Cbemkal Senses Vol.12 no.l pp.

77-87, 1987

Identification of compounds in bovine cervico-vaginal mucus

extracts that evoke male sexual behavior

W.R.Klemm, G.N.Hawkins and E.De Los Santos

Department of Veterinary Anatomy, Texas A&M University, College Station,
TX 77843, USA

Abstract. Cervico-vaginal mucus was collected from 140 cows at or near estrus. Samples were partially
purified, evaporated, and reconstituted in water for testing over a 2-year period to determine if any extracts

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would sexually excite bulls. Extracts warmed to body temperature were randomly presented in a flat dish
to bulls that were loosely confined in a stanchion, with no cows around. Certain types of extracts evoked
linked behaviors comparable to those exhibited naturally with estrus females (olfactory inspection, licking
of sample, flehmcn, penile contraction, preputial secretion). Extracts were also examined by gas chromato-
graphy — mass spectrometry. Analysis identified over 20 compounds: alcohols, diols, alkenes, ethers, diethers,
ketones, primary amines, aromatic alkanes. Nine compounds were found in samples that had been validated
as being sexually stimulating; six of these were validated as positive with another bull when randomly tested
after 1 year of storage. None of these compounds to our knowledge has been reported as present in estrous
mucus and one or more of them may be considered as putative sex pheromones.

Introduction
The physical and chemical properties of bovine cervico-vaginal mucus have been of
long-standing interest to reproduction researchers because the mucus enhances con-
ception by facilitating viability and transport of sperm. The secretions generally become
more abundant at estrus and become viscous enough to permit pulling the mucus into
long strands (5 — 12 cm) before it breaks (Paleologou, 1979; Panigahi, 1964). Estrous
mucus also produces a 'fern-like' crystalline pattern when smears are dried on a glass
slide (Noonan et al., 1975); ferning seems to correlate with Na or Cl content (Aboul-
Ela et al., 1983).
These physical changes in mucus are only loosely correlated with estrus, and have
not shown sufficient correlation to be used in diagnostic tests for estrus, even though
several have been well studied (e.g. viscosity, conductivity, pH) (Foote, 1975). Estrus
detection is a major problem in cattle operations where bulls are not present, as with
artificial insemination and embryo transplant operations (Foote, 1975).
Estrus detection, by bulls under field conditions, seems to involve the sensory detec-
tion of a pheromone (Hart et al., 1946; Hradecky et al., 1983; Jacobs et al., 1980,
1981; Macmillan and Fielden, 1964; McGrath etal., 1981; Paleologou, 1977a,b; and
Sambraus and Waring, 1975). Discovery of the pheromone could expedite develop-
ment of a reliable field test to detect estrus in both beef and dairy cattle. At present,
there is no practical, reliable way to detect estrus other than the mounting behavior
performed by bulls under natural conditions (Foote, 1975; Paleologou, 1977a,b;
Williamson et al., 1972). Herdsmen, even well-trained observers, are often wrong in
their diagnoses of estrus (Kiser et al., 1977; Paleologou, 1977a; Williamson et al., 1972).
Some literature does describe chemical composition of the reproductive tract secre-
tions which are the most likely source of pheromone, but that research has not focused

© IRL Press Limited, Oxford, England 77

W.R.Kkmm, G.N.Hawkins and E.De Los Santos

on volatile compounds, despite the fact that bulls can remotely sense something in the
mucus of estrous cows which is sexually arousing. Under field conditions, visual stimuli
probably do play a role in stimulating male sexual behavior, but our in vitro tests with
cervico-vaginal mucus clearly show that there is an olfactory component, independent
of vision, in the stimulation of male reproductive behavior, supporting the reports of
others (Hart et al., 1946; Izard and Vandenbergh, 1982; Jacobs et al., 1980; Paleologou,
1977a,b; Sambraus and Waring, 1975; W.R.Klemm, G.H.Hawkins and E.De los Santos
in preparation). Similar evidence for a pheromone has been indicated by studies where
dogs or rats were trained to detect estrus by smelling cervico-vaginal secretions or urine
(Kiddy et al., 1978; Ladewig and Hart, 1981).
Cervico-vaginal mucus contains a great deal of glycoprotein (Friess, 1972; Kandukuri

