Urine-Derived Key Volatiles May Signal Genetic Relatedness in Male Rats
Advance Access publication October
Urine-Derived Key Volatiles May Signal Genetic Relatedness in Male Rats
Yao-Hua Zhang 0 1
Jian-Xu Zhang 1
0 Graduate School, Chinese Academy of Sciences , Beijing 100039 , China
1 State Key Laboratory of Integrated Management of Pest Insects and Rodents in Agriculture, Institute of Zoology, Chinese Academy of Sciences , 1-5 Beichenxi Road Beijing 100101 , China
Olfactory cues play a vital role in kin recognition and mate choice of the rat. Here, using 2 inbred strains of rats, Brown Norway (BN) and Lewis, as models to simulate kinship via genetic distance, we examined whether urine-derived volatiles are genetically determined, and, if so, how they code for olfactory information and the degree of genetic relatedness in mate choice. Binary choice tests showed that BN females preferred the urine odor of Lewis males over that of BN males, suggesting that they avoided males genetically similar to themselves and were able to assess this olfactorily. Gas chromatography-mass spectrometry analysis revealed that the composition of urine-derived volatiles was more similar within strains than between strains and suggests that odortypes may reflect genetic relatedness. Our data further show that BN males had lower ratios of 2-heptanone and 4-heptanone and higher ratios of dimethyl sulfone and 4-ethyl phenol than Lewis males. When we supplemented BN and Lewis male urine to make each similar, the preferences of BN females were reversed. We conclude that some urine-derived volatiles covary in relative abundance with degree of genetic relatedness, and this relationship may play a key role in chemical signaling and genetic identity in this species.
chemosignal; genetic identity; mate choice; rat; urine
Kin recognition and discrimination allow animals to
distinguish between kin and nonkin using conspecific cues (Hepper
1991b). Several cue-based mechanisms are responsible such
as social familiarity via prior association during early
development (e.g., siblings and parents) and genetically
determined-phenotype matching (Hepper 1991a; Mateo
2003). In rodents, body odor is an important cue mediating
sociosexual behavior (Brown 1985; Hepper 1991b). There
is considerable evidence to support phenotype matching
in a variety of rodent species, such as golden hamsters
(Mesocricetus auratus), Belding’s ground squirrels
(Spermophilus beldingi), and beavers (Castor canadensis)
(Holmes 1986; Sun and Mu¨ ller-Schwarze 1997, 1998; Mateo
and Johnston 2000; Mateo 2002). Animals with multiple
paternity or maternity may best assess their relatedness to
unfamiliar conspecifics by comparing their own odortypes
released from specialized scent glands with those of
unidentified individuals (so-called phenotype matching)
(Mateo and Johnston 2000).
Kin recognition and discrimination are important for mate
choice and allow for the optimization of inbreeding and
outbreeding (Hepper 1991a). Unrelated individuals are likely to
possess different genotypes, and animals avoid breeding with
close relatives to ensure offspring heterozygosity (Pusey and
Wolf 1996). It has been well documented in mice that females
express mate preferences for genetically dissimilar males
(Roberts and Gosling 2003). Early studies with inbred
congenic mouse lines showed that females chose males with
major histocompatibility complex (MHC) types different from
themselves, without prior experience (Penn and Potts 1998).
Although manipulating artificial housing has revealed that
MHC-dependent familial imprinting provides a more
effective mechanism to avoid mating with kin, wild mice do not
show behavioral imprinting on maternal MHC haplotypes
(Penn and Potts 1998; Sherborne et al. 2007).
Olfactory cues are to some extent genetically determined.
This odor–genes covariance means that odors emitted by
rodents can be used by conspecifics to assess genetic relatedness
(Todrank and Heth 2003; Todrank et al. 2005). Closer
genetic relationships between conspecifics are reflected by
greater similarities in their odors and provide a basis for
phenotype matching. Specifically, individuals with traits that
most closely match an animal’s template are its closest kin
(Mateo 2003; Busquet and Baudoin 2005). Specific
genotypes can be reflected by urine volatile composition in mice,
and MHC may provide the main source of variation in odors
used for individual recognition (Yamazaki et al. 1999). For
example, MHC-determined urinary odor is composed of
a mixture of volatile carboxylic acids relative to the
characteristic of the odortype, though no genotype-unique
compound has been detected (Singer et al. 1997). Modification
of a single gene causes a significant change in the composition
of urine-borne pheromones and elicits a distinct spatial pattern
of glomerular activation within the main olfactory bulb
(Schaefer et al. 2001; Novotny et al. 2007; Zhang et al.
2010). Volatile compounds comprising social odors can reflect
genetic distances and relatedness in rodents and have been also
found in interspecies (e.g., Mus domesticus and M. spicilegus),
interstrain (e.g., ICR/CD-1, Kunming and C57B/6 mice), and
family membership (e.g., beavers) (Sun and M u¨ller-Schwarze
1998; Zhang, Rao, et al. 2007; Soini et al. 2009).
