To date, there are no published studies that explore the mechanism behind the reduction in aggression observed in response to used nest material or whether odors exist that can promote affiliative behaviors in mice. Providing nesting material is becoming standard practice for laboratory mice and its transfer during cage cleaning helps reduce aggression although it does not completely eliminate it. In order to understand what in the nest is specifically effective at altering mouse behavior, we must have better insight into the chemical signals deposited there and where they come from. Once these specific signals have been identified, further research can examine methods to develop compounds that could then be added to mice environments to help reduce aggression. Additionally, there are no reports that quantitatively analyze the VOC contents of murine plantar sweat, which has historically been suggested as the source of nesting material odor deposits. Therefore, the first aim of this experiment was to quantify compounds deposited within the nests of mouse strains known to exhibit different aggression levels and link them to plausible sources. Our working hypothesis was that the compounds present on the nests would exhibit strain specific properties. We predicted that chemical analyses of the nests from historically peaceful mice would contain VOCs in different proportions than those from the nests of historically aggressive males; in particular, they would contain higher levels of VOCs originating in plantar sweat and lower levels of VOCs originating in urine. To do this, we used three strains known for varying aggression levels: SJL (high aggression), C57BL/6 (moderate aggression), and AJ (low aggression). Our second aim was to determine whether these VOC profiles are related to mouse social behavior. Our working hypothesis was that VOC profiles from the nest and sweat correlate with social behavior in group housed males, with the assumption that behavior is affected similarly across strains. We predicted that these odor profiles would be associated with lower rates of aggressive behavior and/or higher rates of affiliative behavior. In contrast, profiles from urine would be associated with higher rates of aggression. Social behavior was taken as a cage level measure, while odor profiles were taken from individuals based on dominance rank in the tube test [35].
The only VOC with a high negative loading on urine PC2 was 6-hydroxy-6-methyl-3-heptanone, a MUP ligand that accelerates puberty in female mice [49]. Although it did not directly relate to aggression, it was positively correlated with allo-grooming and was produced more by B6 mice than SJL and AJ. This result was unexpected since male mouse pheromones from urine have been shown to promote aggression between males [25, 50]. However, to our knowledge, 6-hydroxy-6-methyl-3-heptanone has not been directly tested for effects in males. Based on this data, it may have a role promoting affiliative behavior.
1st Studio Siberian Mouse Hd 114 78
Download: https://tinurli.com/2vDPxE
Compounds were identified or tentatively identified by matching retention times and mass spectra with standard compounds when available (Sigma-Aldrich Chemical Co.) and with spectra through NIST Mass Spectral Search Program for the NIST/EPA/NIH Mass Spectral Library (Version 2.0 a, 2002). Additionally, in-house (Novotny Laboratory) synthesized mouse urinary pheromone compounds and the in-house spectral database were utilized for identifications.
Reviewer #1: In this study, Barabas and colleagues collected body odours (urine, saliva, and plantar sweat, as well as nest odours collected from nesting material) from 3 mouse strains, in an attempt to provide a factual basis to the empirical observation that reusing bedding material reduces aggression (through olfactory signals) upon cage changing in mouse husbandry. Compounds were identified by GCMS and their abundance correlated to various behaviours (mostly pertaining to social/aggressive interactions). Some odour profiles were correlated with affiliative behaviours. Please note, the manuscript would have been clearer if the figure legends had featured together out of the main text body.
Table 1: browsing through the compounds detected, I found a couple of surprises, I checked that methyldihydrojasmonate has also been detected in human saliva (DOI: 10.1007/s10886-010-9846-7), however can the authors explain why limonene can be detected in mouse saliva?
Reviewer #2: Previous studies have shown that transferring soiled nest material when groups of male mice are transferred into a clean cage can reduce their aggression. The main aim of this study was to quantify volatile organic compounds (VOCs) in nest material soiled by male mice, identify the likely sources of nest odor compounds (from urine, saliva or plantar glands), and establish whether differences in aggression levels between strains correlate with levels of different VOCs (or combinations). A second aim was to determine the relationship between VOC profiles and mouse social behavior (more specifically, aggressive and affiliative behavior), particularly to test the hypothesis that VOCs in soiled nest material and plantar gland secretion (here referred to as sweat) reduce aggression and promote positive (affiliative) social interactions.
In this study, Barabas and colleagues collected body odours (urine, saliva, and plantar sweat, as well as nest odours collected from nesting material) from 3 mouse strains, in an attempt to provide a factual basis to the empirical observation that reusing bedding material reduces aggression (through olfactory signals) upon cage changing in mouse husbandry. Compounds were identified by GCMS and their abundance correlated to various behaviours (mostly pertaining to social/aggressive interactions). Some odour profiles were correlated with affiliative behaviours. Please note, the manuscript would have been clearer if the figure legends had featured together out of the main text body.
Table 1: browsing through the compounds detected, I found a couple of surprises, I checked that methyldihydrojasmonate has also been detected in human saliva (DOI: 10.1007/s10886-010-9846-7), however can the explain why limonene can be detected in mouse saliva?
Previous studies have shown that transferring soiled nest material when groups of male mice are transferred into a clean cage can reduce their aggression. The main aim of this study was to quantify volatile organic compounds (VOCs) in nest material soiled by male mice, identify the likely sources of nest odor compounds (from urine, saliva or plantar glands), and establish whether differences in aggression levels between strains correlate with levels of different VOCs (or combinations). A second aim was to determine the relationship between VOC profiles and mouse social behavior (more specifically, aggressive and affiliative behavior), particularly to test the hypothesis that VOCs in soiled nest material and plantar gland secretion (here referred to as sweat) reduce aggression and promote positive (affiliative) social interactions.
Reviewer #1: I thank and congratulate the authors for their work to improve this manuscript. This report is very interesting and I am looking forward to reading up on future developments on the question of mouse aggression and plantar sweat. I would however suggest not to forget other odorant sources (e.g. urine or saliva), volatile compounds evaporate and can evade detection, and rarely biologically work in isolation (ie they are naturally occurring as part of mixtures, even though the literature glorifies single molecules like PEA, TMT and the likes).
The study is designed to look for VOC candidates that might have common effects in regulating aggression across mouse strains. I think it is important to clarify this underlying assumption, as differences between chosen strains in the odors used to regulate aggression, or in sensitivity to these VOCs, would result in a lack of association using this design. An example is mentioned in the Discussion (though not in this context) where strains differ in expression of a scent protein that stimulates aggression (lines 445-450), but nonetheless strain differences in expression do not correspond to differences in aggression but to strain lineage, which deserves further comment.
I thank and congratulate the authors for their work to improve this manuscript. This report is very interesting and I am looking forward to reading up on future developments on the question of mouse aggression and plantar sweat. I would however suggest not to forget other odorant sources (e.g. urine or saliva), volatile compounds evaporate and can evade detection, and rarely biologically work in isolation (ie they are naturally occurring as part of mixtures, even though the literature glorifies single molecules like PEA, TMT and the likes).
You are correct that altering aggression was our focus, but we did not know what to expect in terms of behavior diversity in the cage. Affiliative behaviors were included since previous work on laboratory mouse social behavior primarily does not take place in the home cage and little is published on what influences these behaviors in the laboratory and how that would relate to aggression. Thus, we did not want to risk overlooking social differences that may have been something other than agonistic behavior. Additionally, both promoting affiliative behavior and reducing aggression would benefit mouse welfare. This explanation has been added to the intro (line 70-76). 2ff7e9595c
Comments