Rats can discriminate between the urine odors of genetically identical mice maintained on different diets

Physiology& Behavior,Vol. 51, pp. 1079-1082, 1992

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Rats Can Discriminate Between the Urine Odors of Genetically Identical Mice Maintained on Different Diets H E A T H E R M A C I N T O S H S C H E L L I N C K , A N N M A R I E W E S T A N D R I C H A R D E. B R O W N I

Department o f Psychology, Dalhousie University, Halifax, Nova Scotia, Canada B 3 H 4J1

Received 21 August 1991

SCHELLINCK, H. M.. A. M. WEST AND R. E. BROWN. Rats can discriminate between the urine odors of genetically identical mice maintained on different diets. PHYSIOL BEHAV 51(5) 1079-1082, 1992.--Male Long-Evans hooded rats were tested in a habituation--dishabituationprocedurefor their abilityto discriminatebetween the urine odors of male C57BL/6J mice maintained on two different diets. There were discriminable differences between the urinary odors of two individual mice maintained on different diets. The rats did not dishabituate when presented with urine odors from two individuals on the same diet or two odor samples from the same individual. These results indicate that individual urinary odors of geneticallyidentical mice are influenced by diet. We discussthe hypothesisthat diet may act together with genetic differences,commensal bacteria, and hormonal changes to convey olfactory information used for individual recognition. Individual odors

Urine

Diet

Mice

Mus musculus

URINE odors are used for individual recognition by many species of mammals (7,15). In both rats (12,23,26) and mice (2,31,32) these individual urinary odors have been linked to genetic differences at the major histocompatibility complex (MHC). Congenic strains of rats and mice, which are genetically identical except for the genes at one or more MHC loci, can be discriminated by their urinary odors (2,9,10,32). Likewise, rats are able to discriminate between individual mice by their urine odors (2,3,24). The MHC genes code for glycoproteins which are incorporated into the cell membrane of nearly all cells in the body. Because of the extremely polymorphic nature of these MHC genes, it is unlikely that two nonidentical individuals would have the same MHC type (18). Thus, it is conceivable that the MHC could impart an odor of individuality to each animal and that this odor would reflect its genetic constitution. Since this MHCdependent individual odor is expressed in the urine (26,31), it could be used as a mechanism for phenotype matching in kin recognition (l 6,29,30). A genetic basis for individual odors provides a consistent mechanism for indicating individuality which is not subject to variation over time. However, factors other than the genes of the MHC also influence the urinary odors of Requests for reprints should be addressed to Richard E. Brown.

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rodents. For example, the sex chromosomes and the background genes (i.e., non-MHC regions) contribute to the individual odors of mice (4,34). In rats, commensal bacteria are also important in determining the unique urinary odor. The urine odors of MHC congenic rats reared in a germfree environment can not be consistently discriminated (25,27). Thus, it would appear that, in rats, bacteria interact with the genetic differences at the MHC to produce individually distinct urinary odors (11). To account for this finding, we hypothesized that the gastrointestinal bacteria act on dietary products to produce a pool of volatile metabolites which are carried to the urine by the class I MHC antigens (27). Dietary changes alter the urine odors of guinea pigs (1) and the feces odors of rats, mice, and gerbils (5,13,14,28). The urine ofgermfree rats is qualitatively and quantitatively different from that of conventionally reared rats (17) and since some urinary components are formed by the action of gastrointestinal bacteria on dietary products (22), we hypothesized that dietary changes might alter individual urinary odors independently of genetic differences. To test this, we examined the effects of diet on individual odors in a strain of genetically identical mice.

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,S'uhject,~ The subjects were 24 male Long-Evans hooded rats purchased from Charles River (Quebec) when 70-90 days of age. They were housed individually in 25 × 19.5 × 18.5 cm Wahman hanging cages in a colony room on a 12:12 reversed light:dark cycle, with lights off from 0800 h to 2000 h. Purina lab chow and water were provided ad lib throughout the experiment. Each subject was tested when about 120-150 days of age.

diet. All tests were conducted under dim white light during the dark phase of the light-dark cycle.

Statistical Analysis A randomized blocks ANOVA was used to examine ditti:rences in time spent sniffing the odor samples over the nine trials, and Newman-Keuls post hoc tests were used to determine which means differed from each other. RESUL'|S AND DISCUSSION

Urine Donor~ The urine donors were C57BL/6J mice born in our laboratory to mated brother/sister pairs of mice purchased from the Jackson Laboratory (Bar Harbor, ME). The urine donors thus had an H-2 b MHC (H-2) type. The urine donors were weaned at 21 days of age and housed in all-male littermate groups until 70 days of age when they were housed individually in 29 × 18 × 12.5 cm plastic cages with wood chip bedding. All mice were housed in a colony room on a 12:12 reversed light:dark cycle, with lights off from 1130 h to 1930 h. Urine donors and their parents were maintained on one of two different diets, either Purina lab chow (Diet P) or Hagan hamster food (Diet H). The Purina diet had 25.3% crude protein, 4.8% fat, 58.1% carbohydrate, 1.02% calcium, and 0.84% phosphorous. The Hagen diet had 14.7% crude protein, 15.9% fat, 65.2% carbohydrate, 0.28% calcium, and 0.42% phosphorous. The Purina diet has an energy value of 4072 calories per gram, while the Hagen diet has an energy value of 4802 calories per gram.

Urine Collection Urine samples were collected daily from six different individual male mice on each diet during the light phase of the L: D cycle. Samples were obtained by holding the mouse over a collecting vial and gently stroking the abdomen above the bladder. This method guaranteed fresh urine samples which were not contaminated by feces. Samples were identified by donor and collection date and stored in 1.5 ml Canlab collection vials and frozen at - 2 0 ° Celsius until required.

