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ANALYSIS OF ULTRASONIC COMMUNICATION
DURING SEXUAL BEHAVIOR IN DEER MICE

(PEROMYSCUS MANICULATUS BAIRDI)

By

Steven M. Pomerantz

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Psychology and Neuroscience Program

1981

ABSTRACT
ANALYSIS OF ULTRASONIC COMMUNICATION

DURING SEXUAL BEHAVIOR IN DEER MICE
(PEROMYSCUS MANICULATUS BAIRDI)

By

Steven M. Pomerantz

The present study investigated the role of ultrasonic
communication by deer mice during sexual behavior. In sexual
behavior tests with sexually receptive females, males that copulated
always produced 35-kHz ultrasonic vocalizations, whereas males failing
to copulate seldom produced ultrasonic vocalizations. There was no
evidence to indicate that females produced ultrasounds.

Hormonal status of the female influenced male ultrasound
production. Males vocalized with sexually receptive females, but
rarely vocalized with ovariectomized females. Moreover, sexually
receptive females that were anesthetized elicited male ultrasonic
calling.

A courtship role for precOpulatory ultrasounds was indicated
by the finding that males always produced ultrasounds prior to the
onset of copulation. Furthermore, during the period when males were
producing precopulatory ultrasounds, increases in male solicitation
rate, female locomotor activity, and in the proximity maintained

between the male and female were observed. Thus, prec0pulatory

Steven M. Pomerantz

ultrasounds were associated with both elevated levels of male sexual
behavior and female proceptive behavior (i.e., female behavior that
facilitates cepulation). Male ultrasounds do not appear to play an
important role during copulation, since vocalization rate declined
during this period, while other measures of male and female sexual
behavior remained high. Following ejaculation, male ultrasonic
calling resumed despite substantial decreases in other male sexual
behaviors. Also, females were very active during this period,
indicating that postejaculatory vocalizations may serve to sustain
proceptive behavior of the female during a time when the male is in a
withdrawn state.

Gonadal hormones influenced male ultrasonic calling. Testos-
terone, as well as two of its major metabolites, dihydrotestosterone,
and estradiol, restored ultrasound production in long-term castrated
males. Combined treatment with subthreshold dosages of both .
dihydrotestosterone and estradiol activated male ultrasonic calling
and male copulatory behavior. This observation supports the
hypothesis that testosterone may stimulate all aspects of male sexual
responding in deer mice by being metabolized to both its reduced
metabolite, dihydrotestosterone, and its aromatized metabolite,
estradiol.

In conclusion, these experiments suggest that ultrasonic

vocalizations by male deer mice are an integral component of their

sexual behavior repertoire.

ACKNOWLEDGEMENTS

I offer’many thanks to Dr. Lynwood Clemens for his support and
good-nature during my graduate career. I also wish to thank Dr.
Lauren Harris, Dr. John Johnson, Jr., and Dr. John King for their
helpful comments and suggestions regarding my graduate work.

I also gratefully acknowledge the expert technological assis-
assistance of Evan Fox, Richard Mark, and Michael Vandenberg.
Insightful editorial comments were made by Dr. Gary Dohanich and Dr.
Anthony Nunez. My thanks go out to all the members of Dr. Clemens'
laboratory. I also recognize the secretarial contribution made
by Ms. Elizabeth Keith.

Finally, I wish to affectionately recognize my parents and

friends for their encouragement and support of my academic ambitions.

TABLE OF CONTENTS

Page

LIST OF FIGURES O O O O O O O O O 0 O O O O O O O O O O O O O O —. iv
LIST OF TABLES O O O O O O O O O O O O O O O I O O O O O O O O C v

INTRODUCTION 0 I O O O O O O I O O O O O O I O O O O O O O O O O 1

 

Communication During Rodent Reproductive Behavior . . . . . 2
Stimuli Capable of Eliciting Ultrasounds . . . . . . . . . . 8
Behavior of the Ultrasound Producer and Endogenous Factors
Influencing Ultrasound Production . . . . . . . . . . . . 14
Behavioral Responses to Ultrasonic Vocalizations . . . . . 27
Objectives of the Present Study . . . . . . . . . . . . . 34
GENERAL METHODS O o o o o o e e ‘ o o o o o o o o o o o o o o o o 36
EXPERIMENT 1 O O O O O I O O O O O O O O O O O O O O O O O O O 39
EXPER IMMT 2 O O O O O O O O O O O O O O O 0 O O O O O O O O O 45
EXPERIMENT 3 O O O O O O O O O O O O O O O O O O O O O O O O O 49
EHERIMENT 4 O 0 O O O O I O O O O O O O O O O O O I O O O O 0 6o

EXPERIMENT 5 O O O O O O O O O O O O O O O O O O O O O O O O I 74
SUWRY AND CONC IOUSIONS O 0 O O I O O O O O O O O O O I O O O O 85
REFERENCE NOTES 0 O O O O O O O O O O O O O O O O O O O O O O O 87

LIST OF REFERENCES 0 O O O O O O O O O O O O O O O O O O O O O %

iv

LIST OF FIGURES
Figure Page

1 Mean (iSEM) vocalization frequency and vocalization rate
of male deer mice before copulation (PVIL), during
copulation (EL), and after ejaculation (PEI) . . . . . . 51

2 Mean (:SEM) solicitation frequency, solicitation rate,
and % locomotor activity by vocalizing and copulating
male deer mice during different temporal periods of the
sexual behavior test . . . . . . . . . . . . . . . . . . 56

3 Percentage of castrated male deer mice exhibiting
ultrasonic vocalizations and mounting behavior following
treatment with 1 ug EB/day, 2 ug EB/day, 3 ug EB/day,
or OIL . . . . . . . . . . . . . . . . . . . . . . . . . 63

4 Percentage of castrated male deer mice exhibiting
ultrasonic vocalizations, mounting, intromission, and
ejaculation following treatment with 50 ug DHTP/day,
100 ug DHTP/day, or 200 ug DHTP/day . . . . . . . . . . . 65

5 Percentage of castrated male deer’mice exhibiting
ultrasonic vocalizations, mounting, intromission, and
ejaculation following treatment with 1 ug EB/day, 50 ug
DHTP/day, 1 ug EB + 50 ug DHTP/day, or 200 ug TP/day . . 68

6 Mean GtSEM) solicitation frequency, solicitation rate,
1 locomotor activity, and % proximity observed during
different temporal periods of the sexual behavior test
in sexually receptive female deer mice tested with
vocalizing and copulating males . . . . . . . . . . . . . 78

LIST OF TABLES
Table Page
1 Rodent Ultrasonic Calls During Heterosexual Encounters . . 5

2 Relationship Between Male Copulation and Ultrasonic
vocalizat ions 0 O O O O I O O O O O O I 0 O O O O O O O O 41

3 Comparison of Male Solicitation Frequency, Solicitation
Rate, and Locomotor Activity in 3 Groups of Male
Deer Nice 0 O O O O O O O O O O O O O O O O O I I O O O O 54

4 Vocalization Performance of Castrated Male Deer Mice
Given Various Daily Hormone Treatments . . . . . . . . . 70

5 Influence of Male Behavior on Female Proceptive Behavior
in Deer nice 0 O O I O O O O O O O O O O O O O O O O O O 76

INTRODUCTION

Coordination of male and female sexual behavior is a key
requirement for successful reproduction. At appropriate times, both
the male and female must be sufficiently motivated and able to perform
sex-specific and species-specific sexual behaviors. Obviously, a
mating pair does not achieve the synchrony observed in their
reproductive behavior by accident or good fortune, instead coordinated
patterning of sexual activity is facilitated by communication between
the sexual partners.

Individuals of both sexes transmit various signals that advertise
their species identity, sexual identity, sexual arousal, sexual and
social status, location, and other information important to a
prospective mate. The receiver must accurately perceive and integrate
the incoming messages so that an apprOpriate and effective response
can be made and the communication process continued. Thus, social
signalling occurring in a reproductive context means that both members
of a mating pair are assuming and exchanging the roles of sender and
receiver of messages. In this fashion, natural selection would be
expected to operate on both sexes of the communicating dyad to yield
"co-evolved, bidirectional signal production and signal reception"

(Green & Marler, 1979)-

Communication is not restricted to one modality. Depending on
the species and the ecological constraints on that species, various
social signals may be appropriate. As noted by Kelly (1980), ”Species
diversity in signalling modalities may be viewed as adaptations for
effective communication in different habitats” (p.111). Certain
signals are better suited for communication in certain situations than
others. For example, acoustic and chemical signals are generally
able to be transmitted and received over longer distances than visual

or tactual signals (Marler, 1976).

Communication During Rodent Reproductive Behavior

Various modes of communication are important for rodent
reproductive behavior (Note 1). Although tactile and olfactory
signalling will be briefly covered in this discussion, the major
emphasis of this review will be on the role of ultrasonic
communication (>20 kHz) in rodent sexual behavior.

Somatosensory stimulation provided by both partners is extremely
important for reproduction. Tactile stimulation of the female's
flanks, rump, and tailbase by the male has been found to be both
necessary and sufficient for eliciting the lordosis reflex (Pfaff,
1980). Lordosis, in turn, promotes genital stimulation of both sexes;
thereby, faciltating vaginal entry of the male's penis, sperm
transport following ejaculation, and subsequent fertilization of the
ova (Adler, 1978). A

Tactile stimulation is obviously essential for directing

copulatory mounts by the male. However, prior to the initiation of

copulation, messages must be sent which serve to bring the sexes
together. Whether by glandular secretion, marking, or urination,
males and females of all rodent species studied send chemical signals.
There is good evidence to suggest that these chemosignals function to
attract and arouse conspecific mates (Bronson & Caroom, 1971; Caroom &
Bronson, 1971; Doty, 1972; Johnston, 1974; 1975; 1979; Murphy, 1973)-
Moreover, the possibility that the hormonal status of the animal is
being communicated by these chemosignals seems likely, since both the
chemical composition of the odors and the behaviors necessary for odor
emission have been found to be regulated by gonadal hormones (Bronson,
1971; Johnston, 1977; 1979; Thiessen, Friend & Lindzey, 1968). The
importance of olfactory stimulation was further demonstrated by
studies in which olfactory impairment was produced experimentally.
Complete bilateral olfactory bulbectomy of males in many different
rodent species resulted in severe disruption or elimination of mating
(reviewed by Murphy, 1976). In females, preventing olfactory
stimulation apparently does not interfere with performance of
lordosis, but deficits in precopulatory or courtship behavior have
been noted (reviewed by Murphy, 1976).

The ability of muroid rodents to produce and hear sounds above 20
kHz is well-established (Brown & Pye, 1976; Nyby & Whitney, 1978).
Ultrasonic vocalizations occur in a variety of social settings. For
example, studies demonstrated that ultrasonic communication is
important during mother-infant interactions (Allin & Banks, 1971;
1972; Bell, 1974; Bell, Nitschke, Gorry, & Zachman, 1971; Bell,
Nitschke, Zachman, 1972; Colvin, 1973; Smotherman, Bell, Starzec,

Elias, & Zachman, 1974). Separation from the home nest, cold stress,

rough handling by the dam, and presence of novel conspecific odors are
all stimuli which elicit ultrasound production from infant rodents.
These ultrasonic calls, in turn, signal the mother to modify her
maternal behavior (reviewed by Bell, 1974; 1979; Noirot, 1972; Sales &
Smith, 1978).

Among adult rodents evidence suggests that ultrasonic signalling
infuences animal aggressive behavior, territoriality, alarm behavior,
and reproductive behavior (reviewed in Nyby & Whitney, 1978). The
present discussion will concentrate on the importance of ultrasonic
communication occurring within the context of reproductive behavior.
Occassionally, it will be necessary to discuss and compare the role of
adult ultrasounds in this social context with that found in other
social contexts.

Table 1 summarizes the heterosexual situations in which
ultrasounds have been detected in 13 rodent species, the sex or sexes'
producing vocalizations, and the physical characteristics of the
vocalizations. Depending on the species, ultrasounds have been
detected during one or all of the following time periods of a mating
sequence: (1) precopulatory or courtship - time from introduction of
mating pair until first mount; (2) copulatory - time from first mount
until ejaculation; and (3) postejaculatory - time from ejaculation
until the next mount of the following copulatory species (Nyby a
Whitney, 1978). Species differences with respect to the sex of the
animal producing ultrasounds have also been noted. In species such as
golden hamsters and collared lemmings, both sexes participate in
ultrasound production (Brooks & Banks, 1973; Floody, Pfaff, & Lewis,

1977), while in species such as house mice, rats, and Mongolian

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gerbils, the male is primarily responsible for ultrasonic vocalization
(Holman, 1980; McIntosh, & Barfield, 1980; Sales, 1972; Whitney,

Coble, Stockton, & Tilson, 1973).
Stimuli Capable of Eliciting Ultrasounds

Ultrasonic vocalizations generally do not occur spontaneously,
but appear as an animal's response to a particular social situation.
In sexual encounters, signals being sent by one sex have been found to
directly elicit or influence ultrasonic emmission of the opposite sex.
Comparative research has revealed interesting similarities and
differences between species concerning the nature and manner in which

environmental stimuli elicit ultrasound production.

