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Stability of chemical communicants of gender in guinea pig urine
BEHAVIORAL AND NEURAL BIOLOGY
32~ 364-375 (1981)
Stability of Chemical Communicants of Gender in Guinea Pig Urine 1 JUDITH L. WELLINGTON Monell Chemical Senses Center, Philadelphia, Pennsylvania 19104
GARY K. BEAUCHAMP Monell Chemical Senses Center and Department of Otorhinolaryngology and Human Communication, University of Pennsylvania, Philadelphia, Pennsylvania 19104 AND
AMOS B. SMITH,III Monell Chemical Senses Center and Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Male guinea pigs tested in a two-choice preference test spent significantly more time with female than male urine after the urines had b e e n aged up to 40 days. When the animals were not able to contact the urine samples the investigation times were greatly attenuated. These results support the hypothesis that the chemical signals of gender found in guinea pig urine are of low volatility. They further indicate that signals persist for remarkably long periods, at least under laboratory conditions.
Chemical signals serve to transmit a variety of messages among conspecific organisms. Based primarily on examples from insects, Wilson and Bossert's theory (1963) argued that fade time for a message, the amount of time between when the chemical is released and when it no longer is sufficiently strong to serve its function, should be related to the type of message transmitted. For example, alarm substances and trail substances would be expected to dissipate more quickly than sexual attractants. For mammals, similar arguments are intuitively appealing (Johnston i This research was supported by NSF Grants BNS 76-01642 and BNS 79-06234. A. B. Smith, III, is a Camille and Henry Dreyfus Teacher Scholar, 1978-1983, and recipient of a National Health Career Development Award, 1980-1985. The authors thank B. R. Criss, C. Wojciechowski, and M. Bobman for excellent technical assistance. 364 0163 - 1047/81/070364-12502.00/0 Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
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& Schmidt, 1979). The persistence of odor trails followed by dogs has often been noted (McCartney, 1968). Experimental data for the fade time for chemical messages between mammals have only recently begun to accumulate. Mtiller-Velten (1966) reported that the fear scent of house mice fades within 24 hr. Lydell and Doty (1972) found that the preference of male rats for estrous female urine relative to anestrous female urine disappeared after 24 hr of storage. The lack of persistence of the information is reasonable in these cases given the presumed functional significance of the signals. Other studies are more difficult to interpret. Johnston (1974) reported that the attraction of male hamsters to female vaginal secretion collected on cotton swabs was greatly reduced when the secretion had been allowed to stand for 12 hr prior to testing. In these tests the hamsters were not permitted to contact the secretion. Subsequently, Johnston and Lee (1976) and Johnston and Schmidt (1979) found that hamster vaginal secretions which had been placed on a substrate by the animals retained their attractiveness, relative to no odor controls, for at least 100 days. Flank marks were attractive for 40 days. In these experiments, the animals were permitted to contact and lick as well as sniff the aged secretion. Johnston and Schmidt also found that in two-choice tests, male hamsters responded more strongly to fresh flank and vaginal marks than to 1-day-old marks. The animals did not discriminate 1-day-old marks from those 10 days old. Several recent studies in which urine has been used also indicate a persistence of information in aged stimuli. Evans, Mackintosh, Kennedy, and Robertson (1978) reported that the aggression-reducing signal in the urine of female mice persists for at least a week; older samples were not tested. Female mouse urine elicits ultrasounds from male mice (Nyby & Zakeski, 1980) for at least 30 days. Again no outside limit was determined. The adaptive significance of such persistence of information is unclear. Two recent reports have not documented persistence of odors. Jones and Nowell (1977) determined that the aversive component in male mouse urine was absent when urine was aged 3 days or more. Similarly, Epple, Alveario, Golob, and Smith (1980) found that saddle-back tamarins distinguished male scent marks from those of females when the marks were fresh, 1- or 2-day-old but not 3- or 4-day-old marks. Tamarins also discriminated fresh marks from 1-day-old marks in choice tests. The published data on the persistence of messages found in mammalian secretions cannot be easily fitted into the ecological framework suggested by Wilson and Bossert (1963). This is due in part to the fact that the exact functional significances of many mammalian chemical messages are unclear since, in general, only sniffing or investigation times spent with a sample are measured and compared to times spent with appropriate
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WELLINGTON, BEAUCHAMP, AND SMITH
control substances. However, one can often infer from such assays what information is transmitted by a sample. For example, if material from individuals of class A is preferred to or discriminated from material from individuals of class B, then one can conclude that information differentiating these classes is contained in the material. One then can ask w h e t h e r animals respond differently to aged samples from the two classes. Thus the persistence of the information can be determined while the function(s) may remain unknown. A variety of types of information is available in guinea pig (Cavia sp.) urine. For example, we found that fresh anestrous female guinea pig urine is preferred to fresh male urine or to water in two-choice preference tests (Beauchamp, 1973) and single-sample tests (Beauchamp & BeriJter, 1973). Males also spend more time with fresh urine from a strange intact male than with their own (Beauchamp, 1973). Thus, information on gender and individual identity is available in urine. In an earlier study, we reported that female guinea pig urine was no longer more attractive than male urine or water after the female urine had aged at room temperature in open vials for 48 hr or more (Beauchamp & Ber~ter, 1973). We concluded that information was lost over this aging period. However, other behavioral observations suggest a longer activity for urinary signals. Male and female guinea pigs often deposit a small volume of urine on the ground when they mark; urine is also sprayed on other individuals or substrates (Beauchamp, Wellington, Wysocki, Brand, Kubie, & Smith, 1980; Rood, 1972). Observations of animals in social groups suggested that urine marks that were several days old elicited persistent investigation. We therefore instituted a series of experiments to explore the response of male guinea pigs to urine samples collected from several classes of donors and aged for varying lengths of time.
