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The temporal patterning of 40–60 kHz ultrasonic vocalizations and copulation in the rat (Rattus norvegicus)
BEHAVIORAL AND NEURAL BIOLOGY 29, 349--358 (1980)
The Temporal Patterning of 40-60 kHz Ultrasonic Vocalizations and Copulation in the Rat
(Rattus norvegicus) TRACY K . MCINTOSH AND RONALD J. BARFIELD 1
Department of Biology, Livingston College, Rutgers University, New Brunswick, New Jersey 08904 Ultrasonic vocalizations are associated with copulatory behavior in the rat; however, the precise temporal relationship of these vocalizations to mating events is not known. In this study, 40-60 kHz ultrasonic vocalizations were recorded during sexual behavior and related to the periodicity of events that occur during mating. The occurrence of these ultrasounds was correlated with indices of sexual motivation. Ultrasonic vocalizations serve both to reflect and affect the arousal levels of the mating pair. The patterning of vocalizations suggests that these sounds play an important role in the coordination of male and female activity thereby facilitating the integration of mating behavior of the species.
In the course of copulation, both male and female rats emit ultrasonic vocalizations with the main energy around 40-60 kHz (Sales, 1972b). High rates of vocalization seem to be associated with hormonal and behavioral readiness of both the male and female to copulate (Geyer, McIntosh, & Barfield, 1978). Recent studies indicate that these vocalizations serve a communicatory function during copulation. Specifically, female rats previously exposed to 50-kHz vocalizations show increased solicitation behavior (darting and hopping) when subsequently placed with a male (Geyer et al., 1978); and, additionally, exposure to these vocalizations resulted in a dramatic increase in the proceptive behavior of female rats in the presence of a castrated male (Mclntosh, Barfield, & Geyer, 1978). After ejaculating, the male rat enters a refractory period. This refractory period is characterized by two phases. First, there is an absolute refractory period during which the male is incapable of reinitiating sexual 1 This research was supported by a National Institute of Child Health and Human Development Research Grant HD 04484 to Ronald J. Barfield. Tracy K. Mclntosh was a predoctoral trainee on a National Institute of Mental Health Interdisciplinary Training Grant MH 13006-05. Hormones used in this study were generously donated by Schering Corporation, Bloomfield, N.J. We would like to thank Dr. Benjamin Sachs and Dr. Norman Adler for their helpful comments on the manuscript. 349 0163-1 047/80/070349-10502.00/0 Copyright© 1980by AcademicPress, Inc. All rights of reproductionin any form reserved.
350
MCINTOSH AND BARFIELD
activity. Following this, there is a relative refractory period in which copulatory behavior may be resumed in response to a supranormal arousing stimulus (Beach & Holz-Tucker, 1949). During the postejaculatory period, the male rat emits 22-kHz vocalizations for a period which appears to conform directly to the length of the absolute refractory period (Barfield & Geyer, 1972, 1975). Recently, Anisko, Suer, McClintock, and Adler (1978) found that preejaculatory 22-kHz ultrasonic vocalizations occur during the copulatory sequence. These authors related the occurrence of these preejaculatory 22-kHz calls to specific copulatory events. Brown (1979) has suggested that these preejaculatory 22-kHz vocalizations may function to inhibit female agonistic behaviors during copulation, thereby facilitating sexual responsiveness. Ultrasonic vocalizations of 40 to 60 kHz are also associated with a variety of activities that occur during the copulatory cycle. We have no information, however, on the precise relationship of these ultrasonic vocalizations to the specific sequential events during mating. The purpose of this study was to examine the 40- to 60-kHz ultrasonic vocalizations produced by rats as they occur in a temporal relationship to specific events in the copulatory cycle. METHODS Forty sexually experienced male Long-Evans hooded rats were selected as subjects and caged singly under reversed lighting conditions (dark phase; 9:30-21:30). The animals were housed in individual galvanized steel- and wire-mesh cages measuring 30 × 18 × 18 cm. Food (Purina Rat Chow) and water were available ad libitum. All subjects were screened for sexual behavior two weeks before behavioral testing and were thus accustomed to the testing situation. All tests were performed in glass-walled aquaria (40 × 26 × 29 cm) during the dark phase under red illumination. The male was allowed to adapt to the test chamber for at least 5 min prior to the introduction of a receptive female (the "Introductory period"). The females were ovariectomized Long-Evans rats injected with 50/zg of estradiol benzoate 54 hr before the test and 500/xg of progesterone 6 hr before testing. Each male was tested for sexual behavior with a receptive female. All tests were terminated after 20 min if no copulatory activity occurred following the introduction of the female. The following behavioral events were scored manually on a push-button activated Rustrak event recorder: (1) mounts, intromissions, and ejaculations by the male; (2) the occurrence of 40- to 60-kHz vocalizations during and following mating; (3) 22-kHz postejaculatory vocalization; (4) darting and hopping by the female. The following measures were derived from the raw data: Intromission Frequency (IF, the number of intromissions prior to ejaculation);
351
ULTRASONIC VOCALIZATION AND MATING
