Animal Reproduction Science 197 (2018) 87–92

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Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

Identification of putative volatile sex pheromones in female

T
domestic dogs (Canis familiaris)
⁎
Michał Dzięcioła, , Martyna Woszczyloa, Antoni Szumnyb, Tadeusz Jezierskic,
Robert Kupczyńskid, Ewa J. Godzińskae, Barbara Pieczewskaa, Wojciech Niżańskia
a
Wroclaw University of Environmental and Life Sciences, Department of Reproduction, 50-366, Wrocław, Plac Grunwaldzki 49, Poland
b
Wroclaw University of Environmental and Life Sciences, Department of Chemistry, 50-375, Wrocław, C.K. Norwida 25, Poland
c
Institute of Genetics and Animal Breeding of Polish Academy of Sciences, Department of Animal Behavior, Jastrzębiec, 05-552, Magdalenka, Poland
d
Wroclaw University of Environmental and Life Sciences, Department of Environment Hygiene and Animal Welfare, Wrocław University of
Environmental and Life Sciences, 51-630, Wrocław, Poland
e
Laboratory of Ethology, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, Pasteur St. 3, PL, 02-093, Warsaw, Poland

A R T IC LE I N F O ABS TRA CT

Keywords: More than hundred urine samples, vaginal secretions, vulval and anal imprints as well as anal sac
Pheromones secretions, collected during various phases of the ovarian cycle, were evaluated with the HS-
Dogs SPME/GC–MS technique. The results indicate there were differences among samples of urine
Estrus collected during particular phases of the ovarian cycle. Unequivocally, the amount of carbonyl
Volatile compounds
aromatic compounds, such as acetophenone (hypnone) and benzaldehyde, increased during the
Urine
period of proestrus and estrus of the estrous cycle. This was accompanied by increases in me-
thylketones, e.g., 2-octanone, 2-pentanone and 3-hexanone. Simultaneously, amounts of sulfide
compounds (1-methylotiopropane; 1-methylotiobutane, 1-methylotipentane and dimethyl tri-
sulfide) decreased during the period of estrus and abruptly increased in diestrus. These ob-
servations suggest a possible dual mechanism of interaction between males and females during
and subsequent to the mating period, including the existence of both attracting and repelling
signals. No significant changes were detected in samples other than urine. Further studies in-
cluding a proteomic approach as well as behavioral assessments are suggested to identify the
complete range of estrual semiochemical signals and to verify the semiochemical activities of
identified substances.

1. Introduction

Specific chemical compounds secreted during estrus, are involved in semiochemical communication and are important for de-
tection of sexually active females by male dogs. In response to subconsciously detected sex pheromones, specific behavioural, as well
as physiological reactions, of the recipient can be observed (Rekwot et al., 2001; Kustritz, 2005; Dzięcioł et al., 2012b).
Studies dedicated to the identification of putative sex pheromones have been conducted in many species including mammals
(Signoret, 1991; Achiraman and Archunan, 2005; Sankar and Archunan, 2008; Ponmanickam et al., 2010; Rajanarayanan and
Archunan, 2011; Būda et al., 2012). According to Goodwin et al. (1979), the substance suspected to be the primary sex pheromone in
domestic dogs, was methyl paraben (methyl 4-hydroxybenzoate). Further studies, however, did not confirm this hypothesis and the

⁎
Corresponding author.
E-mail address: michal.dzieciol@upwr.edu.pl (M. Dzięcioł).

https://doi.org/10.1016/j.anireprosci.2018.08.016
Received 1 June 2018; Received in revised form 26 July 2018; Accepted 15 August 2018
Available online 17 August 2018
0378-4320/ © 2018 Published by Elsevier B.V.
M. Dzięcioł et al. Animal Reproduction Science 197 (2018) 87–92

most recent research unambiguously indicates a lack of semiochemical activities of methyl paraben, as well as its absence in female
secretions during estrus (Kruse and Howard, 1983; Schultz et al., 1985; Dzięcioł et al., 2014, 2017).
A survey of research studying the mechanism of action of pheromones indicates the possibility of a complex pattern determined
by the emitted signal in females during estrus. Additionally, in observations of male-female interactions during courtship, complex
features of the semiochemical signal pathway may indicate multiple products are being released by different glands in different
locations that signal the sexual state (Rekwot et al., 2001; Kashiwayanagi, 2003; Pageat and Gaultier, 2003; Brennan and Keverne,
2004; Sankar and Archunan, 2008).
Because there are no actual studies identifying and confirming the composition of the semiochemical signals released by the
female domestic dog (Canis familiaris) in estrus, the present study focused on the identification of these compounds in this species.