GENERAL METHOD The subjects were sexually mature, male domestic guinea pigs (Cavia porcellus) bred at the Monell Center. The animals were weaned and housed individually at 3 weeks of age. They were maintained on commercial guinea pig chow and water ad libitum. Urine was collected from four adult females and four adult males housed in wire-bottom cages by placing a clean sheet of aluminum foil beneath the donor's cage. Urine that was free from fecal or food contamination was collected every 2-6 hr, pooled according to sex, and frozen. Previous work indicated that defrosted urine was not differentiated from fresh urine (Beauchamp, unpublished). Estrous state was not determined for female donors since previous studies indicated that males do not discriminate estrous from non-estrous female urine (Beauchamp & Ber~iter, 1973). Animals were tested in their home cages (55 × 50 x 35 cm high). For single sample
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tests (Experiment 3a) a sample placed on a single clean glass plate was presented to each test animal. For two-choice tests (all other experiments) two plates were presented simultaneously, 10 cm apart, to each test animal. In all tests, general behavioral observations as well as the amount of time the animal spent with his nose within 1.0 cm of the sample during the 2.0-min (single-sample) or 4.0-min (two-choice) tests were recorded. The observer was not aware of the predicted results. EXPERIMENT 1
Method In this experiment, discrimination between male and female urine samples that had been dried and aged for various lengths of time were examined. Urine was placed on clean glass test plates and was allowed to dry. The plates were stored in an air-conditioned room (65°-75°F, approximately 50% humidity) for times ranging from 5 hr to 92 days. Ten male subjects were given a series of two-choice tests comparing male to female urine stored for equal amounts of time (e.g., female urine aged I day was compared to male urine aged 1 day). The tests were conducted sequentially; the first test compared urines aged 5 hr, the second 24 hr, the third 3 days, and so forth. At least 48 hr separated each test. The times spent investigating the female and male sample for each length of aging were compared using separate one-tailed t tests since the tests were not counterbalanced and previous experiments (Beauchamp, 1973) allowed us to predict that female urine should be preferred to male urine. Results and Discussion Males spent significantly more time (p < .05) investigating female urine than male urine when the urines were aged up to 40 days (Fig. 1). Males 70
A
[]OO
~ 20
8 5 hr.
24 hr.
3 da.
da.
14 da,
22 da.
40 da.
61 da.
92 da.
Aged
FIG. 1. Responses of male guinea pigs to choices between male and female urines which had been aged on glass plates for times ranging from 5 hr to 92 days.
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WELLINGTON, BEAUCHAMP, AND SMITH
did not show a significant preference for female urine as compared to male urine when the samples were aged 61 or 92 days. Since previous experiments (Beauchamp, 1973) have demonstrated that males continue to prefer female to male urine after many repeated tests, this lack of preference is not due to loss of interest in the samples. The results of Experiment 1 are in agreement with recent work with several other species indicating that information in chemical signals is, in some cases, remarkably persistent (see citations under introduction). However, these data contrast with those we previously reported, which indicated that when urine was stored in open vials for 48 hr or longer female urine was no longer more attractive to adult male guinea pigs than was male urine or water (Beauchamp & Bertiter, 1973). We hypothesized three possible reasons for these differing results: (1) differences in methods of storing urine (dry vs wet); (2) differences in methods of presenting urine (dry vs wet); and (3) differences in methods of testing (single-sample presentation vs choice test). In the present experiments urine was aged and tested in a dry state, whereas in the earlier published study, it was aged and tested as an aqueous solution. Perhaps bacterial action degraded the attractive components and/or produced volatile components which masked the attractive components. Dry storage should depress bacterial activity yielding a urine sample that would be more attractive than that stored wet. Additionally, during the drying process, volatile masking compounds would evaporate more rapidly from the urine, which could result in aged dry urine being more attractive than aged wet urine. In the following experiment (2), the first two of the three hypotheses were examined. Urine was aged for approximately 20 to 40 days either wet or dry and then tested in either a wet or dry state. EXPERIMENT 2
In the first of three phases of this experiment (2a), we tested responses of male guinea pigs to choices between water and female urine aged either wet or dry for approximately 20 days. The second phase (2b) was similar except that the choice involved aged female urine vs aged male urine. In the third phase (2c) comparisons were made between female urine aged wet and female urine aged dry. The urines were either both wet or both dry at the time of the test. Method 2a. Female urine was collected and pooled. A 0.1-ml sample of urine was pipetted onto each of 40 plates and stored at room temperature. A 9-ml sample of urine was placed into a vial and stored at room temperature. After 19 days the sample stored in the vial was divided into four equal portions.
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On each test day, 10 samples of urine stored on plates were tested. Half of these were reconstituted with 0.1 ml deionized water before testing. Ten samples of urine stored in vials were also prepared. First the urine was diluted to its original volume (2.25 ml) with deionized water. Then ten 0.1-ml samples were pipetted onto glass plates. Of these, half were allowed to dry for 3 hr before testing while the rest were tested immediately (wet). This resulted in four sample types of female urine: stored dry, tested dry; stored dry, tested wet; stored wet, tested dry; and stored wet, tested wet. Twenty male guinea pigs were tested in a two-choice test comparing aged urine to water. The four sample pairs were presented in a counterbalanced fashion, 19, 21, 24, and 26 days after aging began. The data were subjected to a three-way analysis of variance with repeated measures. 2b. Urine from adult male and female guinea pigs was collected and pooled by sex. Half of the urine for each sex was frozen and stored at - 60°C. The other half was prepared as follows. Twenty 0.1-ml samples of each male and female urine were placed on glass plates to dry. Additionally, 9 ml of male urine and 9 ml of female urine were set out in open vials. After 18 days, frozen samples were thawed and again twenty 0.1-ml samples of male and female urine were placed on glass plates to dry and 9 ml of male and 9 ml of female urine were set out in open vials. Urine stored in vials was diluted to the original volume on Day 39 before testing. On Days 39, 42, 44, and 46 from the beginning of the experiment, 20 adult males were presented with two-choice tests. Again, four urine treatments (aged dry or wet, stored 20 or 40 days) were presented in a counterbalanced fashion over the 4 test days. However, in these tests, female urine was compared to male urine treated in the same way. The data were subjected to a three-way analysis of variance with repeated measures. 2c. Two 12-ml samples of pooled adult female urine were placed in open vials and stored at room temperature. Additional urine was pipetted onto 38 glass plates with a 100-~1 pipet. After 19 days, one of the samples stored in vials was diluted to 12 ml, placed on glass plates, and tested immediately. Nineteen of the urine samples that had been stored dry were dissolved in 0.1 ml of water each. Nineteen male guinea pigs were given a direct two-choice comparison of female urine that had been stored dry vs female urine that had been stored wet. Two days later, the same animals were given the same two-choice comparison test. This time both urines were dry for testing. The data were analyzed with separate t tests. Results and Discussion Female urine was preferred to water and to male urine when the urines had been stored for 20 days, regardless of how the urine was stored (wet
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WELLINGTON, BEAUCHAMP, AND SMITH 701
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Fze. 2. Responses of male guinea pigs to choices between water and female urine which had been aged for 20 days. For aging, urine was stored in vials (wet) or on glass plates (dry).