Mount Latency (ML, time from the introduction of the female to the first mount); Intromission Latency (IL, time from the introduction of the female to the first intromission); Ejaculation Latency (EL, time from the first intromission to ejaculation); Intercopulatory Interval (ICI, a measure of copulatory rate derived from dividing EL by the total number of intromissions); Vocalization Latency (VL, time from ejaculation to the beginning of the 22-kHz vocalization); Vocalization Termination (VT, time from ejaculation until the end of the 22-kHz vocalization); Postejaculatory Interval (PEI, time from ejaculation until the first intromission of the next copulatory series). An ultrasonic microphone fixed at the top of the testing chamber and connected to a Holgate ultrasonic receiver was used to monitor the vocalizations. The ultrasonic receiver was tuned to monitor only the 40to 60-kHz vocalizations that occur during mating. The sensitivity of this instrument allows for fine discriminative tuning thereby precluding the possibility that any other frequency would be monitored. An oscilloscope was connected in parallel with the ultrasonic receiver for further ultrasound verification. RESULTS
Copulation to ejaculation occurred in 28 of the 40 tests. As seen in Table 1, during the 5-min introductory period, vocalization rates in tests that resulted in ejaculation were not significantly different from those tests in which ejaculation was not reached. Following the introduction of the female into the test cage, however, the successful copulators had significantly shorter latencies to vocalize than did the nonejaculators (t test; t (38) = 5.57; p < .001). On the other hand, in the first 20 min of behavioral testing, significantly more vocalizations were observed in the tests inTABLE 1 N u m b e r o f Vocalizations Occurring in the Introductory Period and during Copulation
Vocalizations in introductory period
L a t e n c y to vocalize following introduction of the female (sec)
No. o f vocalizations during mating ( l s t 20 min)
T e s t s resulting in ejaculation ( N = 28, m e a n +- SEM)
46.8 --- 7.8
4.9 ± 1.1
95.5 ± 16.1
T e s t s without ejaculation ( N = 12, m e a n ± SEM)
53.4 ± 11.6
27.3 ± 9.5
301.9 ± 43.9
p*
N.S.
< .001
< .001
* S t u d e n t ' s t test.
352
MCINTOSH AND BARFIELD 300.
200' :E
I00.
•e
• • i
o|e ~ o , 200
|
i
i 400
j
| 600
EL
(SEC)
| O0
I000
FIG. I. Correlation between the number of 40- to 60-kHz vocalizations produced during mating and the ejaculation latencies (EL) for all tests resulting in ejaculation ( N = 28) (correlation coefficient, r = .58, p < .01).
volving nonejaculators than in the tests involving active maters (t test; t (38) = 5.80; p < .001). Rates of vocalization during copulation appear to inversely reflect sexual arousal during the copulatory sequence. Figure 1 shows that there is a positive correlation between latencies to ejaculation and the number of vocalizations produced during the 28 mating tests (correlation coefficient, r = .58; p < .01). An argument may be made that the slower maters vocalized more during mating because they had a longer time period in which to produce the vocalizations. An analysis of the first 10 rain of each mating test, however, demonstrated that there was an even greater positive correlation between EL and the number of vocalizations during the first 10 min of copulation (correlation coefficient, r = .61; p < .01). Furthermore, ICIs (a measure of copulatory rate) were found to be positively correlated with the number of vocalizations produced during mating (Fig. 2) (correlation coefficient, r = .60; p < .01). These data taken 200-
$
•
IO0Z
e
....: Icl (sic)
Correlation between the number of 40- to 60-kHz vocalizations produced during mating and the intercopulatory interval (ICI) for all tests resulting in ejaculation ( N = 28) (correlation coefficient, r, = .59, p < .01). FIG. 2.
ULTRASONIC VOCALIZATION AND MATING
353
TABLE 2 O c c u r r e n c e of 50-kHz Vocalizations prior to and following M o u n t s and Intromissions Behaviouralevent Mount
In~i'omission
p*
M e a n No. of vocalizations (+_ SEM) in 10 sec before
1.6 --- 0.4
4.8 _+ 0.6
< .001
M e a n time (+- SEM) without vocalizations after (sec)
5.8 -+ 1.1
28.5 _+ 4.1
< .001
Note. N u m b e r of tests = 28. * t T e s t for related m e a s u r e s .
together indicate that the faster maters vocalized significantly less than the slower copulators during mating. Immediately prior to mounts or intromissions, 40- to 60-kHz vocalizations occur in bursts or clusters. The results shown in Table 2 demonstrate that the rate of vocalizations in 10 sec preceding intromissions is significantly greater than that preceding mounts (t test for related measures; t (27) = 4.25; p < .001). A unique and noticeable period of silence often followed each mount or intromission during mating. Table 2 also demonstrates that the length of this period was significantly greater following intromissions than following mounts (t test; t (25) = 5.97; p < .001), occupying approximately 95% of the Interintromission Interval. Figure 3 indicates a distinct clustering of vocalizations immediately preceding ejaculations. A significantly greater amount of 40- to 60-kHz vocalizations occurred within the 10-sec period immediately preceding ejaculation than during a period 20 or 30 sec prior to ejaculation (one-way ANOVA; F (1.54) = 20.98;p < .001). In addition, the amount ofvocaliza-
~P
!
30~20 TIME
20-10
i
10" |JAC
(SEC)
FIG. 3. T h e n u m b e r of 40- to 60-kHz vocalizations occurring in s u c c e s s i v e 10-sec intervals beginning 30 sec prior to ejaculation. Bars indicate S E M (N = 28).
