Animal Learning & Behavior/997,25 (2), /93-/99

Odors of individuality originatingfrom the major histocompatibility complexare masked by diet cues in the urine of rats

HEATHER MACINTOSH SCHELLINCKDalhousie University, Halifax, Nova Scotia, Canada

BURTON M. SLOTNICKAmerican University, Washington, D.C.

and

RICHARD E. BROWNDalhousie University, Halifax, Nova Scotia, Canada

Male Long-Evansrats were trained to discriminate between the urine odors of two congenic strainsof rats (PVG and PVG.Rl) in an olfactometer on a go/no-go operant task with water reinforcement.These odor donors differ genetically at only one locus of the major histocompatibility complex (MHC).The ability of the subjects to transfer their training to discriminate between novel individuals of thesame MHC strain on the same and different diets was then examined. The subjects correctly general-ized their original training to new samples from previously undiscriminated individuals of the sameMHC type on the same diet without a significant drop in performance. A change in diet disrupted theperception ofthe MHC-related odor. In a second experiment, rats were trained to discriminate betweenthe odors of pairs of genetically identical PVG or PVG.Rlrats on different diets and were tested for gen-eralization using the odors of rats of the other strain (PVG.Rl or PVG) maintained on the same diet.Changing the strain of the odor donor did not disrupt the learned discrimination. The results of thesetwo experiments confirm the ability of the MHC to generate constant cues in the urine but also revealthat variable diet cues disrupt the perception of these cues. The relationship between the MHC, diet,and other factors in determining individual odorsis discussed.

The urine odors ofmammals contain information aboutthe species, sex, age, dominance status, reproductive sta-tus, and individual identity of each animal. These odorsare determined by genetic, hormonal, bacterial, anddietary factors (Brown, 1979; Brown & Schellinck,1992). Some of these factors, such as genetic differences,are constant over time. Odors related to bacteria and hor-mone levels may also be consistent for long periodsof time, but other cues, such as those provided by diet,may change over time. It has been hypothesized that theconstant cues provide an odor of individuality (Brown,1979; Halpin, 1986, 1991). In mice and rats, the majorhistocompatibility complex (MHC) is recognized as a ge-netic locus which is important in determining these indi-vidually unique odors (Beauchamp, Gilbert, Yamazaki,& Boyse, 1986; Brown, Roser, & Singh, 1990; Ya-mazaki, Beauchamp, Bard, & Boyse, 1990). However,the sex chromosomes (Yamazaki et aI., 1986) and autoso-mal genes (Beauchamp, Yamazaki, Duncan, Bard, &Boyse, 1990; Eggert, Holler, Luszyk, & Ferstl, 1995;

Correspondence and reprint requests should be addressed to R. E.Brown, Department of Psychology, Dalhousie University, Halifax, NS,Canada B3H 4Jl (e-mail: rebrown@is.dal.ca).

Monahan, Yamazaki, Beauchamp, & Maxson, 1993;Schellinck, Monahan, Maxson, & Brown, 1993) alsoprovide consistent cues in the urine which may influenceodors of individuality.

To demonstrate that the MHC is a source of constantcues for the individual odor ofmice, Yamazaki and his col-leagues showed that mice trained to discriminate betweenthe odors of urine samples from MHC-congenic mice ina Y-maze could generalize their learning to novel odorsamples from previously undiscriminated individuals ofthe same strains (Yamaguchi et aI., 1981; Yamazaki et aI.,1990; Yamazaki et aI., 1983). Wehave used a habituation-dishabituation task and discrimination training in an op-erant olfactometer to investigate the role of the MHC indetermining the origin of individual odors in rats (Brown,Singh, & Roser, 1987; Schellinck, Brown, & Slotnick,1991; Singh, Brown, & Roser, 1987). Although these ex-periments demonstrated that the odors ofMHC-congenicrats are discriminable, we have not established that ratsuse a consistent odor cue to make this discrimination. Anypair ofurine odors may contain many odor-related cues be-tween which rats could learn to discriminate. If the MHC-related odor is constant across time or across all rats ofthat MHC type, rats should be able to learn to discriminateone pair ofMHC-related odors and then transfer this learn-

193 Copyright 1997 Psychonomic Society, Inc.

194 SCHELLINCK, SLOTNICK, AND BROWN

ing to other pairs ofthe same odor types, as has been dem-onstrated in mice.