or dry) or presented (wet or dry). In Experiment 2a (Fig. 2) males spent more time with female urine than they did with water (F(1, 19) = 28.0, p < .001). No other significant effects were found. Similarly, analysis of the results of experiment 2b (Fig. 3) revealed that the male subjects spent more time with female urine than with male urine (F(1, 19) = 8.6, p < .01). This was the only significant effect. In Experiment 2c (Fig. 4) no preference was seen when males were given a choice of urine stored wet and urine stored dry no matter how the urine was presented. These data demonstrate that guinea pig urine aged either dry or wet retains its attractive components for at least 20 days. In addition, males can discriminate female from male urine even when the urines have been aged for 40 days. These data thus supplement those of Experiment 1 in demonstrating the persistence of the active components. It is remarkable that even when aged wet, in which case bacterial activity could be
~;~99 03 40
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Urine
~ ddUrine
Wet Dry 20 days
Wet
DW
40 days
FIG. 3. Responses of male guinea pigs to choices between male and female urines which had been aged 20 and 40 days. For aging, urine was stored in vials (wet) or on glass plates (dry).
STABILITY OF COMMUNICANTS OF GENDER
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6O
~5o kkl ÷1 I
40
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._= -~ 20 r-
8 ffl 10
Stored Tested
Dry Wet Dry
Dry Wet Wet
FIG. 4. Responses of male guinea pigs to 20-day-old female urine when tested in a choice between urine aged dry and urine aged wet.
expected to be most vigorous, female urine still retains its attractiveness to the male guinea pig. These data also eliminate storage (wet vs dry) differences as an explanation for the different findings of Experiment 1 compared to the Beauchamp and Bertiter (1973) study. Experiment 1 shows that urine is still attractive after 40 days whereas Beauchamp and Bertiter reported it to lose attractiveness after 2 days. If storage differences were the explanation we would predict that in Experiment 2c, urine stored dry would be preferred to that stored wet. This was not the case. In the next experiment, we examined the third possible alternative, that the difference is due to use of a single-sample method compared with an apparently more powerful pair-wise comparison as used in the studies reported here. EXPERIMENT 3
Males were given a series of single-sample presentations of water, male urine, fresh female urine, and urine aged 48 to 96 hr. If our previous use of single-sample tests was responsible for the findings of rapid loss of activity, we should have been able to repeat the results and observe a loss (relative to fresh female urine) of responsiveness to the aged urine. In fact, the aged urine should not have been investigated more than water. In this experiment, we also presented a freeze-dried aged urine to the animals. Freeze-drying should remove noxious volatile compounds produced during aging and thus it might be predicted that aged urine that has been freeze-dried would be preferred to aged urine that is untreated. In Experiment 3b, female urine which had been aged for 96 hr was presented in a two-choice test compared with water.
372
WELLINGTON, BEAUCHAMP, AND SMITH
Method 3a. Female urine was collected, pooled, and centrifuged at 5000 g and the supernatant was divided into three equal samples. One sample was further divided, half was frozen for storage at - 60°C and half was freezedried, reconstituted with water, and stored at -60°C. The other two samples were stored at room temperature for 48 and 96 hr, respectively. At the end of room temperature storage, half of the sample was stored at -60°C and the other half was freeze-dried, reconstituted with deionized water, and stored at -60°C. Seventeen males were tested with single-sample presentations of eight stimulus types in a counterbalanced fashion over a period of 2½ weeks, each test separated by 48 to 72 hr. The stimuli included deionized water, male urine, and six types of female urine: (1) fresh; (2) freeze-dried; (3) stored for 48 hr; (4) stored for 48 hr and then freeze-dried; (5) stored for 96 hours; and (6) stored for 96 hr and then freeze-dried. 3b. Female urine was collected, pooled, placed in a vial, and stored at room temperature for 96 hr before testing. Eighteen animals were given a two-choice test of aged urine vs deionized water. Results and Discussion
For the single-sample test (3a) analysis of variance indicated significant differences among the samples (F(7, 112) = 2.79, p < .01, Fig. 5). The Newman-Keuls procedure was used to compare means. These analyses revealed no effect of freeze-drying (p > .05). Males spent significantly more time with fresh urine and urine aged 48 hr than with water (p < .01) but no difference was found in the response to female urine aged 6O
+J ~4o
+
03
>
-~, 20 "0 t-
lO (/3
H~O
d
fd
I
48 hr
fd 9 6 hr fd 48 hr (2 96 hr 0
FIG. 5. Responses of male guinea pigs to single sample presentations of water (H20); male urine (6); female urine (2); freeze-dried female urine (fd 2); female urine aged for 48 hr (48 hr 2); female urine aged for 48 hr and then freeze-dried (fd 48 hr 9); female urine aged 96 hr (96 hr 9); female urine aged for 96 hr and then freeze-dried (fd 96 hr 9).