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et al., 1977) in which the amino acid composition changes with phase of the estrous
cycle (Hayashi and Matsukawa, 1979). Vestibular gland secretions contain a substan-
tial amount of sialic-acid-bearing glycoprotein (Freiss, 1972). In cervical mucus, sialic
acid content is minimal at estrus (Hamana et al., 1971). The pH of cervico-vaginal
mucus is most acid (— 6.5 — 6.7) at estrus, and at this time the water content is greatest,
while total protein is lowest (Mori et al., 1979; Paleologu, 1979; Schilling and Zust,
1968). Mucus has a distinct decrease in peroxidase activity at estrus, followed by a
marked rise during the next 3 days (Foulkes et al., 1981). Cervico-vaginal mucus at
estrus has increased sodium, chloride and magnesium, while calcium, potassium and
lipids are decreased then (Paleologu, 1979). Electrical conductivity is greatest at estrus
(Mori et al., 1979; Stan, 1969).
The source of the mucus may be the glands of the cervix or the major vestibular
glands (Bartholin's glands) in the vestibule, all of which hypertrophy at estrus (Friess,
1972) in response to estrogens (Herrick, 1951; Kroes et al., 1970).
We have taken an approach to estrus detection which is based on the fact that bulls
have no difficulty in knowing when cows are in estrus; a capability which presumably
derives from the fact that estrous cows give off a volatile pheromone from their cer-
vico-vaginal mucus and urine which signals the presence of estrus. Such a pheromone
should be detectable by chemical means, and thus might lead to a simple chemical test
for estrus. We therefore have begun a program of chemical analysis of purified com-
pounds in cervico-vaginal mucus from estrous cows.

Methods

Sample collection
Samples were collected generally by rectal massage of the reproductive tract or from
an infusion tube placed within the vagina prior to artificial insemination procedures
on 140 cows. Insemination technicians reported that these cows had shown some signs
of estrus within the previous 24 h or, in a few cases, were presumed to be in estrus
because of the elapsed time since prostaglandin injection. Several cattle operations pro-
vided the samples, generally under field conditions where immediate freezing and ship-
ment in a frozen state were not possible. Many of the samples also had a degree of
fecal contamination. Samples were kept frozen at 0°C upon receipt in our laboratory
until extraction began. Extracts were kept frozen except when being used for testing
in the bull assay and for mass spectrographic analysis.

78
 Bovine cervico-vaginal mucus extracts

Sample extraction
Samples were purified in several ways, inasmuch as we could not know which ap-
proaches would be effective. Some samples were processed by liquid partitioning with
chloroform — methanol — water. Others were loaded onto open-column packings con-
taining either cyanosilane, C18 reverse-phase packing, or ion exchange packings (6-ml
cartridges, Baker Co.), using solvent systems recommended by the manufacturer. In
each instance of partial purification, the solvents were evaporated at 35°C to remove
organic solvent, and the residue was reconstituted in 30 ml of water for testing on bulls;
this same concentration was used for mass spectrometry analysis after the bull assay.

Sample extraction — liquid partitioning

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The typical partitioning procedure consisted of dissolving mucus samples (generally
5—40 ml per cow) in four volumes of chloroform:methanol (4:1). This mixture
commonly created three immiscible layers. The lower two layers contained organic
solvent and were separately rotary-evaporated and then reconstituted with water.

Sample extraction — open-column chromatography

The loading solvent, the fl fraction, which passed through the column as the column
was loaded was saved for behavioral testing along with the solvent, f2, which was used
for elution. Whenever a loading or eluting solution contained organic solvent, the solu-
tion which passed through the column was rotary-evaporated to remove noxious organic
odors.

Silica gel. Samples were extracted in 10 volumes of chloroform:methanol (1:1), filtered,

and loaded through the column. After allowing 5 ml of methanol to flush the column,
30 ml of phosphate buffer solution was used for elution.

C18 reverse-phase. Samples processed in this way were first extracted by 4 volumes
of chloroform-methanol (1:1) or in 6 volumes of water. The aqueous solution, or the
chloroform — methanol extract, reconstituted in water after rotary evaporation, was load-
ed onto the column packing at a rate of 1 drop/s. The eluent (f2) was comprised of
5 ml of methanol followed by 20 ml of chloroform — methanol (1:1).

Anionic exchange. Mucus was extracted with water, pH was adjusted to 10 with 0.5 M
NaOH, and loaded through a quaternary amine cartridge. Elution was produced with
30 ml of acetic acid solution at a pH of 5.4.

