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A meta-analysis of sex differences in neonatal rodent ultrasonic vocalizations and the implication for the preclinical maternal immune activation model

Biology of Sex Differences volume 16, Article number: 4 (2025) Cite this article

Abstract

As the earliest measure of social communication in rodents, ultrasonic vocalizations (USVs) in response to maternal separation are critical in preclinical research on neurodevelopmental disorders (NDDs). While sex differences in both USV production and behavioral outcomes are reported, many studies overlook sex as a biological variable in preclinical NDD models. We aimed to evaluate sex differences in USV call parameters and determine if USVs are differently impacted based on sex in the preclinical maternal immune activation (MIA) model. Results indicate that sex differences in USVs vary with developmental stage and are more pronounced in MIA offspring. Specifically, developmental stage is a moderator of sex differences in USV call duration, with control females emitting longer calls than males in early development (up to postnatal day [PND] 8), but this pattern reverses after PND8. MIA leads to a reduction in call numbers for females compared to same-sex controls in early development, with a reversal post-PND8. MIA decreased call duration and increased total call duration in males, but unlike females, developmental stage did not influence these differences. In males, MIA effects varied by species, with decreased call numbers in rats but increased call numbers in mice. MIA timing (gestational day ≤ 12.5 vs. > 12.5) did not significantly affect results. Our findings highlight the importance of considering sex, developmental timing, and species in USVs research. We discuss how analyzing USV call types and incorporating sex as a biological variable can enhance our understanding of neonatal ultrasonic communication and its translational value in NDD research.

Plain English Summary

This study looks at how young rodents in their first couple weeks of life communicate using high-pitched sounds called ultrasonic vocalizations (USVs), particularly in the context of when they are separated from their mothers. These vocalizations are often measured in preclinical research aimed at understanding neurodevelopmental disorders (NDDs), such as autism. We evaluated whether there are differences between male and female rodents in how they produce these sounds and how they respond following exposure to an infection while gestating, a model known as maternal immune activation (MIA). Our findings showed that sex differences in vocalizations depend on the age of rodents and are more noticeable in those affected by MIA. In the early days of development, female rodents made longer calls than males, but this pattern reversed as they grew older. For females exposed to MIA, the number of calls decreased, while males showed different patterns depending on whether they were rats or mice. The timing of when the mother experienced immune activation did not significantly change the results. Overall, this research emphasizes the need to consider sex, age, and species when studying these vocalizations. Understanding these factors can help improve preclinical research on early communication in relation to NDDs.

Highlights

Sex modulates ultrasonic vocalizations (USV) in maternal immune activation (MIA).

Developmental stage moderates sex differences in USV call duration.

MIA reduces call numbers in females early (< / = PND8), but not later development.

MIA effects in males vary by species: call numbers decrease in rats, increase in mice.

Including sex as a variable enhances translational value in preclinical research.

Introduction

Communication plays an essential role in survival across diverse mammalian taxa, including whales, primates, mustelids, bats, and rodents, which use high-frequency (i.e., ≤ 20 kHz) ultrasonic vocalizations (USVs) for conspecific communication [2, 4, 11, 18, 27, 44, 47, 55, 60]. Rodents are highly altricial species born with the inability to see, hear, or thermoregulate, and as such, neonates rely on maternal care [12, 25, 82]. Consequently, rodent pups emit USVs, which intensify in stressful conditions, such as in periods of maternal separation [15, 23, 25, 42]. As the earliest measure of social communication and one of the first feasible behavioral tests for neonates, examining USV production in response to maternal separation is critical in preclinical research of neurodevelopmental disorders (NDDs), such as autism spectrum disorder (ASD).

Zippelius and Schleidt [88] first classified neonate USVs as “whistles of loneliness,” expressed in response to maternal separation. Such USVs are believed to be innate signals that improve survivability by eliciting maternal retrieval [25]. The ventral pouch of the larynx, supported by a dorsally bent rostro-ventral component of the thyroid cartilage, contributes to the production of USVs and is developed in utero [70]. Mouse pups emit USVs shortly after birth, although they are born deaf, with the ear canal not opening until PND10-11 [5, 25, 32, 56]. Furthermore, cross-fostering studies show that mice fail to mimic USVs from other genetic strains [46]. Together, this research suggests that pup USVs are inherent with a key role in survival.

Three types of vocalizations have been described in infant pups: (1) low-frequency (below 10 kHz) or ‘wiggling calls’ that trigger maternal licking and are produced when pups try to reach their mother’s nipple [24], (2) broadband or ‘pain calls’ with frequencies between 4 to 40 kHz inhibit adult biting or injury and are emitted during postpartum cleaning of pups (Haack et al., 1983) and (3) isolation or distress calls (between 30 and 90 kHz) which prompt maternal retrieval and approach behaviours [23]. These isolation-induced USVs have increasingly received attention as a tool for assessing early communication delays in preclinical rodent models of NDDs [74].

Relevance of USVs in NDDs

One of the most well-established preclinical models of ASD is the maternal immune activation (MIA) model, which consists of maternal gestational infection either via a viral mimic (e.g., polyinosinic:polycytidylic acid (poly I:C)) or bacterial agents (e.g., lipopolysaccharide (LPS)). MIA offspring show increases in ASD-like behaviors, including social communication/interaction delays and repetitive behaviors (e.g., [41]). As MIA is a risk factor for ASD in humans [43, 64], and the core symptoms of ASD are recapitulated with this model, it is now one of the most studied environmental (non-genetic based) models of ASD [66].

In preclinical studies, including the MIA model, social communication delays are measured by USV communication during maternal separation; this is one of the earliest viable behavioral assessments for social communication in neonates and often predict later ASD-like phenotypes [16, 68]. However, there are considerable inconsistencies in the direction of change and interpretation of USV data. For instance, many report that MIA increases USV emissions [17, 48], while others find decreases in MIA offspring (e.g., Carlezon et al. 2019; [54]), and others report no differences (e.g., [50, 77]). Yet, the interpretation is often similar—whether increases or decreases in USV number or duration, these alterations are described as evidence of delays or deficits in neonatal communication.

Similarly, methods for collecting and analyzing USVs vary widely, including differences in analysis metrics (i.e., number, duration, frequency, classifications/call types), the species/strain of rodents used, the embryonic day (E) of MIA induction, the type of MIA (bacterial vs viral), as well as variation in the developmental stage of USV testing. Furthermore, many studies do not consider sex as a biological variable, and instead either only include males, pool the sexes, or do not report the sex of the subjects. In the current meta-analysis, we found that less than one-third of the 32 MIA studies identified for inclusion reported USV data by sex (see Fig. 1A). Given the 4:1 male bias in ASD, it is critical to include sex in preclinical work to improve the translational value of findings. This is especially important in USV research, as there are significant effects of sex and sex-by-environment interactions in rodent USV production.