STABILITY OF COMMUNICANTS OF GENDER
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96 hr compared to water (p > .05). Fresh female urine elicited more investigation than did male urine (p < .01). Fresh urine, 48- and 96-hr aged urines all differed from each other (p < 0.01). Male urine was investigated for a longer period than was water (p < 0.05). The results of Experiment 3b were different than those of 3a. Males spent significantly more time with female urine aged 96 hr (X = 38.2 ___ 7.4 sec) than with water (X = 7.6 +__ 1.9 sec; t(18) = 3.61; p < .01) when the two stimuli were presented simultaneously. These data illustrate the different results obtained by the two testing methods. Use of the single-sample method revealed that by 96 hr, female urine was not investigated any longer than was water or male urine. These results are in general agreement with our earlier report (Beauchamp & BeAter, 1973). Comparison of these results with those of Experiment 3b illustrates the differences obtained when results from simultaneous choice tests are contrasted with results from successive presentation of similar stimuli. In simultaneous two-choice tests finer distinctions are possible presumably because the animal can compare and contrast the two substances very rapidly. Clearly, the method of testing has a profound influence on the results obtained and hence on their interpretation. Experiment 3a also revealed no difference between freeze-dried and untreated aged urine. This negative result must be treated with caution, however, as the single-sample method, as we have illustrated here, is not powerful in revealing differences between samples. EXPERIMENT 4 The previous studies have demonstrated that information on the gender of the urine donor is remarkably persistent. This suggests that the compounds that transmit the information are of low volatility. Experiment 4 was carried out to test the effect of limiting the access of the animals to the sample such that they could not contact the urine. Method Male and female urine was collected, pooled by sex, and frozen until use. Urine was pipetted onto glass test plates and dried for 3 hr. The plates were placed 0.5 cm under a screen for presentation. Nine males were given a two-choice test of male vs female urine. The data were analyzed by a t test. Results and Discussion When the male guinea pigs could not contact urine dried for 3 hr the investigation time was greatly depressed (compared with Experiment 1) and they did not appear to discriminate male from female urine O{ = 7.6 +__ 3.0; /~" ~ = 5.2 _+ 2.0; t(8) = 0.76). In contrast, males
374
WELLINGTON, BEAUCHAMP, AND SMITH
clearly discriminated male from female urine dried 5 hr when contact with the urine was possible (Experiment 1). GENERAL DISCUSSION
The persistence of chemical signals for gender in guinea pig urine is remarkable. Males discriminated male urine from female urine that had been aged for as long as 40 days. Studies using other methodologies could reveal an even longer persistence. For volatile compounds in urine and/or glandular secretions the temporal pattern of release of the compounds is dependent on the surface on which the mark is placed, the relative humidity and the carrier solvent. Regnier and Goodwin (1977) demonstrated good predictability of their experimental results by physiochemical theories of volatility applied to a volatile chemosignal. In contrast, in this study we found that the presence of a solvent (water) during aging had little effect on the persistence of the chemical signals in guinea pig urine. One explanation for this finding is that nonvolatile compounds in the urine are the chemical cues for gender. Our chemical and behavioral studies support this interpretation. In Experiment 4 we found that when males were presented with urine that had been dried for 3 hr and then placed 0.5 cm under a screen that prevented contact with the urine, they did not discriminate male from female, and investigation times were greatly reduced. This is consistent with the hypothesis that the signals are of low volatility. Furthermore, compounds with molecular weights of approximately 800 g/mole appear to be involved in the communication of sexual identity in the guinea pig (Beauchamp et al., 1980). Finally, we have shown that a nonvolatile dye added to urine is taken into the vomeronasal organ of male guinea pigs during investigation thereby providing a potential route for sensory reception of nonvolatile signals (Wysocki, Wellington, & Beauchamp, 1980). The persistence of a signal for long periods such as those reported here as well as those reported for hamsters (Johnston & Schmidt, 1979) and mice (Nyby & Zakeski, 1980; Evans et al., 1978) could be the direct result of evolutionary selection for large, complex compounds to communicate information. Compounds of higher molecular weight and complexity can carry more information than simple small molecules. The persistence of these molecules may be a secondary evolutionary effect. However, the persistence may also have functional implications. It remains to be determined what role such persistence plays under normal conditions. REFERENCES Beauchamp, G. K. (1973). Attraction of male guinea pigs to conspecific urine. Physiology and Behavior, 10, 589-594. Beauchamp, G. K., & Berfiter, J. (1973). Source and stability of attractive components in guinea pig (Cavia porcellus) urine. Behavioral Biology, 9, 43-47.
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Beauchamp, G. K., Wellington, J. L., Wysocki, C. J., Brand, J. G., Kubie, J. L., & Smith, A. B., III. (1980). Chemical communication in the guinea pig: Urinary components of low volatility and their access to the vomeronasal organ. In D. MiillerSchwarze and R. M. Silverstein (Eds.) Chemical Signals: Vertebrates and Aquatic Invertebrates. New York: Plenum. Epple, G., Alveario, M. C., Golob, N. F., & Smith, A. B., III. (1980). Stability and attractiveness related to age of scent marks of saddle-back tamarins (Saguinus fuscicollis). Journal of Chemical Ecology, 6, 735-748. Evans, C. M., Mackintosh, J. H., Kennedy, J. F., & Robertson, S. M. (1978). Attempts to characterize and isolate aggression reducing olfactory signals from the urine of female mice Mus musculus L. Physiology and Behavior, 20, 129-134. Johnston, R. E. (1974). Sexual attraction function of golden hamster vaginal secretion. Behavioral Biology, 12, 111-117. Johnston, R. E., & Lee, N. A. (1976). Persistence of the odor deposited by two functionally distinct scent marking behaviors in golden hamsters. Behavioral Biology, 16, 199-210. Johnston, R. E., & Schmidt, T. (1979). Responses of hamsters to scent marks of different ages. Behavioral and Neural Biology, 26, 64-75. Jones, R. B., & Nowell, N. W. (1977). Aversive potency of male mouse urine: A temporal study. Behavioral Biology, 19, 523-526. Lydell, K., & Doty, R. L. (1972). Male rat odor preferences for female urine as a function of sexual experience, urine age and urine source. Hormones and Behavior, 3, 205-212. McCartney, W. (1968). Olfaction and Odours. Berlin: Springer-Verlag. Mfiller-Velten, H. (1966). lJber den Angstgeruch bei der Hausmaus (Mus musculus L.) Zeitschrift fiir vergleichende Physiologie, 52, 401-429. Nyby, J., & Zakeski, D. (1980). Elicitation of male mouse ultrasounds: Bladder urine and aged urine from females. Physiology and Behavior, 24, 737-740. Regnier, F. E., & Goodwin, M. (1977). On the chemical and environmental modulation of pheromone release from vertebrate scent marks. In D. Miiller-Schwarze & M, M. Mozell (Eds.), Chemical Signals in Vertebrates. New York: Plenum. Rood, J. P. (1972). Ecological and behavioral comparisons of three genera of Argentine cavies. Animal Behavioral Monographs, 5, 3-83. Wilson, E. O., & Bossert, W. H. (1963). Chemical communication among animals. Recent Progress in Hormone Research, 19, 673-710. Wysocki, C. J., Wellington, J. L., & Beauchamp, G. K. 0980). Access of urinary nonvolatiles to the mammalian vomeronasal organ. Science, 207, 781-783.
