Behavioural Brain Research 172 (2006) 169–172

Short communication

Are oestrus odours species specific? Olivier Rampin a , Nathalie J´erˆome a , Christine Briant b , Franck Bou´e c , Yves Maurin a,∗ a

Analyse & Mod´elisation en Imagerie Biologique, NOPA, UMR 1197, Institut National de la Recherche Agronomique Universit´e Paris Sud, Bˆat 325, F-78352 Jouy-en-Josas, France b Physiologie de la Reproduction & des Comportements, Institut National de la Recherche Agronomique, F-37380 Nouzilly, France c Unit´ e de Recherche sur la Rage et les maladies e´ mergentes, Agence Fran¸caise de S´ecurit´e Sanitaire des Aliments, BP 9, F-54220 Malzeville, France Received 24 February 2006; received in revised form 5 April 2006; accepted 7 April 2006 Available online 5 June 2006

Abstract Adult male rats were exposed to faeces odours of three animal species (rat, fox and horse). They displayed erections in the presence of faeces from oestrous females (whatever the species). In addition, fox faeces (whatever the gender or hormonal status) elicited an expected freezing reaction. It is suggested that oestrous female faeces of these three species share common odorants which depend on the hormonal status and characterize female receptivity. © 2006 Elsevier B.V. All rights reserved. Keywords: Hormonal status; Sex behaviour; Penile erection; Freezing; Faeces; Rat; Horse; Fox

In mammals, odours are major triggers for behaviours related to either species or individual survival (e.g. sexual partner selection or predator avoidance [1,3,5,11,13]. Olfactory cues, addressed to conspecifics (reproductive behaviour) or to other species (predatory/defensive behaviours), are present in faeces, urine and in various body secretions [18]. Thus, the odour of oestrous female rats attracts male conspecifics [12] and that of their urine sexually arouses them [9], while the odour of predator faeces triggers defensive reactions in rats and other rodents [1]. Such defensive behaviours have been studied in various prey species in response to the odours of their respective predators [1], while, to our knowledge, the reproduction related behavioural responses to olfactory sexual stimuli have only been studied between conspecifics. Yet, reproductive behaviour includes many aspects (attraction, sexual arousal, copulation. . .) that no information indicates as being solely triggered by olfactory stimuli from conspecifics. Are olfactory cues which characterize sexual receptivity devoid of significance for non conspecifics? Here we show that exposure of male rats to faeces of oestrous

∗

Corresponding author. Tel.: +33 1 34 65 24 14; fax: +33 1 34 65 25 05. E-mail address: [email protected] (Y. Maurin).

0166-4328/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2006.04.005

females from different species (fox, horse and rat) elicited penile erections (an index of sexual arousal [14]). In addition, as previously described [19], all fox faeces elicited a defensive reaction characterized by freezing episodes [13]. Two sets of 40 Brown-Norway rats (20 males and 20 females, Elevage Ren´e Janvier, France) were used, at 6 months interval. They were housed in our animal facility, under a reversed 12-h light–dark cycle (lights on at 15:30) at 21 ± 2 ◦ C. Rodent pellet chow (Usine d’Aliment Rationnel, France) and tap water were available ad libitum. During the first 3 weeks, rats were acclimatized: (i) to the reversed light–dark cycle; (ii) to the observer through daily manipulation; and (iii) to the test arena through five stages of 10 min in it. They were then sexually trained for two more weeks, since it is known that sexually experienced rats exhibit a much higher attraction for oestrous females that naive ones do [4]. Sexual training was performed according to Sachs [15], except that we used naturally receptive females (taken among the 20 females), as determined by vaginal smears. Training was performed between 7:00 and 13:00. The 12 best responders (i.e. rats which had ejaculated each time they had been put into the presence of a receptive female) were selected for the odour test. The experimental setup of the test was as follows: the floor of the test arena (a 40 cm × 30 cm × 30 cm