Fig. 1
figure 1

Lack of integration of sex as a biological variable and limited USV call parameters in MIA research. A Less than a third of MIA studies, analyzed USVs by sex, with the majority of studies only including males or pooling the sexes in their analysis. Only 1 study analyzed USVs in females alone. B The most commonly reported USV call parameter is call number (49% of papers), while total call duration (10%), average call duration (17%), and call frequency (12%) are each reported in less than a fifth of studies

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USV Production Varies by Sex and Environmental Condition

USV production and structure differ between pup sexes, with males generally producing more USVs of longer durations, lower frequencies, and lower amplitudes than females [16, 52]. Such differences may contribute to signal saliency and subsequent maternal retrieval, as dams allocate more attention to male pups than females [3]. For example, all male litters receive more maternal care than all female litters [1], and when in stressful conditions, mothers produce female-biased litters to optimize their fitness [28]. Neuroendocrinological mechanisms, such as hypothalamic-pituitary axis (HPA) activation and co-expression of FOXP1 and androgen receptors in the striatum, are involved in mediating sex differences in USV variation [8, 21, 30].

Pup USVs are also impacted by their rearing environment and developmental stage [84]. For instance, pups reared in larger, environmentally enriched housing produce fewer USVs with shorter durations and lower frequencies than those in standard, under-stimulating conditions [7, 84]. Moreover, pup USV production gradually rises following birth, peaks at PND8 in mice [15, 74] and PND10 in rats [37], then decreases until stabilizing in puberty. Environmental differences affect HPA activation and can modulate USV deficits in preclinical models of ASD, including the MIA model [7, 21, 84, 87, 89]. Thus, sex and sex-by-environment interactions influence USVs in rodents, and developmental stage, species, and strain may modulate sex differences in USVs. As such, depending on these various factors, sex differences may be overestimated, underestimated, or ignored in experimental research [89], leading to misinterpretations of USV data and underrepresentation of sex as a mediating factor in translational rodent models of NDDs.

Present study objectives

We conducted meta-analyses to assess whether (1) there are sex differences in neonatal isolation-induced USVs, (2) MIA alters neonatal isolated-induced USVs, and (3) USVs of males and females are differentially affected by MIA. Within the meta-analyses assessing these three main questions, we also assessed whether the timing of MIA, type of MIA (viral vs bacterial vs other- e.g., valproic acid), developmental stage (i.e., postnatal day of USV recordings), or species (rats vs mice) were moderators of sex and/or MIA differences in neonatal USV isolation calls.

Methods

Literature search

We conducted a search for studies that evaluated sex differences in neonatal USVs in response to brief maternal separation (referred herein as "baseline studies") and those that assessed neonatal USVs in MIA preclinical models (referred herein as "MIA studies"). The search was performed using two major databases, PubMed, and Google Scholar. For baseline studies, we used keywords such as (“pup ultrasonic vocalizations” OR “neonate ultrasonic vocalizations” OR “isolation-induced ultrasonic vocalization”), combined with (“sex differences” OR “sex”). For MIA studies, additional keywords included (“maternal immune activation” OR “MIA” OR “poly ic” OR “lipopolysaccharide” OR “valproic acid” OR “animal model” OR “ASD”). This systematic review followed PRISMA protocol [63], Fig. 2). The complete search strings are available in the supplementary material.

Fig. 2
figure 2

PRISMA Flowchart detailing the identification and screening of identified records for the systematic review and meta-analysis

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Criteria for study inclusion

At the screening phase, papers were selected based on the following criteria according to the objectives of this systematic review: (i) Studies with one or more neonatal USV parameters evaluated (e.g., call number, total call duration, average call duration, and call frequency) in male and/or female offspring; (ii) USVs recorded prior to or during the weaning developmental stage (i.e., PND 3—21), and (iii) MIA model studies with intervention occurring during any phase of the gestational period along with appropriate controls (e.g., vehicle injection). The abstracts of all PubMed and Google Scholar records for baseline (n = 368) and MIA studies (n = 410) were evaluated for inclusion in the meta-analysis. Of 778 studies, only 23 baseline and 35 MIA independent studies included information on neonatal isolation-induced USVs recorded in rodent offspring. We excluded 4 of the 23 baseline studies [15], Hahn et al., 1997 & 1998; and Thornton et al., 2005), as sexes were pooled in the analyses and reporting of neonatal USV outcomes.

Extraction of study characteristics and USV parameters data

Study characteristics specific to species, strain, sex of offspring, age at which USVs were recorded, duration of USV recording, type of MIA immunogen used (e.g., poly I:C, LPS, valproic acid), dosage, gestational day of MIA induction, and frequency of administration were extracted. For USV parameters, we extracted the mean, SEM or SD, and sample size for male and female offspring (both treatment and control groups in the case of MIA studies) from each study for Hedges’s g calculation to correct for small sample bias [80]. We contacted corresponding authors of studies with insufficient statistical information or unclear data. If authors did not respond or could not provide the requested information, data were extracted from graphs using Webplot Digitizer [71]. Three of the 35 MIA studies [22, 38, 83] were excluded because the descriptive mean and SEM/SD were not provided or data could not be extracted from violin plots. When sample sizes were reported as ranges, the most conservative (i.e., lower value) was used to calculate the effect size.

Meta-analysis

Data for the meta-analysis was analyzed using the “metafor” package in R version 4.2–0 [81]. Some studies provided multiple measures for the same USV parameters (such as call number, mean call duration, total call duration, and call frequency) based on the developmental stage of USV recording. To address this, a three-level multilevel model was used to nest measures from the same study, correcting for the likely correlation between measures from the same study with planned subgroup analyses. First, to investigate potential sex differences in neonatal USVs in response to brief maternal separation, control samples of male and female offspring from MIA studies (n = 9 papers) were combined with the initial 19 baseline studies (total n = 28). Here, the model included species (mice vs. rats) and developmental stages (early vs. late PND) as moderators. The peak of USV production in rodents occurs around PND 8 [74], and as such PND 8 and below were classified as the early neonatal period, and anything above PND 8 as late. Additional moderators included, the gestational timing of MIA induction (early vs. late MIA), with gestational day (GD) 12.5 as the cut-off for early MIA and anything above GD 12.5 as late MIA, and the type of MIA immunogen used (i.e., viral injection: poly I:C vs. bacterial: LPS vs. other).

Results

Are there sex differences in neonatal USVs in response to brief maternal separation?