32~ 364-375 (1981)
Stability of Chemical Communicants of Gender in Guinea Pig Urine 1 JUDITH L. WELLINGTON Monell Chemical Senses Center, Philadelphia, Pennsylvania 19104
GARY K. BEAUCHAMP Monell Chemical Senses Center and Department of Otorhinolaryngology and Human Communication, University of Pennsylvania, Philadelphia, Pennsylvania 19104 AND
AMOS B. SMITH,III Monell Chemical Senses Center and Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Male guinea pigs tested in a two-choice preference test spent significantly more time with female than male urine after the urines had b e e n aged up to 40 days. When the animals were not able to contact the urine samples the investigation times were greatly attenuated. These results support the hypothesis that the chemical signals of gender found in guinea pig urine are of low volatility. They further indicate that signals persist for remarkably long periods, at least under laboratory conditions.
Chemical signals serve to transmit a variety of messages among conspecific organisms. Based primarily on examples from insects, Wilson and Bossert's theory (1963) argued that fade time for a message, the amount of time between when the chemical is released and when it no longer is sufficiently strong to serve its function, should be related to the type of message transmitted. For example, alarm substances and trail substances would be expected to dissipate more quickly than sexual attractants. For mammals, similar arguments are intuitively appealing (Johnston i This research was supported by NSF Grants BNS 76-01642 and BNS 79-06234. A. B. Smith, III, is a Camille and Henry Dreyfus Teacher Scholar, 1978-1983, and recipient of a National Health Career Development Award, 1980-1985. The authors thank B. R. Criss, C. Wojciechowski, and M. Bobman for excellent technical assistance. 364 0163 - 1047/81/070364-12502.00/0 Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
STABILITY OF COMMUNICANTS OF GENDER
365
& Schmidt, 1979). The persistence of odor trails followed by dogs has often been noted (McCartney, 1968). Experimental data for the fade time for chemical messages between mammals have only recently begun to accumulate. Mtiller-Velten (1966) reported that the fear scent of house mice fades within 24 hr. Lydell and Doty (1972) found that the preference of male rats for estrous female urine relative to anestrous female urine disappeared after 24 hr of storage. The lack of persistence of the information is reasonable in these cases given the presumed functional significance of the signals. Other studies are more difficult to interpret. Johnston (1974) reported that the attraction of male hamsters to female vaginal secretion collected on cotton swabs was greatly reduced when the secretion had been allowed to stand for 12 hr prior to testing. In these tests the hamsters were not permitted to contact the secretion. Subsequently, Johnston and Lee (1976) and Johnston and Schmidt (1979) found that hamster vaginal secretions which had been placed on a substrate by the animals retained their attractiveness, relative to no odor controls, for at least 100 days. Flank marks were attractive for 40 days. In these experiments, the animals were permitted to contact and lick as well as sniff the aged secretion. Johnston and Schmidt also found that in two-choice tests, male hamsters responded more strongly to fresh flank and vaginal marks than to 1-day-old marks. The animals did not discriminate 1-day-old marks from those 10 days old. Several recent studies in which urine has been used also indicate a persistence of information in aged stimuli. Evans, Mackintosh, Kennedy, and Robertson (1978) reported that the aggression-reducing signal in the urine of female mice persists for at least a week; older samples were not tested. Female mouse urine elicits ultrasounds from male mice (Nyby & Zakeski, 1980) for at least 30 days. Again no outside limit was determined. The adaptive significance of such persistence of information is unclear. Two recent reports have not documented persistence of odors. Jones and Nowell (1977) determined that the aversive component in male mouse urine was absent when urine was aged 3 days or more. Similarly, Epple, Alveario, Golob, and Smith (1980) found that saddle-back tamarins distinguished male scent marks from those of females when the marks were fresh, 1- or 2-day-old but not 3- or 4-day-old marks. Tamarins also discriminated fresh marks from 1-day-old marks in choice tests. The published data on the persistence of messages found in mammalian secretions cannot be easily fitted into the ecological framework suggested by Wilson and Bossert (1963). This is due in part to the fact that the exact functional significances of many mammalian chemical messages are unclear since, in general, only sniffing or investigation times spent with a sample are measured and compared to times spent with appropriate
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WELLINGTON, BEAUCHAMP, AND SMITH
control substances. However, one can often infer from such assays what information is transmitted by a sample. For example, if material from individuals of class A is preferred to or discriminated from material from individuals of class B, then one can conclude that information differentiating these classes is contained in the material. One then can ask w h e t h e r animals respond differently to aged samples from the two classes. Thus the persistence of the information can be determined while the function(s) may remain unknown. A variety of types of information is available in guinea pig (Cavia sp.) urine. For example, we found that fresh anestrous female guinea pig urine is preferred to fresh male urine or to water in two-choice preference tests (Beauchamp, 1973) and single-sample tests (Beauchamp & BeriJter, 1973). Males also spend more time with fresh urine from a strange intact male than with their own (Beauchamp, 1973). Thus, information on gender and individual identity is available in urine. In an earlier study, we reported that female guinea pig urine was no longer more attractive than male urine or water after the female urine had aged at room temperature in open vials for 48 hr or more (Beauchamp & Ber~ter, 1973). We concluded that information was lost over this aging period. However, other behavioral observations suggest a longer activity for urinary signals. Male and female guinea pigs often deposit a small volume of urine on the ground when they mark; urine is also sprayed on other individuals or substrates (Beauchamp, Wellington, Wysocki, Brand, Kubie, & Smith, 1980; Rood, 1972). Observations of animals in social groups suggested that urine marks that were several days old elicited persistent investigation. We therefore instituted a series of experiments to explore the response of male guinea pigs to urine samples collected from several classes of donors and aged for varying lengths of time.