Rats (Rattus norvegicus) have also proven to be capable of
discriminating between unfamiliar kin and nonkin via
olfaction (Hepper 1983; Hepper 1991a). In particular, rats can
distinguish among kin of different degrees of relatedness
(Hepper 1987). Rats spend greater amount of time
investigating less closely related conspecifics in the following order:
cousins are investigated more than half siblings, which are
investigated more than full siblings, and both unfamiliar
and genetically unrelated rats are investigated most (Hepper
1987). Rats therefore possess a genetic identifier which is in
direct proportion to their relatedness and can be used to
discriminate degrees of kinship during phenotype matching
(Hepper 1987). Such kin recognition may be primarily based
on olfactory cues (Hepper 1983; Hopp et al. 1985). As in
mice, voided urine, including volatile compounds derived
in bladder urine and discharged from preputial glands, in
rats serves as the major source of odor (Zhang, Liu, et al.
2008;Zhang, Sun, et al. 2008). However, little is known about
genetically determined odor volatiles in this species, except
for some work on pheromones or putative pheromones
associated with gender, social hierarchy, pup–mother
relations, and alarm (Brouette-Lahlou et al. 1991; Beynon
and Hurst 2004; Gutie´rrez-Garc´ıa et al. 2007; Pohorecky
et al. 2008; Zhang, Sun, et al. 2008; Osada et al. 2009).
We posit that genetically mediated volatiles of rats could
provide olfactory information on genetic relatedness and
function as a genetic identifier for phenotype matching.
The genes of inbred strains such as in mice are homozygous
at nearly all loci, and the attraction of mice to the urinary
odors of other mice is subject to a ‘‘parent-of-origin’’ effect
which causes them to prefer the urine of unrelated strains to
the same strain as their mothers (Beck et al. 2000; Isles et al.
2001). Here, we used 2 inbred strains of rats as a model to
simulate kinship via genetic distance. We conducted
combined binary choice tests and gas chromatography–mass
spectrometry (GC-MS) to clarify whether kin recognition
and inbreeding avoidance in rats is based on genetically
determined odor similarity and dissimilarity. We used female
Brown Norway (BN) rats of an inbred strain as odor
recipients to investigate olfactory sex preferences for male urine of
2 inbred strains, BN and Lewis. We then used GC-MS to
look for chemical differences between strains for subsequent
use in behavioral experiments.
Materials and methods
Ten male and 10 female BN rats and 8 male and 8 female
Lewis rats were used as urine and preputial gland donors.
Female BN rat donors were used as recipients. All animals
were purchased at 20 weeks of age (Vital River Laboratory
Animal Technology Co. Ltd). Males were housed
individually and females in groups of 3–4 in plastic cages (37 · 26 ·
17 cm). The room had a reversed 14:10 h light:dark
photoperiod (lights on at 19:00) and was maintained at 23 ± 2 C.
Food (standard rat chow) and water were provided ad
libitum. We determined estrous cycles via vaginal smears for
several days before behavioral testing and found that all
females had an estrous cycle of 4 days, despite asynchrony;
therefore, on a given test day, randomly selected recipient
females would have covered all stages of the estrous cycle.
Scent collection and sample preparation
We placed rats in a clean mouse cage (25 · 15 · 13.5 cm)
fitted with a wire grid 1 cm above the floor of the cage to
collect urine for behavioral and chemical assay. Urine was
absorbed immediately after it was voided using a disposable
glass capillary and transferred to a vial in ice. Urine
deposited next to feces was not collected. Animals were euthanized
via cervical dislocation, and paired preputial glands were
immediately dissected. Urine and preputial glands were
individually sealed in vials and kept at –20 C prior to use.
To characterize the composition of urine samples, we
mixed 250 lL dichloromethane (purity >99.5%; DIMA
Technology, Inc.) with 250 lL urine, stored it at 4 C for
12 h, and then used the bottom phase (the layer with
dichloromethane) for chemical analysis. To extract
compounds from preputial gland secretion (PGS), the gland
tissue was weighed, extracted in a volume of
dichloromethane that reflected an extract concentration of 1 mg/3 lL
solvent, and stored at 4 C for 12 h. We then removed the
tissue and used the remaining solution for GC-MS analysis.
In order to supplement the urine of each strain of male with
putative signal compounds to simulate the other, we first
diluted each compound with dichloromethane to a
manageable concentration. We then transferred certain amounts of
solution to a clean vial, uncovered the vial, and allowed it to
vaporize for 5 min. We then proportionally added the urine
samples of one strain mixed equally from 10 males for BN
rats or 8 males for Lewis rats into the vial to simulate the
other. In detail, according to the results from the GC-MS
analysis as described below, to simulate Lewis male urine,
we added BN male urine with certain amounts of
4-heptanone, 2-heptanone, dimethyl sulfone, and 4-ethyl phenol
to produce authentic concentrations equal to those in Lewis
males, and we replenished BN urine with certain amounts of
4-heptanone and 2-heptanone to produce ratios equal to
those in Lewis male urine. Likewise, dimethyl sulfone and
4-ethyl phenol were added into Lewis male urine to simulate
BN male urine.
The responses of female BN rats to scented glass rods (20 cm
long, 4-mm diameter) were measured in their home cages in
a separate dim room during the dark phase of the light cycle
(Lai et al. 1996; Zhang, Sun, and Novotny 2007; Zhang, Liu,
et al. 2008). For each test, we kept one test rat in the home
cage while temporarily moving its cage mates into an
identical holding cage. One end of the glass rod was painted with
2 lL of urine; the other end was held by the tester. Two scented
glass rods were simultaneously presented to a subject. We
recorded the total time spent investigating for 3 min after the
subject first sniffed or licked the rod tip (Zhang, Liu, et al. 2008).