In each multilevel meta-analysis model, no significant effect size of sex differences in neonatal USVs in response to brief maternal separation was observed for call number (g = − 0.01 [− 0.13, 0.12], p = 0.983; SFig 1), mean call duration (g = − 0.03 [− 0.30, 0.25], p = 0.851; SFig 2), total call duration (g = 0.21 [− 0.04, 0.47], p = 0.098; SFig 3) and call frequency (g = 0.15 [− 0.11, 0.41], p = 0.259; SFig 4). We also tested whether sex differences in neonatal USVs in response to brief maternal separation were influenced by developmental stage (early vs. late PND) and species (rats vs. mice). A moderator effect was observed for mean call duration (Q = 6.57, p = 0.037), with a significant difference between late PND vs. early PND (g = − 0.67 [− 1.18, 0.16], p = 0.010; Table 1).

Table 1 Results from moderator analyses for developmental stage and species
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Sub-group analyses were conducted to understand the significant interaction between developmental stage and sex differences in mean call duration. While these analyses did not reach statistical significance, the direction of the sex difference is reversed depending on developmental stage, with females having a higher mean call duration than males in early PND groups (g = 0.36 [− 0.11, 0.82], p = 0.130;), but males having higher mean call duration than females in late PND groups, (g = − 0.31 [− 0.64, 0.02], p = 0.067). No other moderators were significant (p > 0.05).

Does maternal immune activation (MIA) influence USVs in response to brief maternal separation (irrespective of sex)?

Across studies, MIA significantly influences mean call duration (g = − 0.26 [− 0.45, − 0.07], p = 0.006; Fig. 3A) and call frequency (g = 0.37 [0.01, 0.73], p = 0.043; Fig. 3B), but not call number (g = − 0.19 [− 0.50, 0.11], p = 0.220; Fig. 4) or total call duration (g = 0.17 [− 0.32, 0.66], p = 0.500; Fig. 5). The results of potential moderators: developmental stage (Early vs. Late PND), species (Rats vs. Mice), timing of MIA (Early vs. Late MIA), and type of MIA (Viral vs. Bacterial vs. Other) are reported in Table 2: call number (Q = 10.89, p = 0.054), mean call duration (Q = 1.99, p = 0.851), total call duration (Q = 18.95, p < 0.001), and call frequency (Q = 13.60, p = 0.009).

Fig. 3
figure 3

Meta-analysis results indicate that maternal immune activation (MIA) decreases neonatal USV mean call duration, and increase call frequency in response to maternal separation compared to vehicle controls

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Fig. 4
figure 4

Meta-analysis results indicate that maternal immune activation does not significantly influence neonatal USV call number

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Fig. 5
figure 5

A Meta-analysis results indicate that maternal immune activation does not

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Table 2 Results from moderator analyses for developmental stage, species, timing of MIA, and type of MIA for models that include one or both sexes
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Sub-group analyses were conducted to understand the influence of significant moderators (see table notes, Table 2). These results suggest that call number is reduced with MIA in early developmental stages, while MIA does not differ from controls in later development. Call duration shows a reversal with age, such that MIA offspring had shorter, but non-significant, total call duration than controls in early PND groups, but had longer, non-significant, total call duration than controls in late PND groups. Species differences suggest that MIA reduces total call duration in rats, but increases total call duration in mice. Subgroup analyses of MIA type (bacteria, viral vs other [i.e., VPA]) indicated that while MIA offspring had significantly higher call frequency than controls in other MIA treatments (g = 0.61 [0.31, 0.90], p < 0.001), this difference was non-significant (and in the reverse direction) for viral MIA offspring compared to controls (g = 0.06 [−0.36, 0.47], p = 0.786).

Potential publication bias was also evaluated via funnel plots (Fig. 5a–d) and tested using Kendall’s rank correlations: call number (τ = − 0.06, p = 0.510), mean call duration (τ = − 0.20, p = 0.260), total call duration (τ = − 0.08, p = 0.765), and call frequency (τ = 0.16, p = 0.542).

Does MIA affect USVs in males and females differently?

Multilevel meta-analysis models were conducted by comparing neonatal USVs between control and MIA male rodents and by comparing between control and MIA female rodents, allowing us to assess whether MIA influences USVs more in males than females. Among male rodents, MIA significantly influences mean call duration (g = − 0.41 [− 0.66, − 0.17], p = 0.001; Fig. 6A) and total call duration (g = 0.78 [0.25, 1.31], p = 0.004; Fig. 6B), but not call number (g = − 0.27 [− 0.63, 0.08], p = 0.126; Fig. 7) and call frequency (g = 0.31 [− 0.34, 0.95], p = 0.349; SFig. 5). Among female rodents, MIA does not significantly influence call number (g = − 0.05 [− 0.36, 0.27], p = 0.762; SFig. 6), mean call duration (g = − 0.23 [− 0.58, 0.12], p = 0.203; SFig. 7), and call frequency (g = 0.05 [− 0.28, 0.37], p = 0.766; SFig. 8).

Fig. 6
figure 6

Meta-analysis results indicate that MIA decreases neonatal USV mean call duration (A) and increases total call duration (B) among male rodents compared to same-sex controls

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Fig. 7
figure 7

Meta-analysis results indicate that neonatal USV call number does not significantly differ between control and MIA male rodents

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We then assessed whether sex was a significant moderator in combined analyses of male and female data. Sex was found to significantly moderate mean call duration (g = 0.06 [0.04, 0.08], p < 0.001), but not call number (g = − 0.13 [− 0.34, 0.09], p = 0.253) and call frequency (g = 0.19 [− 0.17, 0.55], p = 0.298).

The results of potential moderators (Table 3 for males, Table 4 for females): for males, species differences were found, such that MIA reduced call numbers in rats, while in mice MIA increased call numbers compared to controls. For females, MIA reduced USV call number in early development (< / = PND8), but increased call number relative to controls in later development (> PND8).

Table 3 Results from moderator analyses for developmental stage, species, timing of MIA, and type of MIA for models comparing control and MIA male rodents
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Table 4 Results from moderator analyses for developmental stage, species, timing of MIA, and type of MIA for models comparing control and MIA female rodents
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Discussion

The present meta-analyses revealed that developmental stage is a significant moderator of sex differences in USVs, and sex differences in USVs are more pronounced in the preclinical MIA model. While USVs differed between MIA and control offspring when data were pooled across sexes, these differences should be interpreted with caution: Analyses by sex revealed that sex differences in USVs depend on developmental stage and species. Moreover, the majority of differences were found in mean or total call duration; this is an important consideration for future research, as many studies evaluating early communication delays via USVs report only call number (see Fig. 1B), without any measure of call duration. The differences in mean call duration, but not always in total call duration or call number, may also suggest that there are group differences in call types (i.e., flat, two-step, chevron, etc., see Fig. 8A). As such, we recommend that in studying USVs in MIA (or other preclinical models), researchers consider developmental stage, species, sex, and expand their analysis to include additional USV parameters, including call type. Below, we detail the significance of these findings, as well as provide recommendations for non-invasive visual sexing of neonates, and for automated scoring software that allows for accurate and relatively quick analyses of USV call parameters and call type classification.