Our experiments have shown that different methods oftesting for odor discrimination may lead to different re-sults when the same pairs of odors are used. For exam-ple, rats in a habituation-dishabituation task do not dis-criminate between urine odors from two individuals ofthe same inbred strain, nor do they discriminate betweentwo urine samples from the same individual. In the op-erant olfactometer, however, thirsty rats can learn tomake both of these discriminations in order to get a waterreward (Schellinck & Brown, 1995). In another experi-ment, rats trained to discriminate between the urine odorsof two individual rats did not respond to probe trials ofthe odors ofthese rats after their gut bacteria were elim-inated. This led us to believe that removal of these bacte-ria made the urine odor unrecognizable. Yet, when thesame odors were presented in a discrimination learningtask, the discrimination was learned significantly fasterthan the discrimination between two novel odors. This"savings-effect" indicates that the odors of the bacteria-depleted rats were recognized (Schellinck & Brown,1995). Thus, lack ofgeneralization from one odor to an-other does not necessarily mean that the odors were notrecognized, and it suggests that the outcome of general-ization trials may be difficult to interpret. In the presentexperiments, therefore, we used a transfer-of-trainingparadigm to assess whether rats would be able to learn torecognize odor cues in the urine of MHC-congenic ratsand to transfer this recognition to other individuals of thesame strain.

The influence of variable cues, such as those providedby a change in diet, on the discrimination of geneticallybased MHC-related urine odors was also investigated.Rats found it easier to discriminate between the urineodors of MHC-congenic mice on different diets thanto discriminate between the urine odors of MHC-con-genic mice on the same diet (Brown, Schellinck, & West,1996). Moreover, it was easier for rats to remember thedifference between the urine odors ofgenetically identicalmice on different diets than it was for them to rememberthe difference between the urine odors of MHC-congenicmice on the same diet. This result was contrary to our hy-pothesis that genetic and dietary cues would contributeadditively to the discriminable odors found in the urine ofmice.

We proposed two hypotheses to account for the find-ing that diet-related cues were remembered more easilythan the MHC-related cue. First, the constant genetic cuemay have been masked by the diet cues. Second, as therats were discriminating the odors ofmice rather than theodors of rats, they may have been more attentive to di-etary than to MHC cues. In the present study, therefore,we used the transfer-of-training paradigm to investigatethe influence of diet on the ability of rats to maintain alearned discrimination ofurine odors from MHC-congenicrats. We also examined the role of genetic cues in dis-rupting the performance of rats that had learned to dis-

crimmate between the urine odors of genetically identi-cal rats on different diets.

EXPERIMENT 1

MethodSubjects. Long-Evans male rats (n = 6), purchased from Charles

River Canada (St. Constant, Quebec) at a weight of 250-275 g,were individually housed in 48 X 27 X 16 em polycarbonate cageswith stainless steel grid covers and woodchip bedding and main-tained on a reversed 12:12-h light:dark cycle, with lights off at 9 a.m.Ten days prior to odor discrimination training, their water intakewas restricted to 10-15 ml per day for the duration ofthe experiment.

Urine donors. The urine donors were PVG (n = 8) and PVG.Rl(n = 7) male rats purchased from Harlan Olac Ltd., Shaw's Farm,Blackthorn, England, at 6 weeks of age. They were housed underthe same conditions as were the test subjects. These two strains ofrats differ genetically at only one locus of the class Ia locus of theMHC, and individuals of the same strain are identical to each other(Brown et al., 1990).