House Mice

Adult males produced a greater number of ultrasounds when
placed with an adult female than when placed with another adult male
(Whitney et a1., 1973). Investigations into the signals from adult
females which are necessary or sufficient for eliciting male
ultrasounds revealed that males vocalized in response to the
presentation of soiled bedding from an adult female (Whitney, Alpern,
Dizinno, & Horowitz, 1974). Thus, chemical cues contained in the
soiled bedding were sufficient to elicit male ultrasounds whereas,
visual, auditory, or movement signals apparently were not necessary.
Subsequently, it was discovered that female mouse urine contained the
necessary ultrasound-eliciting properties, but male mouse urine did

not (Nyby, Wysocki, Whitney, & Dizinno, 1977b).

Experiments designed to test factors which might influence the
signal value of female urine revealed that female urine probably
acquires its ultrasound-eliciting properties when the female reached
puberty (Nyby, Wysocki, Whitney, Dizinno, & Schneider, 1979).
Neonatal or adult castration of male mice did not improve the abilty
of their adult urine to stimulate significant ultrasound production.
Also, neither androgen administration to neonatal females nor adult
castration of females adversely affected the abilty of their adult
urine to elicit ultrasounds. The importance of the pituitary in
mediating the ultrasound-eliciting cues in female urine was
demonstrated by the observation that female urine from
hypohysectomized females failed to stimulate male ultrasound
production. Recent evidence suggests that neither FSH nor LH alone,
but rather a combination of FSH and LH is necessary for female urine
to acquire its signal value (Nyby, personal communication).

The signal value of female urine may be learned by males. Adult
males that were separated from females at weaning did not initially
emit ultrasounds in response to female urine (Dizinno, Whitney, &
Nyby, 1978). Males that were similarly isolated from females at
weaning, but as adults provided with brief exposure to a female (e.g.
one 3-minute exposure), subsequently emitted ultrasounds in the
presence of female urine (Dizinno et al., 1978; Nyby et al., 1977b).
Moreover, in these socially experienced males that had acquired the
ability to produce ultrasounds, repeated testing with female urine
resulted in extinction of the ultrasound response (Dizinno et al.,
1978). Thus, experiential factors influence the ultrasonic

:responsiveness of males. In fact, using classical conditioning

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paradigms, artificial odors could acquire signal value (Nyby, Whitney,
Schmitz, & Dizinno, 1978). This was demonstrated by placing perfume
on the mother prior to weaning or on adult females used to provide the
male with his first heterosexual experience. When the males were
tested for ultrasound production to the perfume, only males that had
previously encountered perfume as adults on adult female mice emitted
ultrasounds. These data support the notion that female urine acquires
its signal value as a result of adult experience rather than through
neonatal imprinting.

Paradoxically, some results of male ultrasounds in tests using
stimulus animals appear to contradict the results obtained using
urinary stimuli alone (Nyby, Wysocki, Whitney, Dizinno, Schneider, a
Nunez, 1981). Adult castrated males, neonatally castrated adult
males, hyp0physectomized males, prepubertal females, and
hypophysectomized females all elicited ultrasonic vocalizations,
whereas urine from these same stimulus animals was ineffective in
stimulating ultrasounds (Nyby et al., 1979). The authors suggest that
the strategy of the male is to produce courtship vocalizations to
another mouse regardless of sexual, hormonal, or chemosensory status.
Depending on the ensuing behavioral interactions, ultrasounds will
either be inhibited (e.g., during aggression) or maintained (e.g.,
during sexual behavior). One criticism of this research on the signal
value of stimulus animals is that the tests were only 3 minutes in
duration. Therefore, failure to find the same ultrasound-eliciting

Draperties in stimulus animals as were found using urinary stimuli

could be related to a failure to give the males sufficient time to

11

recognize behavioral differences which might have existed among the

different stimulus animals.

Laboratory Rats

Male rats produce two types of ultrasounds. Fifty kHz
vocalizations occur during courtship and copulatory behavior, whereas
22-kHz vocalizations occur during a variety of social situations
including aggression, following ejaculation, and occassionally
following a period of courtship or preejaculatory 50-kHz calling
(Barfield, Auerbach, Geyer, & McIntosh, 1979). In contrast to male
mice, stimulation of both 50- and 22—kHz male rat calls is greatly
influenced by the hormonal state of the female (Geyer & Barfield,
1978). Males tested with ovariectomized females exhibited low rates
of calling. Estrogen treatment of ovariectomized stimulus females led
to a higher rate of production of both 50- and 22-kHz calls, but
maximal production was observed when males were tested with females
receiving both estrogen and progesterone.

Olfactory cues being produced by receptive females are apparently
important in eliciting male ultrasounds. Males vocalized in the
presence of an anesthetized receptive female or the soiled cage
shavings from an estrus female (Geyer & Barfield, 1978). In contrast,
anesthetized ovariectomized (OVX) females were not effective in
eliciting ultrasounds. An interesting finding in this study was that
50-kHz ultrasonic vocalizations were emitted for a longer period by
males tested with an awake OVX female than’by males tested with an
anesthetized estrous female. These results indicate that in addition

to olfactory signals, other signals (e.g. visual, auditory, tactile)

12

which females emit during behavioral interactions with males play a
significant role in maintaining male ultrasound production. The
influence of signals other than olfactory cues appears particularly
important for male preejaculatory 22-kHz vocalizations. These 22-kHz
vocalizations were produced by males in response to females that were
exhibiting decreased lordosis intensity and increased aggressiveness

toward the male (Brown, 1979).

Golden Hamsters

In golden hamsters, both males and females produce ultrasonic
vocalizations. Following presentation of a stimulus male, estrous
females emitted ultrasounds (Sales, 1972). Moreover, olfactory cues
provided by either male cage shavings or an anesthetized male
stimulated female ultrasound production (Floody, Pfaff, & Lewis,
1977). Also, auditory stimuli elicited female ultrasonic signalling
(Floody & Pfaff, 1977b), as demonstrated by the observation that
estrous females vocalized in response to synthetic tape recordings of
male calls. Experience was found to influence female calling.
Estrous females given repeated social experience with males began to
anticipate the presence of the male by increasing their rate of
calling immediately prior to the introduction of the male (Floody et
al., 1977).

Male hamsters that produced ultrasonic vocalizations to an
estrous female were observed to continue vocalizing after the removal
of the female from the test cage (Floody & Pfaff, 1977s). However,
physical characteristics of the call differed in the two situations.

In the female's presence, male vocalizations tended to have lower

13

minimum frequencies, higher maximum frequencies, longer durations, and
fewer rapid frequency changes than when the male was alone following
the female's removal. Similar to rats, male hamster ultrasounds are
influenced by the hormonal state of the female (Floody et al., 1977).
Males that were paired with estrous females vocalized significantly
more often than males paired with diestrous females. Also, similar to
both rats and house mice, olfactory cues provided by anesthetized
females elicited male vocalizations (Floody et al., 1977). However,
males did not differentially vocalize to anesthetized females which
were in different hormonal states, as they had done with awake females
in different hormonal states. These results indicate that males may
discriminate between estrous and nonestrous females on the basis of
behavioral cues (e.g., female ultrasonic vocalizations, lordosis, Or

aggression).

Other Species

In other rodent species that produce ultrasounds during
heterosexual encounters (see Table 1), research on cues that elicit
ultrasounds is not extensive. In general, males appear to vocalize in
response to females. Also, as has been found in other species,
associative learning may be important in determining the stimuli that
elicit male vocalizations. For example, after repeated testing, male
collared lemmmings would begin producing ultrasounds with the approach
of the experimenter and, occassionally, would attempt to mount the
experimenter's gloved hand (Brooks & Banks, 1973). Estrous female
collared lemmings, in contrast, did not appear to need any cues to

elicit ultrasonic calling, but rather they spontaneously vocalized in

14

their cage (Brooks & Banks, 1973). In other words, these females seem

to advertise their behavioral receptivity ultrasonically.

Behavior of the Ultrasound Producer and Endogenous Factors

Influencing Ultrasound Production

By observing the behavior of vocalizing animals which occurs in
association with ultrasound emission, additional information can be
obtained concerning the cues that trigger ultrasound production. For
example, if an animal vocalizes following investigation of another
stimulus animal, but not while alone, it is reasonable to assume that
cues from the stimulus animal triggered ultrasound production.
However, it is important to recognize that internal as well as
external referents trigger ultrasonic vocalizations. Consequently,
all the necessary external cues that elicit ultrasounds may be
present, but still an animal may refrain from vocalizing.
Alternatively, there are situations in which no apparent external
referents are present but, nonetheless, an animal vocalizes to
advertise some aspect of its internal state. Green and Marler (1979)
hypothesized that an animal utilizes both internal and external
referents to form a "signal-generating assessment". On the basis of
this assessment a signal and the behavior accompanying it may or may
not be produced. In this section, endogenous factors which influence
ultrasound production are discussed, as well as the behaviors that are
associated with ultrasounds. Each species will be discussed

separately; however, since relatively little research has been done on

15

neural mechanisms involved in ultrasound production, information on

neural mechanisms from the different species will be pooled.

House Mice

In house mice, male ultrasounds were more prevalent during
courtship periods than during any other stage of a sexual encounter
(Nunez & Bean, unpublished observations; Sales, 1972). Precopulatory
ultrasonic pulses occurred during male approaches toward the female
and male anogenital investigations of the female. In contrast,
ultrasounds were rarely detected than males were involved in
maintenance activities such as eating, drinking, or self-grooming.
During the initial stages of cOpulation, ultrasounds often occurred
simultaneously when the male mounted the female. As c0pulation
progressed, male ultrasonic vocalizations declined during
intromissions and ceased by the time the male ejaculated (It was not
determined whether, following an ejaculation, males vocalize before
initiating subsequent mating sequences).

Interestingly, the production of courtship ultrasounds seems to
be influenced by the dominance status of the male (Nyby, Dizinno, &
Whitney, 1976). Socially dominant males responded quicker to the
presence of a female with ultrasounds and called significantly more
often than subordinate males. This finding is especially important in
.light of reports which indicate that in house mice, dominant males
sired vastly more offspring than subordinate males (DeFries &
McClearn, 1970). Quite possibly, dominant males communicate both
their social status and their sexual motivation by emitting

ultrasounds. Consequently, females can select males on the basis of

l6

ultrasound production. Female responses to male ultrasounds will be
discussed later.

As might be expected from a behavior that is closely associated
with sexual behavior, male house mice ultrasounds are strongly
influenced by gonadal hormones. Both the latency to the first
ultrasound and the number of ultrasounds produced by adult males
declined following castration (Dizinno & Whitney, 1976; Nunez, Nyby, &
Whitney, 1978). Subsequent testosterone (T) administration to
castrated males restored ultrasonic vocalizations. Furthermore, a
recent study by Nunez et al., (1978) indicates that T may exert its
stimulatory effects in part by aromatization (conversion) to estradiol
(E). E restored ultrasonic vocalizations in a significant preportion
of castrated males. However, E treatment did not consistently support
ultrasound rates comparable to those achieved by T-treated males. In
contrast, dihydrotestosterone (DHT), a reduced androgenic metabolite
of T, did not stimulate ultrasound production when administered alone
and, when administered in combination with E, did not significantly
enhance the call rates over animals receiving E alone.

In house mice the effect of sex hormones on ultrasonic
’vocalizations appears to be predominantly "activational" rather than
"organizational". This conclusion is based on the finding that
females, which were ovariectomized as adults and treated with T,
emitted many more ultrasounds to stimulus females than either sham or
OVX females treated with OIL (Nyby, Dizinno, & Whitney, 1977a). In
fact, T-treated OVX females exhibited a level of ultrasound production
comparable to T-treated castrated males. Evidently, adult females

possess the potential to exhibit ultrasounds, but normally do not

l7

vocalize because they lack the necessary hormonal substrate as adults.