Apparatus and Procedure The habituation-dishabituation apparatus described previously (8) was used in this experiment. This was a 38.5 X 32 × 17.5 cm opaque plastic box covered by a 35.5 x 32 X 17.5 cm wire mesh top. A 5.5 cm circle of filter paper (Whatman #1) was taped to the center of one side of the mesh top and 0.10 ml of water or urine was placed in the center of the filter paper from a syringe. The time spent rearing and sniffing the stimulus odor was recorded over a 2-min trial with a stopwatch. After each odor presentation, the mesh top was removed, cleaned with water, and air-dried before being reused. Between subjects, the testing chamber was cleaned with a 70% ethyl alcohol solution. Each subject was tested once in a series of nine 2-min odor presentation trials, three with water, three with urine sample 1, and three with urine sample 2. Each group of eight subjects received one of three different urine odor pairs. These were urine from: (A) two male mice on different diets; (B) two male mice on the same diet; and (C) two samples from the same male mouse. In the first two tests, half of the subjects received urine samples in the order AB, and half in the reverse order. In the third test, half of the subjects received urine from mice on the Purina diet and half received urine from mice on the Hagen

Rats could discriminate between the urine odors of C57BL/ 6J male mice on two different diets [Fig. I(A)]. There were significant differences among the nine means for all eight subjects, F(8, 63) = 4.53, p < .001, and more time was spent sniffing the odor samples on trials 4 and 7 than on any other trials (p < .05). Hence, the two urinaD' odors were discriminable. Rats did not dishabituate when presented with the urine odors of individual mice on the same diet [Fig. I(B)]. There were significant differences among the nine means for all eight subjects, F(8, 63) = 9.50, p < .001, but only trial 4 significantly differed from any other trial (p < 0.05). Thus, subjects dishabituated when presented with one sample of urine after the water presentations, but did not respond differently to the second urine odor from a

10 9 8 7 ~, 6

A. Different Diets

g5 ~- 3 2 1

0 9 8 7 "~ 6 q) " 5

B. Same Diets

3 2 1

0 9

C. Same individuals

8

7 5 3 2 1

%

0 l Water

Urine I

Urine 2

FIG. 1. Mean time (s) -+ SEM spent rearing and snitfing urine odors from (A) two mice on differentdiets; (B) two mice on the same diet; and (C) two urine samples from the same individual mouse.

DIET INFLUENCES URINE ODORS OF MICE

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different individual maintained on the same diet. Likewise, rats did not increase their sniffing time when presented with the second of two urine samples from the same individual maintained on either Diet P or Diet H [Fig. I(C)]. There were significant differences among the nine means for all eight subjects, F(8, 63) = 23.90, p < .001. However, only on trial 4 did the investigative time significantly differ from any other trial (p < 0.05). The results indicate that male Long-Evans hooded rats are able to discriminate between the urinary odors of genetically identical mice maintained on different diets, and that this discrimination can be made regardless of day-to-day variations in the odors of individuals which may occur due to physiological or environmental changes (25). This result indicates that diet should be taken into account when considering the factors involved in the production of unique individual odors. The two diets used differ in their five main nutrients and in their energy value. It is possible that these differences produce variations in the metabolites excreted in the urine, thus contributing to individual differences in urinary odors and conveying information on individuality. It is well known that specific nutrients can create a distinctive urine odor. For example, in humans, the consumption of asparagus produces the odor of rotten cabbage in the urine (21) and disorders of amino acid metabolism such as phenylketonuria and maple syrup urine disease result in characteristic urine odors (20). The elimination of these urine odors after dietary changes which reduce the intake of the specific proteins involved further emphasizes the role of diet in creating distinctive urinary odors. It is unlikely that a dietary factor alone could provide a consistent mechanism for individual recognition. Indeed, the results of previous studies suggest that individual urinary odors are produced primarily by genetically controlled metabolic variations (2,4,32). An environmental factor, such as diet, would vary according to season and locale in which an individual resides. Genetic components would not be subject to such variability.

However, odor production may be strongly affected by diet (1,14) and variations in diet may convey information of value in the recognition of individuals. Differences in diet may also be useful in the identification of kin because individuals would be more familiar with an odor which reflects a diet similar to their own. Individual recognition may be influenced by other nongenetic factors as well. For example, sex hormones alter an animal's odor (6,7) but individual rats can be discriminated whether they are gonadally intact or gonadectomized (8). In order for olfactory cues to be used for individual or kin recognition by phenotype matching, the odors should provide a consistent representation of the genetic differences between individuals (19,29,30). Odor differences based on genetic differences between individuals provide a direct mechanism for individual or kin recognition while odor differences based on dietary differences would provide an indirect mechanism for individual or kin recognition (29) and both of these mechanisms may act together. The urinary odor of individuality appears to be determined by a complex interaction of genetic, hormonal, microbiological, and dietary factors. Although there is no evidence that bacteria are essential for the production of the unique urinary odors in mice, as occurs in rats (24,33), the fact that diet alters urine odors as well as feces odors in mice (5,13) indicates that factors other than genetic differences must be active in producing discriminable differences in the urinary odors of individual mice. This suggests that an unchanging genetic component in conjunction with certain environmental factors may be operating to produce urinary odors of individuality. ACKNOWLEDGEMENTS We thank Dr. Derek Anderson of the Department of Animal Science at the Nova Scotia AgriculturalCollege, Truro, NS, Canada, for the feed analysis. This research was supported by the NSERC of Canada grant No. A7441 to REB. HMS was supported by a NSERC postgraduate scholarship.

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