2.3.1. Alarm 22 kHz USV Acoustics

Although studies have confirmed differences between male and female alarm USV productions, most studies have focused on solely the number or overall duration of alarm USVs produced in fear contexts and have overlooked acoustic features or alarm subtypes. In Blanchard's anti-predator USV study, female rats produce more frequent alarm USVs with a higher mean frequency and shorter duration [70]. In the response to a predator, male rats primarily produced negatively accelerated descending frequency alarm USVs, whereas female rats primarily produced linear descending alarm USVs. Another study that evaluated the effects of serotonin transporter deficiency in a fear condition found that female rats produced fewer overall alarm USVs than male rats with a higher frequency modulation and longer USV duration than males [72]. This finding complemented other studies that found higher frequencies of 22 kHz alarm USVs of female rats [70,105,163,164].

In summary, although alarm emission rates have been revealed to be different between sexes, sex differences in the subtypes and acoustic parameters of alarm USVs are relatively unexplored. The sex differences may be differentially regulated by the endocrine system with male rat alarm USVs being affected by sex hormones and female rat alarm USVs having less hormone dependence.

#### 2.3.2. Pup Distress USV Acoustics

As previously described, pups produce distress USVs during approximately the first 3 weeks of life in response to separation/isolation from the dam. This distress signal functions as both a social and survival act and signals the dam to retrieve and care for the pup. Because male pups produce more distress USVs, and these USVs tend to be lower in both mean frequency and amplitude compared to female pups, dams tend to respond to the male pups producing these USVs more so than female pups [17,36]. This sexual dimorphism may be mediated by the *FOXP2* gene, with a general reduction in *FOXP2* protein observed in females compared to males [17]. Although few studies have investigated the sexual dimorphism of distress USVs of typical rat pups, several studies in Table 1 highlight sex differences in distress USVs in various experimental models.

#### 2.3.3. Adult 50 kHz USV Acoustics

While USV acoustic properties have not been extensively studied between sexes, there have been a small number of studies exploring the difference between male and female rat vocalizations in terms of specific acoustic parameters. One study explored the impact of social situations on vocalizations between sexes, which revealed that female rats produced a higher proportion of frequency modulated 50 kHz USVs when exposed to a male partner compared to a female partner [154]. This suggested that female USVs may be indicative of sexual motivation. Other studies found that during rough-and-tumble play, levels of 50 kHz vocalizations was decreased as a result of Cacna1c haploinsufficiency, a gene implicated in social signal processing, which was more robustly noted in males than females [78]. Additionally, studies have explored acoustical parameter differences in male and female rats in isolation. Specifically, during isolation, the mean frequency of 50 kHz USVs was significantly lower in males than in females [61]. While there is work to be carried out in acoustic analysis exploring sex differences, these studies highlight the need for the inclusion of male and female rats into experimental studies as we continue to learn how social settings impact vocalizations produced by both sexes.

2.3.4. Estrous Cycle, Pregnancy, Menopause, and Estropause Effects on USV Acoustics Female Rat Hormone Cycle

The female rat, like other mammals, has an ovarian hormone cycle that begins following sexual maturation (puberty); undergoes cyclical regulation by the hypothalamus, ovaries, and pituitary gland; is ceased during pregnancy; and finally undergoes age-related dysregulation and subsequently infertility [165]. Nevertheless, the estrous cycle and agerelated changes in the rat are uniquely different from other mammals.