Laboratory Rats

Similar to house mice, male laboratory rats produce precopulatory
ultrasounds ( SO-kHz) during solicitations and anogenital
investigations of the female (Sales, 1972). McIntosh and Barfield
(1980) examined the association between male vocalizations and male
copulatory behavior. Although the study is flawed in several respects
related to the data analysis, it represents the first attempt to
investigate the precise temporal relationship of male ultrasonic
vocalizations to events which occur during mating. Males vocalized in
bursts or clusters, termed "vocalization bouts", immediately preceding
(i.e. 10 sec before) most mounts, intromissions, and ejaculations.
The highest number of vocalizations occured before an ejaculation. In
addition, more vocalizations preceded an intromission than preceded a
mount. In contrast to the vocalization bouts, following a mount or
intromission there was a discrete period of time, occupying 90-95% of
the interval between intromissions, during which no 50-kHz
vocalizations were produced. This "vocalization pause" was
characterized by autogenital grooming by the male and, further
supports the notion that males experience a period following an
intromission during which very few sexual behaviors are exhibited
(Sachs & Barfield, 1970).

During capulation (i.e. ejaculation latency-time from first
intromission to ejaculation), males that mated quickly (short
ejaculation latency and short intervals between intromissions)

vocalized less frequently than slow meters. This relationship seems

l8

paradoxical. If it is assumed that vocalization rate is positively
related to male cOpulatory rate, then one would predict fast maters to
exhibit high vocalization rates. McIntosh and Barfield (1980) offer
two explanations to account for this curious relationship between
rates of vocalization and rates of copulation:

(1) It is possible that high levels of precopulatory vocaliza-

tions are associated with both sexual arousal and the facilita-
tion of male-female investigatory responses, but that once
copulation has begun, fewer vocalizations are necessary to main-
sexual excitation between the 'aroused' maters and the

female ... (2) The fact that the males who took longer to
ejaculate produced more vocalizations than the faster maters
suggests that the females may have been behaving differently in
in these tests. Indeed female solicitation rates (amount of
darting/unit time) were found to be much lower in those tests
involving slower meters. The possibility exists then, that the
behavior of the female is directly influencing ultrasonic
vocalization production of the male. The increase in 40— to
60-kHz vocalizations in those tests involving slower maters
might therefore reflect increased arousal levels in sexually ex-

cited animals in response to an uncooperative mating partner.

(p.354-355).
Regarding the first hypothesis, there are, unfortunately, no data
presented on whether precopulatory vocalization rate varied from the
vocalization rate observed during copulation (i.e. ejaculation
latency). However, the alternative hypothesis is intriguing in that
it demonstrates the complexity involved in behavioral analysis of
communication. Evidently, male ultrasound performance is not a "fixed
action pattern" which is simply elicited by certain cues. Rather
males appear to adjust and modulate their ultrasound and sexual
behavior performance as a result of the quality of the female response
to previous ultrasounds. The topic of responses to ultrasonic
vocalizations will be discussed in detail later.

Much research has been done on the 22-kHz vocalizations of male

rats. Males produce this call in a variety of social situations

19

including submission in aggressive encounters (Lore, Flannelly, &
Farina, 1976; Sales, 1972b; 1979)., before and after ejaculation
(Anisko, Suer, McClintock, Adler, 1978; Brown, 1979; Barfield & Geyer,
1972; 1975; Pollack & Sachs, 1975) and, occassionally, following bouts
of 50-kHz precOpulatory calling (Geyer & Barfield, 1978; Geyer,
Barfield, & McIntosh, 1978a). Recently, Adler and Anisko (1979)
reviewed these studies and suggested that although the specific
meaning or social function of the 22-kHz call varies in different
social contexts, the message or information provided by the call may
be similar. By emitting this call, the male rat conveys information
about his motivational state; specifically, that he is in a withdrawn,
refractory, or helpless condition.

Adler and Anisko (1979) tested the idea that in a variety of
behavior contexts a male rat in a "helpess state" will produce 22-kHz
calls. They found that 22-kHz calling followed both ejaculation and
inescapable shock treatment. Moreover, after ejaculation, males
behaved in a fashion similar to males that were in a state of learned
helplessness (Maier & Seligman, 1976).

Physiological evidence also supports the notion that during the
postejaculatory period of 22-kHz calling, male rats have entered a
refractory state. They exhibited a high-amplitude, slow wave EEG
activity characteristic of sleep (Kurtz & Adler, 1973; Barfield &
Geyer, 1975), reductions in locomotion and general activity (Dewsbury,
1967), and increases in urination (Anisko, Adler, & Suer, 1979).

These are all signs of parasympathetic activity which indicate that

the animal is in a "vegetative" state (Adler, 1974).

20

Although the state of males producing 22—kHz calls after defeat
in an agonistic encounter may be similar to that following
ejaculation, it does not seem apparent that similar motivational
states underly the pre- and postejaculatory calling of males. Before
ejaculation, males are active rather than being socially withdrawn.
Nevertheless, males emitted 22-kHz calls prior to ejaculation, after
they had already had several ejaculations with a female (Anisko et
al., 1978; Brown, 1979). These preejaculatory vocalizations were
associated with an increase in mounts without intromissions and
appeared to occur in response to decreased lordosis intensity and
increased aggressiveness of the female. Thus, it has been suggested
that even in this situation, the 22-kHz vocalizations resulted from
both the frustration and conflict (i.e. helplessness) that males
experienced in mating with uncooperative females (Adler & Anisko,
1979).

Very little research has been done on hormonal factors mediating
male rat ultrasonic calling. Similar to house mice, castration
results in a decline in both SO-kHz and 22-kHz vocalizations (Geyer et
al., 1978a; Parrott, 1976). There is no information on gonadal
hormone replacement and restoration of 50-kHz vocalizations.
Interestingly, T activated postejaculatory 22-kHz vocalizations in
both castrated males and females exhibiting ejaculatory patterns
(Barfield & Krieger, 1977; Parrott & Barfield, 1975). However, the
calls of the T-treated females tended to be shorter and less reliably
produced than the calls of T-treated males. The full capacity to
exhibit male-like postejaculatory 22-kHz calls appears to depend on

:perinatal exposure to androgens, since females given T neonatally

21

exhibited ejaculatory reflexes and postejaculatory vocalizations which

were indistinguishable from normal males (Barfield & Geyer, 1975).

Golden Hamsters

Although both male and female hamsters emit ultrasounds ( 35-
kHz), the physical characteristics of these calls differs between
the sexes (Floody & Pfaff, 1977a). Male calls tended to have fewer
abrupt changes in amplitude and frequency than female calls. Also,
the mean duration of male ultrasonic calls was nearly twice that of
female calls.

During initial hetrosexual contact before the onset of
copulation, both sexes exhibited high rates of ultrasonic production
(Floody et al., 1977). Female ceased ultrasound production once they
assumed lordosis and the rate of ultrasound production by males
declined dramatically. Unfortunately, hamster vocalizations have yet
to be monitored throughout a copulatory session. It would be
interesting to determine whether male hamsters changed their rate of
production as sexual behavior progressed in a manner similar to male
rats and house mice. Regarding this suggestion, it is important to
realize that although sexual dimorphism in physical characteristics of
hamster ultrasonic vocalizations was noted in individual sound
spectograms, male and female ultrasounds monitored over a long period
of time appeared identical. Thus, any consideration of the
relationship between vocalization rate and sexual behavior would be
confounded by the contribution of both sexes to the vocalization

paramater. However, this problem might be able to be solved by using

:methods of devocalization that have recently become available.

22

Gonadal hormones strongly influence male hamster ultrasonic
vocalizations (Floody, 1981; Floody, Walsh, & Flanagan, 1979b).
Castration resulted in a decline in ultrasound production; this
decline was reversed by T-propionate (TP) treatment (Floody et al.,

1979b). Castrated males given TP vocalized in several situations: (1)
when placed with a receptive female in lordosis; (2) following the

female's removal; and (3) in response to synthetic ultrasounds.

Recent preliminary data indicate that E, an aromatized metabolite
of T, was effective in maintaining ultrasound production in castrated
males, but was not nearly as effective as T (Floody & Merkle,
unpublished). In contrast, DHT, a reduced metabolite of T, did not
maintain ultrasound production. At present these data are not
conclusive since only one dose of each hormone was used.

Nevertheless, the results are consistent with other studies which
suggest that aromatization of androgens plays a greater role in the
hormonal control of male mating behavior in hamsters than reduction of
androgens (DeBold & Clemens, 1978; Whalen & DeBold, 1974). It is
important to realize; however, that reduced androgens may be involved
in male hamster ultrasonic vocalizations since it was recently
demonstrated that DHT and E can act synergistically to control male
hamster copulatory behavior (DeBold & Clemens, 1978).

Similar to males, female hamster ultrasound production is
regulated by their endocrine state. Female hamsters produced more
ultrasounds to male stimuli during estrus than during other periods of

the estrous cycle (Floody et al., 1977). The importance of ovarian
steroids in controlling female ultrasonic vocalizations was more

convinicingly demonstrated in a study in which OVX females were tested

23

with various steroid hormone treatments (Floody, Merkle, Cahill, &
Shapp, 1979a). Daily treatment with E alone maintained moderates
rates of ultrasonic emission that were greater than the rates observed
in progesterone (P) or oil-treated females. Maximal call rates were
activated by combined treatment with E for 3 days followed by 1 day of
P. In the same study, E or P alone failed to stimulate the female
lordosis response, whereas the combination of E + P resulted in
maximal levels of receptivity. Although ultrasound production and
lordosis in female hamsters seem similarly mediated by ovarian
hormones, there appear to be subtle differences in the control of the
two behaviors. E alone is necessary and sufficient for ultrasound
production, whereas E stimulated lordosis only when it was combined

with P.

Other Species

In other species, ultrasonic communication during sexual
encounters is genarally correlated with courtship and copulatory
behaviors (Sales & Pye, 1974). Two species, collared lemmings and
gerbils, have been studied fairly extensively.

In a monograph by Brooks and Banks (1973), ultrasounds were
detected in 70% of tests between male and estrous female collared
lemmings. Sexual behavior occurred regardless of whether there was
ultrasonic communication. However, animals in tests with ultrasounds
tended to exhibit shorter latencies to initiate both male (mounting)
and female (lordosis) sexual behaviors. Although both sexes generally
vocalize during sexual encounters, females were observed to emit the

first ultrasound in 80% of these encounters. Possibly female collared

24

lemmings advertise their sexual receptivity ultrasonically. Such a
notion is supported by the finding that 21 out of 24 estrous females
produced ultrasounds spontaneously while in their home cage, whereas
spontaneous calls were never detected from diestrous females. In
addition to calling, estrous females typically initiated sexual
behavior by soliciting, attacking, and mounting the male. Following
the occurrence of these "male-like" behaviors by the female, males
usually became sexually aroused and started to produce ultrasounds
while chasing, grooming, and mounting the female. Once the pair began
capulating, males continued to vocalize at high rates and females
ceased ultrasound production. Immediately upon ejaculation, all
ultrasonic communication stopped. Interestingly, after an unspecified
period of time following ejaculation, females were reported to
reinitiate sexual activity by emitting ultrasounds while solicting the
male.

Ultrasound production in Mongolian gerbils is sexually dimorphic
'with males being primarily responsible for vocalization (Holman, ‘
1980). Males produce three physically different types of ultrasounds
during sexual interactions. Low intensity, frequency upswing
‘vocalizations were produced during precopulatory and c0pulatory
jperiods. Frequency modulated vocalizations were produced only during
«copulation. Finally, a high-intensity unmodulated tone was emitted
after ejaculation.

Recently, the influence of neonatal gonadal hormones on the
Organization of these sexually dimorphic gerbil vocalizations was
Studied (Holman, 1981). Males castrated on the day of birth showed

severe deficits in ultrasound responding despite the fact that they

25

were given TP as adults. Furthermore, adult females, which were
administered TP, both on the day of birth and as adults, exhibited all
3 types of "male" ultrasounds when paired with receptive females.
These results clearly demonstrate that gonadal hormones can influence

the organization of ultrasound behavior in Mongolian gerbils.

Neural Mechanisms Involved in Ultrasound Production

Since in all rodent species studied, ultrasonic vocalizations are
elicited by exposures to conspecific odors of the Opposite sex,
several investigators have attempted to determine whether ultrasound
production is affected by disruption of olfactory systems. In both
male house mice (Bean; in press) and female hamsters (Kairys,
Magalhaes, & Floody, 1980) ultrasound responding to stimulus animals
was suppressed by olfactory bulbectomy. Additionally, house mice
exhibited a reduction in ultrasonic vocalizations after receiving
vomeronasal tract cuts, but not intranasal ZnSO flush which eliminates
only main olfactory bulb imput (Bean, in press). These results not
only confirm the prediction that olfactory stimulation is essential

. 1

for eliciting ultrasound production, but also suggest that the
vomeronasal system mediates the ultrasonic response to chemosignals by
serving as the primary system involved in the perception of these
signals.

