In this section
Making sense of scents: reducing aggression and uncontrolled variation in laboratory mice
Professor Jane Hurst, Department of Veterinary Clinical Science, University of Liverpool
Scents are the primary means of communication in mice. They underlie most aspects of their social behaviour and are particularly important in mediating aggressive interactions and status differentiation among males. This competitive aggression can be a major welfare concern and source of uncontrolled variation among laboratory mice. Scents also play an invisible role in priming reproductive physiology and development, with additional consequences for immunocompetence, introducing another potential source of uncontrolled variation that could influence many types of experiment. Here, I provide a brief explanation of how wild mice use scents to recognise each other and to control competitive interactions. I then discuss the consequences of this for aggression among laboratory mice and recommend ways to minimize problems through management practices. Known reproductive priming effects are also summarised to show how exposure to scents and cage group size can influence sex hormone levels, reproductive cycling and development. Careful consideration of husbandry and experimental design can also reduce this source of variability.
The sensory world of mice is quite
different from ours. Watch mice for a few minutes and
you soon realise that they are largely led by their noses.
Their primary means of communication is through their
characteristic, pungent scents. Because humans gain information
primarily through vision and hearing, we have little
understanding of the information that other animals gain
through scents. As mouse scents mediate a wide range of
behavioural responses including competitive aggression among
males, as well as influencing physiology through priming
effects, it is important to understand and control these
invisible signals as part of good husbandry and experimental
Competitive scent communication among wild house mice
Although we usually think of the
natural environments for rodents as grassland, woodland
and hedgerows, the preferred habitats of house mice are farms,
grain stores, domestic and catering premises where food is
concentrated and readily available and buildings provide
protection from harsh weather and predators.
Although there are specialised scent glands on the body (particularly around the face), urine is a main source of scents in mice and is used to liberally scent mark their home area (faeces are not used for scent communication in this species). Mouse urine contains a complex mixture of small volatile odorants that are deliberately excreted in urine as chemical signals (pheromones). Some of these are bound to a high concentration of non-volatile proteins termed major urinary proteins or MUPs. The combination of volatile and non-volatile pheromone components provides information on both the identity and current status of the urine owner (see Table 1). Urine is not simply evacuated but deposited as scent marks in many small streaks and spots on the substrate throughout their home area. Although all residents add to these scent marks to advertise their membership of the resident group, the dominant territory owner marks at a much higher rate.
Like most territorial mammals, mice use scent marks to advertise territory ownership. Scent marks are the physical proof of the owners ability to defend the scent marked area, as only a successful territory holder can ensure that the area is suffused with his fresh scent. Dominant territory owners seek out and attack other males that deposit competing scent marks in their territory and counter-mark by depositing their own urine nearby. Other males use these scent marks to recognise the territory owner and will usually flee if they meet the owner or may avoid entering a scent-marked area if the owner has defeated them in the recent past. This considerably reduces the need for aggressive defence by the owner. However, when a male does not defend his territory very effectively, other mice detect the presence of competitor scent marks and are themselves more likely to challenge the owner for dominance of the territory.
Because scent marks remain in the environment, they provide a continuous record of the outcome of competitive challenges between males. This is used by females to select high quality mates. Volatile pheromones in the urine of dominant territory owners are strongly attractive to females. Females prefer males with exclusively scent-marked territories or those that have successfully counter-marked challenges from other males. They avoid inbreeding by preferring mates that smell dissimilar from their parents. Subordinate males, in response to repeated defeat by a territory owner, reduce the concentration of volatile male pheromones in their urine that provoke male aggression. They also greatly reduce their scent marking to avoid competing with the territory owner, but must continue to deposit a small number of scent marks to be recognised and tolerated as a familiar resident. The cost of this is that they are forced to advertise their subordinate status through their scent marks and they are no longer attractive to females.
Pattern of scent marks on the cage floor (traced after 30min) when two males were first singly-housed and subsequently established a dominance relationship and were placed on either side of a mesh barrier to measure scent marking behaviour. The subordinate male suppressed scent marking and deposited urine in a large pool away from the dominant male. Traces were obtained by Dr Caroline Payne.
