Enhancing bull sexual behaviour using oestrus-specific molecules identified in cow urine

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Theriogenology 83 (2015) 1381–1388

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Theriogenology

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Review article

Enhancing bull sexual behavior using estrus-specificmolecules identified in cow urine

Chrystelle Le Danvic a,*, Olivier Gérard b, Eli Sellem b, Claire Ponsart b,Philippe Chemineau c, Patrice Humblot b,1, Patricia Nagnan-Le Meillour d

aUNCEIA, UMR CNRS/USTL 8576, UGSF, Villeneuve D’Ascq, FrancebUNCEIA, Département R&D, Maisons-Alfort, Francec INRA, CNRS, Université de Tours, Haras Nationaux, UMR 6175 Physiologie de la Reproduction et des Comportements, Nouzilly,Franced INRA, UMR CNRS/USTL 8576, UGSF, Villeneuve D’Ascq, France

a r t i c l e i n f o

Article history:Received 29 April 2014Received in revised form 30 January 2015Accepted 1 February 2015

Keywords:CowEstrusUrineChemical signalBullSexual behavior

* Corresponding author. Tel.: þ33 3 20 43 40 10;55.

E-mail address: [email protected] (C1 Present address: Faculty of Veterinary Medicine

Department of Clinical Sciences, SLU, Po Box 7054Sweden.

0093-691X/$ – see front matter � 2015 Elsevier Inchttp://dx.doi.org/10.1016/j.theriogenology.2015.02.0

a b s t r a c t

Deficiencies in bull mating behavior have implications for bovine artificial insemination ac-tivities. The aimof this studywas to identify the compounds present influids emitted bycowsduring estrus, which could enhance bull libido. Chemical analysis of urine samples from cowsled to the characterization of molecules varying specifically at the preestrous and estrousstages. The synthetic counterpart molecules (1,2-dichloroethylene, squalene, coumarin, 2-butanone, oleic acid) were used to investigate the biological effects on male sexual behaviorand sperm production. When presented to males, 2-butanone and oleic acid synthetic mol-ecules significantly loweredmounting reaction timeandejaculation time (�33%and21%after2-butanone inhalation, respectively, P < 0.05). The “squalene þ1,2-dichloroethylene” com-bination induced a 9% increase of sperm quantity (P < 0.05). This study suggests that theidentified estrous-specific molecules could be part of the chemical signals involved in maleand female mating behavior and may be used for a wide range of applications. The identifi-cation of these molecules may have implications for the cattle breeding industry.

� 2015 Elsevier Inc. All rights reserved.

1. Introduction

Production centers collecting semen for bovine artificialinsemination (AI) are regularly confronted with bulls dis-playing reduced or complete absence of sexual behavior, in atemporary or permanentmanner. This concerns bothyoungindividuals, which may be eliminated from the breedingprogramafter a failed sexual function assessment, and adultbulls [1], for which the efficiency of semen collection has ahigh economic impact. Consequences of these disorders are

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numerous for semen collection centers and for breedingcompanies. Significant time losses associated with an in-crease in the risks taken by technicians to excite the bullswith low libido lead to a lower efficiency of workforce. Theaccompanying reduced number of semen doses has conse-quences for genetic improvement. Considering the impactof deficient mating behavior on the availability of bullsemen, new and sustainable management solutions for bullsemen production are critical.

In cattle, as in most of mammals, chemical communi-cation has been described to act either singly or in combi-nation with auditory, visual, or tactile stimuli during theprocess of reproduction [2]. Males in the presence of estrusfemales perform a courtship sequence, which includessmelling behavior (distant or close to the cow), licking, andflehmen. This courtship sequence, resulting in a completesexual behavior with erection, mounting of the female, and