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glass tank) was covered with clean wood chips bedding. The rat was left alone in the arena for 5 min. An aliquot of faeces (placed on a piece of aluminium foil) was introduced into a container which consisted of a plastic tube (diameter: 6 cm, height: 8.5 cm), weighed down at one extremity by a 650 g cylindrical piece of lead and closed at the other one by a metal grid. The container was placed vertically (grid on top) against one of the short walls of the arena, in its middle. The rat was gently placed on the opposite side, its nose facing the other wall. The test lasted for 30 min, during which erections (latency of the first one and number, scored when a sequence of events including all of the following actions: bending forward on the extremity of the hind limbs, bending the head towards the penis, performing genital grooming, displaying forward hip movements, and occasionally including a visible protruding of the penis, lasting altogether a few seconds) and freezing episodes (latency of the first episode, number and duration) were recorded by the observer. The rat was then brought back to its cage, the bedding and the faeces aliquot were discarded, the container and the test arena were washed with ethanol, dried and the test room was ventilated during 15 min. Four to five such tests were carried out every day, each with a different male rat. Rat (Rattus norvegicus) faeces were collected from the group of 40 Brown-Norway animals. Female faeces were collected only at the oestrus and diestrus stages, identified by vaginal smears and by receptive behaviour to a stud male. Animals were isolated in cages with clean bedding for 2 h. Faeces were gathered from the bedding. European fox (Vulpes vulpes) faeces were collected in February and March 2004 and 2005 at the animal facility of AFSSA (Malzeville, France). Hormonal status of the vixens was determined by vaginal smears. Faeces were collected on the inclined plane beneath the metallic cages hosting the animals. Welsh ponies (Equus caballus) faeces were provided by animal facilities at INRA (Nouzilly, France). Hormonal status of the mares was determined by progesterone and total oestrogens assay and by transrectal ovarian echography. Faeces were collected manually, directly inside the rectum. Fox and horse faeces were shipped in dry ice. All faeces were sampled into plastic vials and kept at −20 ◦ C until use. The 12 selected rats and the 11 odour types (faeces of male, oestrous female and diestrous female from the three species, plus food pellet aliquot and empty container) were numbered at random. Each rat was exposed once (at random) to each one of the 11 odours. The observer and the person in charge of data analysis were blind to the odour tested. The whole experiment (acclimatization, sexual training and odour testing) was performed twice, on two different sets of rats, during autumn 2004 and spring 2005. Results were statistically analysed using one-way ANOVA with repeated measures. When the results complied with a normal distribution, equality of variances was tested. In all situations, this test was passed and a Holm–Sidak test was used for the comparison with the result obtained with the empty container, taken as the control value. When normality of the distributions was not satisfied, a Friedman repeated measures ANOVA on ranks was performed. In case of a significant effect (p < 0.05), a Dunn’s test was used to compare the results to the control one.

Fig. 1. Number of erections elicited in male rats by exposure to odours. Columns represent the mean ± S.E.M. value of the number of erections exhibited during the test. The erectile response depended upon the container content (χ2 = 128.48, d.f. = 10, p ≤ 0.001). Post hoc analysis (Dunn’s test in all cases) showed a significantly increased number of erections following exposure to faeces of oestrous rats (n = 4.54 ± 0.55, Q = 5.745, p < 0.05); oestrous vixens (n = 3.08 ± 0.52, Q = 3.590, p < 0.05); and oestrous mares (n = 3.42 ± 0.61, Q = 4.352, p < 0.05), when compared to the value obtained by exposure to an empty container (n = 0.50 ± 0.19), taken as control.

During the 30 min when the sexually experienced male rats were exposed to one among the 11 odours tested (faeces of male or oestrous/diestrous females, food pellet, empty container), the number of erections appeared to depend upon the container content. When exposed to an empty container (control value), male rats exhibited 0.50 ± 0.19 erections. In the presence of either food or faeces from male or diestrous females of the three species, the number of erections was not significantly different from this control value (Fig. 1). At variance, it was significantly increased when the rats were exposed to faeces of oestrous females, whatever their species (rat: 4.54 ± 0.55; vixen: 3.08 ± 0.52; mare: 3.42 ± 0.61). The latency of the first erection was significantly reduced when rats were exposed to faeces of oestrous females (Table 1). Rats also displayed freezing episodes the number of which depended upon the container content (Table 1). When exposed to an empty container (control value), male rats exhibited 5.2 ± 0.7 freezing episodes. In the presence of food or faeces from rat or horse (whatever the gender or hormonal status of the females), the number of freezing episodes was not significantly different from this control value. At variance, it was significantly increased when the rats were exposed to fox faeces (male: 12.7 ± 1.1; oestrous vixen: 13.6 ± 1.5; diestrous vixen: 11.5 ± 1.5). The latency of the first freezing episode also depended upon the container content (Table 1). Indeed, faeces of diestrous vixens induced a shorter latency, as compared to the empty container control value. Since the number of freezing episodes was increased in the presence of fox faeces, while the mean duration of each episode was not changed (Table 1), the total time spent freezing was longer when rats were exposed to fox faeces (Fig. 2).