The urine donors were placed on a diet ofPurina Lab Chow, and,4 weeks later, their urine was collected daily for 1 month by plac-ing them in a 24 X 19.5 X 18.5 em metabolism cagefor 8 h per dayduring the dark phase of the light:dark cycle. The diet was thenchanged to Teklad fat sufficient test diet (TD 69446), and, after 2weeks, their urine was again collected daily for I month. They hadfree access to food and water except during the 8-h urine collectionperiod, when only water was available. The Purina diet had the fol-lowing constituents (in g/kg): crude protein, 253; fat, 48; carbohy-drate, 581; calcium, 10.2; and phosphorous, 8.4. The Teklad dietconsisted of the following (in g/kg): casein, 211; sucrose, 584.5;corn oil, 50.0; cellulose, 104.5;Mineral Mix, 40.0; Vitamin mix, 10.0.

Urine was collected in a stainless steel mesh-covered glass bot-tle placed directly beneath the metabolism cage and then trans-ferred to individually labeled 1.5-ml microcentrifuge tubes (Fisher-brand) and stored at - 30·C. The urine samples from the outbredLong-Evans rats on the Purina diet which provided the training andunknown stimuli were selected from a bank ofurine samples whichhad been collected previously according to the same procedure.

Preparation of odor samples. To eliminate the possibility thatdiscrimination between odor samples was a result ofextraneous dif-ferences in the concentration of the urine or inadvertent contami-nation with feces or foodstuffs, samples collected from the sameindividual for 4-5 days were pooled. Next, aliquots of samples fromtwo or three individuals of the same strain were combined. Suffi-cient combinations of samples were made so that different stimuliwere presented for each daily session. Five different donors con-tributed to the pools to ensure that no more than 2-3 subjects weretrained on combinations from the same donors or received samplesfrom the same individuals for the transfer of training tests.

Apparatus. Odor discrimination was evaluated in two computer-controlled operant olfactometers. Clean air, provided by a GASTcompressor pump, was filtered through activated charcoal and twofritted glass filters as it entered the olfactometer. The rate ofairflowwas regulated by Kontes No. 2needle valves and measured by PorterInstrument Co. flowmeters. The airflow was set at 4,000 cc/minthrough the clean air channel and 120 cc/min through the eight odorstimulus channels. The remainder of the olfactometer consisted ofglass and Teflon ductwork, which carried fresh air as well as thestimulus odors to an odor-sampling tube in the animal test chamber.Two-waysolenoid valves controlled the delivery of clean air to andodorized air from the stimulus jars.

The test chamber was a 26.5 X 17 X 21.5 cm Plexiglas box witha stainless steel grid floor and a removable Plexiglas top. One endof the test chamber contained the odor-sampling tube and a drink-ing tube. When the rat poked its nose into the odor-sampling tube,

MHC AND DIET CUES IN URINE ODORS OF RATS 195

it broke a photobeam, causing the solenoid valves to be activated andan odor to be delivered. The stainless steel drinking tube was con-nected to a water reservoir via a solenoid valve, which was wired tothe grid floor to provide a touch-sensitive circuit. Water reinforce-ment (0.02 ml) was delivered when the rat's licking response com-pleted this circuit. A digital interface built at Dalhousie Universityconnected the olfactometer to a computer which controlled the de-livery of odor stimuli and water reward to the subjects.

Procedure. The odor discrimination training was conducted infour phases: (I) nosepoke training; (2) odor versus no-odor train-ing; (3) two-odor discrimination training using odors from outbredrats; and (4) two-odor discrimination training using odors fromMHC-congenic rats. Following the training procedures, a transfer-of-training phase was initiated in which a number of new odor dis-criminations were introduced within the two-odor discriminationtask learned in phase four.

