red house 142 (Tecniplast, Germany) and a cardboard tube. At every cage change, 5 g of seeds and 5 g of 143 apple were added to the cage as additional enrichment.

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145 Social experience treatments (rearing and housing) 146 We manipulated both paternal exposure and social housing, and we consider both 147 treatments as types of social experience. Females were reared with their father (paternal 148 exposure) or without their father, and weaning was conducted at 21 days after the birth of the 149 pups. After weaning, juvenile mice lived in sibling groups for 14 more days and then female 150 subjects were either socially housed (two female mice per cage) or individually housed (one 151 mouse per cage). Female subjects belonged to one of four different treatments: 9 were 12 253 254 Recording behavior 255 The subject females were video recorded during each trial using an infrared sensitive 256 camera (D-Link, model DCS-3710) positioned above the Y-maze to capture all behaviors.
257 Behavioral analyses were conducted using the Observer software (Observer XT 7.0, Noldus, 258 Netherlands). Recordings were blindly analyzed by two experimenters at half speed. We define 259 "chamber" as a stimulus arm and its corresponding circular arena. The duration of time spent 260 in each zone of either side was recorded and summed to generate our response variables defined 261 as "USV playback chamber" or "background noise chamber".

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We also explored whether housing conditions affected the body mass and estrous cycle 316 of females and whether this would have any implications on female playback preference.
317 Differences in body mass between the various social treatment groups were investigated using 318 a LMM with the response variable body mass. The fixed effects chosen were either "overall 319 social background", "housing treatment" or "paternal exposure" and the random effect was age 320 (to control for the variation in body mass not attributed to housing condition   In addition to testing the effects of housing, paternal exposure, and estrous cycle on 533 female proclivity to investigate male USVs, we also investigated whether these factors interact.
534 We found that a higher proportion of IH females were in receptive estrous stages than non-535 receptive estrous stages, although this was not the case for SH females. Amongst SH females, 536 those in non-receptive estrous stages showed greater USV preferences than those in receptive 537 stages, but there was no effect of sexual receptivity on USV preference amongst IH females.
538 There was no significant interaction between housing type and estrous stage on female USV 539 preference when investigating the entire sample population.

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Our results provide the first evidence to our knowledge that female house mice are more 566 likely to show attraction towards male USVs if they are socially housed, whereas individually 567 housed females are more likely to avoid them. We found no effect of neonatal paternal 568 exposure on female preference for male USV playbacks, though this result does not rule out 569 sexual imprinting (and may be due to females in our study being exposed to auditory cues of 570 adult males in our colony room). We found that non-receptive females show greater 571 preferences for male playbacks than sexually receptive females, as previously reported