Cationic exchange. Aqueous samples were made acidic (pH 5) with 0.1 ml HC1 and
loaded on the packing. Elution was achieved with 30 ml of water (pH 12); before testing,
the f2 was adjusted to a pH of 8 by addition of 0.1 M HC1.

Cyano normal phase. Chloroform:methanol (1:1) extracts were used for loading die
column, and elution was produced by 5 ml of methanol, followed by 25 ml of water.

Bull assay
Samples were warmed to body temperature and presented randomly, one at a time,
to one of five bulls. The bull assay, which is described in detail elsewhere (W.R.Klemm,
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W.R.Klemm, G.N.Hawkins and E.De Los Santos

80
 Bovine cervfco-vaglnal mucus extracts

G.H.Hawkins and E.De los Santos, in preparation), consisted basically of loosely restrain-
ing a bull in a stanchion in an outdoor pavilion with no females around. Test sessions
were held at 1-week intervals or greater, and in a given session a group of samples
(13—50) was presented randomly to the bull, within inter-trial intervals of at least 1
min. Five different bulls were used on different occasions over the 2 years of these
assays, and each bull showed the full range of responses.
Each sample was presented to the bull in a large saucer. Responses were scored as
neutral (no interest), aversive (active aversion movements), or positively attracting.
If the sample was stimulating to the bull, he approached it, licked it, performed flehmen
reactions, and sometimes showed overt signs of sexual arousal, such as penile contrac-
tion and erection, and preputial secretions (Figure 1). Since all samples were wanned

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to body temperature during processing to remove organic solvents, and were also warmed
just prior to testing in the bull assay, we assume that much of the stimulating volatile
compounds had already escaped by the time of presentation to the bulls. Samples that
were behaviorally positive were randomly presented again, either in the same session
or on another day, and had to produce a positive response the second time in order
to be scored as positive.

Gas chromatography — mass spectrometry

Samples were examined either by direct injection or by head-space gas analysis. The
unit used was an HP 5980 A GC-mass spectrometer with a capillary column. Samples
of 3 ill were heated from 70 to 270°C at a rate of 8° per min. The mass range evaluated
was from 50 to 350, with a resolution better than 1 unit at all masses. Spectra were
stored on a 5988 HP data storage system.

Results

Validation of the behavioral test

Results from a typical test session are summarized in Table I. There was no indication
of spurious positives. Most samples proved to be behaviorally negative, i.e. the bull
showed no interest, not even initial 'inspection' reactions. With positive samples,
however, bulls always orientated to the sample, lowered the head to inspect (smell)
it, licked it, elevated the head, and produced at least a degree of flehmen ('yawn' flehmen
was a rarely seen, incomplete kind of flehmen). Orientation sometimes occurred when
the investigator removed the cap to the sample container some 3 m away.
Several measures were taken to assure that the test was not producing false positive
results. On at least 10 occasions we re-tested the same sample on the same bull in the
same test session, and in every case negative samples were still negative while positive
samples were still positive.

Fig. 1. Sequence of behaviors elicited in bulls when they are presented with estrous mucus or certain ex-
tracts thereof. Dish with 30 ml of extract at body temperature is presented toward the bull's nose. The chain
of linked behaviors begins by the bull's orientation to the sample dish, followed by approach, inspection,
and sniffing (A). The bull then licks the sample (B), followed by stroking the roof of the mouth (not shown)
and then flehmen ( O in which the head is extended and elevated, the upper lip and nostrils are drawn backward
and the lower teeth exposed.

81
W.R.Klemm, G.N.Hawkins and E.De Los Santos

Table I. Representative test results with semi-purified extracts — shown in the sequence of sample prepara-
tion to the same bull.

Date Sample Purification method Behavioral response

5/15/85 MC2 COOH, f2 —

G-8 CN, n —
G-10 CN, fl -
G-4 CN, fl + (+ 1 year ago)
5/22/85 S-7 CN, n -
S-« CN, n -
Stone 1 Silica, f2 -
S-7 Anion ex., fl +