There is clearly a strong association between ultrasonic
vocalizations and sexual behavior. Consequently, neural systems that
control ultrasound production may also be involved in the control of

other reproductive behaviors. In contradiction to this suggestion,

medial preoptic lesions in male mice were found to severely disrupt

26

male copulatory behavior without altering either male anogenital
exploration of the female or male ultrasonic vocalizations (Bean,
Nunez, & Conner, 1981). These data indicate that coupling of
courtship and copulatory behaviors most likely does not occur in the
medial preoptic area.

Nevertheless, there is also evidence that neural sites of overlap
exist, so that integration of courtship and copulatory behaviors may
take place. One such site is the mesencephalic central grey. Gonadal
hormones are heavily concentrated by cells in this region (Pfaff &
Keiner, 1973; Sar & Stumpf, 1977). Furthermore, female hamsters with
lesions in this area exhibited a decline in the incidence of both
ultrasonic vocalizations and lordosis responding (Floody & O'Donohue,
1980). Additionally, male rats were observed to vocalize
ultrasonically following electrical stimulation of the central grey
(Yajima, Hayashi, & Yoshi, 1980). Research on the effects of either
stimulation or lesion of this region on male copulatory behavior have
yet to be performed, but in female rats, electrical stimulation of the
central grey facilitated lordosis responding (Sakuma & Pfaff, 1979a),
whereas lesions disrupted lordosis (Sakuma & Pfaff, 1979b).

Since research on the neural control of ultrasonic vocalizations
is so recent, there are obviously large gaps in our knowledge of this
area. A productive research strategy which has recently begun is to
determine the motor pathways necessary for ultrasound output and then
proceed to anatomically trace the brain sites and pathways which are
necessary for ultrasound production. Studies on house mice
(Pomerantz, Nunez, & Bean, in preparation), rats (Roberts, 1975;

Wetzel, Kelly, & Campbell, 1980), and gerbils (Thiessen, Kittrell, &

27

Graham, 1980) have all confirmed that the inferior laryngeal nerves
innervating the larynx are primarily responsible for the motor output
resulting in ultrasonic vocalizations. Bilateral transection of these

nerves resulted in a complete elimination of ultrasounds. Even

unilateral transection produced dramatic deficits in ultrasound
responding. The inferior laryngeal nerves originate in a discrete
region of the nucleus ambiguus (Wetzel et al., 1980) that has been
found to receive direct projections from the mesencephalic central
grey (Jurgens a Pratt, 1979). At present, it is uncertain whether the
central grey is the only region of the brain sending fibers directly
to the nucleus ambiguus. Nonetheless, the central grey does receive
afferents from other brain sites which have been implicated in the
control of sexual behavior (Conrad a Pfaff, 1976a; 1976b; Jurgens 8
Pratt, 1979; Pfaff, 1980). These areas include.the corticomedial and
basolateral amygdala, septal area, medial preoptic area, anterior
hypothalamus, and ventomedial hypothalamus. It seems that with this
anatomical evidence the door has now been Opened for future
physiological studies on the neural mechanisms involved in ultrasound

production.
IBehavioral Responses to Ultrasonic Vocalizations

Hypotheses regarding the functions of a particular communicatory
signal are developed by studying the consequences which follow
performance of that signal. These consequences include changes in the
behavior of both the producer and receiver of the signal. Behavioral

consequences of ultrasound production for the vocalizing animal were

28

previously discussed. This section considers the effects of
ultrasound production on the responses made by recipient animals.
Bell (1974) has suggested that the primary effect of ultrasounds on
recipient animals is to change their level of arousal. However, many
other investigators have argued that ultrasonic vocalizations may

trigger a much more specific behavioral response than a genaral

alteration in arousal.

House Mice

Several researchers have proposed that ultrasonic vocalizations
serve a courtship function in this species (Sales, 1972; Whitney et
al., 1973). This proposal is based on several indirect lines of
evidence. First, as was discussed previously, male ultrasounds
occurred primarily before the onset of cOpulation and waned as
copulation progressed (Sales, 1972). Additionally, male ultrasounds
may facilitate copulation by inhibiting female aggression (Sales,
1972). This suggestion is especially relevant if most matings in
nature occur during postpartum estrus (Whitney et al., 1973), a time
‘when females are very protective of their pups and extremely
aggressive toward strange males (Noirot, Goyens, & Buhot, 1975).
Conceivably, by mimicking infant pup ultrasounds, adult males reduce
the tendency of females to attack them and, consequently, increase the
jprobability that females will approach and investigate them.
13nfbrtunately, no studies have been published on the responses of
either estrous females or postpartum estrous females to male
tzltrasounds. However, preliminary observations by Nunez and Bean

(personal communication) indicated that postpartum estrous females did

29

not reduce their aggression in the presence of vocalizing males.
Furthermore, infant pups were not observed producing ultrasonic
vocalizations until after the postpartum estrous period of the dam
(Noirot, 1966). These observations undermine the notion that adult
male mice utilize ultrasonic vocalizations chimerically so that they
can mate with postpartum estrous females.

Conclusive statements regarding the function of adult male mice
ultrasounds await further research. Nevertheless, as noted by Marler
(1976), the responses to signals are often quite subtle. In fact,
”some signals function not so much to impose a qualitative change on
the behavior of the respondents, but rather to select a particular
class of respondents that may already be predisposed to perform the
response in question." (p.61). Such may be the case for house mice
ultrasonic communication. Recently, it was discovered that females in
natural and artificially induced estrus, but not OVX females,
exhibited a preference for vocalizing males over non-vocalizing males
(Nunez, Bean & Pomerantz, in preparation). If one also considers that
only socially dominant male mice produce ultrasonic vocalizations
(Nyby et al., 1976), then the adaptive significance of ultrasonic
communication in house mice becomes apparent. Dominant males may use
‘ultrasonic vocalizations to communicate their sexual intentions to the
:females that are present. Estrous females, in turn, can identify,

approach, and remain near males that are both sexually interested in

'them and reproductively fit (DeFries & McClearn, 1970).

 

30

Laboratory Rate

The results of several studies support the idea that 50-kHz
vocalizations of male rats promote behaviors of the female that
facilitate mating. Estrous females, which were exposed to 50-kHz
vocalizations immediately before testing, exhibited a shorter latency
to dart (solicit) and a higher rate of darting than estrous females
that were not provided this pretest experience (Geyer, McInntosh, &
Barfield, 1978b)., Additionally, vocalization priming of the female
resulted in shorter male intromission latencies.

Other studies have attempted to elucidate more directly the
function of SO-kHz vocalizations by studying the effects of deafening
females or muting males (Barfield, Auerbach, Geyer, & McIntosh, 1979;
McIntosh, Barfield, & Geyer, 1978; Thomas, Howard, & Barfield, 1981).
After being deafened, estrous females exhibited a reduction in darting
responses to males that were tethered (Barfield et al., 1979).
However, deafening did not interfere with the latency to return and
accept intromissions from a tethered male. A recent study
investigated the mating choice of females during tests with two
tethered males, one of which was devocalized (Thomas et al., 1981).
More solicitations were directed toward the intact vocalizing male
than toward the muted test partner. However, ultrasonic vocalizations
did not influence the number of visits or the duration of visits to
either the vocalizing or muted male. These findings indicate that the
principle function of 50-kHz male rat vocalizations is to facilitate
female solicitations. Interestingly, these vocalizations apparently
do not serve to maintain females in close proximity to vocalizing

males.

31

According to the semiotic theory of communication (Smith, 1979),
although the message of 22-kHz calls by male rats appears to be
similar in different social situations (i.e. declaration of social
withdrawal), the specific meaning or function of the call depends on
the context in which the call is received and the behavioral responses
made by the recipient of the call (Adler & Anisko, 1979; Anisko et
al., 1978). Preejaculatory 22-kHz vocalizations may serve to enhance
receptive behaviors in uncoOperative females (Anisko et al., 1978;
Brown, 1979). However, thus far only data on female behavioral
antecedents (e.g. low lordosis responding, high aggression), and not
female responses to the call, have been collected.

Regarding the postejaculatory 22-kHz call, it has been suggested
that this call maintains the separation between the mating pair during
the postejaculatory interval, while at the same time keeping the
female informed about the location of the male (Barfield & Geyer,
1972). Experimental observations in large enclosures (4 ft by 4 ft)
reported a positive correlation between the amount of ultrasounds
emitted and the amount of spacing between the mating pair (Barfield et
al., 1979). However, since males were relatively immobile while
vocalizing, this effect may have been due to female avoidance of the
male. Nevertheless, no study has yet to demonstrate a direct
one-to-one relationship between 22-kHz vocalizations and inhibition of
female approach and solicitation (Adler & Anisko, 1979 Anisko et al.,
1978).

Conceivably, the postejaculatory 22-kHz call may facilitate
female refractoriness (Adler & Anisko, 1979). This would be adaptive,

since cOpulatory intromissions delivered too soon after ejaculation

32

can disrupt sperm transport (Adler & Zoloth, 1970). Alternatively, if
the potential of mating taking place in a group setting with several
males present is considered, then postejaculatory 22-kHz calls may
serve to inhibit male aggression directed at postejaculatory males.
Indeed, resident males directed less aggression toward vocalizing male
intruders than toward silent males (Lore et al., 1976). Furthermore,
Adler & Anisko (1979) recently demonstrated that resident males
exhibited a longer latency to attack an intruder male which had just
previously ejaculated and was emitting the 22-kHz call than an

intruder which was not given any pretest sexual experience.

Golden Hamsters

Ultrasounds were found to facilitate various female behavioral
responses (Floody & Pfaff, 1977b). Specifically, females exhibited a
preference to approach either female or synthetic ultrasounds played
through a speaker in an arm of a Y-maze. Estrous females exhibited a
stronger preference for both stimuli than diestrous females.
Furthermore, natural female calls were more attractive than the
synthetic calls that were used to mimic male ultrasounds. Clearly, a
similar study needs to be performed where female preference for
natural female versus natural male vocalizations is examined. The
authors suggested that females may respond to female ultrasounds
because males generally are present at a site where female ultrasounds
are being produced. Thus, it would be adaptive for females to
approach a source of female ultrasounds. Several other experiments
provided more convincing evidence of the role male ultrasounds play in

facilitating female reproductive behaviors. First, estrous females

33

were found to increase their rate of ultrasound production after
exposure to synthetic male ultrasounds (Floody & Pfaff, 1977b). Also,
in the absence of males, synthetic ultrasounds activated lordosis by
estrous females (Floody & Pfaff, 1977b). A similar finding on the
functional significance of male ultrasounds was reported by Beach,
Stern, Carmichael, & Hanson (1976). They found that deafening of
estrous females eliminated the attraction and lordosis which they
exhibited toward a caged male.

The responses of males to female ultrasounds has not been as
extensively examined as female responses to male ultrasounds. Estrous
females normally vocalize until lordosis is assumed, at which time
vocalizing ceases. Playback of recorded female ultrasounds stimulated
male calling (Floody et al., 1979b). Furthermore, in a Y-maze, males
also exhibited a preference for the arm in which recordedfemale
ultrasounds were played over the arm without ultrasound (Floody &
Pfaff, 1977b). Apparently, female hamster ultrasounds attract

conspecific males and, also, stimulate them sexually.

Other Species

The functional significance of ultrasonic communication has not
been adequately investigated in other rodent species. In collared
lemmings, although the responses to conspecific ultrasounds were not
directly examined, there is an indication that ultrasonic
vocalizations may facilitate mating. Comparisons of latencies to
first contact, mount, lordosis, and ejaculation revealed that these
scores tended to be shorter in tests with ultrasonic vocalizations

present then in tests without ultrasounds. Obviously, much more

34

comparative work needs to be done on animal responses to conspecific

ultrasonic signals.

Objectives of the Present Study

The preceding review presented a great deal of evidence on
ultrasonic communication during sexual behavior in rodents. However,
the bulk of this evidence dealt with studies on 3 different laboratory
species; house mice, rats, and golden hamsters. Although the
occurrence of ultrasonic vocalizations during sexual encounters has
been verified in other laboratory and wild-trapped species, research
on the causal mechanisms and functional significance of ultrasonic
communication in these species is lacking. There is a need for more
extensive research in these less commonly used rodent species, so that
general principles of ultrasonic communication can be derived.