Fig. 8.
figure 8

A Call type classification (reproduced from [13]). B Sexing fetal and neonatal rodents can be accomplished by visual inspection of anogenital region to identify the dark pigmented spot in the scrotal region of male pups (indicated by red arrows)

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We found that at baseline (i.e., controls, without MIA), developmental stage was a significant moderator of sex differences in USV mean call duration. While subgroup analyses were non-significant, the sign of the effect sizes are reversed, indicating that females emit longer calls earlier on (≤ PND8), but that this sex difference reverses in later development (> PND8). However, it is noteworthy that the majority of studies in the present meta-analyses either excluded females or pooled the sexes (see Fig. 1A), and few studies reported on mean call duration when compared to call number (see Fig. 1B). Given that the strength of meta-analyses is limited by the availability and quality of existing research, more systematic studies are essential to better understand the effects of sex and developmental timing on USV emissions. Nevertheless, the present meta-analysis suggests that researchers should consider developmental timing when studying USVs generally and in translational NDD models, as PND can modulate sex differences in USV emission. It is also notable that how the day of birth is defined varies: PND 0, 0.5 or 1. Yet, the majority of papers do not report how they define the day of birth, which introduces another potential source of variation, when assessing USVs and developmental stage, that should be accounted for in future research.

When assessing the effects of MIA across all studies (i.e., pooling sexes), MIA reduced mean call duration and increased call frequency, and when developmental stage is considered, call number reductions were found to be restricted to early development. Species was also a significant moderator, such that call duration is reduced in MIA rats but increased in MIA mice. However, when sex is considered as a biological variable, it can be discerned that many of these effects are driven by males or females. For example, MIA decreased mean call duration, but increased total call duration in males, but not in females. However, when developmental stage is considered for females, MIA reduced USV call numbers in early development (≤ PND8) relative to controls, but increased in later development (> PND8). Moreover, species differences were found only for males, such that MIA reduced call numbers in rats, while in mice MIA increased call numbers compared to controls. These findings highlight that USVs are affected by MIA in both males and females, but are moderated by developmental stage and species.

Of note, sex differences in the presentation of USV delays underscores the necessity of considering sex as a biological variable in USV and NDD research, especially in light of the sex difference in the clinical manifestations of these conditions. For instance, in ASD, there is a known gender bias in diagnosis, where females who meet the criteria of ASD are less likely to receive a clinical diagnosis [53] or receive a diagnosis later in life [6, 79]. This diagnostic disparity may partly result from variations in symptom presentation. This bias extends into preclinical research, where studies frequently focus on males or fail to account for sex differences, leading to incomplete results and skewed conclusions (e.g., [49, 50, 54, 77]). The present study highlights that neonatal USV production in response to MIA is affected in both males and females, but additional research is necessary to gain a comprehensive understanding of the effects of MIA in the understudied sex—females.

Indeed, most studies in the present meta-analysis were single-sex (17 of 32), and 40% of studies reporting both sexes pooled male and female data (6 of 15 studies) for assessing USVs; although, we note that pooling of the sexes was not always the case for other juvenile behavioral measures in these studies. We hypothesize this may be due to unfamiliarity in sexing neonatal pups or the invasive nature of genotyping and/or tracking into adolescence. However, accurate sexing of neonates, and even fetuses at embryonic day 18, via visual inspection alone is feasible. Our data show fetal sexing accuracy is > 93% (n = 39 of 43), and sexing from PND 4 is > 97% accurate (n = 481 of 492), in C57BL/6 mice. As illustrated in Fig. 8B, males and females show distinct anogenital differences by late embryonic development: males have a noticeable dark spot in the scrotal region, which is absent in females. This pigmentation is the result of higher melanin concentration linked to androgen exposure [85]. It is visible from birth in several dark-fur mouse strains [86] and Long-Evans rats [85], although white-furred strains lack this pigmentation (e.g., A/J, 129X1/SvJ, and C57Bl/6 J-Chr 7A/NaJ, Wolterink-Donselaar et al. Thus, we recommend visual inspection for sexing in dark-furred rodent strains, without genotyping and/or tattooing for tracking, and urge reviewers and editors to accept this method as valid, to promote the inclusion of sex as a biological variable in neonatal rodent studies.

In addition to including sex, expanding USV analysis to include the wide repertoire of USV call types emitted by pups may provide insight into the characteristics of pups’ ultrasounds associated with NDD-like phenotypes. Indeed, USV call types, categories or classifications, since their description by Sales and Smith [72], have become an integral part of qualitative analysis of rodent vocalizations. These spectrographic analyses are based on the frequency modulation and duration of acoustic signals [10] and may provide additional information for quantitative USV parameters, such as call number, duration, and frequency [10, 15, 75]. Before Scattoni et al. [75], USVs were classified into five categories: constant frequency, modulated frequency, frequency steps, composite, and short [10, 72]. Scattoni's taxonomy expanded this framework to include additional call types—complex, harmonics, two-syllable, upward, downward, flat, chevron, short, composite, and frequency steps (Fig. 8A)—allowing for the detection of more subtle differences [15, 75]. Functionally, specific USV call types have been suggested to correlate with later social behavior. For example, infant isolation-induced calls, such as flat and short calls, have been found to correlate with the frequency of social interactions during adolescence [33]. This indicates that analyzing USV call type repertoire in pups can be an additional tool for quantifying the extent of ASD-like traits in rodent models during early development [57].

Several automated software programs (e.g., Avisoft analysis, VocalMat, DeepSqueak, Kaleidoscope Pro) can automatically segment rodent audio files into calls and apply classification algorithms to label vocalizations (for detailed reviews see [19, 29] and [67]). Our lab currently uses Kaleidoscope Pro, in which users can use cluster analyses to recognize a predetermined number of call types. This allows us to accurately sort and classify various calls, significantly accelerating the analysis of USVs. We have successfully classified over 70,000 USV calls with an accuracy rate exceeding 80%, which we can then manually correct to reach near 100% accuracy. We are able to share these training data with interested researchers upon request.

In conclusion, our meta-analyses reveal notable sex differences in USVs, especially in response to MIA. However, these differences vary depending on developmental stage and species. Importantly, none of the analyses showed differences across all USV call parameters, indicating a need for researchers to expand their focus beyond call number to include at minimum call duration and mean call duration. We also recommend incorporating an analysis of USV call types, as the existing studies, while few at this point, have identified sex-specific differences in call types [34, 45]. Additionally, we highlight that visual inspection via the “spot method” [86] is a highly accurate method for determining sex in common laboratory strains (e.g., C57Bl/6 mice and Long Evans rats) and advocate for its use to encourage the inclusion of sex in neonatal research.