In brief, female rats reach sexual maturity at ~3 months of age [166]. The typical estrous cycle of a female rat lasts between 4 and 5 days beginning with proestrus, which is ~14 h and has both high estradiol and progesterone levels (female rat might be receptive during proestrus), estrus (the sexually receptive state) which is ~24–48 h and has low estradiol and progesterone levels, and finally diestrus states which hormone levels begin to rise and the female rat refuses copulation [166,167]. At ~9 months of age female rats will begin to experience irregular cycling for ~1 month and enter estropause that has three stages: constant estrus, persistent diestrus with irregular cycling, and persistent diestrus [165,168]. An important distinction of rat estropause is that rats continue to secrete low-moderate ovarian hormones during constant estrus and elevated ovarian hormone levels during persistent diestrus, which contrasts to humans who experience dramatic loss of ovarian hormones at menopause [165,168]. Because of this difference in ovarian hormone status, the effects of menopause are studied using ovariectomy procedures in the rat model, which more closely mimics menopause of humans by eliminating the primary production of ovarian hormones via removal of the ovaries.

The subsequent sections will summarize the known effects of the ovarian cycle of the female rat on USV production and acoustics. The scant knowledge will be evident in the review.

#### Estrous Cycle, Pregnancy, and USVs

Few studies have investigated the effects of the hormone cycle or pregnancy on USV acoustics. Furthermore, to our knowledge no studies to date have investigated the effects of estropause stages on USV production or acoustics. In the following sections, both the USV production rates and acoustics will be described according to USV type: alarm 22 kHz USVs and 50 kHz USVs.

#### Alarm USVs

Few studies have evaluated the effects of the estrous cycle on alarm 22 kHz USVs. One study evaluated the total duration of alarm USVs produced after a puff of air and found no differences between proestrus and diestrus stages [79]. Nevertheless, although the overall duration of alarm USVs did not differ between the two evaluated estrous states, the entire cycle was not evaluated, and this analysis did not include acoustics. Therefore, the effects of the estrous cycle on alarm USV production and acoustics are unknown.

Although several studies have evaluated how perinatal conditions affect USVs, the effects have primarily been evaluated in the pup offspring rather than the pregnant dams (Table 1). However, two studies have demonstrated that stress affects dam USVs [169,170]. In one study postpartum dams that received brief or long-term separation from their litters, produced more 22 kHz USVs in response to a startle stimulus than control dams [169]. In another study where the non-pregnant female cagemate of a pregnant dam underwent two 30-min stress tests for five consecutive days and then was recorded with pregnant female cagemate, the stressed non-pregnant females produced more 22 kHz USVs during interaction while the pregnant bystander produced more 50 kHz USVs than the stressed non-pregnant females [170]. Therefore, although perinatal models have demonstrated effects on pup USVs, perinatal effects can also affect dam USVs, and currently it is unclear if dam USVs influence the USVs of their pups revealing a large gap in the literature.

#### Adult 50 kHz USVs

In mating contexts, several studies have found that female Long–Evans rats produce more 50 kHz USVs during receptive (proestrus and estrus) stages of the estrous cycle [153,171,172]. Because high rates of 50 kHz USVs are observed at both high hormone (proestrus) and low hormone (estrus) stages, these studies have collectively suggested that 50 kHz USV production rate may serve as a proceptive cue to male rats in mating contexts rather than a hormonal effect [153,171,172].

In a mating context, the USV acoustics of Long–Evans female rats are influenced by the estrous cycle [153]. In general, USV frequency parameters (such as lowest, highest, and median frequencies) are highest during high hormonal states (diestrus II and proestrus) and lowest during low hormonal states (estrus and metestrus) [153]. USV intensity (power) tends to be greatest during low hormonal states (estrus and metestrus), whereas duration and USV complexity (frequency modulation) tend to be greatest during high hormone (diestrus II and proestrus) and receptive states (proestrus and estrus) [153]. Therefore, USV acoustics are influenced by copulation behavior and hormone levels in normal-cycling female rats.

Another study found that hormonal injections influenced USV acoustic parameters of trill and flat-trill 50 kHz USVs during clitoral stimulation [147]. In this study, rats were ovariectomized and treated with estradiol, progesterone, estradiol + progesterone, or a vehicle. The combined estradiol + progesterone treatment significantly increased the rate of USVs as well as the duration and complexity of the USVs [147]. This finding mirrors the previously mentioned study that found USV duration and complexity to be greatest during high hormone/receptive states.