C. Le Danvic et al. / Theriogenology 83 (2015) 1381–13881382

copulation, supports a chemical information exchange be-tween partners during natural estrus [3]. Several bodyfluids are thought to carry this chemical information.Whenestrous and nonestrous fluids were presented to males ofdifferent species, including bulls (saliva, vaginal fluids,milk, feces, serum [4,5]), dogs (vaginal mucus, urine, milk,plasma [6,7]), and rats (urine [8]), the ability to discrimi-nate between the two states is compelling. In the past 10years, investigation of estrous and nonestrous fluids fromcows has led to the characterization of a number of estrus-specific compounds and their effect on bulls. Triethylamine,acetic acid, and 4-propyl phenol, identified in the saliva ofestrus cows, have been shown to increase flehmen fre-quency and mounting behavior in bulls [5]. Acetic acid,1-iodoundecane, and propionic acid, identified as estrus-specific compounds in feces, were shown to inducelonger flehmen and increase the number of mounts whenpresented, alone or in mixture, to bulls [9]. n-Phthalate and1-iodoundecane were identified as estrus-specific com-pounds in cow urine [10], but their biological activity hasnot been found.

On the basis of these findings, we hypothesized thatchemical signals present during estrus may also be impli-cated in the sexual behavior of bulls. The objectives of thisstudy were to identify chemical signals emitted by cowsduring the heat period and to test their effect on sexualbulls’ behavior and sperm production.

2. Materials and methods

2.1. Reagents and chemicals

All chemicals and reagents used in this study(dichloromethane, ethanol, glycerol, 1,2-dichloroethylene(DCE), coumarin, squalene [SQ], oleic acid [OA], 2-butanone) were of analytical grade purity and purchasedfrom Sigma–Aldrich.

2.2. Cows and cycle following

This study was conducted using 32 cows (Bos taurus,Prim’Holstein, andMontbéliarde breeds), housed the INRA-UEPAO (Institut National de la Recherche Agronomique -Unité Expérimentale de Physiologie Animale de l’Orfrasière,Nouzilly, France) andUNCEIA (UnionNationaledesCoopéra-tives d’Elevage et d’Inséminations Animales, Châteauvillain,France) experimental stations. Cows were fed a standarddiet of straw and ad libitum water. Estrus was induced bysynchronization with two PGF2a injections spaced 10 daysapart. Stages of the cycle were determined by immunoassayof plasma progesterone (Ovucheck plasma kit for bovine;BioVet) and LH (LH Detect kit, ReproPharm) concentrationevery six hours and behavioral observations (twice a day),during the interval from the second PGF2a injection to theend of estrus. Five collection sessions in which five, six, oreight cows were followed, have been performed.

2.3. Urine collection

For each animal, urinary samples were collected insterilized glass vials (Supelco) during midstream urination,

three times a day, from 2 days after the second PGF2a in-jection during 1 week, corresponding to preestrous andestrous stages, then 11 days after the second PGF2a injec-tion for the luteal stage. Samples collected from thebeginning of the procedure until first signs of estrus werecategorized as “preestrus samples.” Samples collectedaround the LH peak (when observed) from cows acceptingmount were categorized as “estrus” (between 10 and 48hours). Sampling was done during natural urinations (nomanual bladder stimulations). Vaginal secretions were alsocollected when emitted. The collected samples were frozenwithin the hour and stored up to 3 months at �20 �C untilanalysis.

2.4. Extraction of urinary chemical compounds and gaschromatography–mass spectrometry (GC–MS) analysis

Thawed portions of urine (4 mL) and vaginal mucus(1 mL) were extracted twice by dichloromethane (v:v, pu-rity > 99%) on ice. After a brief mix by vortexing andcentrifugation (2500 rpm for 20 minutes at 4 �C), the upperorganic phase was dried on an anhydrous sodium sulfatecolumn (Sigma–Aldrich) to remove traces of water. Theextract was concentrated to 100 mL under gentle nitrogenstream and stored at �20 �C until chemical analysis. Gaschromatography–mass spectrometry analyses were per-formed on a quadrupole mass analyzer DSQ II (ThermoScientific) coupled to a FOCUS (Thermo Scientific) gaschromatograph. Separation of the extracted urinary com-pounds was carried out on an Equity-5 (30 m� 0.25 mm ID� 0.25 mm) capillary column (Supelco). Heliumwas used asthe carrier gas at a flow rate of 1mL/min. The conditions forthe chromatographic analysis were as follows: the initialtemperature 30 �C was maintained for 5 minutes, and then,it was raised to 280 �C at a rate of 5 �C/min, where it wasmaintained for 10 minutes. Samples (2 mL) were injected insplitless mode (0.5 minutes). The separated compoundswere ionized by electron impact in positive ionmode, usingelectron energy of 70 eV. The injector, the transfer line, andthe source temperature were set to 280 �C, 280 �C, and180 �C, respectively. Gas chromatography profiles of theextracted compounds were compared for each cow duringone estrus period using the GC–MS instrument, whereasthe peak identities were established on the basis of reten-tion time, mass spectra, and comparisons in the NationalInstitute Search Technology (NIST) spectra data bank andwhen available with standard compounds (DCE, coumarin,SQ, OA, and 2-butanone).