I. Nosepoke training. In this initial training procedure, rats learnedto obtain water reinforcement (0.02 ml) by initiating a nosepoke(0.7 sec) into the odor sampling tube and sampling the odor stimu-lus. During the 0.7-sec nosepoke, the odor and the main airflowwere diverted to the exhaust system to allow the odor to mix thor-oughly with the air prior to reaching the rat. The odor, urine froman outbred Long-Evans rat, was then presented within the cleanairstream for 2 sec. The subjects were required to sample the stim-ulus odor for at least 0.25 sec in order to receive water reinforcement.The training procedure was automated and computer-controlled asdescribed in Schellinck et al. (1991).

2. Odor versus no-odor training. As soon as the nosepoke train-ing was completed, odor versus no-odor training began. Subjectsobtained water reinforcement after they sampled the urine odorfrom the same Long-Evans outbred rat (S+) whose odor was usedin nosepoke training. On halfof the trials, no odor was present afterthe rat nosepoked, and on these S - trials, the rat did not obtainwater reinforcement if it licked the drinking tube. Failure to main-tain a nosepoke for 0.7 sec or failure to sample the airstream for0.25 sec resulted in a 4-sec delay before another trial began. Sub-jects were given 200 trials, 100 S+ and 100 S-, per day. For pur-poses of analysis, the trials were divided into 10 blocks of 20 trials(10 S+ and 10 S-).

In this go/no-go paradigm, four types of response are possible: ahit or correct response to the S+ (licking the drinking tube); a missor no response to the S+ (not licking the drinking tube); a correctrejection or no response to the S- (not licking the drinking tube);or a false alarm or incorrect response to the S- (licking the drink-ing tube). Hits and correct rejections each contribute to the correctscore; misses and false alarms contribute to the incorrect score. Thecriterion for determining that a subject successfully acquired theodorlno odor discrimination was a minimum score of 85% correcton the first block of 20 trials and a mean of 85% for the remainingnine blocks of 20 trials. The subjects were tested daily until thislearning criterion was met for one session. Data were collected andrecorded via input to a computer from a digital interface.

3. Two-odor discrimination training with odors from outbredrats. Following successful acquisition of the odor/no-odor discrim-ination, the subjects were trained to discriminate between urineodors from two Long-Evans outbred rats. The parameters describedfor the odor/no-odor training were in effect in this and all subsequentphases of the experiment. Once the subjects reached the criterionfor discrimination learning, they were switched to a 50% partial re-inforcement schedule for the S+ odors. Because the S - odors werenever reinforced, this created a 25% reinforcement schedule over all200 trials. Stimulus presentation was controlled so that a maximumof six nonreinforced S+ trials were presented in succession.

4. TWo-odor discrimination trainingwith odorsfrom MHC-congenicrats. After the subjects had learned to discriminate between theurine odors of outbred rats, they were trained to discriminate be-tween urine odors collected from the two strains ofMHC-congenicrats (PVG and PVG.R I) maintained on a Purina diet. Odors from rats

of one strain were designated as the S+ for 3 of the subjects, andodors from rats of the other strain were designated as the S+ for theother 3 subjects. Only one pair of odors was presented each day. Fol-lowing successful acquisition of the learning criterion, subjectswere placed on the partial reinforcement schedule described inPhase 3.

5. Transfer oftraining tests. The transfer of training tests beganas soon as the learning criterion was met for discriminating betweenodors from MHC-congenic rats. The test sessions were divided intotwo phases. During the first five blocks (100 trials), subjects wererequired to discriminate between S+ and S- urine samples fromMHC-congenic donors used in Phase 4. Then, prior to the start ofBlock 6, the test odors were introduced by exchanging the stimulusjars used on Blocks 1-5 with stimulus jars containing the newodors. The purpose of this exchange was to determine whether thesubjects could generalize their odor discrimination performance tothe newly introduced odor stimuli. The partial reinforcement sched-ule used in Phases 3 and 4 was continued in the transfer oftrainingsession. The S+ odor was not reinforced for the first five presenta-tions on Block 6 for any of the test odor pairs.

The following pairs of odors were presented to each of the sub-jects on Blocks 6-10 ofthe generalization phase ofthe test session.