GENERAL METHOD The subjects were sexually mature, male domestic guinea pigs (Cavia porcellus) bred at the Monell Center. The animals were weaned and housed individually at 3 weeks of age. They were maintained on commercial guinea pig chow and water ad libitum. Urine was collected from four adult females and four adult males housed in wire-bottom cages by placing a clean sheet of aluminum foil beneath the donor's cage. Urine that was free from fecal or food contamination was collected every 2-6 hr, pooled according to sex, and frozen. Previous work indicated that defrosted urine was not differentiated from fresh urine (Beauchamp, unpublished). Estrous state was not determined for female donors since previous studies indicated that males do not discriminate estrous from non-estrous female urine (Beauchamp & Ber~iter, 1973). Animals were tested in their home cages (55 × 50 x 35 cm high). For single sample
STABILITY OF COMMUNICANTS OF GENDER
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tests (Experiment 3a) a sample placed on a single clean glass plate was presented to each test animal. For two-choice tests (all other experiments) two plates were presented simultaneously, 10 cm apart, to each test animal. In all tests, general behavioral observations as well as the amount of time the animal spent with his nose within 1.0 cm of the sample during the 2.0-min (single-sample) or 4.0-min (two-choice) tests were recorded. The observer was not aware of the predicted results. EXPERIMENT 1
Method In this experiment, discrimination between male and female urine samples that had been dried and aged for various lengths of time were examined. Urine was placed on clean glass test plates and was allowed to dry. The plates were stored in an air-conditioned room (65°-75°F, approximately 50% humidity) for times ranging from 5 hr to 92 days. Ten male subjects were given a series of two-choice tests comparing male to female urine stored for equal amounts of time (e.g., female urine aged I day was compared to male urine aged 1 day). The tests were conducted sequentially; the first test compared urines aged 5 hr, the second 24 hr, the third 3 days, and so forth. At least 48 hr separated each test. The times spent investigating the female and male sample for each length of aging were compared using separate one-tailed t tests since the tests were not counterbalanced and previous experiments (Beauchamp, 1973) allowed us to predict that female urine should be preferred to male urine. Results and Discussion Males spent significantly more time (p < .05) investigating female urine than male urine when the urines were aged up to 40 days (Fig. 1). Males 70
A
[]OO
~ 20
8 5 hr.
24 hr.
3 da.
da.
14 da,
22 da.
40 da.
61 da.
92 da.
Aged
FIG. 1. Responses of male guinea pigs to choices between male and female urines which had been aged on glass plates for times ranging from 5 hr to 92 days.
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did not show a significant preference for female urine as compared to male urine when the samples were aged 61 or 92 days. Since previous experiments (Beauchamp, 1973) have demonstrated that males continue to prefer female to male urine after many repeated tests, this lack of preference is not due to loss of interest in the samples. The results of Experiment 1 are in agreement with recent work with several other species indicating that information in chemical signals is, in some cases, remarkably persistent (see citations under introduction). However, these data contrast with those we previously reported, which indicated that when urine was stored in open vials for 48 hr or longer female urine was no longer more attractive to adult male guinea pigs than was male urine or water (Beauchamp & Bertiter, 1973). We hypothesized three possible reasons for these differing results: (1) differences in methods of storing urine (dry vs wet); (2) differences in methods of presenting urine (dry vs wet); and (3) differences in methods of testing (single-sample presentation vs choice test). In the present experiments urine was aged and tested in a dry state, whereas in the earlier published study, it was aged and tested as an aqueous solution. Perhaps bacterial action degraded the attractive components and/or produced volatile components which masked the attractive components. Dry storage should depress bacterial activity yielding a urine sample that would be more attractive than that stored wet. Additionally, during the drying process, volatile masking compounds would evaporate more rapidly from the urine, which could result in aged dry urine being more attractive than aged wet urine. In the following experiment (2), the first two of the three hypotheses were examined. Urine was aged for approximately 20 to 40 days either wet or dry and then tested in either a wet or dry state. EXPERIMENT 2
In the first of three phases of this experiment (2a), we tested responses of male guinea pigs to choices between water and female urine aged either wet or dry for approximately 20 days. The second phase (2b) was similar except that the choice involved aged female urine vs aged male urine. In the third phase (2c) comparisons were made between female urine aged wet and female urine aged dry. The urines were either both wet or both dry at the time of the test. Method 2a. Female urine was collected and pooled. A 0.1-ml sample of urine was pipetted onto each of 40 plates and stored at room temperature. A 9-ml sample of urine was placed into a vial and stored at room temperature. After 19 days the sample stored in the vial was divided into four equal portions.
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On each test day, 10 samples of urine stored on plates were tested. Half of these were reconstituted with 0.1 ml deionized water before testing. Ten samples of urine stored in vials were also prepared. First the urine was diluted to its original volume (2.25 ml) with deionized water. Then ten 0.1-ml samples were pipetted onto glass plates. Of these, half were allowed to dry for 3 hr before testing while the rest were tested immediately (wet). This resulted in four sample types of female urine: stored dry, tested dry; stored dry, tested wet; stored wet, tested dry; and stored wet, tested wet. Twenty male guinea pigs were tested in a two-choice test comparing aged urine to water. The four sample pairs were presented in a counterbalanced fashion, 19, 21, 24, and 26 days after aging began. The data were subjected to a three-way analysis of variance with repeated measures. 2b. Urine from adult male and female guinea pigs was collected and pooled by sex. Half of the urine for each sex was frozen and stored at - 60°C. The other half was prepared as follows. Twenty 0.1-ml samples of each male and female urine were placed on glass plates to dry. Additionally, 9 ml of male urine and 9 ml of female urine were set out in open vials. After 18 days, frozen samples were thawed and again twenty 0.1-ml samples of male and female urine were placed on glass plates to dry and 9 ml of male and 9 ml of female urine were set out in open vials. Urine stored in vials was diluted to the original volume on Day 39 before testing. On Days 39, 42, 44, and 46 from the beginning of the experiment, 20 adult males were presented with two-choice tests. Again, four urine treatments (aged dry or wet, stored 20 or 40 days) were presented in a counterbalanced fashion over the 4 test days. However, in these tests, female urine was compared to male urine treated in the same way. The data were subjected to a three-way analysis of variance with repeated measures. 2c. Two 12-ml samples of pooled adult female urine were placed in open vials and stored at room temperature. Additional urine was pipetted onto 38 glass plates with a 100-~1 pipet. After 19 days, one of the samples stored in vials was diluted to 12 ml, placed on glass plates, and tested immediately. Nineteen of the urine samples that had been stored dry were dissolved in 0.1 ml of water each. Nineteen male guinea pigs were given a direct two-choice comparison of female urine that had been stored dry vs female urine that had been stored wet. Two days later, the same animals were given the same two-choice comparison test. This time both urines were dry for testing. The data were analyzed with separate t tests. Results and Discussion Female urine was preferred to water and to male urine when the urines had been stored for 20 days, regardless of how the urine was stored (wet
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WELLINGTON, BEAUCHAMP, AND SMITH 701
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Fze. 2. Responses of male guinea pigs to choices between water and female urine which had been aged for 20 days. For aging, urine was stored in vials (wet) or on glass plates (dry).