We repeated the test for each animal on another day and
summed the investigation time over both trials for use.
For our habituation–dishabituation tests, we provided
recipients with a urine sample from the same male 4 times
and then introduced a novel sample on the fifth trial. We
allowed 2 min between trials. We measured the time spent
sniffing (within 1 cm of the rod) and licking each rod tip
using stopwatches. To control for experimenter bias, the
experimenter was blind to the nature of the sample. Using these
methods, male urine samples of BN males were randomly
paired with those of Lewis males and then presented to
BN females. Each combination was used only once.
Gas chromatography–mass spectrometry analysis
We used an Agilent Technologies Network 6890N GC
system coupled with 5973 Mass Selective Detector with
the NIST/EPA/NIH Mass Spectral Library (2002 version;
Agilent Technologies 2002). Xcalibur (Windows XP) was
used for data acquisition and processing. The GC was
equipped with a HP5-MS separation capillary column (30 m long,
0.25 mm inner diameter · 0.25-lm film thickness). Helium
was used as the carrier gas (1.0 mL/min). The inject
temperature was set at 280 C. The oven temperature program
was set initially at 50 C, heated by 5 C/min to 100 C,
then ramped by 10 C/min until 280 C, and held for
5 min. Finally, the temperature was increased to 300 C
and held for 10 min postrun to clean the column. Electron
impact ionization was used at 70 eV. Transfer line temperature
was set at 280 C. Scanning mass ranged from 30–450 amu. We
injected a 5 lL at a splitless mode for urine and 3 lL sample in
a split mode (1:10) for PGSs.
Tentative identifications were made by comparing the mass
spectra of GC peaks with those in the MS library (NIST2002).
Thirteen of the tentatively identified compounds, 4-heptanone,
2-heptanone, dimethyl sulfone, 4-methyl phenol, 4-ethyl
phenol, indole, E-b-farnesene, dodecanoic acid, tetradecanoic
acid, hexadecanoic acid, Z9-octadecenoic acid, Octadecanoic
acid, and squalene (all purity >98%; ACROS Organics)
were further confirmed by matching retention times and mass
spectra with the authentic analogs.
Abundance and relative abundance of compounds were
used for quantitative comparisons between groups. The
abundance of a particular compound was quantified by
GC peak area. The peak area of a particular compound
was then converted into a percentage of summed peak areas
from all targeted GC peaks of either urine or PGS, as its
We quantified 4-heptanone, 2-heptanone, dimethyl sulfone,
and 4-ethyl phenol in urine by comparing their GC areas
in the samples with an established standard curve (GC area
vs. concentration). To determine the variability of urine
and PGS among individuals, we calculated relative
standard deviation (RSD) using the formula: RSD = (standard
deviation/mean) · 100, where the data we used for calculation
were the percentage (relative abundances) of volatile peak
areas for the 2 strains (Zhang, Rao, et al. 2007).
The distribution of raw data was examined using a
Kolmogorov–Smirnov test and either parametric or
nonparametric tests were applied to behavioral tests and GC data. If
data were normally distributed, one-way analysis of variance
with Bonferroni post hoc t-tests were used for GC data,
whereas paired-samples t-test were used for behavioral
data and RSDs. If data were not normally distributed, a
Kruskal–Wallis H with post hoc Mann–Whitney U test was
used for chemical data and Wilcoxon sign rank test for
behavioral data and RSDs. All Statistical analyses were conducted
using SPSS (v15.0, SPSS Inc.). Alpha was set at P < 0.05.
We used hierarchical cluster analysis to process GC-MS
data from rat urine and PGS. Hierarchical cluster analysis
is a statistical method for finding relatively homogenous
clusters of cases based on measured characteristics. It sorts
cases into clusters so that the degree of association is strong
between members of the same cluster and weak between
members of different clusters. We used this analysis (average
linkage) with Pearson’s correlation coefficient tests to
examine the similarity of individual volatile profiles.
The procedures of animal care and use in this study fully
complied with Chinese legal requirements and were approved
by the Animal Use Committee of the Institute of Zoology,
Chinese Academy of Sciences.
Preputial gland size
Preputial glands were found to be heavier (absolute weight)
in males than females. The relative weight of the gland was
heavier in Lewis females than males. Between strains, both
the absolute and relative weight of the preputial gland were
heavier for BN than Lewis males (Table 1).
Volatile composition of rat urine and PGS
We characterized 5 early eluting compounds from voided
urine including 2 ketones, 1 sulfone and 2 phenols (Figure 1;
Tables 2 and 3). We detected and identified 25 compounds
from PGS including aldehydes, aliphatic acids, indole, and
some terpenoid polyenes (Figure 2; Table 4).
The relative abundances of some volatile compounds
within strains showed quantitative sexual dimorphism.
For urine, 4-heptanone was found to be male-specific,
2-heptanone was higher in males, and dimethyl sulfone
was higher in females in both BN and Lewis rats (Figure 1;
Table 2). For PGS in BN rats, 8 compounds (3, 8, 9, 12, 20,
22, 23, and 25) were significantly higher and 5 compounds
(2, 5, 6, 15, and 17) lower in males compared with females.