Sales (1972) noted the occurrence of ultrasonic vocalizations
during courtship and cOpulatory behavior of deer mice (Peromyscus
maniculatus). However, this report was based on only 4 observations.
The present study initially sought to replicate these observations and
determine whether ultrasonic vocalization behavior was sexually
dimorphic. Subsequently, an attempt was made to identify causal
mechanisms and adaptive functions of ultrasonic calling during deer
mice mating encounters. In order to achieve these objectives, I
concentrated on the same broad areas which were discussed in the
literature review. To reiterate these are: (1) External or
environmental stimuli which elicit ultrasonic vocalizations; (2)

Behavior of the ultrasound producer and endogenous factors influencing

35

ultrasound production; and (3) Behavioral responses to ultrasonic

vocalizations.

36

GENERAL METHODS

Animals.

The animals used in this experiment were male and female deermice

(Peromyscgs maniculatus bairdi) bred in the laboratory from stock

 

originally trapped in East Lansing, Michigan. All were 3-4 months of
age at the beginning of behavioral testing. Deer mice were housed
individually in plastic cages, 48 X 27 X 13 cm for males , and 27 X 16
X 14 cm for females. Wood shavings were used for bedding and cotton
Nestlets (Ancare Corp.) were provided as nesting material. Food and
water were available at all times. Deer mice were maintained on a
reverse day-night cycle of 16 hr light and 8 hr dark with lights off
at 1030.

Apparatus.

Ultrasonic vocalizations were monitored on a QMC Instruments Ltd.
Mini-Bat Detector (London) tuned to a center frequency of 35 kHz. The
QMC detector transforms ultrasounds into low frequency audible
signals. This device was positioned 25 cm above the floor of the
male's cage. Deer mice ultrasonic vocalizations were clearly
distinguishable from other ultrasounds produced by the animals (i.e.,
scratching, digging, and gnawing about the cage).

Tests for Ultrasonic Vocalizations During Sexual Behavior.

To induce sexual receptivity, intact females were injected
subcutaneously with 60 ug estradiol benzoate approximately 72 hr
before behavioral testing and 600 ug progesterone approximately 6 hr

before testing. For certain experiments untreated ovariectomized

females were used. Sexual behavior tests began with the introduction

37

of the receptive female into the male's home cage approximately 4 hr
after lights off. The room in which the tests were conducted was
illuminated by a 25 watt red bulb.

Male copulatory behavior in P. m. bairdi consists of mounts (with
thrusting), intromissions (with penile insertion), and ejaculation.
The temporal pattern of the behavior is such that after several \
intromissions the male ejaculates. Following ejaculation there is a
period of sexual quiescence (postejaculatory interval) before sexual
behavior resumes. Ultrasonic vocalizations, mounts, intromissions,
and ejaculations were recorded on an Esterline Angus Event Recorder.
Sexual behavior tests were terminated when one of the following
criteria had been satisfied: (1) 15 min after the start of the test
with no intromission; (2) no ejaculation within 15 min of the first
intromission; (3) no intromission within 10 min following ejaculation;
(4) first intromission following ejaculation.

Measures.

From the experimental data the following behavioral measures were
derived: mount latency (ML) - time in seconds from introduction of the
female to the first mount; intromission latency (IL) - time in seconds
from introduction of the female to the first intromission; ejaculation
latency (EL) - time in seconds from intromission to ejaculation; mount
frequency (MF) - number of mounts preceding ejaculation; intromission
frequency (IF) -'number of intromissions preceding ejaculation; mean
interintromission interval (MIII) - mean inteval in seconds between
intromissions including the interval between the last intromission and

ejaculation; postejaculatory interval (PEI) - time in seconds from

ejaculation to the next intromission; vocalization latency (VL) - time

38

in seconds from introduction of the female to the first 35-kHz
vocalization; postvocalization intromission latency (PVIL) - time in
seconds from the production of the first 35-kHz ultrasound to first
intromission; vocalization frequency (VF) - total number of 35-kHz
vocalizations; vocalization rate (VR) - number of 35-kHz vocalizations
per minute. VF and VR were calculated for the periods before
copulation, during cOpulation, and after ejaculation. These three
sampling periods correspond to the PVIL, EL, and PEI, respectively.
Tests for Ultrasounds with an Anesthetized Stimulus Animal.

Intact males were paired with receptive or ovariectomized
females. One member of the male-female pair was selected to be
anesthetized 15 min before the start of testing with an intra-
peritoneal injection of chloral hydrate (1mg/gm body weight) and
returned to their home cage. Testing began with the introduction of
the female (either awake or anesthetized) into the male's cage. The

production of 35-kHz ultrasounds was monitored for 10 min.

39

EXPERIMENT 1

The purpose of this experiment was to investigate whether any
relationship exists between ultrasonic vocalizations and sexual
behavior in deer mice (Part A). Also, a determination was made of the

sexual partner that produced ultrasounds during mating (Parts A, B &

c).

Method

Part A - Relationship Between Sexual Behavior
and Ultrasonic Vocalizations

Subjects and Procedure.

Forty-one male and 41 sexually receptive female deer mice were
used in this experiment. In at least two sexual behavior tests prior
to the start of the experiment, hormone treatment had successfully
stimulated receptivity in these females. Also, before the experiment,
males were given one 30 min unmonitored session with a receptive
female. One week after this session, males and females were observed
in sexual behavior tests, once per week, for two consecutive weeks.

TFor each test the animals were paired with a different partner.

IPart B - Evidence for Sexual Dimorphism of Ultrasonic Calling
Subjects and Procedure.

Subjects were 14 sexually experienced male-female pairs derived
from the animals used in Part A. Deer mice pairs were tested for

ultrasonic vocalizations with one member of the pair being selected to

40

be anesthetized, so that, in 7 pairs, the male was anesthetized and,

in the other 7 pairs, the female was anesthetized. In all cases the

female was was receptive.

Part C - Further Evidence for Sexual Dimorphism of Ultrasonic Calling
Subjects and Procedure.

Four sexually experienced males were paired with 4 receptive
females in 15 min sexual behavior tests. 0n the day following this
test, the inferior laryngeal nerves of each male were bilaterally
sectioned under Metofane (Pitman Moore, Inc). anesthesia. One week
after this surgery the same deer mice pairs were tested for

ultrasounds during sexual behavior.

Results

Part A

During the first week of testing for sexual behavior (Table 2),
the occurrence of male copulation was associated with the occurrence
of ultrasonic vocalizations, whereas failure to copulate was.
associated with the absence of ultrasonic emissions (X2(1)a24.0,
‘p<.OO1). Further, in tests resulting in copulation, precopulatory
ultrasounds were always detected. Males that vocalized and copulated
during the first week of testing, continued to do so during the second
'week. Also, three of the four males that vocalized, but failed to
cepulate in the first week, copulated and emitted ultrasounds in the

second week. During the second week, ultrasounds and male sexual

41

TABLE 2

Relationship Between Male Copulation

and Ultrasonic Vocalizations

 

Ultrasonic Vocalizations

 

Present .Abgggg
Week 1
Number of Males Copulating 22 0
Number of Males not Capulating 4 15
Week 2
Number of Males Copulating 25 0

Number of Males not Copulating 1 15

42

behavior were absent in males that did not exhibit these behaviors in
the first week.

From those tests in which vocalizations were present, VL did not
vary from week 1 to week 2, with the overall mean.(iSEM) for both
weeks of testing being 121: 32 sec. Additionally, overall VR was
similar for weeks 1 and 2 with an overall mean (iSEM) of 3.2::0.6.
More complete analyses of vocalization and cOpulatory behavior data
during these tests are presented in Experiment 3, Part A.

VR during weeks 1 and 2 was compared for each male and each
female to determine whether one sex was more likely responsible for
ultrasound production. For males, the Pearson product-moment
coefficient of VR between weeks 1 and 2 was .7807 (p<.001). Due to
the possibility that the 15 males, which failed to exhibit
ultrasounds, were influencing the analysis, they were eliminated and a
correlation coefficient of .6694 (p<.OO1) was obtained for the
remaining 26 males. The correlation coefficient for females was not
significantly different from 0 if the analysis considered all females
(r--.0196) or only females that were in at least one test with
ultrasounds present (r--.1724). These data provide initial evidence

that males rather than females were producing ultrasonic calls.

Part B

Six out of seven males produced 35-kHz vocalizations when tested
with anesthetized receptive females. Mean (iSEM) VF during the 10 min
test was 16.1i 3.1. No ultrasounds were detected when receptive

females were paired with anesthetized males. These data support the

43

notion that only males produced ultrasounds during sexual behavior

testing.

Part C

Prior to the sectioning of the inferior nerves of males,
ultrasonic vocalizations were detected during all male-female
pairings. Also, c0pulatory behavior occurred in 3 of the 4 pairings.
Following the nerve section, no ultrasounds were detected from any of
the 4 pairs of deer mice.‘ Other than the inability to vocalize, these
males did not appear physically disabled; however, only 1 of 4 males

successfully copulated.
Discussion

The results of this study indicate that male deer mice always
produced 35-kHz ultrasonic vocalizations before the onset of
copulatory behavior. Furthermore, in males failing to exhibit
copulatory behavior, ultrasonic calls were seldom detected. This
strong association between male copulatory behavior and ultrasonic
vocalizations indicates that the 35-kHz calls may be an important
component of the sexual behavior repertoire of male deer mice.

Several observations support the conclusion that males were
responsible for producing the 35-kHz ultrasounds detected during
mating tests. For males, VR was positively correlated across the two
mating tests, whereas for females no such correlation was found.
Second, males vocalized when paired with anesthetized receptive

females, whereas receptive females did not produce ultrasounds when

44

paired with anesthetized males. Finally, and most conclusively, after
male deer mice had their inferior laryngeal nerves transected, no
ultrasonic vocalizations were detected from heterosexual pairs which
in previous tests had exhibited ultrasounds. A similar sexual
dimorphism of ultrasound behavior during mating has been reported for
house mice (Sales, 1972; Whitney et al.,1973), rats (McIntosh &

Barfield, 1980), and Mongolian gerbils (Holman, 1980).

45

EXPERIMENT 2

Experiment 1 established that male deer mice respond to sexually
receptive female deer mice by producing ultrasonic vocalizations. In
Experiment 2, the nature of the female cues which elicit male
vocalizations was investigated. Part A contrasted the ability of
females in artificially induced estrus to elicit ultrasounds against
females‘that were ovariectomized. In Part B, an initial determination
was made of the necessary and/or sufficient female stimuli that

promote male deer mice ultrasonic vocalizations.

Method

Part A
Subjects and Procedure._

Subjects were 8 sexually experienced male deer mice. Eight
female deer mice, 4 of which were ovariectomized (OVX) and 4 of which
were sexually receptive, served as stimulus animals. Males were
observed for ultrasounds during sexual behavior tests, once per week,
for two consecutive weeks. The order of presentation of the two
stimulus conditions was counterbalanced so that males were tested with
a sexually receptive female one week and an OVX female on the other

week.

46

Part B
Subjects and Procedure.

Subjects were 12 sexually experienced male deer mice. Stimulus
females were the same as those used in Part A. Six of the males were
randomly selected to be paired with an anesthetized receptive female,
and the remaining 6 males were paired with an anesthetized
ovariectomized (OVX) female. Ultrasonic vocalizations were monitored
for 10 min from males paired with anesthetized females, either

receptive or OVX.

Results

Part A

The percentage of males exhibiting ultrasounds was significantly
higher when males were paired with behaviorally receptive females than
when males were paired with OVX females (Fisher's exact probability
test, p<.05). Whereas all 8 males vocalized with receptive females,
only 2 out of 8 males vocalized with OVX females. Mean VF of the two
males which vocalized with OVX females was 15.5, as compared to a mean
VF of 34.0 when these same two males were paired with receptive
females. Finally, it should be noted that 7 out of the 8 males
copulated and exhibited precopulatory ultrasonic vocalizations during

their tests with receptive females:

Part B

No males produced ultrasounds when presented with an anesthetized

OVX female. In contrast, similar to the results of Exp. 1, Part B, 5

47

out of 6 males vocalized when paired with an anesthetized receptive
female. Mean (ESEM) VF of these males was 13.61;3.2, comparable to

the results reported in Exp. 1, Part B.

Discussion

The results of these experiments demonstrated that male deer mice
respond to sexual signals of conspecific females with ultrasonic
vocalizations. Part A established that for most males, cues
associated with a sexually receptive female were necessary for
eliciting male ultrasounds, since males generally produced ultrasonic
vocalizations only when females were sexually receptive. Ultrasound
tests with anesthetized females (Part B) also confirmed this finding.
Males only vocalized with receptive females and not with OVX females.
Moreover, these findings were suggestive of the posssibility that
chemical signals produced by receptive females were sufficient to
promote male ultrasound responding.