354
MCINTOSH AND BARFIELD
tion produced immediately prior to an ejaculation was found to be significantly greater than the amount of vocalization preceding intromissions (t test; t (27) = 5.55; p < .001). Although it is true that both male and female rats emit 40- to 60-kHz vocalizations during mating, it seems clear that the majority of vocalizations recorded during copulation in this study were produced by the male. Evidence supporting this comes from the fact that: (1) the vocalization patterns were directly correlated with the mounting patterns of the male (i.e., bursts of vocalizations occurred consistently during the approach preceding both mounts and intromissions), and (2) preliminary testing in our laboratory utilizing devocalized females indicates that a temporal patterning of vocalization, similar to that found in the present experiment, occurs when a normal male copulates with a devocalized female (Mclntosh & Thomas, unpublished).
DISCUSSION It is well known that 40- to 60-kHz ultrasonic vocalizations are associated with mating behavior in a variety of rodents (Sales, 1972a, 1972b). The results from this study demonstrate for the first time that these vocalizations have a precise temporal relationship to the events that occur during mating. Vocalization rates were compared between maters and nonmaters during the introductory period and during the first 20 min of the copulatory test. Results indicated that shorter latencies to vocalize following the introduction of the female occurred in those tests resulting in ejaculation. Thus, vocalizations in the early phase of the copulatory cycle appear to be associated with higher levels of sexual arousal. This is consistent with the hypothesis that ultrasonic vocalizations are reflective of the state of arousal of an animal (Bell, 1974). These data also agree with the findings of Geyer (1976) who discovered that ultrasonic emission rates were highest at the initiation of mating and that high rates of vocalizations were correlated with the sexual readiness of the male. The results relating the amount of 40- to 60-kHz vocalizations to arousal levels during the 28 mating tests (as measured by EL and ICI) reveal that the fast maters emitted significantly fewer vocalizations during the course of mating than did the slower maters. It may be argued that the slower maters were allowed a greater period of time to vocalize; however, analysis of the first 5 and 10 min of each mating test still demonstrated a significantly higher rate of 40- to 60-kHz vocalizations for the slower maters. This is a curious finding; if one assumes that the fast maters are more sexually aroused, it would be expected that more vocalizations per unit time would be produced by those animals exhibiting short ejaculation latencies. There are several factors which may account for the paradoxical relationship between vocalizations and copulation: (1) It is possible that high
ULTRASONIC VOCALIZATION AND MATING
355
levels of precopulatory vocalizations are associated with both sexual arousal and the facilitation of male-female investigatory responses, but that once copulation has begun, fewer vocalizations are necessary to maintain sexual excitation between the "aroused" maters and the female. It has been shown, in fact, that high levels ofprecopulatory vocalizations facilitate female solicitation behavior in both the rat (Geyer et al., 1978; Mclntosh et al., 1978) and hamster (Floody & Pfaff, 1977). (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 these tests. Indeed, female solicitation rates (amount of darting/unit time) were found to be much lower in those tests involving the slower maters. The possibility exists then, that the behavior of the female is directly influencing ultrasonic vocalization production by the male. The increase in 40- to 60-kHz vocalizations in those tests involving slower maters might therefore reflect increased arousal levels in sexually excited animals in response to an uncooperative mating partner. This suggests that sexual "frustration" (also a type of arousal) may be one cause of high levels of vocalization sometimes seen during mating. This hypothesis is supported by the fact that male rats deprived of their mating partner will vocalize at very high rates (Mclntosh & Barfield, unpublished) and male rats separated from females by a wire-mesh divider will also show consistently high levels of vocalizations (Geyer et al., 1978). Interestingly, Brown (1979) found that precopulatory 22-kHz vocalizations occurred primarily when the male subjects were mounting the females more often without achieving intromission. These preejaculatory 22-kHz calls were also found to be associated with low lordosis intensity and high aggressive behavior of the female. Quite possibly a frustration-type of sexual arousal is responsible for the preejaculatory 22-kHz calls observed by Brown as well as the 40- to 60-kHz calls observed in the present study in those tests involving slower maters. During the copulatory sequence, it was observed that a burst of 40- to 60-kHz vocalization would immediately precede most mounts or intromissions. This clustering of vocalizations which we have termed a "vocalization bout" was such a novel and intriguing finding that we wondered whether they might serve an important function during mating. Analysis revealed that the amount of vocalizing was significantly greater just prior to an intromission than just prior to a mount. These vocalization bouts may serve to alert the female as to the male's state of sexual arousal, or to enhance the probability or intensity of lordosis by the female. Floody and Pfaff (1977) found that in hamsters, estrous females would maintain the lordosis posture significantly longer in the presence of recorded ultrasounds than in silence. Additionally, we have found that, in some instances, female rats will show lordosis behavior to ultrasounds with minimal or no physical contact from a male (Mclntosh et al., 1978).