TaskA (same stimuli). The two S+ and S- samples presented onBlocks 1-5 were removed and immediately replaced to determinewhether the switching procedure caused any disruption.

Task B (known stimuli). The new S+ and S- samples were com-posed of novel combinations of urine pools from previously dis-criminated individuals of the same MHC-congenic strains.

Task C (same strain stimuli). The new S+ and S- samples werecomposed of urine pools from previously undiscriminated individ-uals of the same MHC-congenic strains.

Task D (new diet stimuli). The new S+ and S- samples werecomposed ofurine pools from previously discriminated individualsafter their diet had been changed to the Teklad test diet.

Task E (unknown stimuli). The new S+ and S- samples werecomposed of urine pools from two previously undiscriminated out-bred Long-Evans rats.

Each transfer of training task was presented on a different testday. The first three generalization tests (Tasks A, B, and C) werepresented in a quasi-random order such that only 2 ofthe 6 subjectsreceived the same task on the same day. All subjects were testedwith the unknown stimuli (Task E) on the 4th day oftesting and thenew diet stimuli (Task D) on the 5th day of testing.

Statistical analysis. A repeated measures analysis of variancewas used to compare the difference in the number oferrors betweenBlocks 5 and 6 over the five tasks. Newman-Keuls post hoc testswere used to determine which means differed from each other.

ResultsAll subjects were able to reach the criterion for dis-

criminating between the odors from MHC-congenic ratswithin 6-7 training sessions in Phase 4.

The subjects averaged 97% correct on Block 5 of thetransfer-of-training sessions over all five tasks, but whennew stimuli were introduced on Block 6, there were signif-icant differences in performance among the tasks. The pro-cess ofremoving the samples in mid-session and replacingthem immediately (Task A) did not disrupt performance,and rats made virtuallyno errors on Blocks 5 or 6 ofthis task.Likewise, there was very little drop in performance onBlock 6 in Tasks Band C, but the mean scores on Block 6for Tasks D and E were 42% and 45%, respectively.

As shown in Figure 1, there was a significant differencein the number of errors between Blocks 5 and 6 amongthe five tasks [F(4,20) = 12.5, P < .001]. Post hoc analy-

196 SCHELLINCK, SLOTNICK, AND BROWN

14 EXPERIMENT 2

With the procedure described in Experiment 1, it shouldbe possible to determine whether the ability of rats todiscriminate between the urine odors ofgenetically iden-tical rats on two different diets is disrupted when thestrain of the urine donor is switched to that of an MHC-congenic rat on the same diet as that of the previousdonors. If the performance of the rats is disrupted whenthe strain of the odor donor is changed, this would pro-vide evidence that MHC-related odor cues are attendedto as much as the dietary cues.

MethodSubjects. Seven Long-Evans male rats obtained from Charles

River Portage (Portage, MI) at a weight of 250-275 g were main-tained under conditions identical to those described for Experiment I.

Urine donors. The rat urine donors were the same as describedfor Experiment I. Urine from two MHC-congenic strains of micewhich differ at only one locus of the MHC (C57BL/6-H-2Kb/J andC57BL/6-H-2Kbml/ByJ) and which were maintained on a Hagendiet was collected as described in Brown et al. (1996) and used forthe unknown odors.

Apparatus and Procedure. The apparatus was the same as de-scribed for Experiment 1. Phases 1-3 of the procedure were con-ducted as in Experiment I. In Phase 4, the subjects were trained todiscriminate between urine odors from genetically identical rats(PVG or PVG.RI) on two different diets, Purina Lab Chow or theTeklad test diet. Four of the subjects were trained to discriminatebetween rats of the PVG strain on the two different diets. Donorsmaintained on Purina Lab Chow provided the S+ odors for 2 sub-jects; the S- odors were collected from donors maintained on theTeklad test diet. The valence of the stimuli was reversed for the other2 subjects. The remaining 3 subjects were required to discriminatebetween rats of the PVG.RI strain on the two different diets. For2 subjects, the S+ odor was from donors eating a Purina diet and theS- odor was from donors eating a Teklad diet. The S+ and S-odors were reversed for the 3rd rat.