The experimental findings using awake females as stimulus animals
differed in several respects from those reported in similar
experiments with other species. Male house mice did not exhibit
differential rates of ultrasound responding to females in different
ovarian hormone states (Nyby et al., 1981). In both laboratory rats
and hamsters, males continued to vocalize to OVX females or diestrous
females, but not at as high a rate as when tested with females in

estrus (Floody et al., 1977; Geyer & Barfield, 1978). This difference
in the male's response to females in different ovarian hormone states

was quantitative rather than qualitative. In contrast to these

48

species, most male deer mice did not vocalize with OVX females.

In other rodent species (Nyby & Whitney, 1978), olfactory cues
produced by female deer mice may be important for eliciting male
calling. The manner in which these chemosignals were transmitted by
receptive females and sensed by males needs to be more fully explored.
Additionally, it is important to stress that although chemosignals
were sufficient for eliciting male'deer mice ultrasounds, sexually
active females likely performed other behaviors which also facilitated
male vocalizations. This suggestion is supported by the observation
that male deer mice vocalized at a higher rate when paired with an
awake female than when paired with anesthetized receptive females.
Similar results confirming the influence of female activity on male
ultrasound were also reported in laboratory rats and hamsters (Floody

et al., 1977; Geyer & Barfield, 1978).

49

EXPERIMENT 3

The preceding experiment examined external stimuli that are
involved in triggering male deer mice ultrasonic vocalizations. In
this experiment, the behavior of the vocalizing male was considered.
By observing the male's behavior, additional clues concerning the
proximate causes of ultrasonic signalling and the information
contained in the ultrasonic message might be provided. Part A of this
experiment explored the temporal relationships that exist between male
ultrasounds and male copulatory behavior by subjecting the data
collected in Exp. 1 to further analysis. In Part B, the relationship
of male ultrasounds and cOpulatory behavior to other male behaviors

was examined.

Method

Part A
Subjects and Procedure.
Subjects and procedure used were discussed in Exp. 1, Part A.

Data originally collected in that experiment were analyzed.

Part B
Subjects and Procedure.
Subjects in this experiment were 47 male deer mice. Males were

given a 30 min unmonitored session in their home cage with a receptive

female. One week after this session, males were observed for

ultrasounds during a sexual behavior test. Sexually receptive females

50

were used as stimulus animals. The following male behaviors were
recorded in addition to those outlined in the General Methods: male
solicitation - male approach toward female from greater than 1 body
length to less than 1 body length; and male locomotor activity -
amount of time in seconds, male ambulating or running about the cage
(if the male was motionless for less than one second during a
locomotor sequence, the activity was judged continuous).

Measures.

In addition to the measures described in the General Methods the
following measures were derived from the behavioral data: male
solicitation frequency - total number of male solicitations; male
solicitation rate - number of male solicitations per min; and 5 male
locomotor activity - % of time in which male involved in locomotor

activity.
Results

Part A

From tests in which males copulated to an intromission following
ejaculation, comparisons of the VF and VR before copulation, during
copulation, and after ejaculation were made (during the PVIL, EL, and
PEI, respectively). There were no significant differences in
ultrasound performance between weeks 1 and 2; therefore, the data for
each male over the two weeks of testing were averaged with the means
(iSEM) shown in Figure 1. VF varied during the three time periods
(one-way ANOVA, F[2,26]=13.4, p<.01). Planned comparisons revealed

that VF was higher after ejaculation than before capulation

51

[:1 Vocalization Freq.

I Vocalization Rate

 

 

 

32 8.0
28 7.0
no 24 A 6.0
C
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‘5 20 5.0
.2
3 16 4.0
o
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'5 12 3.0
2' s 2.0
4 1.0
0 0
Before During After
Cop. Cop. Ejac.

Figure 1. Mean (:SEM) vocalization frequency and vocalization
rate of male deer mice before copulation (PVIL),
during copulation (EL), and after ejaculation (PEI).

Vocalizations/Min

52

(F[1,26]-4.95, p<.05), and that both of these time periods had a
significantly higher VF than that found during copulation
(F[1,26]-27.0, p<.01). VR also varied during the three time periods
(one-way ANOVA, F[2,26]-17.0, p<.01). Again, VR before c0pulation and
after ejaculation was significantly higher than VR during copulation
(F[1,26]=33.8, p<.01). Moreover, precopulatory and postejaculatory VR
did not differ; indicating that the differences observed in VF during
these two time periods was simply a result of the postejaculatory time
span being longer than the prec0pulatory time period. It should also
be noted that ultrasonic calls monitored during the copulatory period
were detected during the intervals between mounts and intromissions
and, occasionally, were synchronous with ejaculation, but were never
detected during a mount or an intromission.

Relationships between measures of male copulatory behavior and
ultrasonic vocalizations were evaluated by performing Pearson
product-moment correlations. Independent correlation coefficients
were computed using data from the first and second test in which both
sex behavior and ultrasounds were exhibited. A significant negative
correlation existed between VR before copulation and the length of the
PVIL for both the first (ne25, r--.4775, p<.01) and second test (n-22,
r'-.3661, p<.05). VL was not found to be related to precopulatory VR
or to any measures of sex behavior performance. In the males' first
test with ejaculation, VR during c0pulation was not correlated with
measures of male sexual performance (i.e. MF, IF, EL, and MIII).
However, in the males' second test with ejaculation, there was a
highly positive correlation between VR during c0pulation and the

length of the MIII (n=20; r=.6694, p<.01), but no relation with other

53

measures of sexual performance. Finally, VR after ejaculation was
significantly and positively correlated with the length of the PEI in
the males' first test (n=8, r-.6653, p<.05). but not in their second

test.

Part B

On the basis of the sex behavior tests, males could be classified
into 3 independent groups: (1) males exhibiting ultrasounds and
copulatory behavior (N-15); (2) males exhibiting ultrasounds but no '
copulatory behavior (N-16); and (3) males not exhibiting either
ultrasounds or c0pulatory behavior (N-16). Of the two groups of males
producing ultrasonic vocalizations, copulating males had a
significantly higher VR than non-copulating males (t(29)-3.67, p<.01).
More importantly, precopulatory VR of copulating males was
significantly higher than the overall VR of vocalizing, non-copulating
males (t(29)-9.76, p<.01), with the means (iSEM) being 3.3: .4 and
1.3 t .2, respectively.

Solicitation frequency, solicitation rate, and locomotor activity
of the 3 groups of males is presented in Table 3. Solicitation
frequency and solicitation rate varied across the 3 groups such that
the two groups of non-copulating males did not differ, but both groups
had a significantly lower solicitation frequency and rate than
vocalizing and copulating males. The 3 male groups did not differ in
locomotor activity.

Solicitation rate and locomotor activity during the VL in both
groups of vocalizing males were similar to the overall solicitation

rate and locomotor activity observed in non-vocalizing and

54

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55

non-capulating males. In males that vocalized and copulated,
solicitation rate was significantly higher after the VL than during
the VL (Sign test, p<.01). In contrast, in males that vocalized but
did not copulate, solicitation rate did not increase following the
first vocalization. Locomotor activity did not change during the
period following the first vocalization in either group of vocalizing
males.

As shown in Figure 2, in vocalizing, copulating males,
solicitation rate and locomotor activity increased significantly
during the PVIL over the levels observed during the VL (Sign tests,
p<.01 and p<.05, respectively). During the EL, solicitation frequency
was significantly higher than during the PVIL (Sign test, p<.01);
however, since the solicitaion rates did not differ between these two
time periods, the difference observed in solicitation frequency
reflected the fact that the EL time span was longer than the PVIL.
Following ejaculation, both solicitation rate and locomotor activity
declined significantly from the levels observed during the EL (Sign
tests, p<.OO1 and p<.OO1, respectively) to a level comparable to that
observed during the VL. With respect to solicitations during the PEI,
it should be noted that males clustered most of their solicitations
into the period immediately preceding the reinitiation of copulation

(last 10% of the PEI).
Discussion

Ultrasonic signalling in male deer mice in response to the

presentation of receptive females appeared to set in motion a sequence

56

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57

of events which, if performed appropriately, led to reproduction.
Male behavior among c0pulating males did not differ from that
exhibited by non-copulating males until the production of the first
ultrasound. Once they began vocalizing, males that copulated
increased both their rate of soliciting females and their level of
locomotor activity. In contrast, non-copulating males (both
vocalizing and non-vocalizing) maintained a constant low rate of
solicitations and low level of locomotor activity throughout the
testing session. Additionally, the VR of vocalizing, non-copulating
males was much lower than the precopulatory VR observed in c0pulating
males. In sum, the results of this experiment indicated that the
initiation of capulation in deer mice is associated with high levels
of male courtship ultrasonic vocalizations, solicitations, and male
locomotor activity.

The temporal patterning of ultrasounds in male deer mice was such
that VR was highest befbre the onset of copulation and after
ejaculation. In comparison to these two time periods a dramatic
reduction in VB was observed during c0pulation. Sales (1972) also
reported ultrasonic emission during both prec0pulatory and c0pulatory
sequences, but did not measure VR during these two time periods.

To further elucidate the relation between ultrasonic
vocalizations and cOpulatory behavior, correlations based on
.performance of these two behaviors were considered. Several factors
could account for the inverse relationship found between precopulatory
VR and the length of the PVIL. First, precopulatory VR may indicate
the male's readiness to mate. Second, precopulatory ultrasonic calls

may serve a courtship function. High VR by the male could then

58

facilitate the onset of c0pulation by promoting increased levels of

female proceptive and receptive behaviors (Beach at al, 1976; Floody
and Pfaff, 1977b; Geyer et al., 1978b; McIntosh et al.,

Following the onset of c0puletion the VR declined. This decline
seen in VR while the animals were copulating might indicate that
during this period, vocalizations were less critical to the
maintenance of female proceptive and receptive behaviors (Beach at
al., 1976; Floody & Pfaff, 1977b). Additionally, along with the
males' copulatory behavior, their high solicitation rate and high
level of locomotor activity most likely served to sustain female
behavior. It should also be noted that a positive correlation between
VR during c0puletion and the length of the MIII was found in one of
the two tests in Part A. Since this relationship was not found in
both tests it may be spurious, reflecting the number of correlations
that were computed. However, McIntosh and Barfield (1980) reported a
similar positive correlation between VR during copulation and MIII in
rats. Possibly, in males exhibiting long intervals between
'intromissions, it is necessary to make use of the same strategies
during c0puletion, such as high rate of ultrasonic vocalizations, that
were employed during the courtship sequence before c0puletion.

Male deer'mice returned to high rates of vocalization following
ejaculation. Also, after ejaculation, males exhibited a reduction in
solicitation rate and locomotor activity similar to that found in '
laboratory rats (Adler & Anisko, 1979: Dewsbury, 1957)- Thus, the
high rate of postejaculatory calling may serve to maintain the
proximity and sexual activity of the female (Barfield & Geyer, 1975).

so that additional c0pulatory series and ejaculations can be achieved.

59

Female deer mice were found to require more than one ejaculation for
maximal pregnancy initiation (Dewsbury, 1979); therefore, it would
clearly be adaptive for males to perform behaviors which maintain the
copulatory readiness of females. In rats, the 22-kHz postejaculatory
call of males may inhibit potentially aggressive behavior of
conspecifics (Anisko et al., 1978; Barfield & Geyer, 1975). Such a
function for postejaculatory vocalizations is not incompatible with

the one being proposed for deer'mice.

60

EDERIMENT 4

The previous experiments established that ultrasonic
vocalizations by male deer mice are intimately related to other
aspects of male sexual behavior. .Since endogenous factors such as
gonadal hormones have a major controlling influence on male deer mice
copulatory behavior (Clemens & Pomerantz, 1981), it would be expected
that the same sex hormones controlling c0pulation would also exert a
similar influence on male ultrasonic calling. For cells within the
central nervous system and genital tissues, it has been demonstrated
that metabolism of testosterone (T) by aromatase enzymes
(aromatization) leads to the production of estradiol (E) (Callard,
Petro, Ryan, 1978; Lieberburg & McEwen, 1975; Naftolin, Ryan, & Petro,
1972) and T metabolism by Sa-reductase enzymes (So-reduction) results
in the production of Sa-dihydrotestosterone (DHT) and other Sa-reduced
androgens (Bruchovsky & Wilson, 1968; Massa, Justo, & Martini, 1975:
Whalen & Rezek, 1972). Both Scsreduction and aromatization of T were
found to be obligatory for T to reliably stimulate male c0pulatory
behavior in castrated male deer mice (Clemens & Pomerantz, 1982). The
present experiment was designed to test the extent to which T, and two
of its major metabolites, E and DHT, influence male ultrasonic

vocalizations.