356
MCINTOSH AND BARFIELD
Obviously much more detailed analysis of the problem is needed before any firm conclusions can be drawn concerning the communicatory function of these 40- to 60-kHz vocalization bouts. Immediately following a mount or intromission, there is also a distinct period of time in which n o 40- to 60-kHz vocalizations are emitted by either animal. This phenomenon was quite dramatic and occurred in virtually every mating test following each mount or intromission. The vocalization pause following intromissions was of significantly greater length than that following mounts. This period of silence was almost always characterized by genital grooming by the male, and usually ended when the male began to show interest and orientation toward the female. Fluctuations in sexual arousal underlie the temporal patterning of male copulation. The period of sexual inactivity (time-out) following intromissions and mount bouts is probably due to a reduction in sexual arousal (Beach, 1956; Sachs & Barfield, 1970). These time-outs may be shortened and copulation accelerated by treatments that tend to increase arousal (Sachs & Barfield, 1976). The 40- to 60-kHz vocalization pause discovered in this study was found to occupy 90-95% of the Interintromission Interval (III) indicating that cessation of vocalization parallels the time-out following an intromission. The fact that vocalizations ceased following intromissions and recommenced immediately prior to a mounting attempt indicates that this period of silence may reflect a state of reduced sexual arousal. Indeed, the short period of sexual inactivity following a mount is reflected in the brief vocalization pause associated with mounting behavior. This supports Beach's (1956) hypothesis that the male's period of inactivity after an intromission is a minor version of the reduction in sexual responsiveness following an ejaculation. Immediately preceding ejaculation, a cluster of 40- to 60-kHz vocalizations occurs, much like the vocalization bouts preceding mounts and intromissions. Substantial vocalization appears to begin up to 30 sec prior to ejaculation, with the largest amount occurring immediately prior to the ejaculatory response (within 10 sec of ejaculation; see Fig. 3). The amount of vocalization prior to an ejaculation was found to be significantly greater than that preceding intromissions. Assuming that sexual arousal is greatest just prior to ejaculation, this finding substantiates the hypothesis that 40- to 60-kHz vocalizations are correlated with arousal levels of the male during copulation. Anisko et al. (1978) also noted that the majority of preejaculatory 22-kHz vocalizations occurred during the period immediately preceding ejaculation, at a time when the male was actively pursuing the female. These authors found that 70% of the 22-kHz preejaculatory calls were associated with the female's avoidance of the male, while 26% were associated directly with female aggression. Brown (1979) also demonstrated that preejaculatory 22-kHz vocalizations were associated with the
ULTRASONIC VOCALIZATION AND MATING
357
aggressiveness of the female and the pursuit of the female by the male. These findings suggest that preejaculatory 22-kHz vocalizations may occur in response to the changing behavior of the female. In light of this, one must consider the possibility that the preejaculatory 40- to 60-kHz vocalizations recorded in this study are also directly influenced by the behavior of the female during the copulatory cycle. Although the functional significance of ;~0- to 60-kHz ultrasonic vocalizations during mating is not entirely clear, the results of the present study suggest that there is a particular and unique temporal relationship between ultrasounds and copulatory events. These vocalizations, like the 22-kHz preejaculatory vocalizations may be indicative of alterations in the motivational state of the male. There is an indication that these 40- to 60-kHz vocalizations may reflect arousal levels, thereby serving to coordinate mating activity. In addition, the broadcasting of arousal levels may, in the natural situation, help maintain a proximity between male and female. Although the precise communicatory function of these 40- to 60-kHz ultrasounds during mating is presently unknown, these results strongly suggest that 40- to 60-kHz ultrasonic vocalizations are an important and integral part of the copulatory repertoire during the mating sequence. REFERENCES Anisko, J., Suer, S., McClintock, M., & Adler, N. (1978). Relation between 22 kHz ultrasonic signals and sociosexual behavior in rats. Journal of Comparative and Physiological Psychology, 92, 824-829. Barfield, R., & Geyer, L. (1972). Sexual behavior: ultrasonic postejaculatory song of the male rat. Science, 176, 1349-1350. Barfield, R., & Geyer, L. (1975). The ultrasonic postejaculatory vocalization and the postejaculatory refractory period of the male rat. Journal of Comparative and Physiological Psychology, 88, 723-734. Beach, F. (1956). Characteristics of masculine "sex drive." In M. R. Jones (Ed.), The Nebraska Symposium of Motivation, pp. 1-32. Lincoln, Nebr.: Univ. of Nebraska Press. Beach, F., & Holz-Tucker, A. (1949). Effects of different concentrations of androgens upon sexual behavior in the castrated male rat. Journal of Comparative and Physiological Psychology, 42, 433-453. Bell, R. (1974). Ultrasounds in small rodents: Arousal-produced and arousal-producing. Developmental Psychobiology, 7, 39-42. Brown, R. (1979). The 22 kHz pre-ejaculatory vocalizations of the male rat. Physiology and Behavior, 22~ 483-489. Floody, O., & Pfaff, D. (1977). Communication among hamsters by high-frequency acoustic signals. III. Responses evoked by natural and synthetic ultrasounds. Journal of Comparative and Physiological Psychology, 91, 820-829. Geyer, L. (1976). Functional Aspects of Ultrasonic Vocalizations Associated with Copulation in Rats. Unpublished doctoral dissertation, Rutgers--The State University. Geyer, L., Mclntosh, T., & Barfield, R. (1978). Effects of ultrasonic vocalizations and male's urine on female rat readiness to mate. Journal of Comparative and Physiological Psychology, 92, 457-462.