O. Rampin et al. / Behavioural Brain Research 172 (2006) 169–172 Table 1 Latency of the first erection; number, latency and mean duration of the freezing episodes elicited in male rats by exposure to odours Odour

Oestrous rat Diestrous rat Male rat Oestrous vixen Diestrous vixen Male fox Oestrous mare Diestrous mare Stallion Food pellet Empty container

Latency of first erection

424 1026 1659 836 1548 1393 645 1461 1424 1699 1373

± ± ± ± ± ± ± ± ± ± ±

86* 135 85 137* 115 121 130* 117 119 62 134

Freezing episodes Number 8.1 6.7 5.7 13.6 11.5 12.7 5.1 5.2 4.7 3.4 5.2

± ± ± ± ± ± ± ± ± ± ±

Latency 1.0 1.3 0.7 1.5* 1.5* 1.1* 1.0 0.6 0.9 0.6 0.7

315 282 379 257 216 278 414 420 395 386 375

± ± ± ± ± ± ± ± ± ± ±

54 53 64 78 38* 67 65 72 64 72 61

Mean duration 19 17 19 22 24 25 15 17 17 14 18

± ± ± ± ± ± ± ± ± ± ±

2 2 2 2 3 2 2 2 3 2 2

Latencies and mean duration are expressed in seconds. The latency of the first erection depended on the odour [F(10, 230) = 17.536, p < 0.001]. Oestrous females odours shortened the latency of the first erection (rat: t = 6.565, p < 0.005; mare: t = 5.033, p < 0.005; vixen: t = 3.715, p < 0.006; Holm–Sidak method in all three cases). The number of freezing episodes also depended upon the odour (χ2 = 89.418, d.f. = 10, p ≤ 0.001). Fox faeces odour significantly increased it (male fox: Q = 4.352, p < 0.05; oestrous vixen: Q = 4.243, p < 0.05; diestrous vixen: Q = 3.547, p < 0.05; Dunn’s test in all three cases). Latency of the first freezing episode was significantly shortened only for diestrous vixen (χ2 = 28.173, d.f. = 10, p ≤ 0.002; Q = 3.003, p < 0.05, Dunn’s test).

The total duration of freezing episodes was 94.2 ± 11.9 s when the container was empty and 303.9 ± 34.5 s, 281.3 ± 31.5 s and 238.8 ± 26.5 s when it contained faeces of male fox, oestrous and diestrous vixens, respectively.

Fig. 2. Total duration of the freezing episodes elicited in male rats by exposure to odours. Columns represent the mean ± S.E.M. value of the total duration of the freezing episodes exhibited during the test. The freezing response depended upon the container content (χ2 = 96.123, d.f. = 10, p ≤ 0.001). Post hoc analysis (Dunn’s test in all cases) showed a significantly increased freezing duration following exposure to faeces of male fox (304 ± 34 s, Q = 4.526, p < 0.05); oestrous vixens (281 ± 31 s, Q = 4.178, p < 0.05); and diestrous vixens (239 ± 26 s, Q = 3.612, p < 0.05), when compared to the value obtained by exposure to an empty container (94 ± 12 s) taken as control.