Similarly for PGS in Lewis rats, 6 compounds (8, 9, 18, 20,
22, and 23) were significantly higher and 5 (2, 5, 6, 17, and 19)
lower in males compared with females (Figure 2; Table 4).
Between strains and within gender, no strain-specific
compounds in either urine or PGS were detected; but 4
compounds in urine and 9 compounds in PGS differed in
relative abundance, quantified by percent GC areas, between
BN and Lewis rats (Tables 2 and 4). In detail: 4-heptanone
(Lewis vs. BN: 4.51% vs. 2.23%, P = 0.021, z = 2.312) and
2-heptanone (Lewis vs. BN: 76.05% vs. 43.31%, P <
0.001, t = 7.549) derived from male urine were significantly
higher in Lewis male urine and dimethyl sulfone (Lewis: BN:
14.86% vs. 45.42%, P = 0.001, z = 3.361) and 4-ethyl phenol
(Lewis:BN: 3.54%:8.77%, P = 0.074, z = 1.785, marginal
significance) were higher in BN male urine than the other
(Figure 1; Table 2). The PGS constituents 1, 7, and 14 were
higher, but compounds 3, 8, 9, 12, 13, and 25 were lower in
Lewis than in BN rats (Figure 2; Table 4). Abundance as
reflected by GC peak area of 3 urine constituents exhibited
considerable difference between the 2 strains (Table 3). In
addition, the levels of 4-heptanone, 2-heptanone, dimethyl
sulfone, and 4-ethyl phenol were estimated to be 0.26 ±
0.60, 2.86 ± 2.19, 4.68 ± 1.76, and 1.06 ± 0.55 ppm in BN
male urine and 1.06 ± 0.90, 17.39 ± 6.56, 6.89 ± 2.12, and
1.47 ± 1.14 ppm in Lewis male urine, respectively.
We used cluster analysis to test the similarity of urine and
PGS constituents among individual BN and Lewis males. A
dendrogram of urine constituents reveals that the rats could
be divided into 2 groups: a cluster composed of 9 individuals
(8 Lewis and 1 BN rat); a cluster of 7 individuals (all BN).
The cluster distance among Lewis rats was less than 5
(Figure 3). The dendrogram for PGS did not show such
a classification (Figure 4).
Intrastrain variation in volatile compounds
In males of the 2 strains, the majority of volatile compounds
in urine and PGS displayed extremely high interindividual
RSDs. Most volatile compounds from the preputial gland
showed higher interindividual than intraindividual RSDs,
quantified by 6 duplicates of one sample (Tables 5 and 6).
Discrimination between individuals and preference
Habituation–dishabituation tests showed that BN females
habituated to repeated exposure to BN male urine
(P = 0.005, Z = 2.803, N = 10). The time spent investigating
the sample then increased when presented with a novel
sample (P = 0.005, Z = 2.805, N = 10, Figure 5a).
Binary choice tests revealed that BN females were more
attracted to BN male urine than to BN female urine (P =
0.038, t = 2.073, N = 9, Figure 5b) and that female BN rats
showed a significant preference for Lewis male urine over BN
male urine (P = 0.001, t = 5.233, N = 9, Figure 6a).
According to the authentic levels of urine constituents in
2 strains mentioned above, to simulate the rat urine of
the other, 0.80 ppm 4-heptanone, 14.53 ppm 2-heptanone,
2.21 ppm dimethyl sulfone, and 0.41 ppm 4-ethyl phenol
were added to BN male urine so that levels were equal to
those of Lewis males. After replenishing the male urine with
Body weight (BW) (g)
PG weight (mg)
Relative PG weight (mg/100 g BW)
BN males (n = 6)
BN females (n = 6)
Lewis males (n = 8)
Lewis females (n = 8)
synthetic analogs, we found that BN females responded
equally to pure BN male urine and adjusted BN male urine
(P = 0.150, t = 1.591, N = 9). Meanwhile, Lewis male
urine was also more attractive than adjusted BN male urine
(P = 0.038, t = 2.073, N = 9, Figure 6a).
Because of interstrain differences in the ratio of the 4
compounds, we needed to supplement the urine from each strain
of male. We added 2-heptanone and 4-heptanone to BN
male urine to simulate Lewis male urine using the formula:
where, x and y represented the percentage of 4-heptanone
and 2-heptanone added to BN male urine. Therefore, 1.21
ppm 4-heptanone and 11.21 ppm 2-heptanone were added
to BN male urine, increasing the proportion of 4-heptanone
and 2-heptanone to 4.51% and 76.05%, respectively.