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McIntosh, T., Barfield, R., & Geyer, L. (1978). Ultrasonic vocalizations facilitate sexual behaviour of female rats. Nature (London), 272, 163-164. Sachs, B., & Barfield, R. (1976). Functional analysis of masculine copulatory behavior in the rat. In J. Rosenblatt, R. Hinde, E. Shaw, and C. Beer (Eds.), Advances in the Study of Behavior, Vol. III. New York: Academic Press. Sachs, B., & Barfield, R. (1970). Temporal patterning of sexual behavior in the male rat. Journal of Comparative and Physiological Psychology, 73, 359-364. Sales (nee Sewell), G. (1972). Ultrasound and aggressive behaviour in rats and other small mammals. Animal Behaviour, 20, 88-100. (a) Sales, G. (1972). Ultrasound and mating behaviour in rodents with some observations on other behavioural situations. Journal of Zoology, 168, 149-164. (b)
The Temporal Patterning of 40-60 kHz Ultrasonic Vocalizations and Copulation in the Rat
(Rattus norvegicus) TRACY K . MCINTOSH AND RONALD J. BARFIELD 1
Department of Biology, Livingston College, Rutgers University, New Brunswick, New Jersey 08904 Ultrasonic vocalizations are associated with copulatory behavior in the rat; however, the precise temporal relationship of these vocalizations to mating events is not known. In this study, 40-60 kHz ultrasonic vocalizations were recorded during sexual behavior and related to the periodicity of events that occur during mating. The occurrence of these ultrasounds was correlated with indices of sexual motivation. Ultrasonic vocalizations serve both to reflect and affect the arousal levels of the mating pair. The patterning of vocalizations suggests that these sounds play an important role in the coordination of male and female activity thereby facilitating the integration of mating behavior of the species.
In the course of copulation, both male and female rats emit ultrasonic vocalizations with the main energy around 40-60 kHz (Sales, 1972b). High rates of vocalization seem to be associated with hormonal and behavioral readiness of both the male and female to copulate (Geyer, McIntosh, & Barfield, 1978). Recent studies indicate that these vocalizations serve a communicatory function during copulation. Specifically, female rats previously exposed to 50-kHz vocalizations show increased solicitation behavior (darting and hopping) when subsequently placed with a male (Geyer et al., 1978); and, additionally, exposure to these vocalizations resulted in a dramatic increase in the proceptive behavior of female rats in the presence of a castrated male (Mclntosh, Barfield, & Geyer, 1978). After ejaculating, the male rat enters a refractory period. This refractory period is characterized by two phases. First, there is an absolute refractory period during which the male is incapable of reinitiating sexual 1 This research was supported by a National Institute of Child Health and Human Development Research Grant HD 04484 to Ronald J. Barfield. Tracy K. Mclntosh was a predoctoral trainee on a National Institute of Mental Health Interdisciplinary Training Grant MH 13006-05. Hormones used in this study were generously donated by Schering Corporation, Bloomfield, N.J. We would like to thank Dr. Benjamin Sachs and Dr. Norman Adler for their helpful comments on the manuscript. 349 0163-1 047/80/070349-10502.00/0 Copyright© 1980by AcademicPress, Inc. All rights of reproductionin any form reserved.
350
MCINTOSH AND BARFIELD
activity. Following this, there is a relative refractory period in which copulatory behavior may be resumed in response to a supranormal arousing stimulus (Beach & Holz-Tucker, 1949). During the postejaculatory period, the male rat emits 22-kHz vocalizations for a period which appears to conform directly to the length of the absolute refractory period (Barfield & Geyer, 1972, 1975). Recently, Anisko, Suer, McClintock, and Adler (1978) found that preejaculatory 22-kHz ultrasonic vocalizations occur during the copulatory sequence. These authors related the occurrence of these preejaculatory 22-kHz calls to specific copulatory events. Brown (1979) has suggested that these preejaculatory 22-kHz vocalizations may function to inhibit female agonistic behaviors during copulation, thereby facilitating sexual responsiveness. Ultrasonic vocalizations of 40 to 60 kHz are also associated with a variety of activities that occur during the copulatory cycle. We have no information, however, on the precise relationship of these ultrasonic vocalizations to the specific sequential events during mating. The purpose of this study was to examine the 40- to 60-kHz ultrasonic vocalizations produced by rats as they occur in a temporal relationship to specific events in the copulatory cycle. METHODS Forty sexually experienced male Long-Evans hooded rats were selected as subjects and caged singly under reversed lighting conditions (dark phase; 9:30-21:30). The animals were housed in individual galvanized steel- and wire-mesh cages measuring 30 × 18 × 18 cm. Food (Purina Rat Chow) and water were available ad libitum. All subjects were screened for sexual behavior two weeks before behavioral testing and were thus accustomed to the testing situation. All tests were performed in glass-walled aquaria (40 × 26 × 29 cm) during the dark phase under red illumination. The male was allowed to adapt to the test chamber for at least 5 min prior to the introduction of a receptive female (the "Introductory period"). The females were ovariectomized Long-Evans rats injected with 50/zg of estradiol benzoate 54 hr before the test and 500/xg of progesterone 6 hr before testing. Each male was tested for sexual behavior with a receptive female. All tests were terminated after 20 min if no copulatory activity occurred following the introduction of the female. The following behavioral events were scored manually on a push-button activated Rustrak event recorder: (1) mounts, intromissions, and ejaculations by the male; (2) the occurrence of 40- to 60-kHz vocalizations during and following mating; (3) 22-kHz postejaculatory vocalization; (4) darting and hopping by the female. The following measures were derived from the raw data: Intromission Frequency (IF, the number of intromissions prior to ejaculation);