Two olfactory systems
In addition to the main olfactory system that detects airborne volatiles, most mammals, reptiles and amphibia have a secondary olfactory system termed the vomeronasal system, although this is non-functional in humans. This secondary system is activated when animals make contact with a scent source and allows them to detect involatile as well as volatile scent components. The main olfactory system responds to a very broad range of odorants in the environment (eg food odours) while the vomeronasal system responds specifically to pheromones used for communication. In mouse urine, volatile pheromones are bound to involatile proteins that delay their release from scent marks. When mice detect an interesting scent in the air through their main olfactory system, their immediate response is to approach the scent source and sniff very closely. A vascular pumping mechanism is then activated that draws stimuli into paired vomeronasal organs which sit inside bony capsules just inside the nostrils. Contact investigation thus provides animals with much more information through the vomeronasal system than available from airborne odorants detected through the main olfactory system and many of the behavioural and priming effects of social odours depend on the ability to contact the scent. However, airborne volatiles are essential to detect the presence of a scent and to determine whether a scent is novel and requires further close contact investigation.
Table 1. Scent information in mouse urine
Many genetic and non-genetic factors contribute to each animals volatile scent profile (Table 1). To be useful, animals need to be able to distinguish between information about the scent owners identity and its current status. The proteins and peptides in urine provide each individual with a genetically determined identity signature that indicates their species, sex and individual identity, in addition to other information such as the owners kinship and MHC type, which remain fixed for life. The major urinary proteins (MUPs) and MHC peptides are so highly polymorphic among outbred wild mice that they provide each individual with an identity signature that is essentially unique. Mice can also learn to recognise the complex volatile profile of familiar individuals so that they can be recognised quickly from a distance from airborne scents. However, this volatile profile is less stable as it is also influenced by current status and environmental factors. Changes in the volatile scent profile induce further close contact investigation, which allows mice to confirm the identity of the scent owner through their involatile genetically-fixed identity signature.
The pattern of major urinary proteins (MUPs) expressed by different individual males captured from two populations. MUPs have been separated by isoelectric focusing according to their charge and stained with Coomassie Blue. Each individual has a different pattern. Gels run by Dr Caroline Payne.
The aggressive competition for breeding opportunities among males and their use of scents to advertise status and competitive ability have a number of consequences for aggression and welfare among laboratory mice. When males are confined together in cages, it is particularly important to avoid situations that provoke intolerant and injurious aggression through careful husbandry practices and to be aware of experimental treatments that might provoke an increase in aggression.
Aggression and scent communication among laboratory mice
The inbreeding of many strains of laboratory mice means that all individuals of the same sex and strain share the same genetically-determined scent signature. The effect of this is to generally lower levels of aggression between group-housed males of the same strain, as all males share the same signature as highly familiar group members. Problems of aggressive intolerance are thus usually greater among outbred males, although aggression within inbred strains can be increased by exposure to scents from a different strain or from the opposite sex. Some inbred strains are also genetically predisposed to high levels of aggression (eg SJL and BALB/c strains).
From both a management and welfare perspective, it is important to house animals in social groups whenever possible. Although female mice show little overt aggression (except towards intruders when pregnant or lactating), males of many laboratory strains can be highly aggressive under some circumstances. Apparently spontaneous outbreaks of intolerant aggression can occur even in relatively low aggression strains, resulting in serious wounding or even fatalities. Management practices can considerably reduce these problems by avoiding the stimuli that provoke aggression in all but completely non-aggressive strains.
In addition to the many influences of scent on behaviour,
urinary scents from other mice in the local population prime
the reproductive physiology and development of each individual
according to the opportunities for reproduction. These effects
can be stimulatory or inhibitory depending on the sex of both
urine owner and recipient and their familiarity (Table
Table 2. Reproductive priming effects of uringe and soiled bedding in laboratory mice
Cells shaded green are stimulatory effects; those shaded lilac are inhibitory. See Koyama (2004) for a detailed review.