2.5. Conditioning of estrus-specific molecules and usedcombination

The commercially available identified molecules (DCE,coumarin, SQ, OA, and 2-butanone) were solubilized, aloneor in mixture in a nonpressurized aerosol dispenser(Hyteck Aroma-Zone, France). Eachmolecule was diluted ata final concentration of 25 pg/mL in glycerol–water solu-tion (50/50, v:v). The concentrationwas chosen on the basisof the estimated quantity of the more abundant compound,coumarin. Quantity was deduced from peak area in thechromatograms of the GC–MS analysis. All solutions were

C. Le Danvic et al. / Theriogenology 83 (2015) 1381–1388 1383

prepared freshly and stored at 4 �C until use. The fourdifferent tested combinations were coumarin, SQ þ DCE, 2-butanone, and OA. Glycerol–water (50/50, v:v) solutionwasused as the control. Squalene and DCE were tested incombination, as they were both increasing during estrus inurinary samples of the same cows.

2.6. Experimental design and effect of treatments on bullsexual parameters

To test the biological activity of identified estrous-specific molecules, assays were carried out using bulls(aged 4–5.5 years) in four different French semen produc-tion centers. Coumarin was tested at “CREAVIA29” (Plou-nevezel, France), SQ þ DCE at “CREAVIA56” (Locminé,France), 2-butanone at “Ain Génétique Service” (Ceyzériat,France), and OA at “COOPELSO” (Soual, France). In eachcenter, 10 adult dairy bulls (Bos taurus, Prim’Holstein, andNormande breeds) were used, equally housed, and fedbefore and during the experiment. Bulls were selected at 2to 3 months of age and had no previous contact with fe-males. They were accustomed to the artificial rubber vaginaused for semen collection from the age of 10 to 12 months.Before study, semen was collected twice (Ain GénétiqueService and COOPELSO) or thrice (CREAVIA29 and CREA-VIA56) weekly, one ejaculate each time, under AI centers’routine conditions. Within each semen collection center,bulls were divided into two groups, normal or low libido,according to their previous sexual behavior on the basis of abull handler’s appreciation. The term libido refers here tothe willingness and eagerness of the bull to mount andejaculate [11]. The first group (G1, n ¼ 5 bulls) correspondsto animals whose reactivity to the teaser and time toejaculation (Et) are consistent with those of all stud bulls.This group was called the “normal” libido group. The sec-ond group (G2, n ¼ 5 bulls), called the “weak libido” group,included animals with slow or very slow reactivity to theteaser and a longer time to ejaculate.

In each center, the study was divided into two periods.First, 2 weeks were dedicated to ensure the distributionbetween the two bull groups. During this period, or adap-tation period, all bulls only received the control solution.The four followingweeks, called the “experimental period,”were designed as following. In each center, one groupreceived the control solution during 1 week, whereas thesecond group received one of the four treatments. Groupsand solutions were switched everyweek so that each groupwas alternately “control” and “treated” in a way each bullcould be considered as its own control in subsequentanalyses.