351
ULTRASONIC VOCALIZATION AND MATING
Mount Latency (ML, time from the introduction of the female to the first mount); Intromission Latency (IL, time from the introduction of the female to the first intromission); Ejaculation Latency (EL, time from the first intromission to ejaculation); Intercopulatory Interval (ICI, a measure of copulatory rate derived from dividing EL by the total number of intromissions); Vocalization Latency (VL, time from ejaculation to the beginning of the 22-kHz vocalization); Vocalization Termination (VT, time from ejaculation until the end of the 22-kHz vocalization); Postejaculatory Interval (PEI, time from ejaculation until the first intromission of the next copulatory series). An ultrasonic microphone fixed at the top of the testing chamber and connected to a Holgate ultrasonic receiver was used to monitor the vocalizations. The ultrasonic receiver was tuned to monitor only the 40to 60-kHz vocalizations that occur during mating. The sensitivity of this instrument allows for fine discriminative tuning thereby precluding the possibility that any other frequency would be monitored. An oscilloscope was connected in parallel with the ultrasonic receiver for further ultrasound verification. RESULTS
Copulation to ejaculation occurred in 28 of the 40 tests. As seen in Table 1, during the 5-min introductory period, vocalization rates in tests that resulted in ejaculation were not significantly different from those tests in which ejaculation was not reached. Following the introduction of the female into the test cage, however, the successful copulators had significantly shorter latencies to vocalize than did the nonejaculators (t test; t (38) = 5.57; p < .001). On the other hand, in the first 20 min of behavioral testing, significantly more vocalizations were observed in the tests inTABLE 1 N u m b e r o f Vocalizations Occurring in the Introductory Period and during Copulation
Vocalizations in introductory period
L a t e n c y to vocalize following introduction of the female (sec)
No. o f vocalizations during mating ( l s t 20 min)
T e s t s resulting in ejaculation ( N = 28, m e a n +- SEM)
46.8 --- 7.8
4.9 ± 1.1
95.5 ± 16.1
T e s t s without ejaculation ( N = 12, m e a n ± SEM)
53.4 ± 11.6
27.3 ± 9.5
301.9 ± 43.9
p*
N.S.
< .001
< .001
* S t u d e n t ' s t test.
352
MCINTOSH AND BARFIELD 300.
200' :E
I00.
•e
• • i
o|e ~ o , 200
|
i
i 400
j
| 600
EL
(SEC)
| O0
I000
FIG. I. Correlation between the number of 40- to 60-kHz vocalizations produced during mating and the ejaculation latencies (EL) for all tests resulting in ejaculation ( N = 28) (correlation coefficient, r = .58, p < .01).
volving nonejaculators than in the tests involving active maters (t test; t (38) = 5.80; p < .001). Rates of vocalization during copulation appear to inversely reflect sexual arousal during the copulatory sequence. Figure 1 shows that there is a positive correlation between latencies to ejaculation and the number of vocalizations produced during the 28 mating tests (correlation coefficient, r = .58; p < .01). An argument may be made that the slower maters vocalized more during mating because they had a longer time period in which to produce the vocalizations. An analysis of the first 10 rain of each mating test, however, demonstrated that there was an even greater positive correlation between EL and the number of vocalizations during the first 10 min of copulation (correlation coefficient, r = .61; p < .01). Furthermore, ICIs (a measure of copulatory rate) were found to be positively correlated with the number of vocalizations produced during mating (Fig. 2) (correlation coefficient, r = .60; p < .01). These data taken 200-
$
•
IO0Z
e
....: Icl (sic)
Correlation between the number of 40- to 60-kHz vocalizations produced during mating and the intercopulatory interval (ICI) for all tests resulting in ejaculation ( N = 28) (correlation coefficient, r, = .59, p < .01). FIG. 2.
ULTRASONIC VOCALIZATION AND MATING
353
TABLE 2 O c c u r r e n c e of 50-kHz Vocalizations prior to and following M o u n t s and Intromissions Behaviouralevent Mount
In~i'omission
p*
M e a n No. of vocalizations (+_ SEM) in 10 sec before
1.6 --- 0.4
4.8 _+ 0.6
< .001
M e a n time (+- SEM) without vocalizations after (sec)
5.8 -+ 1.1
28.5 _+ 4.1
< .001
Note. N u m b e r of tests = 28. * t T e s t for related m e a s u r e s .
together indicate that the faster maters vocalized significantly less than the slower copulators during mating. Immediately prior to mounts or intromissions, 40- to 60-kHz vocalizations occur in bursts or clusters. The results shown in Table 2 demonstrate that the rate of vocalizations in 10 sec preceding intromissions is significantly greater than that preceding mounts (t test for related measures; t (27) = 4.25; p < .001). A unique and noticeable period of silence often followed each mount or intromission during mating. Table 2 also demonstrates that the length of this period was significantly greater following intromissions than following mounts (t test; t (25) = 5.97; p < .001), occupying approximately 95% of the Interintromission Interval. Figure 3 indicates a distinct clustering of vocalizations immediately preceding ejaculations. A significantly greater amount of 40- to 60-kHz vocalizations occurred within the 10-sec period immediately preceding ejaculation than during a period 20 or 30 sec prior to ejaculation (one-way ANOVA; F (1.54) = 20.98;p < .001). In addition, the amount ofvocaliza-
~P
!
30~20 TIME
20-10
i
10" |JAC
(SEC)
FIG. 3. T h e n u m b e r of 40- to 60-kHz vocalizations occurring in s u c c e s s i v e 10-sec intervals beginning 30 sec prior to ejaculation. Bars indicate S E M (N = 28).