Data availability

Data is provided within the manuscript or supplementary files.

References

  1. Alleva E, Caprioli A, Laviola G. Litter gender composition affects maternal behavior of the primiparous mouse dam (Mus musculus). J Comp Psychol. 1989;103(1):83–7. https://doiorg.publicaciones.saludcastillayleon.es/10.1037/0735-7036.103.1.83.

    Article  CAS  PubMed  Google Scholar 

  2. Arch VS, Narins PM. “Silent” signals: Selective forces acting on ultrasonic communication systems in terrestrial vertebrates. Anim Behav. 2008;76(4):1423–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.anbehav.2008.05.012.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Asaba A, Okabe S, Nagasawa M, Kato M, Koshida N, Osakada T, Mogi K, Kikusui T. Developmental social environment imprints female preference for male song in mice. PLoS ONE. 2014;9(2): e87186. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pone.0087186.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Au, W. W. L. (1993). The Sonar of Dolphins. Springer Science & Business Media.

  5. Baker BH, Rafikian EE, Hamblin PB, Strait MD, Yang M, Pearson BL. Sex-specific neurobehavioral and prefrontal cortex gene expression alterations following developmental acetaminophen exposure in mice. Neurobiol Dis. 2023;177: 105970. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.nbd.2022.105970.

    Article  CAS  PubMed  Google Scholar 

  6. Begeer S, Mandell D, Wijnker-Holmes B, Venderbosch S, Rem D, Stekelenburg F, Koot HM. Sex differences in the timing of identification among children and adults with autism spectrum disorders. J Autism Dev Disord. 2013;43(5):1151–6. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s10803-012-1656-z.

    Article  PubMed  Google Scholar 

  7. Binder MS, Bordey A. Semi-natural housing rescues social behavior and reduces repetitive exploratory behavior of BTBR autistic-like mice. Sci Rep. 2023;13(1):1. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41598-023-43558-0.

    Article  CAS  Google Scholar 

  8. Bowers JM, Perez-Pouchoulen M, Edwards N, Mccarthy M. Foxp2 mediates sex differences in ultrasonic vocalization by rat pups and directs order of maternal retrieval. J Neurosci. 2013;33:3276–83. https://doiorg.publicaciones.saludcastillayleon.es/10.1523/JNEUROSCI.0425-12.2013.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Bradshaw J, McCracken C, Pileggi M, Brane N, Delehanty A, Day T, Federico A, Klaiman C, Saulnier C, Klin A, Wetherby A. Early social communication development in infants with autism spectrum disorder. Child Dev. 2021;92(6):2224–34. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/cdev.13683.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Branchi I, Santucci D, Vitale A, Alleva E. Ultrasonic vocalizations by infant laboratory mice: a preliminary spectrographic characterization under different conditions. Dev Psychobiol. 1998;33(3):249–56. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/(sici)1098-2302(199811)33:3%3c249::aid-dev5%3e3.0.co;2-r.

    Article  CAS  PubMed  Google Scholar 

  11. Brudzynski, S. M. (2018). Handbook of Ultrasonic Vocalization: A Window into the Emotional Brain. Academic Press.

  12. Brust V, Schindler PM, Lewejohann L. Lifetime development of behavioural phenotype in the house mouse (Mus musculus). Front Zool. 2015;12(Suppl 1):S17. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/1742-9994-12-S1-S17.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Burke FF (2023) Hyperandrogenization and late gestational infection result in an autism-like phenotype in a rodent model. Masters thesis, Memorial University of Newfoundland

  14. Capas-Peneda S, Saavedra Torres Y, Prins JB, Olsson IAS. From mating to milk access: a review of reproductive vocal communication in mice. Front Behav Neurosci. 2022;16: 833168.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Caruso A, Marconi MA, Scattoni ML, Ricceri L. Ultrasonic vocalizations in laboratory mice: strain, age, and sex differences. Genes Brain Behav. 2022;21(5):e12815.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Caruso A, Ricceri L, Scattoni ML. Ultrasonic vocalizations as a fundamental tool for early and adult behavioral phenotyping of Autism Spectrum Disorder rodent models. Neurosci Biobehav Rev. 2020;116:31–43. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.neubiorev.2020.06.011.

    Article  PubMed  Google Scholar 

  17. Choi GB, Yim YS, Wong H, Kim S, Kim H, Kim SV, Huh JR. The maternal interleukin-17a pathway in mice promotes autism-like phenotypes in offspring. Science. 2016;351(6276):933–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Clausen KT, Malmkvist J, Surlykke A. Ultrasonic vocalisations of kits during maternal kit-retrieval in farmed mink. Mustela vison Appl Anim Behav Sci. 2008;114(3):582–92. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.applanim.2008.03.008.

    Article  Google Scholar 

  19. Coffey, K. R., Marx, R. E., & Neumaier, J. F. (2019). DeepSqueak: a deep learning-based system for detection and analysis of ultrasonic vocalizations. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology, 44(5), 859–868. HYPERLINK "https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41386-018-0303-6" https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41386-018-0303-6

  20. Connors EJ, Migliore MM, Pillsbury SL, Shaik AN, Kentner AC. Environmental enrichment models a naturalistic form of maternal separation and shapes the anxiety response patterns of offspring. Psychoneuroendocrinology. 2015;52:153–67. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.psyneuen.2014.10.021.

    Article  CAS  PubMed  Google Scholar 

  21. Coutellier L, Friedrich A-C, Failing K, Würbel H. Variations in the postnatal maternal environment in mice: Effects on maternal behaviour and behavioural and endocrine responses in the adult offspring. Physiol Behav. 2008;93(1):395–407. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.physbeh.2007.09.008.

    Article  CAS  PubMed  Google Scholar 

  22. Cristancho AG, Tulina N, Brown AG, Anton L, Barila G, Elovitz MA. Intrauterine inflammation leads to select sex-and age-specific behavior and molecular differences in mice. Inter J Mol Sci. 2022;24(1):32.

    Article  Google Scholar 

  23. Ehret G. Infant rodent ultrasounds – a gate to the understanding of sound communication. Behav Genet. 2005;35(1):19–29. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s10519-004-0853-8.

    Article  PubMed  Google Scholar 

  24. Ehret G, Bernecker C. Low-frequency sound communication by mouse pups (Mus musculus): wriggling calls release maternal behaviour. Anim Behav. 1986;34(3):821–30.