For all trials, at the beginning of each session of semencollection, each bull received two sprays (1 mL) of the so-lution in each nasal cavity. Sprays were directly applied tothe nasal cavities to avoid variation due to diffusion in theair. Then, the reaction time (Rt) and the Et were recordedunder the same conditions by the same technician whileusing a chronometer. The reaction time corresponds to theinterval between the moment at which the teaser is pre-sented to the bull (the bull stands approximately 1.5 mbehind the teaser) and the bull’s spontaneous first falsemount. The time to ejaculation is the addition of sexual

preparation time to Rt. Sexual preparation time corre-sponds to the scheme recommended by Amann and Alm-quist [12], which consists in one false mount followed bytwo minutes of active restraint and then two other falsemounts until the thrust leading to ejaculation. All sampleswere collected by means of an artificial vagina. Productionparameters were assayed. The volume of ejaculate wasestimated from a direct reading in a graduated collectiontube, and the total concentration (billion spermatozoa/mL)was measured using a spectrophotometer (IMV Technolo-gies, L’Aigle, France). Within each center, dispensers werelabeled with a code, so that stockmen used them blindly toavoid human bias.

All procedures used in this study (biological samplecollection, bull assays) were in accordance with the Guidefor the Care and Use of Agricultural Animals in AgriculturalResearch and Teaching (Federation of Animal Science So-cieties, 1999).

2.7. Statistical analysis

In each center, a comparison of each group of bulls, i.e.,G1 and G2, was performed during the adaptation period tomake sure that differences related to libido could beobserved (mean comparison, Student test). A first ANOVAon the overall population was performed using a GlobalLinear Model procedure (Statistical Analysis Software 9.1;SAS Institute Inc.) and involved Rt, Et, and production pa-rameters, i.e. volume, concentration, and total number ofspermatozoa. The following variation factors were takeninto account: week, bull, treatment (control vs. treatment)and two-by-two interactions. A second intragroup GLMprocedure analysis was performed to see if effects of thetreatment were similar according to the bulls’ initial libido.Group and the interaction group by treatment were addedto the previous variation factors taken into account for theanalysis. Multiple comparisons and individual contrastswere tested using the Scheffe option available under SAS.

3. Results

3.1. Identification of estrous-specific compounds

Identification of estrous-specific compounds was theresult of the differential analysis by GC–MS of urinecomposition of the same cow at three stages of its cycle:preestrous (follicular stage), estrous, and luteal stage. Onlycows presenting LH surge followed by a progesterone risewere taking into account (18 of 32). Compounds wereselected on the basis of their appearance, disappearance, orincrease in relative quantity at the estrous stage, assumingthat the observed variation compared to the background ispart of their biological activity. A strong variability wasobserved in the chemical profiles among animals. Thechromatograms shown in Figure 1 indicate that for cows0630 and 0832, the relative quantity of coumarin wasgreater in preestrous urinary samples than those obtainedat the time of ovulation. For cow 5260, there was noobvious change throughout the cycle. For each cow, onlyone chromatogramwas obtained for the vaginal mucus thatis secreted only at the estrus stage. 2-Butanone was

Fig. 1. Heterogeneity of coumarin profile for three cows. For each cow, seven samples were collected over the duration of the cycle. *Indicates ovulation time. NI,nonidentified compounds.

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identified in this mucus samples except for one cow (datanot shown). Despite this variability, six compounds wereselected for their presence or increase around the ovulationtime. Five of them were accurately identified by compari-son with the NIST data bank (Table 1 and Fig. 2) andcommercially available counterparts: coumarin, 2-butanone, OA, SQ, DCE. For the sixth compound, 6-amino-undecane, identificationwas based only the NIST data bankcomparison as it is not commercially available. This lastcompound was not used for bull assay.

3.2. Initial comparison of libido groups

Performances of the two groups (G1 and G2) werecompared on the basis of recorded libido parameters dur-ing the adaptation period for each center. Results wereconsistent with expectation for three of the four trials.Indeed, Rt and Et were significantly higher for G2 than forG1 at CREAVIA29, CREAVIA56, and Ain Génétique Service(Table 2). In those centers, bulls of the group G2 took longerto excite and give semen (between 4 and 12 minutes more

Table 1Number of cows displaying identified chemicals in their urine and vaginalmucus at the three stages of estrus cycle (N ¼ 32 cows).