In a mating context, an ovariectomy affects the rate of USV production but has minimal effects on USV acoustics when compared to USVs of normal-cycling rats. The elimination of the cycle via ovariectomy overall reduces the number of USVs produced during mating contexts [153,171,172]. Studies found that ovariectomized Long–Evans female rats produced fewer 50 kHz USVs in a mating context than receptive age-matched females [153,171,172]. Additionally, in ovariectomized rats the USV acoustic parameters of frequency, complexity, intensity, and duration did not differ from control rats when compared across the estrous cycle [153]. Nevertheless, this reduction in USV rate can be counteracted with estradiol + progesterone injections [151,173]; however, estradiol or progesterone alone does not increase the number of 50 kHz USVs in ovariectomized Long–Evans rats in mating contexts [173].

In a non-mating social context, ovariectomized Sprague–Dawley rats receiving estradiol produced fewer 50 kHz USVs than ovariectomized Sprague–Dawley rats without hormone treatment [174]. Although the study's authors predicted a higher 50 kHz USV production rate in rats receiving estradiol treatment, the decreased USV production may be indicative of improved social memory of the estradiol treatment group. Additionally, combined estrogen and progesterone treatments may be required to enhance social USVs. In a similar non-mating social context, ovariectomized Long–Evans female rats produce a similar number of USVs with similar acoustics to age-matched normal-cycling rats [153]. These results indicate that the estrous cycle influences the rate and acoustics of 50 kHz USVs during mating contexts more than non-mating social contexts.

In social isolation, the estrous cycle has less influence on USV parameters [153]. In isolation, female rats in estrus produced the most USVs with the lowest frequency parameters, greatest intensity, and complexity [153]. Female rats in metestrus produced the USVs with the least complexity, and shortest duration [153]. These results indicate that both the behavior and hormonal levels of the ovarian cycle influence USV acoustics of the normal-cycling female rat.

#### Ovarian Hormone Summary

In summary, the effects of the estrous cycle and ovarian hormones require further study to determine their influence on the female rat USV. To date, in the normal-cycling female rat, the estrous cycle is known to influence the number and acoustics of the 50 kHz USV in social contexts (primarily mating contexts). Additionally, the ovariectomy is known to reduce the number of 50 kHz USVs produced during mating contexts but not the acoustic parameters. Collectively, studies demonstrate an effect of ovarian hormones on 50 kHz USVs.

#### **3. Conclusions**

Biological and acoustic sex differences are apparent in the rat laryngeal mechanism. The type and acoustic features of USVs are different between male and female rats and are dependent on age, strain, and experimental models. Additionally, rat vocal folds are also sexually dimorphic which may contribute to the observed USV production and acoustic sex differences. This sexual dimorphism has been partially attributed to sex hormones; however, few studies of the laryngeal mechanism have investigated the role of hormones in influencing USV production and acoustic features. With recent advances in technology (such as DeepSqueak [138]) which simplifies and reduces the time burden of USV analysis, sexual dimorphism of the rat larynx can be further explored.

**Author Contributions:** Conceptualization, C.L. and M.R.C.; methodology, C.L.; resources, C.L., C.K.B. and N.E.S.-H.; data curation, C.L.; writing—original draft preparation, C.L., C.K.B. and N.E.S.- H.; writing—review and editing, C.L. and M.R.C.; visualization, C.L.; supervision, C.L. and M.R.C.; project administration, C.L.; funding acquisition, M.R.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by National Institutes of Health, grant numbers: T32DC009401- 12 (PI: Thibeault), F31DC018726 (PI: Broadfoot), and R01DC018584 (PI: Ciucci). The APC was funded by R01DC018584(PI: Ciucci).

**Acknowledgments:** Thank you to the members of Ciucci's lab who provided valuable feedback and editing for this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