Male scents on females: Male scents generally stimulate female reproductive physiology, bringing females into oestrus so that they are ready to mate. Young females may attain first oestrus up to 10 days earlier than normal when exposed to unfamiliar male scents (the familiar scent of their father has no such effects). If anoestrus adult females are allowed contact with male urine or soiled bedding, this initiates the oestrus cycle so that most females come into oestrus three days after exposure. This induction and synchrony of oestrus in adult females may be useful for timed matings in the laboratory or to ensure that females are at the same stage of the oestrus cycle in experiments. All of these stimulatory effects are induced by male pheromones acting through the vomeronasal system. Nasal contact with a male or his urine is therefore important to induce these effects and it is unlikely that airborne volatiles in the animal unit will have significant effects. However, contact may occur when soiled substrate is kicked out of cages and falls into those below, when animals of the opposite sex share soiled handling facilities, or by accidental transfer of their sticky urine via hands or equipment.
Importantly, contact with unfamiliar male urine can block pregnancy in a newly conceived female if she encounters the scent before the embryos implant into the wall of the uterus, returning her to oestrus so that she is available to mate again. However, continued contact with fresh scent from the male she has mated with helps to protect the pregnancy by maintaining prolactin levels so that embryos implant successfully. Urine from a male of the same inbred strain as the sire will not be detected as unfamiliar and will not block pregnancy as this shares the same individual identity cues as the sire.
Male scents on males: Less is known about reproductive priming effects on males, but scents from adult males generally have inhibitory effects on reproductive behaviour and sperm in other males, in addition to stimulating aggression. Urine from aggressive dominant males appears to be particularly potent in suppressing young and subordinate males.
Female scents on females: The effects of female scents depend on whether these come from reproductively active females or from non-reproductive females that live under crowded conditions. In crowded groups, non-reproductive females produce a pheromone (2,5-dimethylpyrazine) which affects other females by delaying puberty in young females and inhibiting oestrus cycling in adults. The greater the density of females and the more prolonged the exposure to this pheromone, the stronger the inhibitory effect. In wild populations, offspring survival is considerably reduced in overcrowded nest sites so this delays investment in offspring until females find more favourable conditions. In the laboratory, caged groups of more than three females generally produce this inhibitory pheromone but those in groups of 2-3 continue to cycle normally. Wild females prefer to nest communally with one or two other females as this increases offspring protection and survival. Correspondingly, urine from pregnant, lactating or singly housed oestrus females stimulates reproductive cycling in other females (see Table 2) and females are strongly attracted to nest with familiar companions. Single housing of females can lead to severe stereotypies and other abnormal behaviour which may have a significant impact on their responsiveness and variability in experiments.
Female scents on males: Exposure of males to female scents increases androgen production, stimulating faster maturation and increasing the aggressiveness of adult males. Bedding soiled by females also increases the number of sperm, particularly if the bedding comes from group-housed females. However, exposure to 2,5-dimethylpyrazine, which is produced at high levels by crowded non-reproductive females, has a negative influence, suppressing maturation of male reproductive organs and possibly suppressing their immunocompetence.
Controlling contact with priming pheromones is thus an important management consideration to reduce variability in physiology. The number of females housed in the same caged group is a particularly important consideration the physiological status of those housed in small groups or singly will differ considerably from those housed in larger groups. Group size should therefore be considered carefully as part of the experimental design. The induction of oestrus by male scents may be a useful management tool not only in husbandry but also to synchronise female status in experiments.
Many members of my research group and collaborators have contributed to our understanding of scent communication in mice, but Prof Rob Beynon, Dr Charlotte Nevison, Julia Lacey, Dr Mike Thom, Dr Richard Humphries, Dr Duncan Robertson, Dr Nick Malone, Dr Caroline Payne, Sarah Cheetham, Dr Samantha Gray and Dr Tracey Rich particularly contributed to the data and ideas summarised in this article. Thanks also to Dr Mike Thom for the image of an inbred mouse against a mirror and Dr Caroline Payne for images of status-dependent scent marking and MUP patterns.
Scent communication in mice
Reproductive priming pheromones
All views and opinions expressed in this article are those of the author and do not necessarily reflect the views and opinions of the NC3Rs.
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