Compound Urine Vaginal mucus

Pre-estrus Estrous Post-estrus Estrous

Coumarin 15 4 1 (trace) 06-Amino-undecane 7 1 1 (trace) 0Squalene 11 4 1 41,2-Dichloroethylene 8 4 0 02-Butanone 3 0 0 24Oleic acid 12 10 1 (trace) 7

for Rt and between 7.5 and 16.5 minutes for Et). As nosignificant difference between the two libido groups wasobserved at COOPELSO, molecule effect for the libido groupwas not assessed for the trial at this center. Semen pro-duction parameters were comparable between the two li-bido groups except for the trial performed at CREAVIA29(data not shown). In this trial, the volume of the ejaculate(5.2 � 1.6 mL vs. 6.4 � 3 mL, P < 0.05) and the number ofspermatozoa per ejaculate (6.3 � 3.4 billions of spermato-zoa vs. 8.9 � 5.3 billions of spermatozoa, P < 0.05) werehigher for the “weak libido” group.

3.3. Effect of treatments on bull sexual parameters

Treatment effects were analyzed for all bulls in eachcenter, regardless of the libido group they were initiallyassigned to. Libido and semen production parameters weremeasured and compared after inhalation of the controlsolution or the tested molecules (coumarin, SQ þ DCE, 2-butanone, OA). Among the four tested combinations, 2-butanone (Ain Génétique Elevage) and OA (COOPELSO)induceda significantdecrease of libidoparameters (Table 3).Thirty-twopercentage and24%of timewere savedonRt andEt, respectively (P<0.05), after inhalationof 2-butanone.Upto 5 minutes per bull was saved on Et during this trial. Adecrease of 31% and 18% on Rt and Et was observed afterinhalation of OA. Similar trends with results close to sig-nificance were found with SQ þ DCE (CREAVIA59; �25% onRt, P¼ 0.08 and�13% on Et, P¼ 0.06; Table 3). Interestingly,the number of spermatozoa and the concentration perejaculate were increased by 9% (11.1 � 2.4 vs. 10.2 � 2.2billions of spermatozoa, P < 0.05) and 6% (1.56 � 0.22 vs.1.47 � 0.2 billions of spermatozoa/mL, P < 0.05),

Fig. 2. Comparison of mass spectrum of urine molecules (Exp) with standard spectrum (Std) of the NIH library. (A) Oleic acid, (B) squalene, (C) coumarin,(D) 6-amino-undecane, (E) 2-butanone, and (F) 1,2-dichloroethylene.

C. Le Danvic et al. / Theriogenology 83 (2015) 1381–1388 1385

respectively, with the SQ þ DCE combination (CREAVIA56;Table 3). No significant effects on libido or production pa-rameters were seen after treatment with coumarin (CREA-VIA29). The individual bull effect was significant for all theanalyzed parameters, meaning that behavior after treat-ment is specific to each animal. No interaction between“bull” and “treatment” effectswas observed (except for Et inthe experiment involving coumarin [CREAVIA29]). Thus,improvement of libido or production parameters could beattributed to the treatment.

When taking in consideration groups, no significant ef-fect could be observed on Rt or Et after coumarin (CREA-VIA29) or SQ þ DCE (CREAVIA56) treatments (Table 4).These treatments did not influence libido parameterswhatever the libido group. In contrast, production param-eters were positively impacted in G1 and not in G2 afterthese two treatments. Treatment with coumarin (CREA-VIA29) affects production parameters in bulls presenting anormal libido with an increase in the concentration andnumber of spermatozoa per ejaculate (þ11% and þ15%,respectively, P � 0.05). Similar effects on concentrationwere observed after SQþDCE (CREAVIA56;þ10%, P< 0.05).In the experiment involving 2-butanone (Ain Génétique

Service), libido parameters were significantly improvedwhatever the initial libido group (Table 4). However, Rtsaved time was significantly higher in bulls presentingnormal libido (�55% vs. �26%). A significant increase ofspermatozoa concentration of 10% (P < 0.05) in bulls pre-senting weak libido (G2) was also observed after this 2-butanone treatment.