Similarly, when adding dimethyl sulfone and 4-ethyl
phenol to Lewis male urine, to make it similar to BN male urine,
we let x and y be the percentage of dimethyl sulfone and
4-ethyl phenol added to Lewis male urine using the formula:
Consequently, 30.62 ppm dimethyl sulfone and 5.02 ppm
4-ethyl phenol were added to Lewis male urine so that
their ratio was equal to those in BN male urine (Tables 2
Further 2-choice tests showed that BN females responded
equally to Lewis male urine and BN male urine following
supplementation (P = 0.655, t = 0.465, N = 9). No preference
was found between BN male urine and Lewis male urine
supplemented with dimethyl sulfone and 4-ethyl phenol
(P = 0.415, t = 0.678, N = 9). The attractiveness of BN male
urine was significantly increased after supplementation (P =
0.015, t = 2.429, N = 9), and Lewis male urine was more
attractive than Lewis male urine following supplementation
(P = 0.051, t = 1.955, N = 9). Moreover, females showed a
significant preference for supplemented BN male urine over
Lewis male urine (P = 0.015, t = 2.429, N = 9, Figure 6b).
Figure 1 Representative GC profile of dichloromethane extract from male
urine. GC conditions are described in Materials and methods section.
Numbered GC peaks correspond to compounds in Tables 2 and 3. Peak 1, 2,
3, 5 are 4-heptanone, 2-heptanone, dimethyl sulfone and 4-ethyl phenol,
Compounds 1 2 3
Six duplicate of
Compounds 1 2 3
Six duplicate of
Figure 2 Representative GC profile of dichloromethane extract from male
preputial gland (top: 10–24 min; bottom: 24–33 min). GC conditions
are described in Materials and methods section. Numbered GC peaks
correspond to compounds in Table 4. Peak 5, 6, 8, 9, 10, 13, 18 are
Eb-farnesene, E,E-a-farnesene, dodecanoic acid, tetradecanoic acid,
hexadecanal, hexadecanoic acid, and squalene, respectively.
Previous behavioral work suggests a correlation between
genes and social odor and that odor can be used by
conspecifics during olfactory assessment of genetic relatedness by
rodents (Hepper 1983; Hepper 1991a; Todrank and Heth
2003; Todrank et al. 2005). It therefore appears that rats
can distinguish among kin of different relatedness (Hepper
1987). Inbred mice strains are approximately identical in
genotype and homozygous at nearly all loci (Beck et al. 2000).
The similarity in volatile composition of individual scent
reflects this genetic similarity (Singer et al. 1997; Beauchamp
and Yamazaki 2003). Here, we used 2 inbred strains of rats as
a model to simulate kinship and showed that inbred BN
female rats had a normal preference for male urine over female
urine of their own strain (Figure 5b) and chose Lewis male
urine over BN male urine (Figure 6a). Because female
recipients represented all stages of the estrous cycle, we do not
think that endocrine state affected female behavior in our
experiments. That is, we posit that females show a preference
for a specific odor regardless of their estrous state. Our
findings suggest that urine-borne volatiles are capable of
conveying olfactory cues to females to assess genetic relationships
during mate choice and avoid inbreeding. Much evidence
shows that animals avoid breeding with close relatives (Pusey
and Wolf 1996) and that females may ensure the
heterozygosity of their offspring via odortype matching when
choosing mates (Brown 1997). These results from inbred female
rats support the ‘‘good-genes-as-heterozygosity’’ hypothesis
that females, especially inbred females, may choose mates
that are genetically dissimilar and result in offspring of
greater heterozygosity (Isles et al. 2001; Ilmonen et al.
2009). Inbred females may gain more benefits from this
strategy than outbred females (Ilmonen et al. 2009).
Chemically, we characterized 30 compounds from urine
and PGS of BN and Lewis rats and found neither
strainspecific nor sex-specific compounds, except male-specific
4-heptanone. However, quantitatively, 2-heptanone was
richer in males than in females, and dimethyl sulfone and
2 preputial gland–secreted sesquiterpens, E-b-farnesene
and E,E-a-farnesene, were richer in females (Figures 1
and 2, Tables 2, 3, and 4). These results are consistent with
those previously reported in Sprague-Dawley rats (Zhang,
Sun, et al. 2008; Osada et al. 2009) and suggest that rats
of different strains may share similar odor volatiles coding
for chemical signals. Moreover, the volatiles are species
shared. Some of these compounds such as 2-heptanone,
dimethyl sulfone, and/or 4-heptanone have also been found in
the urine of other mammals including minks, dogs, and
rabbits (Zhang YH, Zhang JX, unpublished data).
Six duplicate of
0.03a 0.02 0.03 0.01
0.01c 0.07c 0.02 0.01
0.01a 0.01 0.04 0.00
0.02 0.04 0.03 0.02
0.01c 0.03c 0.02 0.01
0.01c 0.06c 0.04 0.01
0.05a 0.07 0.09 0.01
0.08a,c 0.04c 0.31 0.07
0.03a,c 0.02c 0.34 0.07
0.04 0.28 0.09 0.03
0.03 0.05 0.04 0.00
0.02a 0.01 0.05 0.00
0.17a 0.35 1.05 0.20
0.03a 0.03 0.04 0.01
0.02 0.04 0.04 0.00
0.14 0.09 0.53 0.06
0.02c 0.07c 0.07 0.01
1.20c 2.16c 70.27 1.72
1.87c 1.22c 9.62 0.77
0.14c 0.04c 0.31 0.03
0.20 0.07 2.42 0.08
0.14c 0.27c 1.35 0.11
0.41c 0.09c 0.55 0.05
0.96 0.34 12.22 0.66
0.31a 0.06 0.45 0.04
The means in a row marked by the same superscript letters show significant differences (P < 0.05, using one-way ANOVA with Bonferroni post hoc t-test or
Kruskal–Wallis H with post hoc Mann–Whitney U test). The compound marked by asterisks are definitively identified. RT, retention time.