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HK4 C18, f2 + (yawn flehmen)
A Raw mucus (known - ) -
S-6 CN, fl +
S-7 CN, fl -
S-9 C18, fl -
Silica, fl + (yawn flehmen)
G-4 CN, f2 +
G-3 CN, fl -
S-10 CN, fl -
S-7 Anion ex., f2 -
S-8 C18, fl -
S-5 CN, n -
S-3 Anion ex., f2 -
S-2 CN, n -
S-10 CN, f2 -
G-2 CN, n -
HK1 Anion ex., f2 -
S-8 C18, T2. -
HK-2 C18, f2 -
S-5 CN, fl -
G-5 CN, fl -
HK-2 C18, fl -
G^ CN, fl +
HK-3 Silica, fl -
G-3 CN, n -
S-2 CN, fl +
HK-1 Silica, fl -
HK-3 Silica, f2 -
G-5 CN, f2 -
G-2 CN, fl -
Stone 1 Silica, f2 -
S-9 C18, f2 -
HK-1 Anion ex., fl -
S-3 Anion ex., f2 -
HK-4 C18, fl -
S-3 Anion ex., fl -
S-4 Silica. (2

Another indication of test validity is the consistency of results with the same bull
to the paired fractions from the same original mucus sample (Table II). Best results
were seen with anionic exchange fl and cyano fl fractions in that bulls found the f 1
fractions to be positive and the f2 fractions negative.
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 Bovine cervico-vaginal mucus extracts

Table II. Incidence of reactions to the paired fraction ot a known positive sample — both fractions tested
on same bull.

Purification No. of positive Response to paired fraction

fractions Positive Negative

Silica, f2 3 0 3
C18, fl 2 1 1
C18, (2 3 1 2
Anionic ex., fl 5 0 5
Cationic ex., fl 4 2 2
Cationic ex., f2 2 2 0
Cyano, fl 15 0 15

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Cyano, f2 1 1 0

Table m . Summary of purification methods that produced positive fractions.

Procedure Sample and + fraction

Liquid partitioning 85 G-3, aqueous

85 G-6, aqueous
Silica gel HK1, f2
S-l, fl
S-4, fl
C18 S-8, fl
85, S-4, fl
HK2, f2
HK4, f2
MCI, f2
Anionic exchange S-3, fl
S-7, fl
MCI, fl
Cationic exchange G-14, fl
G-14, f2
MCI and MC2, fl
MCI, MC2andMC3: all f2
Cyano S-2, S-6, S-7, S-10, S-5
G-2, G-3, GA, G-5, G-6,
G-8, G-10, G-ll, 85 S-6:
All fl
G-4, f2

A final indication of test validity is to test the same fraction at different times on
a different bull. Of 43 samples tested in 1984 that were negative, 41 were found negative
when tested in 1985 on a different bull. Of 21 positive samples in 1984, 12 were still
positive in 1985, despite having been warmed and tested once before and having been
stored for 1 year at only 0°C.

Purification procedures that yield positive samples

Of the 254 sample tests on bulls, 49 produced positive responses. Several of the purifica-
tion procedures were able to preserve behaviorally stimulating properties in one, but
83
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Table IV. Compounds identified in semi-purified extracts.

Compound Mass numbers (m/z) Formula

Alcohols
'1-heptanol 50, 55, 58, 59, 73, 101, 116 CH3(CH,)5CH,OH
4-undecanol, 7 ethyl, 2 methyl 73, 154, 181, 214 (CHJ)JCHCH,CHOH(CH,),C(CH,),H(CH,)J
2-dodecanol 73, 168, 186 CHJCHOH(CHJ),CH3
l-butoxy-4 butanol 73, 113, 128, 146 CHJ(CH,)JCH2OCHJ(CH2),CH2OH
non-2-ene-4-one-1 -ol 71, 123, 156 CH,OHCH,CHCOCH,(CH,)jCH,
deca-2-ene-4-one-1 -ol 71, 152, 170 CHJOHCHJCHCOCH^CH^CHJ

Diols
2
1,3-dihydroxypentane 43, 45, 47, 59, 61, 75, 89, 104 CHJCHJCHOHCH,CHOH
? unsaturated diol 71, 122, 134, 211,223,246,276 C,^2tO2
2,4-dihydroxyheptane 56, 58, 76, 83, 85, 98, 117, 132 CHjCH2CHOH(CH2)2CHOHCH,
1,2-cyclopentanediol, 3 methyl 95,97, 112, 115, 130
Alkenes
1-decene, 8-methyl 82, 139, 154 CH2CH(CH2),CH(CHj)CH2CH,
2-undecene, 4,5-dimethyl 126, 152, 182
1-tetradecene 70, 139, 181, 196 CH^CH^CH,
1 -tridecene 70, 137, 167, 182 CH^CH^.CH,
1-hexene 3,5,5-trimethyl 55, 81, 126 CHJCHCHCHJCHJCCCHJJJ
3,6,8-tridecenoic acid 73, 102, 121, 133, 207
Ethers/diethers
1-ethyoxydccane 41, 141, 186 (CHJCO)CH2(CH2),CH3
1,3-dimethoxypropane 45, 59, 73, 104 CHJCKCHJ),
OR
1
1,2-dimethoxypropane CH5O(CHj)2COCHj
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Compound Mass numbers (m/z) Formula