4. Discussion

Urine is a highly complex fluid, the composition ofwhich is dependent on the metabolism, the external tem-perature, and of endocrine status [13]. Temperature wasconsidered to only have a limited impact on the studybecause no relationship was observed between the collec-tion period of the year (April or October) and the variationin chemical composition. All cows received the same stan-dard diet for all trials performed, to minimize the impact ofmetabolism. A variation in chemical urinary profiles wasobserved during estrus cyclewith compounds apparition orincrease during preestrus and estrous. Thiswas expected, asthe estrous cycle is under hormonal control. But, interest-ingly, our work highlights that this chemical composition

Table 2Libido assessment on the basis of average reaction time and time toejaculation (in seconds) during the adaptation period for both libidogroups.

Treatment N n Libido parameters

Rt Et

CREAVIA29 (N ¼ 89 ejaculates)Normal libido (G1) Control 5 42 139 � 357 378 � 361Weak libido (G2) Control 5 47 895 � 1041 1368 � 1176P value <0.05 <0.05

CREAVIA56 (N ¼ 60 ejaculates)Normal libido (G1) Control 5 30 89 � 180 323 � 280Weak libido (G2) Control 5 30 320 � 302 780 � 478P value <0.05 <0.05

Ain Génétique Service (N ¼ 40 ejaculates)Normal libido (G1) Control 5 20 89 � 153 337 � 192Weak libido (G2) Control 5 20 730 � 672 1058 � 791P value <0.05 <0.05

COOPELSO (N ¼ 27 ejaculates)Normal libido (G1) Control 5 15 10 � 11 30 � 17Weak libido (G2) Control 5 12 18 � 16 43 � 19P value NS NS (0.07)

Values are expressed as round mean � standard deviation.Abbreviations: Et, time to ejaculation; N, number of bulls; n, number ofejaculates; NS, not significant; Rt, reaction time.

C. Le Danvic et al. / Theriogenology 83 (2015) 1381–13881386

variation is different between the cows with differentmolecules and periods of apparition in relation to estrous.Chemical communication in mammals, as in insects, is acomplex process, which implicates chemical mixturesproduced and released into the environment by an animalaffecting the behavior or physiology of others. The globalchemical profiles of an animal result in the combination ofits individual odor signatures with its emitted pheromonalcompounds. This complex chemical bouquet largely par-ticipates to the interindividual heterogeneity [14]. It is this

Table 3Effect of the treatment on reaction time and time to ejaculation during the expe

N n Libido parameters

Rta Eta

C (tested at CREAVIA29)Control 10 59 546 � 967 1181Treatment 10 60 464 � 782 1050P value NS NS

SQ þ DCE (tested at CREAVIA56)Control 10 60 310 � 383 707 �Treatment 10 60 232 � 342 615 �P value NS (0.08) NS (0.

B (tested at Ain Génétique Service)Control 10 40 807 � 913 1232Treatment 10 40 549 � 601 935 �P value <0.05 <0.05

OA (tested at COOPELSO)Control 10 39 16.7 � 14.7 35.6 �Treatment 10 38 11.5 � 11.5 29 � 1P value <0.05 0.05

Values are expressed as round mean � standard deviation.Abbreviations: B, butanone; C, coumarin; Conc, concentration of ejaculate; Et, tnumber of spermatozoa in the ejaculate; NS, not significant; OA, oleic acid; Rt, reejaculate.

a Rt and Et were expressed in seconds.b Volume is expressed in mL.c Concentration is expressed in billion of spermatozoa/mL.d Number of spermatozoa per ejaculate is expressed in billions.

complex background, which makes identifying chemicalcues or pheromones so challenging in many organisms andcould explain why very few mammalian pheromones havebeen identified (e.g., rabbit [15], elephant [16]).