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tion produced immediately prior to an ejaculation was found to be significantly greater than the amount of vocalization preceding intromissions (t test; t (27) = 5.55; p < .001). Although it is true that both male and female rats emit 40- to 60-kHz vocalizations during mating, it seems clear that the majority of vocalizations recorded during copulation in this study were produced by the male. Evidence supporting this comes from the fact that: (1) the vocalization patterns were directly correlated with the mounting patterns of the male (i.e., bursts of vocalizations occurred consistently during the approach preceding both mounts and intromissions), and (2) preliminary testing in our laboratory utilizing devocalized females indicates that a temporal patterning of vocalization, similar to that found in the present experiment, occurs when a normal male copulates with a devocalized female (Mclntosh & Thomas, unpublished).
DISCUSSION It is well known that 40- to 60-kHz ultrasonic vocalizations are associated with mating behavior in a variety of rodents (Sales, 1972a, 1972b). The results from this study demonstrate for the first time that these vocalizations have a precise temporal relationship to the events that occur during mating. Vocalization rates were compared between maters and nonmaters during the introductory period and during the first 20 min of the copulatory test. Results indicated that shorter latencies to vocalize following the introduction of the female occurred in those tests resulting in ejaculation. Thus, vocalizations in the early phase of the copulatory cycle appear to be associated with higher levels of sexual arousal. This is consistent with the hypothesis that ultrasonic vocalizations are reflective of the state of arousal of an animal (Bell, 1974). These data also agree with the findings of Geyer (1976) who discovered that ultrasonic emission rates were highest at the initiation of mating and that high rates of vocalizations were correlated with the sexual readiness of the male. The results relating the amount of 40- to 60-kHz vocalizations to arousal levels during the 28 mating tests (as measured by EL and ICI) reveal that the fast maters emitted significantly fewer vocalizations during the course of mating than did the slower maters. It may be argued that the slower maters were allowed a greater period of time to vocalize; however, analysis of the first 5 and 10 min of each mating test still demonstrated a significantly higher rate of 40- to 60-kHz vocalizations for the slower maters. This is a curious finding; if one assumes that the fast maters are more sexually aroused, it would be expected that more vocalizations per unit time would be produced by those animals exhibiting short ejaculation latencies. There are several factors which may account for the paradoxical relationship between vocalizations and copulation: (1) It is possible that high
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levels of precopulatory vocalizations are associated with both sexual arousal and the facilitation of male-female investigatory responses, but that once copulation has begun, fewer vocalizations are necessary to maintain sexual excitation between the "aroused" maters and the female. It has been shown, in fact, that high levels ofprecopulatory vocalizations facilitate female solicitation behavior in both the rat (Geyer et al., 1978; Mclntosh et al., 1978) and hamster (Floody & Pfaff, 1977). (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 these tests. Indeed, female solicitation rates (amount of darting/unit time) were found to be much lower in those tests involving the slower maters. The possibility exists then, that the behavior of the female is directly influencing ultrasonic vocalization production by the male. The increase in 40- to 60-kHz vocalizations in those tests involving slower maters might therefore reflect increased arousal levels in sexually excited animals in response to an uncooperative mating partner. This suggests that sexual "frustration" (also a type of arousal) may be one cause of high levels of vocalization sometimes seen during mating. This hypothesis is supported by the fact that male rats deprived of their mating partner will vocalize at very high rates (Mclntosh & Barfield, unpublished) and male rats separated from females by a wire-mesh divider will also show consistently high levels of vocalizations (Geyer et al., 1978). Interestingly, Brown (1979) found that precopulatory 22-kHz vocalizations occurred primarily when the male subjects were mounting the females more often without achieving intromission. These preejaculatory 22-kHz calls were also found to be associated with low lordosis intensity and high aggressive behavior of the female. Quite possibly a frustration-type of sexual arousal is responsible for the preejaculatory 22-kHz calls observed by Brown as well as the 40- to 60-kHz calls observed in the present study in those tests involving slower maters. During the copulatory sequence, it was observed that a burst of 40- to 60-kHz vocalization would immediately precede most mounts or intromissions. This clustering of vocalizations which we have termed a "vocalization bout" was such a novel and intriguing finding that we wondered whether they might serve an important function during mating. Analysis revealed that the amount of vocalizing was significantly greater just prior to an intromission than just prior to a mount. These vocalization bouts may serve to alert the female as to the male's state of sexual arousal, or to enhance the probability or intensity of lordosis by the female. Floody and Pfaff (1977) found that in hamsters, estrous females would maintain the lordosis posture significantly longer in the presence of recorded ultrasounds than in silence. Additionally, we have found that, in some instances, female rats will show lordosis behavior to ultrasounds with minimal or no physical contact from a male (Mclntosh et al., 1978).