2. Material and methods

2.1. Ethics statement

The research was conducted in accordance with regulations on animal experimentation and with the guidelines for the use of
animals in research. The 2nd Local Commission for Animal Experimentation in Wrocław, Poland issued a statement approving the
experimental protocol (permission no. 17/2017).

2.2. Animals

The animals used in the experiments were dogs of the Beagle breed belonging to the Local Experimental Kennel, as well as some
patients of the Clinic of Reproduction. All animals (n = 32) included in the experiment were previously examined by a clinician and
assessed as being healthy. Because presence of additional, artificial odors could influence the results, as well as disturb the process of
mating (in a case of patients use), neither cosmetics nor anti-parasitic collars were used around the time of estrus in females.
Furthermore, to exclude the risk of affecting the results, all urine samples were also assessed for kidney, bladder and lower urinary
tract diseases. Only samples from healthy females were used for further examination.

2.3. Collection of materials

For chemical analysis, samples from the vulva, vagina and anal regions as well as urine were collected from females at various
stages of the ovarian cycle (anestrus, proestrus, estrus and diestrus). The exact stage of the estrous cycle was determined on the basis
of owners’ reports, clinical examination, laboratory tests (vaginal cytology and measurement of progesterone concentrations) and
evaluation of adult male behavior towards females (Kustritz, 2005, 2006). Progesterone concentration in peripheral blood was
determined by the enzyme-linked fluorescence assay (ELFA; mini VIDAS® Biomerieux, France; Brugger et al., 2011).
Evaluation of male reactions towards females confirming the extent of a male’s attraction to a female was based on having typical
sequences of male behavior including approaching, sniffing, and licking the bitch, and increased salivation and attempts to copulate
by the male (Raymer et al., 1986; Kustritz, 2005; Dzięcioł et al., 2012a).
Samples were collected from the vulva and anal region, as an imprint with using a piece of sterile cotton gauze, held firmly for
about 10 s. Furthermore, a small amount of anal sac secretion was collected on the gauze through the routine medical method of
gently squeezing the glands. The vaginal secretions were obtained by introducing the cotton swab into the vagina for about 10 s. As
the last procedure, in all cases, samples of urine were collected using a sterile steel ladle, during natural, spontaneous urination.
Immediately after collection, all samples were placed in a fresh, clean, odor-free glass bottle and stored at -20 °C (Kustritz, 2006). All
samples were analyzed in the same apparatus with the same laboratory conditions. In case of sample collection with the use of cotton
gauze, always the blind samples were collected (without biological material) and analyzed together with the primary samples
(containing biological material).

2.4. Chemical analysis

2.4.1. Analysis of volatile compounds in the samples of urine, vagina, vulva and anal region
Isolation of aromatic compounds from the urine, vulva and anal region was performed using the headspace solid phase micro-
extraction (HS-SPME) procedure. A manual SPME holder (Supelco, Bellefonte, PA, USA) with a vivinylbenzene/carboxen/poly-
dimethylsiloxane (DVB/CAR/PDMS, 50/30 μm, coating 1 cm) fiber (Supelco) was used to extract headspace volatiles from urine
samples. Prior to use, the fiber was conditioned at the gas chromatography (GC) injection port at a temperature of 250 °C for 1 h. For
each extraction, 2 to 5 mL of liquid was placed in a 40 mL glass vial with a plastic screw cap and a Teflon-coated septum (Supelco).
Solid sodium chloride (1 g) and beta-Ionone (0.5 μg) were added to the sample and sonication occurred for 5 min. For an internal
standard, beta-Ionone was used, after assessing and assuring that it was absent in fresh samples. Beta-ionone was selected as the
standard because it easily separates from other volatiles, is stable at high temperatures and does not react with water. After
homogenization of the contents, the vial was maintained in a water bath at 60 °C during equilibration (10 min) and extraction
(40 min) and was partially submerged so that the liquid phase of the sample was in the water. Subsequently, the SPME fiber was
removed from the vial and inserted into the GC/mass spectrometry (MS) injector (250 °C) for 10 min.
The GC/MS analysis was performed using a Varian Saturn Ion Trap 2000 GC/MS/MS System with CP-3800 GC (Walnut Creek, CA,