Pairwise comparison revealed that many volatiles differed
significantly between strains (Tables 2, 3, and 4). Moreover,
the dendrogram from the cluster analysis revealed that
urinederived volatiles could better reflect genetic similarity and
dissimilarity than PGS-produced volatiles in rats and that
this similarity may reflect a closer genetic relationship
(Figures 3 and 4). As for urine-derived volatiles, individuals
shared more similarities within strain than between strain
(Figure 3). As in mice, urine-derived volatiles are more
sensitive to genetic shifts than PGS-produced volatiles (Zhang,
Rao, et al. 2007). Some scent volatiles have been
demonstrated to be capable of coding for olfactory genetic
information in mice (Singer et al. 1997; Schaefer et al. 2001;
Novotny et al. 2007). Our pairwise comparison revealed
that urine-derived 4-heptanone, 2-heptanone, dimethyl
sulfone, and 4-ethyl phenol differed in relative and/or
absolute abundance for BN and Lewis males and might code
for such genetic information in voided urine. Singer et al.
(1997) demonstrated that urinary volatiles covary in
relative concentrations with genotypes and contribute to
unique individual odors (odortypes) in mice. The relative
concentration or ratio of urine components is a vital index
for putative chemical signal components and our results
also support this notion.
Replenishing BN male urine with 4-heptanone, 2-heptanone,
dimethyl sulfone, and 4-ethyl phenol at absolute levels similar
Figure 3 Dendrogram for average linkage hierarchical clustering of urine
data (the relative abundances of volatile compounds for BN and Lewis male
rats). Each sample is represented by the initial letter of the strain name
affixed with a number (L stands for Lewis male urine and B stands for BN
male urine, eight individuals in each group).
Figure 4 Dendrogram for average linkage hierarchical clustering of PGS
data (the relative abundances of volatile compounds for BN and Lewis male
rats). Each sample is represented by the initial letter of the strain name
affixed with a number (L stands for Lewis male PGS and B stands for BN
male PGS, six individuals in each group).
to Lewis male urine did not result in a difference in BN
female response (Figure 6a). Although we have regulated all
between-strain different compounds of BN male urine to Lewis
male urine levels, the responses of BN females remained the
same. Because variation in the relative abundance of the 4
compounds proved to determine the chemical signals between
strains, we speculate that 4-methyl phenol and other
GCundetected compounds in urine might affect the ratio of the
4 compounds in spiked BN male urine. This meant that they
could not reach the ratios exactly as those present in Lewis male
Taking relative concentration into consideration,
2heptanone and 4-heptanone were found at a higher ratio
Table 5 Individual variation (RSD) of relative abundance of the urine
volatiles of 2 strains of rats
Six duplicate of
RSD was calculated using the formula RSD = (standard deviation/mean) ·
100, where mean and standard deviation (SD) are the average of each
volatile peak area (in percentage) and their SD, respectively. Wilcoxon
matched-pair signed-rank test for RSDs between same compounds of each
in Lewis males, whereas dimethyl sulfone and 4-ethyl phenol
were higher in BN males. These compounds may form the
main part of the odortype for these strains. We focused
on only those constituents which had lower relative ratios
and spiked BN and Lewis male urine with synthetic analogs.
The ratios of 2-heptanone and 4-heptanone in spiked BN
urine were the same as those for Lewis, but the ratios of
dimethyl sulfone and 4-ethyl phenol were changed and not
equal to either BN or Lewis. However, individual variation
in BN urine does exist (mean dimethyl sulfone abundance
was 14.86% ± 4.50%, 4-ethyl phenol was 3.54% ± 4.30%),
so our simulation should be acceptable. Furthermore,
although the ratios of 4-heptanone and 2-heptanone were
changed, spiked Lewis male urine was similar to BN male
urine. We therefore succeeded in simulating BN male urine
through the addition of both dimethyl sulfone and 4-ethyl
phenol to Lewis male urine, and BN females preferred pure
Lewis male urine over rescented urine from Lewis males.
Similarly, we successfully simulated Lewis male urine by
adding 4-heptanone and 2-heptanone to BN male urine.
BN females did not differ in their response for pure BN male
urine and rescented Lewis male urine and showed a
preference for rescented BN male urine over rescented Lewis male
urine (Figure 6b). Behavioral tests revealed that these
preferences were based on preferences for another strain rather
than preference for novel stimuli (if it were because of novel
stimuli, preference should always have been for spiked
urine). Hence, our data show that the relative concentration
of scent volatile compounds may be reliable indicators when
screening for potential genetically determined chemosignals.
In addition, the large interindividual variation of volatile
compounds detected in rat urine and PGS indicates that
urine-borne volatiles might code for individual information
in the analog form, as in mice (Zhang, Rao, et al. 2007).
