Research reportSexual responses of male rats to odours from female rats in oestrus are not affected by female germ-free status
Introduction
Male rats use odours to detect female rats in oestrus [1,2], but olfactory cues are not necessary for successful copulation [3]. Reproduction in rats born and raised in sterile isolators (germ-free rats) has not been reported to be associated with any difficulty, despite changes to the olfactory capacity of rodents kept in this manner [4]. We were interested in knowing if female germ-free rats were a source of oestrous odours similar to those detected by male rats in conventional females [1,5]. No published results of the composition of volatiles emanating from germ-free rodents could be found, so we set out to perform a comparative GC–MS (gas chromatography–mass spectrometry) analysis of faeces samples collected from male and female germ-free rats as well as male rats kept in the same type of housing and feeding, but not kept germ free.
We have previously shown that male rats respond with an increased number of penile erections when exposed to the odours of faeces not only from female rats in oestrus, but also oestrous faeces from vixens and mares [6]. Several attempts have been made to identify specific odorants associated with oestrus in mammals [[7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18]], but little agreement is evident in the literature, and the smell of oestrus is likely to consist of a mixture of odorants in different proportions [11].
Many animal species use odours in their social interactions with conspecifics. It is well-known that individual odours can change dependent on the type of food eaten [19,20], and rodents can distinguish between the odours from healthy and diseased conspecifics [21]. However, the role of microbes in animal olfactory communication is largely unknown [22]. Some animal odours arise from processes carried out by their microbiota [23,24], in particular associated with scent-marking [25,26]. In birds, the uropygial (preen) gland can harbour bacteria [27] and odours emanating from this gland contains sexual olfactory information in the females of some bird species [28,29]. Similarly, the Indian mongoose uses anal pocket bacteria metabolites as an odorant signature [26]. However, in some mammalian species, intestinal and vaginal microbiota are stable across the oestrous cycle [30,31] indicating little microbial influence on the odorant signals emitted. Olfactory capacity is also influenced by the microbiota [4], and Champagne-Jorgensen et al. [32] recently found that social odour recognition in mice showed long-term deficits if the mice had been treated with low-dose penicillin during their adolescence. Overall, these findings raise the question as to what extent odours associated with oestrus and detected by male rats are a product of or influenced by the microbiota of the females. One way to test this is to present conventionally housed male rats to faeces from germ-free female rats in oestrus.
Using GC–MS, we first examined the importance of microbiota as a source of odours in rats (experiment 1). The second aim was to study the behavioural responses, in particular the number of penile erections, of conventionally housed and sexually experienced male rats exposed to faeces from conventional and germ-free female rats in oestrus, as well as various other odours as positive and neutral controls (experiment 2).
Section snippets
GC–MS analyses of rat faeces (experiment 1)
This experiment aimed to make an overall comparison of volatiles in faeces from conventional and germ-free rats. We exploited a situation where a group of male Fischer rats were housed in an isolator in the germ-free animal facility without being kept germ free. No females were housed conventionally in the isolators, but faeces samples from both male and female germ-free rats were collected at the same time. Unfortunately, stage in the oestrous cycle at the time of faeces collection was unknown
Behavioural responses of conventional male rats to faeces from germ-free female rats (experiment 2)
As stated in the Introduction, male rats are able to detect the reproductive state of female rats via their odours [6]. Given the low abundance of volatiles found in germ-free faeces, we wanted to investigate if conventionally raised male rats, which had experience with conventionally raised female rats in oestrus, could detect oestrous odours in faeces from germ-free female rats. The available germ-free females were of the Fischer strain, and the males used for odour detection were Brown
General discussion
The results of the GC–MS analysis of rat faeces revealed a significantly reduced odorant composition for the germ-free samples, with significantly lower abundance and reduced variety of odorants compared to samples from conventionally housed rats. Despite this, faeces from germ-free female rats in oestrus were able to evoke penile erections in conventionally housed male rats with sexual experience with conventional females.
Germ-free rats differ from conventionally reared conspecifics in a
Declarations of interest
None.
Acknowledgements
The authors are grateful to the staff in the two animal houses for looking after the rats, to Isabelle Pellerin (UPMC, Paris VI) for her help with carrying out the GC–MS analyses, and to Olivier Rampin for his useful comments on an earlier version of this paper. This work was supported by a Credits Incitatifs grant from the Department of Animal Physiology and Livestock Systems (Phase), Inra, France.
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