61

Method

Subjects and Procedure

Hale deer mice that exhibited ultrasounds and copulatory behavior
in previous experiments were selected for use in the present
experiment. Following their successful sexual behavior test, 72 males
were castrated under Metofane anesthesia. Six weeks after castration,
males were pretested for sexual behavior with a receptive female.
Hales were matched according to whether they exhibited ultrasounds in
this pretest and were assigned to one of several different daiLy
hormone treatment groups. Hormone treatment began the day after the
pretest and continued for two weeks. In Part A, the daily treatments
used were: 1 ug estradiol benzoate (EB, n-e); 2 ug EB (N'9); 3 ug EB
(n-7); and sesame oil (OIL, fl-8). In Part B, the daily treatments
were: 50 ug dihydrotestosterone prOpionate (DHTP, N=8); 100 ug DHTP
(n-s); and 200 ug DHTP (N-B). In Part C, the daily treatments were: 1
ug EB + 50 ug DHTP (n-s) and 200 ug TP (n-a). Hormone treatments were
dissolved in .02 ml sesame oil and injected subcutaneously. Hales

received two sexual behavior tests with a receptive female, one per

week, beginning one week after the start of hormone treatment. During
the course of the experiment, one male receiving 50 ug DHTP/day and
one male receiving 200 ug DHTP/day died.
Measures

All measures were the same as in the General Methods, with the
exexception that overall VR was defined as the number of 35-kHz
vocalizations/min starting with the production of the first

ultrasound. This redifined measure was used so that VR in

62

non-copulating males could be compared to the precopulatory VR of

copulating males.

Results

Part A

Percentages of castrated males exhibiting ultrasonic
vocalizations and mounting are represented in Figure 3. On the first
week, 1 ug EB EB/day activated male ultrasound production to a greater
extent than any other treatment (x2(1)-4.85, p<.05). By the second
week, calling was stimulated to a greater extent in castrated males
receiving 1 ug EB/day or 2 ug EB/day than in males receiving either 3
ug EB/day or OIL (x2(1)-13.69, p<.01). It should be noted that males
receiving 3 ug/day were noticeably lethargic during testing and
non-testing periods.

Regarding measures of ultrasound performance, vocalization
latencies of males receiving 1 ug EB/day and 2 ug EB/day were similar,
with a mean.(tSEH) of 416::95 sec and 5981:106 sec, respectively.
Similarly, vocalization rates of the two groups did not differ, with
the mean (iSEH) of the 1 ug EB/day group being 1.01:.4 and the mean
(iSEH) of the 2 ug EB/day group being 1.11:.5. Measures of ultrasound
performance were not analyzed in males receiving 3 ug EB/day or OIL,
since differences from the other groups in performance would only
reflect differences in the percentage of males vocalizing.

Although a few EB-treated animals exhibited mounting behavior

(most notably males receiving 2 ug EB/day), none of the EB treatment

Figure 3.

Week I

‘0-

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20-

 

40'

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20-

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63

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TR

EATMENT

Percentage of castrated male deer mice exhibiting
ultrasonic vocalizations and mounting behavior following
treatment with 1 ug EB/day, 2 ug EB/day, 3 ug EB/day,

or OIL.

64

groups were significantly different from OIL controls (Figure 3).

Also, no males exhibited intromission or ejaculation.

Part B

Figure 4 illustrates the effects of DHTP treatment on male
ultrasonic calling and male copulatory behavior. Doses of 100 ug
DHTP/day and 200 ug DHTP/day restored ultrasounds in all males. The
200 ug DHTP/day dosage activated ultrasounds after only one week of
treatment, whereas two weeks were necessary for the 100 ug DHTP/day
dosage to be completely effective. DHTP also activated male
copulatory behavior in a dose-dependent manner (Chi-square test for
number of males mounting, x2(2)-7.14 p<.05). Chi-square tests for the
number of males in different groups exhibiting intromission and
ejaculation were not significant; however, intromission frequency of
males receiving 200 ug DHTP/day was significantly higher than males
receiving 100 ug DHTP/day (Mannewhitney U test, U89, p<.05).

Comparison of measures of ultrasound performance revealed that
males receiving 200 ug DHTP/day had a significantly shorter VL (U-B.
p<.OO1) and higher VR (U-Z, p<.OO1) than males receiving 100 ug
DHTP/day. Mean (iSEM) VL of the two groups was 57::16 sec and 414i:91
sec, respectively; and mean CtSEM) VR of the two groups was 4.41:.2

and 1.51;.3, respectively.

65

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TREATMENT

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67

Part C

Males treated with 1 ug EB/day from Part A and males treated with
50 ug EB/day from Part B were included in the analyses for this
experiment. Figure 5 shows the percentages of castrated males
exhibiting ultrasonic vocalizations, mounting, intromission, and
ejaculation. Only males receiving 50 ug DHTP/day did not vocalize in
week 1. However, by week 2, the percentage of males exhibiting
ultrasonic vocalizations did not vary across treatments. Regarding
copulatory behavior, combined treatment with 1 ug EB + 50 ug DHTP/day
was as effective as 200 ug TP/day; and both of these treatments were
significantly more effective than 1 ug EB/day or SO ug DHTP/day (for
mounting, x2(1)‘7.96, p<.O1; for intromission, x2(1)-8.65, p<.O1; for
ejaculation, x2(1)-9.79, p<.01).

Measures of vocalization performance in the different treatment
groups are compared in Table 4. VL of males receiving combined
treatment of EB + DHTP was significantly shorter than males receiving
TP, EB, or DHTP alone. Overall VB of TP and EB + DHTP groups did not
differ; however, overall VR of the EB +DHTP group was significantly
higher than males receiving DHTP alone and approached significance
when compared to males receiving EB alone (0-17, p<.065). The failure
of the latter comparisdn to reach significance could be a result of
the fact that males receiving EB + DHTP did not vocalize at a high
rate during copulation. Moreover, VR of the EB + DHTP group during
the PVIL was significantly higher than the overall VR of males
receiving EB alone (U=6, p<.01). Finally, it should be noted that

males treated with TP or EB + DHTP performed similarly to intact

68

 

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70

TABLE 4

VOCALIZATION PERFORMANCE OF CASTRATED MALE DEER

MICE GIVEN VARIOUS DAILY HORMONE TREATMENTS

1 ug EB 50 ug DHTP
a

MEASURES

b c
VL (sec) 415 j; 95 739 i 77
VR (overall) 1.0 i 0.4 0.4 i 0.2"
VR during PVIL ---- .....
VR during EL ---- --..-
VR during PEI ----- ---__
N 8 7

“for all measures mean (3; SEM)
bp<:.05, two-tailed ManneWhitney U test
cp<:.01, two-tailed Mann-Whitney U test

1 ug EB +

50 ug DHTP

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71

males, in that their VR was lower during the EL than during the PVIL

and PEI (Kruskall Wallis one-way ANOVA, H'42.6 p<.OO1).

Discussion

Results of this experiment demonstrated that TP, as well as two
of its major metabolites, DHTP and EB, restored the performance of
ultrasonic vocalizations in long-term castrated male deer mice.
Additionally, both T? and its reduced metabolite, DHTP, were able to
restore the complete copulatory pattern. In contrast, the aromatized
metabolite of TP, EB, was only minimally effective in restoring
mounting behavior. Finally, evidence was obtained which indicates
that aromatized and reduced metabolites of T may synergize to control
male sexual behavior in this species.

As indicated previously (Clemens & Pomerantz, 1981), TP and DHTP
were equipotent in facilitating male sexual behavior. These results
lead to the possibility that Se-reduction of TB to DHTP may contribute
to the activation of male sexual behavior in deer mice. Such a notion
was supported by the finding that TP activation of male copulatory
behavior in castrated male deer mice was inhibited by simultaneous
administration of a compound that blocked Se-reduction (Clemens &
Pomerantz, 1982).

Despite the production of ultrasonic vocalizations by castrated
males receiving EB, replication of the facilitation of mounting
behavior by EB was not seen (Clemens & Pomerantz, 1981). There were
several differences in the methodology used in the present study which

may account for the failure of EB to restore mounting behavior.

 

72

First, in the earlier Clemens & Pomerantz study, 3 weeks of EB
treatment was required for a significant percentage of castrated males
to mount females. In the present experiment, castrated males were
only treated for two weeks. Secondly, the mean mount latency of
EB-treated males in the Clemens & Pomerantz study was longer than the
15 min test used in the present experiment. Nevertheless, in the
present experiment, EB did stimulate vocalizations and did synergize
with a subthreshold dosage of DHTP to activate male sexual behavior.
These results stongly suggest that E does play a significant role in
mediating male sexual behavior of deer mice. This hypothesis is
further supported by the finding that TP activation of male copulatory
behavior in castrated males was prevented by simultaneous
administration of an aromatization inhibitor (Clemens & Pomerantz,
1982).

Although a significant percentage of castrated males receiving 1
ug EB/day exhibited ultrasounds and a small percentage of castrated
males receiving 50 ug DHTP/day vocalized, males in both of these
groups did not vocalize as soon after the introduction of the female,
or at as high a rate as males receiving a combined dosage of both of
these hormones. The synergistic action of EB and DHTP on male sexual
behavior supports the notion that in deer mice, T activates male
reproductive behavior by being metabolized to Se-reduced androgens and
estrogens. That is to say, T may be acting as a prohormone (Clemens
& Pomerantz, 1982).

In all species thus far investigated, T has been found to
activate male ultrasonic vocalizations (Floody et al., 1979; Nunez et

al., 1978; Parrott & Barfield, 1975)- Similar to deer mice, E

73

facilitated male ultrasonic vocalizations in house mice (Nunez et al.,
1978) and hamsters (Floody, 1981). In contrast to deer mice, DHT did
not influence ultrasound production in either of these species.
Generally, species differences and similarities in the abilty of DHT
or E to mediate ultrasonic vocalizations were also observed in the
ability of these hormones to mediate copulatory behavior (Floody,
1981; Luttge, 1980). It remains to be determined whether species
differences in the ability of metabolites of T to activate male sexual
behavior reflect varying degrees of reliance on T metabolism by

5e—reduction and/or aromatization.

Frrnl

74

EXPERIMENT 5

The functional significance of a particular behavior is often
analyzed by observing the consequences of performing that behavior.
In the previous experiments it was suggested that ultrasound
production by male deer mice may be involved in and possibly facili-
tate c0pulation by a mating pair. By observing the consequences of
male vocalizations on female behavior, the final experiment was
designed as an initial attempt to address the fuctional significance
of ultrasonic vocalizations. Beach (1976) coined the term "proceptive
behavior" for female "appetitive responses evoked by stimuli from
males which initiate or increase the probability of masculine sexual
behavior directed at the female." (p.116). If, as previously
suggested, ultrasonic vocalizations serve a courtship function, than

male ultrasounds should facilitate female proceptive behavior.

Method

Subjects and Procedure

Subjects in this experiment were 55 sexually receptive female
deer mice. For all females, hormone treatment stimulated behavioral
receptivity in two consecutive, weekly, sex behavior tests conducted
prior to the start of the experiment. Two groups of stimulus males
were derived from behavior testing conducted before the start of the
experiment. One group consisted of males which c0pulated during a 30
min. session with a receptive female (N-30), while the other group

included males that did not copulate during two consecutive, weekly,

75

30 min sessions (N-ZS). In the eXperiment, females were observed
during sexual behavior tests with males from one of the two stimulus
male groups. In addition to those behaviors outlined in General
Methods, the following female proceptive behaviors were recorded: (1)
female solicitation - female approach toward the male from greater
than 1 body length to less than 1 body length; (2) female locomotor
activity - amount of time in seconds female spent ambulating or
running about the cage (Note 2); and (3) proximity - amount of time in
seconds male and female within 1 body length of each other (Note 3).
Measures

In addition to the measures described in General Methods, the
following measures were derived from the behavioral data: female
solicitation frequency - total number of female solicitations; female
solicitation rate - number of female solicitations per min; % female
locomotor activity - S of time in which female involved in locomotor
activity; and % proximity - % of time in which proximity was

maintained between the male and female.