    Article  Google Scholar 

  25. Ehret G. Frequency and intensity difference limens and nonlinearities in the ear of the house mouse (Mus musculus). J Comp Physiol. 1975;102:321–36.

    Article  Google Scholar 

  26. Esposito G, Venuti P. Understanding early communication signals in autism: a study of the perception of infants’ cry. J Intellect Disabil Res. 2010;54(3):216–23. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/j.1365-2788.2010.01252.x.

    Article  CAS  PubMed  Google Scholar 

  27. Feng AS, Narins PM, Xu C-H, Lin W-Y, Yu Z-L, Qiu Q, Xu Z-M, Shen J-X. Ultrasonic communication in frogs. Nature. 2006;440(7082):333–6. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/nature04416.

    Article  CAS  PubMed  Google Scholar 

  28. Firman RC. Exposure to high male density causes maternal stress and female-biased sex ratios in a mammal. Proc R Soc B. 2020;287(1926):20192909. https://doiorg.publicaciones.saludcastillayleon.es/10.1098/rspb.2019.2909.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Fonseca, A. H., Santana, G. M., Bosque Ortiz, G. M., Bampi, S., Dietrich, M. O. (2021). Analysis of ultrasonic vocalizations from mice using computer vision and machine learning. eLife, 10, e59161. https://doiorg.publicaciones.saludcastillayleon.es/10.7554/eLife.59161

  30. Fröhlich H, Rafiullah R, Schmitt N, Abele S, Rappold GA. Foxp1 expression is essential for sex-specific murine neonatal ultrasonic vocalization. Hum Mol Genet. 2017;26(8):1511–21. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/hmg/ddx055.

    Article  CAS  PubMed  Google Scholar 

  31. Gaub S, Ehret G. Grouping in auditory temporal perception and vocal production is mutually adapted: the case of wriggling calls of mice. J Comp Physiol. 2005;191(12):1131–5. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s00359-005-0036-y.

    Article  Google Scholar 

  32. Geal-Dor M, Freeman S, Li G, Sohmer H. Development of hearing in neonatal rats: air and bone conducted ABR thresholds. Hear Res. 1993;69(1–2):236–42. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/0378-5955(93)90113-f.

    Article  CAS  PubMed  Google Scholar 

  33. Granata LE, Valentine A, Hirsch JL, Brenhouse HC. Infant ultrasonic vocalizations predict adolescent social behavior in rats: Effects of early life adversity. Dev Psychobiol. 2022;64(3): e22260. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/dev.22260.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Granata L, Valentine A, Hirsch JL, Honeycutt J, Brenhouse H. Trajectories of mother-infant communication: an experiential measure of the impacts of early life adversity. Front Hum Neurosci. 2021;15: 632702. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fnhum.2021.632702.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Granon, S., Faure, A., Chauveau, F., Cressant, A., & Ey, E. (2018). Why should my mouse call me? Acoustic communication in mouse models of social disorders: ultrasonic vocalizations as an index of emotional and motivational states. In Handbook of Behavioral Neuroscience (Vol. 25, pp. 423–431). Elsevier.

  36. Grimsley JM, Monaghan JJ, Wenstrup JJ. Development of social vocalizations in mice. PLoS ONE. 2011;6(3): e17460.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Gulia KK, Patel N, Radhakrishnan A, Kumar VM. Reduction in ultrasonic vocalizations in pups born to rapid eye movement sleep restricted mothers in rat model. PLoS ONE. 2014;9(1):e84948. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pone.0084948.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Guma E, Snook E, Spring S, Lerch JP, Nieman BJ, Devenyi GA, Chakravarty MM. Subtle alterations in neonatal neurodevelopment following early or late exposure to prenatal maternal immune activation in mice. NeuroImage Clin. 2021;32:102868.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Haack, B., & Ehret, G. (2009). Categorical perception of mouse pup ultrasound by lactating females.

  40. Haack, B., Markl, H., & Ehret, G. (2009). Sound communication between parents and offspring.

  41. Holy TE, Guo Z. Ultrasonic songs of male mice. PLoS Biol. 2005;3(12):e386.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Haida O, Al Sagheer T, Balbous A, Francheteau M, Matas E, Soria F, et al. Sex-dependent behavioral deficits and neuropathology in a maternal immune activation model of autism. Transl Psychiatry. 2019;9(1):124.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Hammerschmidt K, Radyushkin K, Ehrenreich H, Fischer J. Female mice respond to male ultrasonic ‘songs’ with approach behaviour. Biol Let. 2009;5(5):589–92.

    Article  CAS  Google Scholar 

  44. Han VX, Patel S, Jones HF, Dale RC. Maternal immune activation and neuroinflammation in human neurodevelopmental disorders. Nat Rev Neurol. 2021;17(9):564–79.

    Article  PubMed  Google Scholar 

  45. Hasiniaina AF, Scheumann M, Rina Evasoa M, Braud D, Rasoloharijaona S, Randrianambinina B, Zimmermann E. High frequency/ultrasonic communication in a critically endangered nocturnal primate, Claire’s mouse lemur (Microcebus mamiratra). Am J Primatol. 2018;80(6): e22866. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/ajp.22866.

    Article  PubMed  Google Scholar 

  46. Kentner AC, Scalia S, Shin J, Migliore MM, Rondón-Ortiz AN. Targeted sensory enrichment interventions protect against behavioral and neuroendocrine consequences of early life stress. Psychoneuroendocrinology. 2018;98:74–85. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.psyneuen.2018.07.029.

    Article  PubMed  Google Scholar 

  47. Kikusui T, Nakanishi K, Nakagawa R, Nagasawa M, Mogi K, Okanoya K. Cross-fostering experiments suggest that mice songs are innate. PLoS ONE. 2011;6(3):e1772. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pone.0017721.

    Article  CAS  Google Scholar 

  48. Kim S, Kim H, Yim YS, Ha S, Atarashi K, Tan TG, Huh JR. Maternal gut bacteria promote neurodevelopmental abnormalities in mouse offspring. Nature. 2017;549(7673):528–32.

    Article  PubMed  PubMed Central  Google Scholar 

  49. Kruger M. Ultrasonic Hearing in Cats and Other Terrestrial Mammals. Acoustics Today. 2021;17:18. https://doiorg.publicaciones.saludcastillayleon.es/10.1121/AT.2021.17.1.18.

    Article  Google Scholar 

  50. Lammert CR, Lukens JR. Modeling autism-related disorders in mice with maternal immune activation (MIA). Methods Mol Biol. 2019;1960:227–36. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/978-1-4939-9167-9_20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Lan XY, Gu YY, Li MJ, Song TJ, Zhai FJ, Zhang Y, Zhan JS, Böckers TM, Yue XN, Wang JN, Yuan S, Jin MY, Xie YF, Dang WW, Hong HH, Guo ZR, Wang XW, Zhang R. Poly(I:C)-induced maternal immune activation causes elevated self-grooming in male rat offspring: Involvement of abnormal postpartum static nursing in dam. Front Cell Dev Biol. 2023;11:1054381. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fcell.2023.1054381.