Once the subjects reached the criterion of 85% correct on eachblock of trials in a session on a 50% partial reinforcement sched-ule, Phase 5 was initiated in which the following test pairs of odorswere introduced on Block 6 of one of the next four sessions

TaskA (same stimuli). The same S+ and S- urine samples dis-criminated on Blocks 1-5 were presented.

TaskB (known stimuli). The new S+ and S- urine samples werecomposed of novel combinations of urine pools from previouslydiscriminated rats of the same strain on the same two diets.

TaskC (different strain stimuli). The new S+ and S- urine sam-ples were composed of urine pools from previously undiscrirni-nated individuals ofa different MHC type on the same diets. TheseS+ and S- donors had been maintained on the same diets as the S+and S- donors that each subject had previously learned to dis-criminate. The donors of these odor stimuli were MHC-congenic tothe previous donors. Thus, rats trained with PVG donors were givenodors from PVG.RI donors on the same diets and vice versa.

Task D (unknown stimuli). The new S+ and S- urine sampleswere composed ofurine pools from a different species on a com-pletely unknown diet (two previously undiscrirninated MHC-congenic mice maintained on a Hagen diet).

All four tasks were presented in a quasi-random order such thatno more than 2 individuals were tested on the same task in the samesession. As in Experiment I, the new odors were introduced by ex-changing the stimulus jars as described for Experiment I and the S+odor was not reinforced for the first five presentations on Block 6,for any of the odor pairs.

UNKNOWNSANE

10

12

4

en

~'"w

KNOWN SANE DIfFERENTSTRAIN DIET

ODOR TYPE

Figure 1. The mean (+SEM) difference between the number oferrors (Block 6 minus Block 5) after the urine odors from MOe-congenic rats on the same diet were replaced with the same sam-ples, known samples, samples from individuals of the samestrains, samples from the same strains fed different diets, or sam-ples from unknown outbred rats.

o-l-

MHC AND DIET CUES IN URINE ODORS OF RATS 197

ResultsAll subjects were able to discriminate between the odors

ofgenetically identical rats maintained on different dietswithin 3-5 sessions. In the transfer-of-training tests, sub-jects scored an average of99.4% correct on Block 5 overall four tasks, but there were significant differences inperformance on Block 6. In Task A, the process ofswitch-ing the same samples in mid-session and replacing themimmediately did not disrupt performance, since none ofthe subjects made any more errors on Block 6 than onBlock 5. There was very little drop in performance onBlock 6 in Tasks Band C, but the mean score on Block 6on Task D was 56%.

As is shown in Figure 2, there were significant differ-ences in the number of errors between Blocks 5 and6 among the four tasks [F(3,18) = 212.4, p < .001]. Posthoc analysis indicated that performance did not differwhen the same samples were reintroduced (Task A); whensamples were from novel pools of urine from knowndonors (Task B); or when samples were from unknownindividuals ofa different MHC-congenic strain which wereon the same diets as the donors on Blocks 1-5 (Task C).Performance dropped significantly on Block 6 when urinesamples from unknown MHC-congenic mice on an un-known diet were introduced (Task D) (p < .01).

Thus, after the rats learned to discriminate betweenthe urine odors of genetically identical rats on differentdiets, they were able to transfer this learning to discrim-inate between urine samples from genetically identicalindividuals of an MHC-congenic strain on the samediet. This result demonstrates that the introduction ofstrain differences does not disrupt an odor discriminationbased on dietary cues. When both the diet and speciescues are changed, however, the discrimination is disrupted(Task D).

10

6

~ 4

iliFF.STRAINSSAME DIET

OOOAT'/PE

Figure 2. The mean (+SEM) difference between the number oferrors (Block 6 minus Block 5) after the urine odors from genet-ically identical rats on different diets were replaced with the samesamples, known samples, samples from individuals of a differentstrain-on the same diets, or samples from unknown MHC-congenicmice on an unknown diet.