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Obviously much more detailed analysis of the problem is needed before any firm conclusions can be drawn concerning the communicatory function of these 40- to 60-kHz vocalization bouts. Immediately following a mount or intromission, there is also a distinct period of time in which n o 40- to 60-kHz vocalizations are emitted by either animal. This phenomenon was quite dramatic and occurred in virtually every mating test following each mount or intromission. The vocalization pause following intromissions was of significantly greater length than that following mounts. This period of silence was almost always characterized by genital grooming by the male, and usually ended when the male began to show interest and orientation toward the female. Fluctuations in sexual arousal underlie the temporal patterning of male copulation. The period of sexual inactivity (time-out) following intromissions and mount bouts is probably due to a reduction in sexual arousal (Beach, 1956; Sachs & Barfield, 1970). These time-outs may be shortened and copulation accelerated by treatments that tend to increase arousal (Sachs & Barfield, 1976). The 40- to 60-kHz vocalization pause discovered in this study was found to occupy 90-95% of the Interintromission Interval (III) indicating that cessation of vocalization parallels the time-out following an intromission. The fact that vocalizations ceased following intromissions and recommenced immediately prior to a mounting attempt indicates that this period of silence may reflect a state of reduced sexual arousal. Indeed, the short period of sexual inactivity following a mount is reflected in the brief vocalization pause associated with mounting behavior. This supports Beach's (1956) hypothesis that the male's period of inactivity after an intromission is a minor version of the reduction in sexual responsiveness following an ejaculation. Immediately preceding ejaculation, a cluster of 40- to 60-kHz vocalizations occurs, much like the vocalization bouts preceding mounts and intromissions. Substantial vocalization appears to begin up to 30 sec prior to ejaculation, with the largest amount occurring immediately prior to the ejaculatory response (within 10 sec of ejaculation; see Fig. 3). The amount of vocalization prior to an ejaculation was found to be significantly greater than that preceding intromissions. Assuming that sexual arousal is greatest just prior to ejaculation, this finding substantiates the hypothesis that 40- to 60-kHz vocalizations are correlated with arousal levels of the male during copulation. Anisko et al. (1978) also noted that the majority of preejaculatory 22-kHz vocalizations occurred during the period immediately preceding ejaculation, at a time when the male was actively pursuing the female. These authors found that 70% of the 22-kHz preejaculatory calls were associated with the female's avoidance of the male, while 26% were associated directly with female aggression. Brown (1979) also demonstrated that preejaculatory 22-kHz vocalizations were associated with the
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aggressiveness of the female and the pursuit of the female by the male. These findings suggest that preejaculatory 22-kHz vocalizations may occur in response to the changing behavior of the female. In light of this, one must consider the possibility that the preejaculatory 40- to 60-kHz vocalizations recorded in this study are also directly influenced by the behavior of the female during the copulatory cycle. Although the functional significance of ;~0- to 60-kHz ultrasonic vocalizations during mating is not entirely clear, the results of the present study suggest that there is a particular and unique temporal relationship between ultrasounds and copulatory events. These vocalizations, like the 22-kHz preejaculatory vocalizations may be indicative of alterations in the motivational state of the male. There is an indication that these 40- to 60-kHz vocalizations may reflect arousal levels, thereby serving to coordinate mating activity. In addition, the broadcasting of arousal levels may, in the natural situation, help maintain a proximity between male and female. Although the precise communicatory function of these 40- to 60-kHz ultrasounds during mating is presently unknown, these results strongly suggest that 40- to 60-kHz ultrasonic vocalizations are an important and integral part of the copulatory repertoire during the mating sequence. REFERENCES Anisko, J., Suer, S., McClintock, M., & Adler, N. (1978). Relation between 22 kHz ultrasonic signals and sociosexual behavior in rats. Journal of Comparative and Physiological Psychology, 92, 824-829. Barfield, R., & Geyer, L. (1972). Sexual behavior: ultrasonic postejaculatory song of the male rat. Science, 176, 1349-1350. Barfield, R., & Geyer, L. (1975). The ultrasonic postejaculatory vocalization and the postejaculatory refractory period of the male rat. Journal of Comparative and Physiological Psychology, 88, 723-734. Beach, F. (1956). Characteristics of masculine "sex drive." In M. R. Jones (Ed.), The Nebraska Symposium of Motivation, pp. 1-32. Lincoln, Nebr.: Univ. of Nebraska Press. Beach, F., & Holz-Tucker, A. (1949). Effects of different concentrations of androgens upon sexual behavior in the castrated male rat. Journal of Comparative and Physiological Psychology, 42, 433-453. Bell, R. (1974). Ultrasounds in small rodents: Arousal-produced and arousal-producing. Developmental Psychobiology, 7, 39-42. Brown, R. (1979). The 22 kHz pre-ejaculatory vocalizations of the male rat. Physiology and Behavior, 22~ 483-489. Floody, O., & Pfaff, D. (1977). Communication among hamsters by high-frequency acoustic signals. III. Responses evoked by natural and synthetic ultrasounds. Journal of Comparative and Physiological Psychology, 91, 820-829. Geyer, L. (1976). Functional Aspects of Ultrasonic Vocalizations Associated with Copulation in Rats. Unpublished doctoral dissertation, Rutgers--The State University. Geyer, L., Mclntosh, T., & Barfield, R. (1978). Effects of ultrasonic vocalizations and male's urine on female rat readiness to mate. Journal of Comparative and Physiological Psychology, 92, 457-462.
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McIntosh, T., Barfield, R., & Geyer, L. (1978). Ultrasonic vocalizations facilitate sexual behaviour of female rats. Nature (London), 272, 163-164. Sachs, B., & Barfield, R. (1976). Functional analysis of masculine copulatory behavior in the rat. In J. Rosenblatt, R. Hinde, E. Shaw, and C. Beer (Eds.), Advances in the Study of Behavior, Vol. III. New York: Academic Press. Sachs, B., & Barfield, R. (1970). Temporal patterning of sexual behavior in the male rat. Journal of Comparative and Physiological Psychology, 73, 359-364. Sales (nee Sewell), G. (1972). Ultrasound and aggressive behaviour in rats and other small mammals. Animal Behaviour, 20, 88-100. (a) Sales, G. (1972). Ultrasound and mating behaviour in rodents with some observations on other behavioural situations. Journal of Zoology, 168, 149-164. (b)