or dry) or presented (wet or dry). In Experiment 2a (Fig. 2) males spent more time with female urine than they did with water (F(1, 19) = 28.0, p < .001). No other significant effects were found. Similarly, analysis of the results of experiment 2b (Fig. 3) revealed that the male subjects spent more time with female urine than with male urine (F(1, 19) = 8.6, p < .01). This was the only significant effect. In Experiment 2c (Fig. 4) no preference was seen when males were given a choice of urine stored wet and urine stored dry no matter how the urine was presented. These data demonstrate that guinea pig urine aged either dry or wet retains its attractive components for at least 20 days. In addition, males can discriminate female from male urine even when the urines have been aged for 40 days. These data thus supplement those of Experiment 1 in demonstrating the persistence of the active components. It is remarkable that even when aged wet, in which case bacterial activity could be
~;~99 03 40
Stored Aged
Urine
~ ddUrine
Wet Dry 20 days
Wet
DW
40 days
FIG. 3. Responses of male guinea pigs to choices between male and female urines which had been aged 20 and 40 days. For aging, urine was stored in vials (wet) or on glass plates (dry).
STABILITY OF COMMUNICANTS OF GENDER
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6O
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40
._= ' ~ 30 a) >
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Stored Tested
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Dry Wet Wet
FIG. 4. Responses of male guinea pigs to 20-day-old female urine when tested in a choice between urine aged dry and urine aged wet.
expected to be most vigorous, female urine still retains its attractiveness to the male guinea pig. These data also eliminate storage (wet vs dry) differences as an explanation for the different findings of Experiment 1 compared to the Beauchamp and Bertiter (1973) study. Experiment 1 shows that urine is still attractive after 40 days whereas Beauchamp and Bertiter reported it to lose attractiveness after 2 days. If storage differences were the explanation we would predict that in Experiment 2c, urine stored dry would be preferred to that stored wet. This was not the case. In the next experiment, we examined the third possible alternative, that the difference is due to use of a single-sample method compared with an apparently more powerful pair-wise comparison as used in the studies reported here. EXPERIMENT 3
Males were given a series of single-sample presentations of water, male urine, fresh female urine, and urine aged 48 to 96 hr. If our previous use of single-sample tests was responsible for the findings of rapid loss of activity, we should have been able to repeat the results and observe a loss (relative to fresh female urine) of responsiveness to the aged urine. In fact, the aged urine should not have been investigated more than water. In this experiment, we also presented a freeze-dried aged urine to the animals. Freeze-drying should remove noxious volatile compounds produced during aging and thus it might be predicted that aged urine that has been freeze-dried would be preferred to aged urine that is untreated. In Experiment 3b, female urine which had been aged for 96 hr was presented in a two-choice test compared with water.
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WELLINGTON, BEAUCHAMP, AND SMITH
Method 3a. Female urine was collected, pooled, and centrifuged at 5000 g and the supernatant was divided into three equal samples. One sample was further divided, half was frozen for storage at - 60°C and half was freezedried, reconstituted with water, and stored at -60°C. The other two samples were stored at room temperature for 48 and 96 hr, respectively. At the end of room temperature storage, half of the sample was stored at -60°C and the other half was freeze-dried, reconstituted with deionized water, and stored at -60°C. Seventeen males were tested with single-sample presentations of eight stimulus types in a counterbalanced fashion over a period of 2½ weeks, each test separated by 48 to 72 hr. The stimuli included deionized water, male urine, and six types of female urine: (1) fresh; (2) freeze-dried; (3) stored for 48 hr; (4) stored for 48 hr and then freeze-dried; (5) stored for 96 hours; and (6) stored for 96 hr and then freeze-dried. 3b. Female urine was collected, pooled, placed in a vial, and stored at room temperature for 96 hr before testing. Eighteen animals were given a two-choice test of aged urine vs deionized water. Results and Discussion
For the single-sample test (3a) analysis of variance indicated significant differences among the samples (F(7, 112) = 2.79, p < .01, Fig. 5). The Newman-Keuls procedure was used to compare means. These analyses revealed no effect of freeze-drying (p > .05). Males spent significantly more time with fresh urine and urine aged 48 hr than with water (p < .01) but no difference was found in the response to female urine aged 6O
+J ~4o
+
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>
-~, 20 "0 t-
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d
fd
I
48 hr
fd 9 6 hr fd 48 hr (2 96 hr 0
FIG. 5. Responses of male guinea pigs to single sample presentations of water (H20); male urine (6); female urine (2); freeze-dried female urine (fd 2); female urine aged for 48 hr (48 hr 2); female urine aged for 48 hr and then freeze-dried (fd 48 hr 9); female urine aged 96 hr (96 hr 9); female urine aged for 96 hr and then freeze-dried (fd 96 hr 9).