DISCUSSION

The ability ofrats that have learned to discriminate be-tween the urine odors of MHC-congenic rats to transferthis training to samples from previously undiscriminatedindividuals of the same strains without a significant dis-ruption in performance has confirmed that differences atthe MHC can generate constant odor cues in rats. Sinceall odor donors were on the same diet and the urine sam-ples were pooled within strains to eliminate adventitiousdifferences, only the MHC cues differed, thus providingthe only discriminable cues for the subjects to attend to.These results corroborate Yamazaki and his colleagues'findings that mice generalize training from a learned dis-crimination between MHC-congenic mice in a Y-mazeto respond to probe trials from unknown mice ofthe samecongenic strains (Yamaguchi et aI., 1981; Yamazaki et aI.,1990; Yamazaki et aI., 1983).

Even though these results indicate that rats can trans-fer training from known odors to unknown odors fromMHC-congenic conspecifics, a change in diet disruptedthe perception of the MHC-related cues to the extent thatthe odors of individuals of the same strains were foundto be as different as odors from previously undiscrimi-nated outbred rats (Figure I). Moreover, the introductionofstrain differences did not disrupt a discrimination basedon dietary cues (Figure 2). These results support our pre-vious finding that differences in diet provide a moresalient stimulus for discriminating urine odors in ahabituation-dishabituation task than do MHC-relatedodors (Schellinck, 1995). These results suggest that ad-ditional factors must be considered when one assesses therole ofMHC-related odor cues in the production ofodorsof individuality and that these factors reflect the signifi-cant effect ofdietary cues in producing discriminable dif-ferences in urinary odors.

The role of dietary cues in providing discriminablebody odors in rodents has been well documented. Ratpups are able to discriminate between the whole bodyodors of their mothers and those of other lactating fe-males only when the two are maintained on different diets(Leon, 1974, 1975). Adult female spiny mice (Acomyscahirinus) prefer to retrieve pups born to mothers main-tained on the same diet as they are fed versus pups bornto mothers on a different diet (Doane & Porter, 1978).Dietary odors are also discriminable in the urine ofguineapigs (Beauchamp, 1976) and mice (Brown et al., 1996;Schellinck, West, & Brown, 1992) as well as the feces ofrats (Leon, 1974, 1975)and mice (Brown & Wisker, 1989)and the whole body and bedding odors ofgerbils (Skeen& Theissen, 1977).

Furthermore, the influence ofdiet can be produced pre-natally. When rat pups had only prenatal experience witha particular diet, they subsequently showed a preferencefor that diet (Hepper, 1988). Rabbits prefer the diet oftheir mothers at birth and at weaning (Hudson & Distel,

198 SCHELLINCK, SLOTNICK, AND BROWN

1995). Preference for diet cues acquired prenatally couldgive cues for later recognition ofkin. The type ofdiet mayalso influence mate choice, since an appropriate dietshould promote good health and contribute to the pro-duction of viable offspring.

The mechanism through which dietary factors pro-duce odor cues has not generally been investigated, al-though Leon (1974) suggested that the sucrose content ofthe Teklad diet may eliminate production of caecal bac-teria and thus eliminate the production of discriminableodors. Bacterial analyses of fecal samples from our urinedonors, however, revealed that the number ofcolonies ofboth gram negative and gram positive bacteria was higherin the feces of the donors when they were on the Tekladdiet than when they were on the Purina diet (Brown &Schellinck, 1995). Thus, we hypothesize that a change inodor resulting from quantitative or qualitative changesin bacteria is likely to be the basis of the differences inodors ofindividuals on these two diets. A general increasein bacteria could have provided quantitative changes inurinary volatiles, thus providing a basis for the discrim-inable differences between the urine odors. Moreover,since the metabolism of different carbohydrates by bac-teria also results in different end products (Dawes, 1992),it seems likely that qualitative differences in volatile urineproducts from bacterial action on the two diets may alsoprovide discriminable odor cues. Such a change in dis-criminability of odors after alterations in bacteria wouldcorroborate our hypothesis that the presence of bacteriais responsible for odors of individuality (Schellinck et al.,1991; Singh et aI., 1990).