Ketones
2-heptanone 73, 83, 96, 99 CHJ(CHJ)4COCH3
1
1 -methoxy-3-pentene 59, 69, 85, 100 CHJCHCHCCH^JOCH,
2
1 -pheny lethanone 69, 77, 87, 104, 105, 120 C,H,CH,OCH
2
l-phenylpentanone 77, 117, 145, 160 C.H^CH^OCH
heptyl-3-one 53, 54, 56, 74, 83, 99, 114 CHJ(CHJ)2CO(CHJ)2CH3
*? 71, 102, 117, 122, 134, 172, 207, 223, 268 C,,H M O
Amines
'2-propanone-l,3-<liol-l-amine 59, 75, 89, 105 C3H7NOj
(serine-like)
1 -pypery 1-2-ethanone 52, 53, 54, 66, 79, 105, 133 C,H4NCH2OCH
71, 93, 122, 134, 136, 194,211 CuHj.N
? Secondary amine C-.H.-N ?
Esters 71, 99. 102, 116, 121, 133, 171, 210, 225
Heptyl methyl ester
Aromatic alkane 52, 56, 89, 105, 121, 136
1-pheny lheptane
77, 91, 162
'These compounds were found in semi-punfied extracts that were behaviorally positive.
These compounds were behaviorally positive when tested blind on two bulls, two occasions 1 year apart.
W.R.Klemm, G.N.Hawkins and E.De Los Santos

not both, of the two major fractions (Table HI). The most consistent results were found
with anionic exchange fl and cyano fl fractions. Unfortunately, these procedures do
not allow concentration of the behaviorally active compounds because they apparently
do not stick to the packing material. The other procedures apparently caused the
behaviorally active compounds to distribute into both fractions, essentially diluting them.
Chemicals identified
Numerous compounds in several major chemical groups have been identified in the
various purified extracts (Table IV). The exact chemical structure of a few of the com-
pounds could not be identified with present equipment. The compounds which at the
present seem most likely to be putative pheromones are the nine compounds that were
present in extracts that gave strong positive reactions in the bull assay. Six of these

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compounds were present in extracts that were still positive even when tested on another
bull over 1 year later.

Discussion
The behavioral assay confirms the evidence in the literature that cervico-vaginal mucus
contains substances that are sexually stimulating to bulls.
The failure to demonstrate positive responses with all samples can be explained by
one or more of the following reasons, (i) Some samples were probably collected after
estrus, which only lasts about 12 h in cattle, (ii) Maximum production of behaviorally
active compounds may occur before estrus, or in the earlier stages, (iii) The behaviorally
active compound is volatile, and loss could have occurred at several steps: the original
collection and delay before refrigeration, loss during transport to the laboratory, loss
during the rotary evaporation of semi-purified extracts, and loss during the heating prior
to behavioral assays.
The extraction and purification procedures have disclosed a wide range of compounds
that have not heretofore been reported as present in cervico-vaginal mucus. One or
more of the nine compounds that were found in behaviorally positive samples may be
the naturally occurring estrus pheromone.
To confirm the identity of the true pheromone(s), synthetic compounds of the type
found in these extracts will have to be tested in the bull assay and/or in tests involving
female cattle that have the compound applied to their vaginal area.

Acknowledgements
We wish to thank the artificial insemination operators who provided samples of cervico-
vaginal mucus: Granada Genetics Inc., Marquez, TX; McKellar Genetics Inc., Mt.
Pleasant, TX; 3X Ranch, Placedo, TX; and McWilliams Ranch, Simms, TX. Thanks
also go to D.L.Bricker and the Texas A&M Center for Chemical Characterization and
Analysis. This research was supported by the U.S.D.A./SEA/CR Project AH-6393
and by the Texas Agricultural Experiment Station.

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Received March 1986; accepted October 1986

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