Male Female Male
Six duplicate of
RSD was calculated using the formula RSD = (standard deviation/mean) · 100, where mean and standard deviation (SD) are the average of each volatile
peak area (in percentage) and their SD, respectively. Wilcoxon matched-pair signed-rank test for RSDs between same compounds of each individual
Specifically, the information could be coded by varying
amounts of shared compounds rather than by unique
compounds. In habituation–dishabituation tests, rats were
capable of discriminating 2 odor stimuli, suggesting that the urine
odor of the extremely inbred BN rats does differ between
individuals (Figure 5a). In agreement with behavior tests, the
high interindividual divergence in volatile composition as
reflected in high RSDs (Tables 5 and 6) may lay the foundation
for learned individual recognition or memorization of
individuals via urine odor and provide information about
individual genotypes despite intrastrain similarities in genotypes
and odortypes of inbred rats. Indeed, previous results from
mice have shown that small changes in genotype (e.g., MHC
genes or Foxn1 gene) cause significant change in the
composition of urine volatiles and consequently change the
responses of recipient mice (Singer et al. 1997; Schaefer
et al. 2001; Novotny et al. 2007; Keller et al. 2009; Zhang
et al. 2010).
Figure 5 (a) Duration of investigation (mean standard error, s) by female
BN rats of male and female BN rat urine samples during a 3 min choice test
(* , P < 0.05, paired t-test ). (b) Discrimination of BN female rats between
different BN male urine samples. Investigation time of the fourth
presentation was lower compared to the first presentation (**, P < 0.01,
Wilcoxon matched-pairs signed-rank test). Investigation time during the test
presentation increased compared with the fourth presentation of the the
habituated sample (##, P < 0.01, Wilcoxon matched-pairs signed-rank test).
In conclusion, female rats may use olfactory cues to assess
relatedness in potential mates and choose mates with
odortypes different from their own during mate choice and
inbreeding avoidance. In particular, urine-derived volatiles
such as 4-heptanone, 2-heptanone, dimethyl sulfone, and
4-ethyl phenol show covariation between relative abundance
and degree of genetic relatedness, and these compounds may
play a key role in chemical signaling, genetic identity, and kin
recognition in the rat.
Figure 6 Duration of investigation (mean standard error, s) by female BN
rats of different male urine samples during a 3 min choice test across two
days. (a) Synthetic analogues added to BN urine according to authentic
levels present in Lewis male urine. (b) Synthetic analogues added to
male urine according to relative abundance in each of the other strains
(*, P < 0.05, paired t-test or Wilcoxon matched-pairs signed-rank test).
We are grateful to Jin-Hua Zhang for assistance with behavioral
tests and Xiaowei Qin for assistance with the GC-MS.
Beauchamp GK , Yamazaki K. 2003 . Chemical signalling in mice . Biochem Soc Trans. 31 : 147 - 151 .
Beck JA , Lloyd S , Hafezparast M , Lennon-Pierce M , Eppig JT , Festing MFW , Fisher EMC . 2000 . Genealogies of mouse inbred strains . Nat Genet . 24 : 23 - 25 .
Beynon RJ , Hurst JL . 2004 . Urinary proteins and the modulation of chemical scents in mice and rats . Peptides . 25 : 1553 - 1563 .
Brouette-Lahlou I , Amouroux R , Chastrette F , Cosnier J , Stoffelsma J , Vernetmaury E. 1991 . Dodecyl propionate, attractant from rat pup preputial gland-characterization and identification . J Chem Ecol . 17 : 1343 - 1354 .
Brown JL . 1997 . A theory of mate choice based on heterozygosity . Behav Ecol . 8 : 60 - 65 .
Brown RE . 1985 . The rodents . II. Suborder Myomorpha . In: Brown RE, Macdonald DW, editors. Social odours in mammals. Oxford: Clarendon Press. p. 345 - 417 .
Busquet N , Baudoin C. 2005 . Odour similarities as a basis for discriminating degrees of kinship in rodents: evidence from Mus spicilegus . Anim Behav . 70 : 997 - 1002 .
Guti e´rrez-Garcı´a AG , Contreras CM , Mendoza-L o´pez MR , Garcı´aBarradas O , Cruz-Sa ´ nchez JS. 2007 . Urine from stressed rats increases immobility in receptor rats forced to swim: role of 2-heptanone . Physiol Behav . 91 : 166 - 172 .
Hepper PG . 1983 . Sibling recognition in the rat . Anim Behav . 31 : 1177 - 1191 .
Hepper PG . 1987 . The discrimination of different degrees of relatedness in the rat-evidence for a genetic identifier . Anim Behav . 35 : 549 - 554 .
Hepper PG . 1991a. Kin recognition . In: Hepper PG, editor. Cambridge : Cambridge University Press . p. 259 - 267 .
Hepper PG . 1991b. Kin recognition cues of vertebrates . In: Hepper PG, editor. Kin recognition . Cambridge : Cambridge University Press . p. 220 - 258 .
Holmes WG . 1986 . Kin recognition by phenotype matching in female belding ground-squirrels . Anim Behav . 34 : 38 - 47 .
Hopp SL , Owren MJ , Marion JR . 1985 . Olfactory discrimination of individual littermates in rats (rattus-norvegicus) . J Comp Psychol . 99 : 248 - 251 .
Ilmonen P , Stundner G , Thoss M , Penn DJ . 2009 . Females prefer the scent of outbred males: good-genes-as-heterozygosity ? BMC Evol Biol . 9 : 104 .