Regardless of the mechanism involved in determiningthe change in odors, dietary cues took precedence overgenetic cues in these experiments. For an odor of indi-viduality to be meaningful, its uniqueness must be per-ceived in spite of changes in variable cues such as thoseprovided by different diets. Until recently (Brown et al.,1996), the influence of the MHC has been examined inisolation, in that only cues provided by the MHC wereavailable for discrimination. Although these experimentsdemonstrated that constant MHC-related cues are avail-able, they have not established that the odors derived fromdifferences at the MHC can still provide information inthe urine in the presence of variable cues.

Thus, our attempt to show that MHC-based cues remainsalient in the context of a change in diet has yielded anull result. There may be several explanations for this.First, if the addition of further constituents to a mixturecreates a new odor, this result should not be unexpected,since the discrimination task would be perceived by therats as a new problem. However, if the individual parts ofa mixture are perceived as independent units, the latterexplanation is not sufficient to explain the disruption inperformance ofthe rats when the diet cues were changed.Although the way in which an odor mixture is perceivedis not well understood, there is some indication thatsome of the individual components of an odor mixturecan be identified. Humans can identify at least four stim-

uli in a mixture, whether the stimuli are simple or com-plex odorants (Laing, 1995). Our own research has re-vealed that rats can detect more than one component inthe urine of a conspecific (Schellinck, 1995).

Second, it is possible that the diets used in this exper-iment may have produced more salient differences and,consequently, more ofa disruption to MHC-related cuesthan diets that are more similar to each other. Since rats areable to discriminate between concentrations of amyl ac-etate that differ by only 0.2% (Slotnick & Ptak, 1977), itseems likely that even minimal changes in urine odor couldhave an impact on odor discrimination. Nonetheless, inthe real world, the diet of a rat may vary from day to day,and it would be appropriate to determine whether dietarycues still overshadow genetic cues in such a situation.

Third, it may be that we are asking the question in aninappropriate context. In an olfactometer, rats are highlymotivated to perform and can make discriminations onthe basis of very subtle differences in odors. Moreover,rats trained in an olfactometer may find that odors pro-vided by dietary cues are more relevant to attend to thancues provided by the MHC. It will be necessary to assessthe response of rats in a social situation in which, for ex-ample, information regarding mate selection or mother-offspring recognition could be derived from the sameodor stimuli. For example, Porter, Mcfayden-Ketchum,and King (1989) examined the huddling response ofwean-ling spiny mice (Acomys cahirinus) and found that theyspent more time huddling with unfamiliar kin or non-kinon the same diets than with non-kin on different diets butspent even more time with familiar kin on the same diets.They concluded that diet and genotype contributed ad-ditively to recognition signatures in this social situation.

In a previous experiment, we have shown that the MHCand dietary odors do not appear to be additive (Brownet al., 1996). Nor does it appear that these cues provideequivalent information. The results of this experimentindicate that the diet odor overshadows the MHC odor. Itis possible that rats can utilize different components ofthe urine odor in different tasks. For example, the MHC-related odor may be used for mate selection and the di-etary odor for food selection. A rat may also act on dif-ferent information in the urine, depending on itsemotional or motivational state. For example, castratedmale rats can be trained to discriminate between theodors ofestrous as opposed to non-estrous females (Carr& Caul, 1962), but, in a preference test, they do not showa preference for the odors ofestrous females (Carr, Loeb,& Dissinger, 1965). If we are to understand the role ofthe MHC-related odor in behavior, it will be important totest the hypothesis that the MHC and dietary cues in theurine odor provide information that can be used in dif-ferent contexts.

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(Manuscript received January 11, 1996:revision accepted for publication May 15, 1996.)