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USA) equipped with a ZB-5MS (30 m × 0.25 mm i.d., 0.25 μm film thickness) capillary column (Phenomenex, USA). Samples were
injected in splitless mode, and helium at 1 mL [min − 1/ min−1] was used as the carrier gas. The oven temperature was maintained
at 60 °C for 3 min, increased to 120 °C at 3 °C [min − 1/ min−1] and then at 300 °C at 15 °C [min − 1/ min−1] and lastly maintained
at 300 °C for 2 min. Mass spectra were recorded in electron impact (EI) ionization mode at 70 eV, scanning the m/z 35–550 range. The
injector was maintained at 250 °C, while the detector temperature was 300 °C.
Compounds were identified by three independent analytical methods: (i) retention indices (RIs), (ii) GC retention times of au-
thentic chemical standards and (iii) mass spectra of compounds (http://massfinder.com/wiki/Terpenoids Library List) and the
NIST14 spectral library collection (http://webbook.nist.gov/chemistry). The RI standards used in this study consisted of a mixture of
aliphatic hydrocarbons ranging from C-5 through C-30 dissolved in hexane. The component quantities were calculated as mean
values from duplicate GC/MS analyses.
Quantification was conducted by GC-FID using a Clarus 580 system (PerkinElmer, Shelton, Connecticut U.S.A.) equipped with an
Elite-5MS (30 m × 0.25 mm i.d., 0.25 μm film thickness) capillary column (PerkinElmer). The beta-ionone was used as an internal
standard (0.5 μg of beta-ionone in sonicated water per urine sample), comparing its concentration with that of other compounds,
assuming a general response factor of 1 for all volatile compounds; no standard curves were constructed for individual compounds
that were detected in the samples. The GC conditions were the same as those for GC/MS. The analyses were conducted using helium
as the carrier gas.

2.4.2. Analysis of fatty acid profiles of urine, the vagina, vulva and the anal region
The lipid extraction was conducted using the previously described method (Maslak et al., 2015). The extracted lipid fraction,
about 50 mg, was subsequently saponified (10 min. at 75 °C) with 5 mL of a 0.5 M solution of KOH/MetOH and subjected to me-
thylation (10 min at 75 °C) in 5 mL 14% (v/v) BF3/MetOH (Sigma-Aldrich, St. Louis, MO, USA). Then, water was added to the
reaction and the methyl esters of fatty acids were extracted with 20 mL of hexane (Avantor Performance Materials, Poland S.A.,
Gliwice, Poland), washed with 10 mL of 10% sodium bicarbonate (UQF Wrocław, Poland) and dried over anhydrous sodium sulfate.
The organic phase was evaporated using reduced pressure and stored at −27 °C until further chromatographical analysis. The FAME
profile was assessed using a gas chromatograph coupled with a mass spectrometer (Shimadzu GCMS QP 2120, Shimadzu, Kyoto,
Japan). Separation was achieved using a Zebron ZB-WAX capillary column with a length of 30 m, an inner diameter of 0.25 mm, and
a film thickness of 0.25 μm (Phenomenex, USA). The GC–MS analysis was performed according to the following parameters: scanning
from 50 to 400 m/z in electronic impact (EI) at 70 eV, mode at 5 scan s[−1/−1] mode. Analyses were conducted using helium as the
carrier gas at a flow rate of 1.0 mL min−1 in a split ratio of 1:10 and with the following program: a) 40 °C for 3 min; b) a rate of 5.0 °C
min−1 from 40 to 160 °C; c) a rate of 30 °C min−1 from 160 to 280 °C. The injector was maintained at 220 °C. Most of the compounds
were identified by using three different analytical methods that were used to compare: i) retention times with authentic chemicals
(Supelco); ii) mass spectra obtained, with the available library (NIST14, similarity indexes > 90%); iii) calculated retention indices
with data presented in the NIST14 library and the NIST webbook (http://webbook.nist.gov, access xxx).