Results

Females were classified as experiencing 1 of 3 experimental
conditions depending on the behavior of the male with which they were
tested. These experimental conditions were: (1) males exhibiting
ultrasounds and copulatory behavior (N=22); (2) males exhibiting
ultrasounds but no copulatory behavior (N=16); and (3) males
exhibiting neither ultrasounds nor copulatory behavior (N=17). As

shown in Table 5, females tested with males that vocalized, regardless

76

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77

of whether the animals c0pulated, solicited males more and exhibited
higher levels of locomotor activity than females tested with
non-vocalizing males. The effects of ultrasounds on female proximity
score appeared confounded with copulation, since only females tested
with c0puleting and vocalizing males had a significantly higher
proximity score than females tested with non-vocalizing males.

A clearer indication of the possible facilitation by ultrasounds
of female proceptive behavior was observed by comparing female
behavior before and after performance of the first ultrasound in both
groups of females tested with vocalizing males. It is noteworthy that
solicitation rate, locomotor activity, and proximity exhibited during
the VL by both groups of females tested with vocalizing males were
very similar to that exhibited by females tested with non-vocalizing
males. Both groups of females experiencing male vocalizations
increased their locomotor activity following production of the first
ultrasound. Additionally, the'proximity score increased significantly
after the VL in females tested with vocalizing and copulating males,
but did not reach significance in females tested with vocalizing and
non-c0pulating males. Solicitation rate remained unchanged in both '
groups of females.

Figure 6 illustrates comparisons of female proceptive behavior
during different temporal“periods in females tested with vocalizing
and copulating males. Females spent a greater percentage of time both
actively moving about the cage and in close proximity to the male
during the PVIL than during the period before the first ultrasound
(Sign tests, p<.OO1). Since solicitation rate did not vary

significantly during these time periods, the increase in solicitation

Figure 6.

 

 

 

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Mean (:SEM) solicitation frequency, solicitation rate,
Z locomotor activity, and Z proximity observed during
different temporal periods of the sexual behavior test
in sexually receptive female deer mice tested with

vocalizing and copulating males.

Note. VL-vocalization latency, PVIL-postvocalization
intromission latency, EL-ejaculation latency, and
PEI-postejaculatory interval.

79

frequency observed during the PVIL was due to the fact that the PVIL
time span was longer than the VL. Levels of female proceptive
behavior remained unchanged after the animals began copulating.
Finally, during the PEI, females spent considerably less time in close
proximity to the male (Sign test, p<.OO1), even though levels of

locomotor activity remained high.
Discussion

After having investigated in separate experiments, sexual
behaviors of male and female deer mice, a strictly behavioral
description of the sequence of events taking place during sexual
behavior tests may prove useful. Generally, after a receptive female
was introduced into the male's cage, she would move toward the male's
nest and occupy it, often displacing the male from the nest in the
process. Initially, she seldom left the nest. However, with the
occurrence of male ultrasonic vocalizations, the female increased her
locomotor activity, circling the male often, and occasionally,
soliciting the male. Concurrent with this increase in female
behavior, male ultrasound production remained high. Also, male
solicitations of the female became much more prevalent. During this
period, the male spent a great deal of time chasing the female.
Normally, she would allow the male to approach and sniff her, before
darting away from him. Often the female bit or barked audibly at the
male pursuing her. Nevertheless, male pursuit of the female

eventually led to copulation.

 

80

During copulation, precopulatory patterns of behavior were
generally maintained, especially male solicitations and female
darting. However, several notable departures from this pattern were
observed. Male vocalizations declined dramatically, often ceasing
completely. Also, male solicitations usually resulted in successful
intromissions with female lordosis. After each intromission, the male
groomed himself before resuming his solicitations and copulation with
the female.

Following ejaculation, the female continued to move about the
cage. However, the behavior of the male changed dramatically. He
became quite inactive, spending most of his time in the corner of the
cage, grooming himself and vocalizing.. It was not until several
minutes after ejaculation that the male stOpped grooming and again
started to solicit the female, so that additional copulatory series

could occur.

It is well established that female locomotor activity increases
during estrus (Dewsbury, 1967; Richter, 1947). This increased
locomotor activity is only one reflection of the many estrogen-induced
changes in female behavior (Wade, 1976). Especially noteworthy in the
present study was the ability of female deer mice to regulate their
activity during estrus, on the basis of whether the male vocalized.
Under natural conditions, when a receptive female enters into the
territory of another male, it would seem adaptive for the female to
seek out a safe place from which she could assess the intentions of
the male toward her. Will he react to her presence in a sexual or

aggressive fashion? Although not directly analogous to the natural

 

81

situation, in the laboratory, female deer mice placed in the male's
cage, markedly increased their locomotor activity and became much more
accessible to the male only after the male began producing
ultrasounds. Conceivably, males signalled both their non-
aggressiveness and also their sexual interest to the female by
producing ultrasonic vocalizations. After the female perceived these
sexual signals by the male, it is quite possible that her reluctance
to move about the cage and exhibit proceptive behavior dissipated.

It is important to note that in many tests, copulation did not
occur despite the fact that males produced ultrasonic vocalizations
and females exhibited proceptive behavior. This may have been due to
the fact that after prouducing their first ultrasound, males that
failed to copulate did not solicit females as frequently as males that
went on to capulate (Experiment 3, Part B). These vocalizing but
non-copulating males appeared to perform the initial requisite sexual
responses; however, they did not progress to more advanced sexual
behaviors. Since males had at most only two previous sessions with
receptive females, their lack of solicitations may have been a result
of their sexual inexperience. Alternatively, their failure to solicit
females may have been a consequence of being rebuffed by the female.
Perhaps by acting aggressively toward males on their initial
solicitations, females may further insure that they mate with a
reproductively fit individual.

During the period from first ultrasound to first intromission,
the distance maintained between the mating pair was reduced from the
level observed before the first ultrasound. This high degree of

proximity seen in the mating pair during the PVIL obviously

 

82

facilitated the initiation of c0pulation. It also appeared to be
correlated with elevated male vocalization rate and male solicitation
rate. In pairs of deer mice in which males vocalized but copulation
did not occur, following the production of the first ultrasound,
proximity scores, as well as male vocalization and male solicitation
rate did not reach the levels attained by copulating pairs. Most
likely, the combination of male vocalizations and male solicitations,
rather than either behavior alone, contributed to the closeness
maintained between the male and female. In other rodent species,
proximity scores and ultrasounds have not been monitored concurrently
during sexual behavior tests. Nevertheless, both female hamsters and
female house mice exhibited a preference to be near an ultrasounding
animal or sound source (Floody a Pfaff, 1977b; Nunez et al., in
preparation). .

The decline in male vocalizations observed during copulation was
not accompanied by any apparent changes in female behavior. Rather,
with the exception of the occurrence of lordosis, females continued to
exhibit the same circling and darting behavior which they had
exhibited immediately preceding copulation. Conceivably, the
c0pulatory behavior itself was a sufficient stimulus to maintain
female behavior through ejaculation. Moreover, in other rodent
species, such as house mice, hamsters, and collared lemmings there are
similar reports that ultrasonic vocalizations waned once the animals
began c0puleting (Brooks and Banks, 1973; Floody et al., 1977; Sales,
1972). These observations have also fostered the notion that
ultrasonic vocalizations of rodents serve primarily a courtship

function (Nyby & Whitney, 1978; Sales 8: Pye, 1974).

83

After male deer mice ejaculated, there was a resumption of
ultrasonic calling. This calling occurred despite an apparent lack of
interest in the female by the male, as evidenced by his substantially
reduced locomotor activity and solicitation rate. Previously, it was
suggested that these postcopulatory vocalizations served to maintain
female arousal, in order that additional c0pulatory series could be
achieved (see Discussion, Experiment 3). The observation that female
locomotor activity remained high during the postejaculatory period
provides further support for this notion. However, since the amount
of time the male and female spent near one another declined during the
PEI, the postejaculatory vocalizations may also be signalling the
female to stay away from the male while he is in a so called
"down-state" (Adler & Aniko, 1979; Barfield & Geyer, 1975). These two
functions for postejaculatory vocalizations are not mutually
exclusive. Furthermore, although no differences in the nature
(features) of the ultrasounds were noted during the different temporal
periods of the sexual behavior test, it is quite possible that the
ultrasound monitoring equipment may not have been able to detect
subtle differences which may have existed.

Finally, in concluding this discussion, it is important to stress
that the functional significance of deer mice ultrasonic communication
was investigated using an indirect approach based on correlational
evidence. Conclusions derived from correlational evidence are at best
tentative, until additional experiments are performed to directly
analyze the consequences of male ultrasounds. Such future work should

examine the experimental effects of muting the male, deafening the

84

female, and varying physical charateristics of male ultrasonic

vocalizations.

85

SUMMARY AND CONCLUSIONS

From the results of the present experiments, the following

conclusions concerning the relationship of ultrasonic vocalizations to

deer mice sexual behavior were derived:

(1)

(2)

(3)

(4)

(5)

(6)

Ultrasonic vocalizations during sexual behavior tests were
sexually dimorphic such that only males produced ultrasounds
(Exp. 1 ).

Males that copulated always produced ultrasonic vocalizations,
whereas males failing to copulate seldom produced ultrasonic
vocalizations (Exp. 1).

Endocrine status of the female influenced male ultrasonic
calling. Males vocalized with sexually receptive females, but
rarely vocalized with ovariectomized females (Exp. 2).

An anesthetized sexually receptive female was a sufficient
stimulus to elicit male ultrasonic calling. Most likely,
olfactory cues triggered ultrasound production in this
situatiion (Exp. 2).

A courtship role for precepulatory ultrasonic vocalizations
was indicated by the observation that males always produced
ultrasonic vocalizations before the onset of c0pulatory

behavior (Exp. 1).

As evidenced by increases in male solicitation rate, female
locomotor activity, and in the proximity maintained between
the male and female, prec0pulatory ultrasonic vocalizations
were associated with both elevated levels of male sexual

behavior and female proceptive behavior (Exp. 3 & Exp. 5).

86

(7) Male ultrasonic vocalizations did not appear to play an impor-

(8)

tant role during copulation, since vocalization rate declined
during this period, while other measures of male and female
sexual behavior remained high (Exp. 3 & Exp. 5).

Following ejaculation, male ultrasonic vocalizations may have
served to sustain the interest of the female in the male
during a time when the male was in a withdrawn state (Exp. 3

a Exp. 5).

(9) Gonadal hormones influenced male ultrasonic calling.

(10)

Testosterone, as well as two of its major metabolites,
dihydrotestosterone and estradiol, restored male ultrasonic
calling in long-term castrated males (Exp. 4).

Combined treatment of castrated males with subthreshold

dosages of both dihydrotestosterone and estradiol activated
male ultrasonic calling, as well as male copulatory behavior.
This observation further supported the hypothesis that
testosterone may stimulate male sexual behavior by being
metabolized to both its reduced and aromatized products

(Exp. 4).

1.

87

REFERENCE NOTES

Communication involving visual cues is apparently of minimal
importance in rodents. No study has yet to find any deficits in
mating behavior following blinding of one of the sexual partners
(Beach, 1942; Hard & Larsson, 1968; Kow & Pfaff, 1976). The possi
bility that gustatory stimulation influences rodent reproductive
behaviors has not been adequately investigated. Both autogrooming
and allogrooming are often observed during sexual behavior testing
(Dewsbury, 1967; 1978); however, the functional significance of
these behaviors remains unclear.

Heretofore, female locomotor activity has not been considered as a
distinct category of proceptive behavior. Rather components of
female locomotor activity such as hopping, darting, and soliciting
are commonly used as measures of proceptive behavior (Beach, 1976;
Madlafousek & Hlinak, 1977). The locomotor activity of female dee
mice often included a category of proceptive behavior that Beach
(1976) termed "alternating approach and withdrawal". Although
solicitations are one form of approach and withdrawal behavior,
female deer mice often approached and withdrew from the male witho
getting within 1 body length of him.

Proximity was judged to be a measure of female proceptive behavior
because pilot observations indicated that female deer mice appear
to exert a greater control over the distance maintained between th
mating pair than males. Females were easily able to prevent males
from getting close to them by fending off, biting, or running away

from soliciting males.

LIST OF REFERENCES

LIST OF REFERENCES

Adler, N. T. The behavioral control of reproductive physiology. In
H. Montagna and H. A. Sadler (Eds.), Reproductive behavior.
New York: Plenum Press, 1974.

Adler, N. T. Social and environmental control of reproductive proces-
ses in animals. In T. McGill, D. Dewsbury, and B. Sachs (Eds.),
Sex and behavior: Status and prospectus. New York: Plenum Press,

1978.

Adler, N. T., & Anisko, J. J. The behavior of communicating: An analy-
sis of the 22 kHz call of rats (Rattus norvggicus). Amer. Zool.,
1979’ E, 493-508.

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