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Thus, rat, fox and horse faeces induced two kinds of behavioural responses in sexually experienced male rats: sexual arousal and defensive reactions, depending on the hormonal status and on the species, respectively. Insofar as (i) the experiments were carried out during the dark phase of the reversed light/dark cycle, under a dim red light; (ii) faeces were deep inside the container (which was covered by a metal grid), the behavioural responses were unlikely to result from visual cues. If such had nevertheless been the case, one would have expected erections to reveal a species difference rather than a hormonal status one, since the samples used for the tests were all alike within the same species. Moreover, the rats could neither touch nor taste the faeces, thus also excluding these two sensory modalities as determinants of the behavioural responses. Altogether, we believe that erections and freezing episodes were very likely elicited by olfactory stimuli only. Familiarity with the test environment might also have biased our results. Indeed, during the training phase, every time the rat was placed in the presence of a sexually receptive female, this occurred into the same arena and in the presence of the same observer, thus possibly turning these various elements into conditioned stimuli. Although this hypothesis may possibly explain that a few erections occurred even in the absence of faeces, it cannot account for the finding that the number of erections was significantly higher than the control value only for faeces of oestrous females. The present results are at variance with those of Kondo et al. [9], who claimed that in male rats, the sexually arousing signal was present in female rat urine and not in their faeces (although these authors observed a few erections in response to the exposure to faeces). Differences regarding the experimental setups (albino/pigmented strains, fresh/frozen faeces, artificial/natural oestrus) might account for the different results concerning the faeces odour. It might also be hypothesized that in our experiments, the erectile effects of oestrous rat faeces result from their contamination by urine insofar as we gathered them from the bedding. The same is true for fox faeces, which were collected on the inclined plane beneath the metallic cages. But urine contamination can be excluded regarding faeces from oestrous mares, which were collected manually intrarectally. Yet these faeces still elicited a comparable number of erections in rats as those of oestrous rats and vixens did. Thus, although we cannot propose a simple explanation for the discrepancy between our results and those of Kondo et al. [9], our present results strongly support the idea that erections were triggered by olfactory cues emanating from faeces. Our results are in favour of a trans-specific olfactory message, characterizing the hormonal status of females, present in their faeces and perceived as sexually stimulating by male rats. It might be argued that previous studies have shown that the hormonal status of cows can be identified by rats [10] or dogs [8]. However, this detection was performed through an operant conditioning paradigm through which rats or dogs (both males and females) were trained to identify oestrous urine or oestrous vaginal secretions and rewarded when the expected (learned and non sexual) response was displayed appropriately. These results do not indicate that oestrous cow odours (i) have a sexual sig-

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nificance for either rats or dogs and (ii) are shared by oestrous females from other species. On the contrary, we show here that male rats which are naive to fox and horse and have never been exposed to their faeces still display sexual responses to olfactory cues from oestrous female faeces. Another notable result was the observation that fox faeces (whether from male or oestrous or diestrous females) significantly increased both the number and total duration of freezing episodes in rats. Freezing is recognized as the initial response to danger, exhibited by preys which have identified a distant predator [13]. For rats, fox is one of these predators and accordingly, fox faeces [19] as well as trimethylthiazoline (TMT), a component of their faeces [6], induce freezing in them. In keeping with these reports, only fox faeces induced freezing episodes, while both conspecific and horse faeces remained ineffective. Moreover, freezing is unlikely to be related to novelty of the stimulus, since horse faeces, that rats have never be exposed to, did not increase the total duration of freezing episodes. Thus, in our experiments, freezing most probably reflects a defensive reaction, behaviour expected from rats exposed to a predator odour. It has been proposed [17] that the behavioural output of conflicting sensory stimuli results from a trade-off between their respective intensities. This may apply notably to the reciprocal antagonistic effects of fear and sexual arousal shown in rodents [1,7] and insects [17]. According to this view, it might be surprising to note that in our experiments, freezing did not antagonize erections. Indeed, the number of erections elicited by faeces of oestrous vixens did not differ from that of erections elicited by oestrous mares or rats. This result may nevertheless be explained by the fact that freezing is an initial response to danger, exhibited when the predator has been identified through a partial stimulus such as its odour [5] but is still far away [13]. Thus, in our experimental situation, the trade-off between the olfactory stimuli emanating from the same faeces, i.e. sexual and predatory, might be strongly in favour of the former. In support of this hypothesis, it has been shown that predator odours are less potent than the presence of the predator itself at eliciting a defensive reaction in rats [2]. On the other hand, the number of erections elicited by oestrous female faeces in the present experiments is very comparable to that of erections elicited either by the administration of apomorphine [16], a potent centrally acting proerectile drug, or the presentation of a oestrous female rat [15], both situations in which a maximal number of erections is observed. Altogether, these results suggest that oestrous vixen faeces simultaneously diffuse a weak predatory signal and a potent sexual one. In conclusion, our results suggest that faeces from oestrous rats, vixens and mares contain a ubiquitous set of odorants which characterize their hormonal status. These odorants are perceived by male rats and exert a powerful sexual arousing effect upon them. The existence of odorants shared by oestrous females (at least in these three species) might lead to reconsider the notion