2.5. Statistical analysis

Data were evaluated with the T-Student test (Statistica 12.0, StatSoft Inc., USA), with the significance level P < 0.05.

3. Results

In the samples collected from the vulva, vagina and anal region, no significant differences in composition were found between
various phases of the ovarian cycle. In the samples of female urine, however, there were significant differences in composition
between the phases of the ovarian cycle. The concentration of some compounds identified in urine was different (P < 0.05) between
compared groups: anestrus compared with proestrus and estrus compared with diestrus. When the animals were approaching
(proestrus) or were receptive to mating (estrus), the concentrations of carbonyl aromatic compounds, such as acetophenone (hyp-
none) and benzaldehyde, significantly increased. This was accompanied by increased concentrations of methylketones, e.g., 2-oc-
tanone, 2-pentanone, and 3-hexanone. Conversely, the sulfide compounds 1-methylotiopropane, 1-methylotiobutane, 1-methyloti-
pentae and dimethyl trisulphide significantly decreased during estrus and were greater during the non-estrual periods (Table 1).
Evaluation of the profiles of fatty acids in urine samples and in the other previously described samples indicated there were no
significant changes in these compounds during the ovarian cycle of dogs (Table 2).

4. Discussion

In domestic dogs, proestrus is the phase when bitches attract males due to the increased release of specific semiochemical
compounds. Increased frequency of urination by the bitch can be observed throughout this time (Kustritz, 2005). For the attracting
effect of males, excretion of highly volatile substances detectable from a great distance seems to be responsible. After approaching
bitches in estrus, however, males appear to confirm their receptivity by close direct contact including sniffing and licking at her urine,
anal gland secretions and the vulva (Rekwot et al., 2001; Kustritz, 2005). This behavior is accompanied by increased salivation by the
males which probably facilitates transportation of the non-volatile semiochemical compounds into the vomeronasal organ, which is
widely considered the organ responsible for pheromone detection (Rekwot et al., 2001).
During proestrus, females are attracted to males, however, will not allow males to mate. A true state of estrus is defined as the

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Table 1
Selected compounds secreted in urine during different phases of ovarian cycle in bitches.

KI exp. – Retention index calculated on the basis series of n-alkanes; KI lit. – Retention index from presented in NIST14 database, Mass Spectral
Library, NIST Mass Spectral Search Program Version 2.0d, Gaithersburg, MD, USA.

Table 2
The average profile of fatty acids (expressed as methyl esters) in vaginal swab collected from females in estrus and anestrus.
Nr Peak Compound tR (min) Area (%) Area (%)
Anestrus Estrus

1 Octanoic acid, methyl ester 13.540 0.401 0.344

2 Decanoic acid, methyl ester 18.790 2.071 2.087
3 4-Decenoic acid, methyl ester, Z- 19.955 0.365 0.411
4 Dodecanoic acid, methyl ester 23.535 0.529 0.563
5 Myristic acid, methyl ester 27.845 2.189 2.220
6 Hexadecanoic acid, methyl ester 31.790 23.794 23.716
7 9-Octadecenoic acid (Z)-, methyl ester 32.200 3.251 3.139
8 Stearic acid, methyl ester 35.410 12.801 12.832
9 Oleic acid, methyl ester 35.690 42.213 42.345
10 Elaidic acid, methyl ester 35.815 3.142 3.204
11 Linoleic acid, methyl ester 36.425 9.244 9.140

tR: retention time of compounds; Area %: percentage area of analyzed FAME (Fatty Acid Methyl Esters) signals.

period when bitches allow mating to occur and is obviously the period associated with when fertilization of the ova will occur. During
the period when bitches allow mating with the male there is also passive acceptance of courtship of the male and frequent copulation
can be observed (Kustritz, 2005).
When evaluating the progress of both the above-mentioned phases of the ovarian cycle, using vaginal cytology (percentage of
cornified epithelial cells) and the progesterone concentration in peripheral blood, a gradual increase in the values for these variables