    Article  PubMed  PubMed Central  Google Scholar 

  52. Lauritsen MB. Autism spectrum disorders. Eur Child Adolesc Psychiatry. 2013;22:37–42.

    Article  Google Scholar 

  53. Lenell C, Broadfoot CK, Schaen-Heacock NE, Ciucci MR. Biological and acoustic sex differences in rat ultrasonic vocalization. Brain Sci. 2021;11(4):459.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Loomes R, Hull L, Mandy WPL. What is the male-to-female ratio in autism spectrum disorder? A systematic review and meta-analysis. J Am Acad Child Adolesc Psychiatry. 2017;56(6):466–74. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.jaac.2017.03.013.

    Article  PubMed  Google Scholar 

  55. Malkova NV, Yu CZ, Hsiao EY, Moore MJ, Patterson PH. Maternal immune activation yields offspring displaying mouse versions of the three core symptoms of autism. Brain Behav Immun. 2012;26(4):607–16. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.bbi.2012.01.011.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Marten K. Ultrasonic analysis of pygmy sperm whale (Kogia breviceps) and Hubbs’ beaked whale (Mesoplodon carlhubbsi) clicks. Aquat Mamm. 2000;26:1.

    Google Scholar 

  57. Meng X, Mukherjee D, Kao JPY, Kanold PO. Early peripheral activity alters nascent subplate circuits in the auditory cortex. Sci Adv. 2021;7(7):9155. https://doiorg.publicaciones.saludcastillayleon.es/10.1126/sciadv.abc9155.

    Article  CAS  Google Scholar 

  58. Möhrle D, Yuen M, Zheng A, Haddad FL, Allman BL, Schmid S. Characterizing maternal isolation-induced ultrasonic vocalizations in a gene-environment interaction rat model for autism. Genes Brain Behav. 2023;22(3): e12841. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/gbb.12841.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Moore CL, Morelli GA. Mother rats interact differently with male and female offspring. J Comp Physiol Psychol. 1979;93(4):677–84. https://doiorg.publicaciones.saludcastillayleon.es/10.1037/h0077599.

    Article  CAS  PubMed  Google Scholar 

  60. Moore CL. The role of maternal stimulation in the development of sexual behavior and its neural basis. Ann N Y Acad Sci. 1992;662:160–77. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/j.1749-6632.1992.tb22859.x.

    Article  CAS  PubMed  Google Scholar 

  61. Mota-Rojas D, Bienboire-Frosini C, Marcet-Rius M, Domínguez-Oliva A, Mora-Medina P, Lezama-García K, Orihuela A. Mother-young bond in non-human mammals: neonatal communication pathways and neurobiological basis. Front Psychol. 2022;13:1064444. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fpsyg.2022.1064444.

    Article  PubMed  PubMed Central  Google Scholar 

  62. Musi B, de Acetis L, Alleva E. Influence of litter gender composition on subsequent maternal behaviour and maternal aggression in female house mice. Ethology. 1993;95(1):43–53. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/j.1439-0310.1993.tb00455.x.

    Article  Google Scholar 

  63. Nolan SO, Hodges SL, Lugo JN. High-throughput analysis of vocalizations reveals sex-specific changes in Fmr1 mutant pups. Genes Brain Behav. 2020;19(2): e12611. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/gbb.12611.

    Article  CAS  PubMed  Google Scholar 

  64. Page MJ, McKenzie JE, Bossuyt PM, Boutron I, Hoffmann TC, Mulrow CD, Shamseer L, Tetzlaff JM, Akl EA, Brennan SE, Chou R, Glanville J, Grimshaw JM, Hróbjartsson A, Lalu MM, Li T, Loder EW, Mayo-Wilson E, McDonald S, McGuinness LA, The PRISMA, et al. statement: an updated guideline for reporting systematic reviews. BMJ (Clinical research ed). 2020;372:71. https://doiorg.publicaciones.saludcastillayleon.es/10.1136/bmj.n71.

    Article  Google Scholar 

  65. Patel S, Dale RC, Rose D, Heath B, Nordahl CW, Rogers S, et al. Maternal immune conditions are increased in males with autism spectrum disorders and are associated with behavioural and emotional but not cognitive co-morbidity. Transl Psychiatry. 2020;10(1):286.

    Article  PubMed  PubMed Central  Google Scholar 

  66. Patten E, Belardi K, Baranek GT, Watson LR, Labban JD, Oller DK. Vocal patterns in infants with autism spectrum disorder: canonical babbling status and vocalization frequency. J Autism Dev Disord. 2014;44(10):2413–28. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s10803-014-2047-4.

    Article  PubMed  PubMed Central  Google Scholar 

  67. Panzenhagen AC, Cavalcanti A, Stein DJ, de Castro LL, Vasconcelos M, Abreu MB, et al. Behavioral manifestations in rodent models of autism spectrum disorder: protocol for a systematic review and network meta-analysis. Syst Rev. 2022;11(1):150.

    Article  PubMed  PubMed Central  Google Scholar 

  68. Premoli M, Memo M, Bonini SA. Ultrasonic vocalizations in mice: relevance for ethologic and neurodevelopmental disorders studies. Neural Regen Res. 2021;16(6):1158–67. https://doiorg.publicaciones.saludcastillayleon.es/10.4103/1673-5374.300340.

    Article  PubMed  Google Scholar 

  69. Premoli M, Pietropaolo S, Wöhr M, Simola N, Bonini SA. Mouse and rat ultrasonic vocalizations in neuroscience and neuropharmacology: State of the art and future applications. Eur J Neurosci. 2023;57(12):2062–96. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/ejn.15957.

    Article  CAS  PubMed  Google Scholar 

  70. Riede T, Borgard HL, Pasch B. Laryngeal airway reconstruction indicates that rodent ultrasonic vocalizations are produced by an edge-tone mechanism. Royal Soc Open Sci. 2017;4(11):170976.

    Article  Google Scholar 

  71. Rilling JK, Young LJ. The biology of mammalian parenting and its effect on offspring social development. Science. 2014;345(6198):771–6. https://doiorg.publicaciones.saludcastillayleon.es/10.1126/science.1252723.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Rohatgi, A. (2023). Webplotdigitizer: Version 4.6, 2022. https://automeris.io/WebPlotDigitizer.

  73. Sales GD, Smith JC. Comparative studies of the ultrasonic calls of infant murid rodents. Dev Psychobiol. 1978;11(6):595–619. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/dev.420110609.