STABILITY OF COMMUNICANTS OF GENDER
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96 hr compared to water (p > .05). Fresh female urine elicited more investigation than did male urine (p < .01). Fresh urine, 48- and 96-hr aged urines all differed from each other (p < 0.01). Male urine was investigated for a longer period than was water (p < 0.05). The results of Experiment 3b were different than those of 3a. Males spent significantly more time with female urine aged 96 hr (X = 38.2 ___ 7.4 sec) than with water (X = 7.6 +__ 1.9 sec; t(18) = 3.61; p < .01) when the two stimuli were presented simultaneously. These data illustrate the different results obtained by the two testing methods. Use of the single-sample method revealed that by 96 hr, female urine was not investigated any longer than was water or male urine. These results are in general agreement with our earlier report (Beauchamp & BeAter, 1973). Comparison of these results with those of Experiment 3b illustrates the differences obtained when results from simultaneous choice tests are contrasted with results from successive presentation of similar stimuli. In simultaneous two-choice tests finer distinctions are possible presumably because the animal can compare and contrast the two substances very rapidly. Clearly, the method of testing has a profound influence on the results obtained and hence on their interpretation. Experiment 3a also revealed no difference between freeze-dried and untreated aged urine. This negative result must be treated with caution, however, as the single-sample method, as we have illustrated here, is not powerful in revealing differences between samples. EXPERIMENT 4 The previous studies have demonstrated that information on the gender of the urine donor is remarkably persistent. This suggests that the compounds that transmit the information are of low volatility. Experiment 4 was carried out to test the effect of limiting the access of the animals to the sample such that they could not contact the urine. Method Male and female urine was collected, pooled by sex, and frozen until use. Urine was pipetted onto glass test plates and dried for 3 hr. The plates were placed 0.5 cm under a screen for presentation. Nine males were given a two-choice test of male vs female urine. The data were analyzed by a t test. Results and Discussion When the male guinea pigs could not contact urine dried for 3 hr the investigation time was greatly depressed (compared with Experiment 1) and they did not appear to discriminate male from female urine O{ = 7.6 +__ 3.0; /~" ~ = 5.2 _+ 2.0; t(8) = 0.76). In contrast, males
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WELLINGTON, BEAUCHAMP, AND SMITH
clearly discriminated male from female urine dried 5 hr when contact with the urine was possible (Experiment 1). GENERAL DISCUSSION
The persistence of chemical signals for gender in guinea pig urine is remarkable. Males discriminated male urine from female urine that had been aged for as long as 40 days. Studies using other methodologies could reveal an even longer persistence. For volatile compounds in urine and/or glandular secretions the temporal pattern of release of the compounds is dependent on the surface on which the mark is placed, the relative humidity and the carrier solvent. Regnier and Goodwin (1977) demonstrated good predictability of their experimental results by physiochemical theories of volatility applied to a volatile chemosignal. In contrast, in this study we found that the presence of a solvent (water) during aging had little effect on the persistence of the chemical signals in guinea pig urine. One explanation for this finding is that nonvolatile compounds in the urine are the chemical cues for gender. Our chemical and behavioral studies support this interpretation. In Experiment 4 we found that when males were presented with urine that had been dried for 3 hr and then placed 0.5 cm under a screen that prevented contact with the urine, they did not discriminate male from female, and investigation times were greatly reduced. This is consistent with the hypothesis that the signals are of low volatility. Furthermore, compounds with molecular weights of approximately 800 g/mole appear to be involved in the communication of sexual identity in the guinea pig (Beauchamp et al., 1980). Finally, we have shown that a nonvolatile dye added to urine is taken into the vomeronasal organ of male guinea pigs during investigation thereby providing a potential route for sensory reception of nonvolatile signals (Wysocki, Wellington, & Beauchamp, 1980). The persistence of a signal for long periods such as those reported here as well as those reported for hamsters (Johnston & Schmidt, 1979) and mice (Nyby & Zakeski, 1980; Evans et al., 1978) could be the direct result of evolutionary selection for large, complex compounds to communicate information. Compounds of higher molecular weight and complexity can carry more information than simple small molecules. The persistence of these molecules may be a secondary evolutionary effect. However, the persistence may also have functional implications. It remains to be determined what role such persistence plays under normal conditions. REFERENCES Beauchamp, G. K. (1973). Attraction of male guinea pigs to conspecific urine. Physiology and Behavior, 10, 589-594. Beauchamp, G. K., & Berfiter, J. (1973). Source and stability of attractive components in guinea pig (Cavia porcellus) urine. Behavioral Biology, 9, 43-47.
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Beauchamp, G. K., Wellington, J. L., Wysocki, C. J., Brand, J. G., Kubie, J. L., & Smith, A. B., III. (1980). Chemical communication in the guinea pig: Urinary components of low volatility and their access to the vomeronasal organ. In D. MiillerSchwarze and R. M. Silverstein (Eds.) Chemical Signals: Vertebrates and Aquatic Invertebrates. New York: Plenum. Epple, G., Alveario, M. C., Golob, N. F., & Smith, A. B., III. (1980). Stability and attractiveness related to age of scent marks of saddle-back tamarins (Saguinus fuscicollis). Journal of Chemical Ecology, 6, 735-748. Evans, C. M., Mackintosh, J. H., Kennedy, J. F., & Robertson, S. M. (1978). Attempts to characterize and isolate aggression reducing olfactory signals from the urine of female mice Mus musculus L. Physiology and Behavior, 20, 129-134. Johnston, R. E. (1974). Sexual attraction function of golden hamster vaginal secretion. Behavioral Biology, 12, 111-117. Johnston, R. E., & Lee, N. A. (1976). Persistence of the odor deposited by two functionally distinct scent marking behaviors in golden hamsters. Behavioral Biology, 16, 199-210. Johnston, R. E., & Schmidt, T. (1979). Responses of hamsters to scent marks of different ages. Behavioral and Neural Biology, 26, 64-75. Jones, R. B., & Nowell, N. W. (1977). Aversive potency of male mouse urine: A temporal study. Behavioral Biology, 19, 523-526. Lydell, K., & Doty, R. L. (1972). Male rat odor preferences for female urine as a function of sexual experience, urine age and urine source. Hormones and Behavior, 3, 205-212. McCartney, W. (1968). Olfaction and Odours. Berlin: Springer-Verlag. Mfiller-Velten, H. (1966). lJber den Angstgeruch bei der Hausmaus (Mus musculus L.) Zeitschrift fiir vergleichende Physiologie, 52, 401-429. Nyby, J., & Zakeski, D. (1980). Elicitation of male mouse ultrasounds: Bladder urine and aged urine from females. Physiology and Behavior, 24, 737-740. Regnier, F. E., & Goodwin, M. (1977). On the chemical and environmental modulation of pheromone release from vertebrate scent marks. In D. Miiller-Schwarze & M, M. Mozell (Eds.), Chemical Signals in Vertebrates. New York: Plenum. Rood, J. P. (1972). Ecological and behavioral comparisons of three genera of Argentine cavies. Animal Behavioral Monographs, 5, 3-83. Wilson, E. O., & Bossert, W. H. (1963). Chemical communication among animals. Recent Progress in Hormone Research, 19, 673-710. Wysocki, C. J., Wellington, J. L., & Beauchamp, G. K. 0980). Access of urinary nonvolatiles to the mammalian vomeronasal organ. Science, 207, 781-783.