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was observed. In contrast, the transition from estrus to diestrus appears to be much more sharply demarcated. A bitch can allow
mating during one day but will have a sudden reduction of the tolerance reflex the following day. Specific changes are detected with
the cytological examination, including the sudden disappearance of cornified epithelial cells and increased amounts of basal cells and
leucocytes. These cell cytology changes are associated with the estrus-to-diestrus changes in the previously described behavioral
responses of females to males (Bouchard et al., 1991). In the case of behavioral events, males quickly recognize the lack of receptivity
of the female and will not attempt to mate. Males seem to discriminate estrus from diestrus mostly by sniffing the female while
bitches in diestrus often do not allow close contact and licking of the vulva. There is no increased salivation in males during contact
with a bitch in diestrus.
Analysis of urine samples collected during different stages of the ovarian cycle indicated significant changes: specific alterations in
concentrations, including increases in some substances and decreases in others. Acetophenone, benzaldehyde and methylketones
were identified as characteristic for the proestrus and estrous phases. Interestingly, at the same time, decreased concentrations of
other substances (sulfide compounds) were observed. These substances are usually recognized by having a distinctive foul odor, and
as concentrations increase the prevalence of the foul odors also increases (Shirasu et al., 2009). It was observed that sulfide com-
pounds that decreased at estrus increased immediately at the end of this phase. This seems to be strictly correlated with the observed
behavioral events, i.e., with a decreasing interest of males in the bitch. This observation may suggest a dual characteristic of the
mechanism of interaction between canine males and females during their mating period, including the attracting effect of some
substances during proestrus and estrus, and a repelling effect due to increased concentrations of sulfide compounds at the beginning
of diestrus.
Fatty acids were recognized as potential semiochemically active compounds in cats and poultry (MacDonald, 1985; Pageat and
Gaultier, 2003; Madec et al., 2008). Sankar and Archunan (2008) suggested that acetic acid and propionic acid were compounds that
increased in the feces of cattle during the estrous phase of the reproductive cycle. In dogs, however, there were no changes in the
present study in concentrations of these substances during the different stages of the ovarian cycle. This is consistent with ob-
servations in other studies where there was investigation in these regards and there were indications that the female attraction effects
of anal gland secretions does not increase during the estrus phase of the reproductive cycle (Doty and Dunbar, 1974; Preti et al.,
1976). The high frequency of inter-individual contacts consisting of sniffing the female anal region during the estrous cycle suggests
the general importance of signals emitted from this body region. The lack of an unambiguous connection between the investigation of
this area by male dogs and sexual behaviors, could suggest the role that the emitted signals have in discrimination of other features,
such as age and gender, which has been previously hypothesized (Doty and Dunbar, 1974; Janssenswillen et al., 2017). In this
context, however, it is worth mentioning that the substances responsible for this kind of interaction are proteins, which were not
investigated in the present study. The role of these non-volatile compounds in stimulating mating behavior can not be under-
estimated, because in another species the lipocalycin protein alone contained in the vaginal discharge was responsible for inducing
mating behavior (Tirindelli et al., 2009). Furthermore, according to Brennan and Keverne (2004), urine proteins could function as a
carrier of pheromones. It is still unknown whether such proteins have pheromonal properties or are only carriers for volatile
pheromones. Further studies should be conducted to assess the role of these substances in the complete array of semiochemical
signals.

5. Conclusions

Specific changes in the composition of urine during particular phases of the ovarian cycle in bitches involve alternating increases
and decreases in the concentrations of some volatile compounds. This suggests a dual character of inter-individual interactions
around the mating period, including attracting and also repelling mechanisms toward the male. Further study including a proteomic
approach, behavioral and physiological assessments are suggested to identify a complete range of the estrual semiochemical signals
and to verify the semiochemical activities of the identified substances.

Conflict of interest statement

The authors declare that no competing interests exist.

Acknowledgements

This study was financially supported byThe National Science Centre (Poland), grant No. UMO-2015/17/B/NZ8/02411, KNOW
project and by statutory research and development activity funds assigned to the Faculty of Veterinary Medicine, Wrocław University
of Environmental and Life Sciences, Poland. Authors would like to thank Dr. Barry Bavister for language correction of the text.

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