of species barrier with respect to the various aspects of sexual behaviour. Acknowledgements We thank Jacqueline Bailly, Blandine Barbier, Marielle Levillain and Michel Munier for help. This work was supported by grants from the Institut National de la Recherche Agronomique, the Haras Nationaux and from R´egion Ile-de-France. References [1] Apfelbach R, Blanchard CD, Blanchard RJ, Hayes RA, McGregor IS. The effects of predator odors in mammalian prey species: a review of field and laboratory studies. Neurosci Biobehav Rev 2005;29: 1123–44. [2] Blanchard RJ, Blanchard DC, Weiss SM, Meyer S. The effects of ethanol and diazepam on reactions to predatory odours. Pharmacol Biochem Behav 1990;35:775–80. [3] Cain DP, Paxinos G. Olfactory bulbectomy and mucosal damage: effects on copulation, irritability, and interspecific aggression in male rats. J Comp Physiol Psychol 1974;86:202–12. [4] Carr WJ, Loeb LS, Wylie NR. Responses to feminine odors in normal and castrated male rats. J Comp Physiol Psychol 1966;62:336–8. [5] Dielenberg RA, McGregor IS. Defensive behavior in rats towards predatory odors: a review. Neurosci Biobehav Rev 2001;25:597–609. [6] Endres T, Apfelbach R, Fendt M. Behavioral changes induced in rats by exposure to trimethylthiazoline, a component of fox odor. Behav Neurosci 2005;119:1004–10. [7] Kavaliers M, Choleris E, Colwell DD. Brief exposure to female odors “emboldens” male mice by reducing predator-induced behavioral and hormonal responses. Horm Behav 2001;40:497–509. [8] Kiddy CA, Mitchell DS, Bolt DJ, Hawk HW. Detection of estrus-related odors in cows by trained dogs. Biol Reprod 1978;19:389–95. [9] Kondo Y, Tomihara K, Sakuma Y. Sensory requirements for noncontact penile erection in the rat. Behav Neurosci 1999;113:1062–70. [10] Ladewig J, Hart BL. Demonstration of estrus-related odors in cow urine by operant conditioning of rats. Biol Reprod 1981;24:1165–9. [11] Larsson K. Impaired mating performances in male rats after anosmia induced peripherally or centrally. Brain Behav Evol 1971;4:463–71. [12] Le Magnen J. Olfacto-sexual phenomenon in white rat. Arch Sci Physiol (Paris) 1952;6:295–331. [13] Misslin R. The defense system of fear: behavior and neurocircuitry. Neurophysiol Clin 2003;33:55–66. [14] Nadler RD, Bartlett ES. Penile erection: a reflection of sexual arousal and arousability in male chimpanzees. Physiol Behav 1997;61:425–32. [15] Sachs BD, Akasofu K, Citron JH, Daniels SB, Natoli JH. Noncontact stimulation from estrous females evokes penile erection in rats. Physiol Behav 1994;55:1073–9. [16] Sachs BD, Akasofu K, McEldowney SS. Effects of copulation on apomorphine-induced erection in rats. Pharmacol Biochem Behav 1994;48:423–8. [17] Skals N, Anderson P, Kanneworff M, L¨ofstedt C, Surlykke A. Her odours make him deaf: crossmodal modulation of olfaction and hearing in a male moth. J Exp Biol 2005;208:595–601. [18] Stoddart MD. The ecology of vertebrate olfaction. London: Chapman & Hall; 1980. p. 234. [19] Vernet-Maury E, Le Magnen J, Chanel J. Emotional behavior of rats; influence of the odour of a predator and a non-predator]. C R Acad Sci (Paris) S´erie D 1968;267:331–4.