    Article  CAS  PubMed  Google Scholar 

  74. Santos S, Ferreira H, Martins J, Gonçalves J, Castelo-Branco M. Male sex bias in early and late onset neurodevelopmental disorders: Shared aspects and differences in Autism Spectrum Disorder, Attention Deficit/hyperactivity Disorder, and Schizophrenia. Neurosci Biobehav Rev. 2022;135: 104577. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.neubiorev.2022.104577.

    Article  CAS  PubMed  Google Scholar 

  75. Scattoni ML, Crawley J, Ricceri L. Ultrasonic vocalizations: a tool for behavioural phenotyping of mouse models of neurodevelopmental disorders. Neurosci Biobehav Rev. 2009;33(4):508–15.

    Article  PubMed  Google Scholar 

  76. Scattoni ML, Gandhy SU, Ricceri L, Crawley JN. Unusual repertoire of vocalizations in the BTBR T+tf/J mouse model of autism. PLoS ONE. 2008;3(8): e3067. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pone.0003067.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Schmidt M, Enthoven L, van der Mark M, Levine S, de Kloet ER, Oitzl MS. The postnatal development of the hypothalamic–pituitary–adrenal axis in the mouse. Int J Dev Neurosci. 2003;21(3):125–32. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/S0736-5748(03)00030-3.

    Article  CAS  PubMed  Google Scholar 

  78. Straley ME, Van Oeffelen W, Theze S, Sullivan AM, O’Mahony SM, Cryan JF, O’Keeffe GW. Distinct alterations in motor & reward seeking behavior are dependent on the gestational age of exposure to LPS-induced maternal immune activation. Brain Behav Immun. 2017;63:21–34. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.bbi.2016.06.002.

    Article  CAS  PubMed  Google Scholar 

  79. Takahashi T, Okabe S, Broin PÓ, Nishi A, Ye K, Beckert MV, Izumi T, Machida A, Kang G, Abe S, Pena JL, Golden A, Kikusui T, Hiroi N. Structure and function of neonatal social communication in a genetic mouse model of autism. Mol Psychiatry. 2016;21(9):1208–14. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/mp.2015.190.

    Article  CAS  PubMed  Google Scholar 

  80. Taylor JL, Smith DaWalt L, Marvin AR, Law JK, Lipkin P. Sex differences in employment and supports for adults with autism spectrum disorder. Autism. 2019;23(7):1711–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1177/1362361319827417.

    Article  PubMed  PubMed Central  Google Scholar 

  81. Turner HMI, Bernard RM. Calculating and synthesizing effect sizes. Contemp Issues Commun Sci Disorders. 2006;33(Spring):42–55.

    Article  Google Scholar 

  82. Viechtbauer W. Conducting meta-analyses in R with the metafor package (Version 42–0). J Stat Software. 2010;36(3):1–48.

    Article  Google Scholar 

  83. Vigli D, Palombelli G, Fanelli S, Calamandrei G, Canese R, Mosca L, Ricceri L. Maternal immune activation in mice only partially recapitulates the autism spectrum disorders symptomatology. Neuroscience. 2020;445:109–19.

    Article  CAS  PubMed  Google Scholar 

  84. Weber EM, Olsson IAS. Maternal behaviour in Mus musculus sp: an ethological review. Appl Anim Beh Sci. 2008;114(1–2):1–22.

    Article  Google Scholar 

  85. Wilkin-Krug L, Macaskill A, Ellenbroek B. Preweaning environmental enrichment alters neonatal ultrasonic vocalisations in a rat model for prenatal infections. Behav Pharmacol. 2022;33:402–17. https://doiorg.publicaciones.saludcastillayleon.es/10.1097/fbp.0000000000000688.

    Article  CAS  PubMed  Google Scholar 

  86. Wilson MJ, Spaziani E. Testosterone regulation of pigmentation in scrotal epidermis of the rat. Z Zellforsch Mikrosk Anat. 1973;140:451–8.

    Article  CAS  PubMed  Google Scholar 

  87. Wolterink-Donselaar IG, Meerding JM, Fernandes C. A method for gender determination in newborn dark pigmented mice. Lab Anim. 2009;38(1):35–8.

    Article  Google Scholar 

  88. Zhao X, Mohammed R, Tran H, Erickson M, Kentner AC. Poly (I:C)-induced maternal immune activation modifies ventral hippocampal regulation of stress reactivity: prevention by environmental enrichment. Brain Behav Immun. 2021;95:203–15. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.bbi.2021.03.018.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Zippelius HM, Schleidt WM. Ultraschall-laute bei jungen mäusen. Naturwissenschaften. 1956;43(21):502–502.

    Article  Google Scholar 

  90. Zuena AR, Zinni M, Giuli C, Cinque C, Alemà GS, Giuliani A, Catalani A, Casolini P, Cozzolino R. Maternal exposure to environmental enrichment before and during gestation influences behaviour of rat offspring in a sex-specific manner. Physiol Behav. 2016;163:274–87. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.physbeh.2016.05.010.

    Article  CAS  PubMed  Google Scholar 

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Funding

This research was supported by a Canadian Institutes of Health Research (CIHR) project grant (495842) and Natural Sciences and Engineering Research Council (NSERC) Discovery Grant (RGPIN-2019-04999) to ASG.

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Author notes

  1. Alison M. Randell and Stephanie Salia co-first authors and contributed equally to this research

Authors and Affiliations

  1. Department of Psychology, Memorial University of Newfoundland and Labrador, St. John’s NL, Canada

    Alison M. Randell, Stephanie Salia, Lucas F. Fowler & Ashlyn Swift-Gallant

  2. Cognitive and Behavioural Ecology Program, Memorial University of Newfoundland and Labrador, St. John’s NL, Canada

    Lucas F. Fowler

  3. Department of Psychology and Counseling, Immaculata University, Immaculata, PA, USA

    Toe Aung

  4. Department of Anthropology, Pennsylvania State University, University Park, PA, USA

    David A. Puts

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A.M.R., S.S. & A.S.-G. conceived the researched question. A.M.R., S.S. & L.F.F. conducted the review of manuscripts and data collection, T.A. & D.A.P. conducted the statistics. T.A., S.S. & L.F.F. prepared figures and tables. All authors contributed to the interpretation and write up of the manuscript.

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Randell, A.M., Salia, S., Fowler, L.F. et al. A meta-analysis of sex differences in neonatal rodent ultrasonic vocalizations and the implication for the preclinical maternal immune activation model. Biol Sex Differ 16, 4 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13293-025-00685-9

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13293-025-00685-9

Keywords

Biology of Sex Differences

ISSN: 2042-6410

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