Isles AR , Baum MJ , Ma D , Keverne EB , Allen ND . 2001 . Genetic imprinting-urinary odour preferences in mice . Nature . 409 : 783 - 784 .
Keller M , Baum MJ , Brock O , Brennan PA , Bakker J. 2009 . The main and the accessory olfactory systems interact in the control of mate recognition and sexual behavior . Behav Brain Res . 200 : 268 - 276 .
Lai SC , Vasilieva NY , Johnston RE . 1996 . Odors providing sexual information in Djungarian hamsters: evidence for an across-odor code . Horm Behav . 30 : 26 - 36 .
Mateo JM . 2002 . Kin-recognition abilities and nepotism as a function of sociality . Proc R Soc Lond B Biol Sci . 269 : 721 - 727 .
Mateo JM . 2003 . Kin recognition in ground squirrels and other rodents . J Mammal . 84 : 1163 - 1181 .
Mateo JM , Johnston RE . 2000 . Kin recognition and the 'armpit effect': evidence of self-referent phenotype matching . Proc R Soc Lond B Biol Sci . 267 : 695 - 700 .
Novotny MV , Soini HA , Koyama S , Wiesler D , Bruce KE , Penn DJ . 2007 . Chemical identification of MHC-influenced volatile compounds in mouse urine . I: quantitative proportions of major chemosignals . J Chem Ecol . 33 : 417 - 434 .
Osada K , Kashiwayanagi M , Izumi H. 2009 . Profiles of volatiles in male rat urine: the effect of puberty on the female attraction . Chem Senses . 34 : 713 - 721 .
Penn D , Potts W. 1998 . MHC-disassortative mating preferences reversed by cross-fostering . Proc R Soc Lond B Biol Sci . 265 : 1299 - 1306 .
Pusey A , Wolf M. 1996 . Inbreeding avoidance in animals . Trends Ecol Evol . 11 : 201 - 206 .
Roberts SC , Gosling LM . 2003 . Genetic similarity and quality interact in mate choice decisions by female mice . Nat Genet . 35 : 103 - 106 .
Schaefer ML , Young DA , Restrepo D. 2001 . Olfactory fingerprints for major histocompatibility complex-determined body odors . J Neurosci . 21 : 2481 - 2487 .
Sherborne AL , Thom MD , Paterson S , Jury F , Ollier WER , Stockley P , Beynon RJ , Hurst JL . 2007 . The genetic basis of inbreeding avoidance in house mice . Curr Biol . 17 : 2061 - 2066 .
Singer AG , Beauchamp GK , Yamazaki K. 1997 . Volatile signals of the major histocompatibility complex in male mouse urine . Proc Natl Acad Sci U S A . 94 : 2210 - 2214 .
Soini HA , Wiesler D , Koyama S , Feron C , Baudoin C , Novotny MV . 2009 . Comparison of urinary scents of two related mouse species, Mus spicilegus and Mus domesticus . J Chem Ecol . 35 : 580 - 589 .
Sun LX , M u¨ ller-Schwarze D. 1997 . Sibling recognition in the beaver: a field test for phenotype matching . Anim Behav . 54 : 493 - 502 .
Sun LX , M u¨ ller-Schwarze D. 1998 . Anal gland secretion codes for relatedness in the beaver , Castor canadensis. Ethology . 104 : 917 - 927 .
Todrank J , Busquet N , Baudoin C , Heth G. 2005 . Preferences of newborn mice for odours indicating closer genetic relatedness: is experience necessary? Proc R Soc Lond B Biol Sci . 272 : 2083 - 2088 .
Todrank J , Heth G. 2003 . Odor-genes covariance and genetic relatedness assessments: rethinking odor-based ''recognition'' mechanisms in rodents . Adv Study Behav . 32 : 77 - 130 .
Yamazaki K , Beauchamp G , Singer A , Bard J , Boyse EA . 1999 . Odortypes: their origin and composition . Proc Natl Acad Sci U S A . 96 : 1522 - 1525 .
Zhang JX , Liu YJ , Zhang JH , Sun LX . 2008 . Dual role of preputial gland secretion and its major components in sex recognition of mice . Physiol Behav . 95 : 388 - 394 .
Zhang JX , Rao XP , Sun LX , Zhao CH , Qin XW . 2007 . Putative chemical signals about sex, individuality, and genetic background in the preputial gland and urine of the house mouse (Mus musculus) . Chem Senses . 32 : 293 - 303 .
Zhang JX , Sun LX , Novotny M. 2007 . Mice respond differently to urine and its major volatile constituents from male and female ferrets . J Chem Ecol . 33 : 603 - 612 .
Zhang JX , Sun L , Zhang YH . 2010 . Foxn1 gene knockout suppresses sexual attractiveness and pheromonal components of male urine in inbred mice . Chem Senses . 35 : 47 - 56 .
Pohorecky LA , Blakley GG , Ma EW , Soini HA , Wiesler D , Bruce KE , Novotny MV . 2008 . Social housing influences the composition of volatile compounds in the preputial glands of male rats . Horm Behav . 53 : 536 - 545 .
Zhang JX , Sun LX , Zhang JH , Feng ZY . 2008 . Sex- and gonad-affecting scent compounds and 3 male pheromones in the rat . Chem Senses . 33 : 611 - 621 .