Despite thisobservedheterogeneity, fewcompoundshavebeen characterized as specific to the period precedingovulation in thepresent study. All of themwerenot identifiedbecause of the complexity of the spectra and the sensibility ofthe available GC–MS. Further studies are needed to charac-terize other variable compounds present during the estrouscycle to complete the urinary estrus signature. Sixcompounds were identified in the present study: coumarin,SQ, 1,2-dicholroethylene, 6-amino-undecane, OA, and 2-butanone. It should be noted that n-phthalate and 1-iodoundecane reported to be present only at the ovulatorystage in urine of Jersey cows [10] were not found as estrous-specific compounds in our study. The di-n-phthalate wasfound several times in our samples but in a nonspecificmanner and sometimes during the luteal stage (data notshown). We attributed the presence of this compound in ourstudy to a contamination from the experimentation duringthe purification procedure. Indeed, phthalates are the mostcommon contaminants found in GC–MS analysis afterchemical extraction of biological samples. Phthalates areplasticizers that can contaminate samples fromvacuum sealsdamaged by high temperatures or vinyl gloves worn by theexperimenter to be protected from the used solvent. The 1-iodoundecane found in Jersey cows in estrus [10] displays asimilar chemical structure to the 6-amino-undecane identi-fied in our study as specific to the preovulatory stage sug-gesting a possible commonmetabolismpathway. Among theidentified compounds, coumarin, SQ, andOAhave previouslybeen described in invertebrates and vertebrates as semi-ochemicals (a chemical substance or mixture used for

rimental period independently of libido groups.

Production parameters

Volb Concc Nb spzd

� 1626 5.5 � 2.5 1.49 � 0.47 8.13 � 4.34� 1357 5.5 � 2 1.43 � 0.45 7.96 � 3.74

NS NS NS

637 7 � 1.5 1.47 � 0.2 10.2 � 2.2518 7 � 1.5 1.56 � 0.22 11.1 � 2.406) NS <0.05 <0.05

� 1309 7 � 2 1.43 � 0.35 9.96 � 2.90726 7 � 2 1.36 � 0.39 9.45 � 2.70

NS NS NS

22 5 � 1.5 1.76 � 0.5 8.79 � 3.46.7 4.5 � 1.5 1.85 � 0.5 8.67 � 3.9

NS NS NS

ime to ejaculation; N, number of bulls; n, number of ejaculates; Nb spz,action time; SQ þ DCE, squaleneþ1,2-dichloroethylene; Vol, volume of the

Table 4Effect of the tested molecules on libido parameters (reaction time and ejaculation time) and production parameters (volume, concentration, and number ofspermatozoa) during the experimental period according to libido groups.

n Libido parameters Production parameters

Rta Eta Volb Concc Nb spzd

C (tested at CREAVIA29)Normal libido (G1, 5 bulls)Control 30 84.7 � 90 316 � 154 5 � 1 1.35 � 0.5 6.73 � 3.1Treatment 30 94.4 � 58 340 � 170 5 � 1 1.50 � 0.47 7.75 � 3.4P value NS NS NS <0.05 0.05

Weak libido (G2, 5 bulls)Control 26 1078 � 1224 2179 � 1965 6.5 � 3 1.51 � 0.4 9.89 � 5Treatment 29 846 � 983 1786 � 1638 6 � 2.5 1.47 � 0.48 8.18 � 4.1P value NS NS NS NS NS

SQ þ DCE (tested at CREAVIA56)Normal libido (G1, 5 bulls)Control 30 45 � 36 271 � 140 7 � 1 1.42 � 0.21 10 � 2.2Treatment 30 38 � 32 255 � 64 7 � 1 1.56 � 0.23 11.1 � 2.3P value NS NS NS <0.05 NS

Weak libido (G2, 5 bulls)Control 30 575 � 389 1143 � 642 7 � 1.5 1.52 � 0.27 10.4 � 2.3Treatment 30 427 � 398 977 � 522 7 � 1.5 1.55 � 0.21 11.1 � 2.5P value NS NS NS NS NS

B (tested at Ain Génétique Service)Normal libido (G1, 5 bulls)Control 20 346 � 305 672 � 388 8 � 3 1.8 � 0.35 14.7 � 3.6Treatment 20 157 � 220 471 � 295 8 � 3 1.85 � 0.63 14.57 � 5.8P value <0.05 <0.05 NS NS NS

Weak libido (G2, 5 bulls)Control 20 1270 � 1079 1793 � 1149 8.5 � 3 1.6 � 0.54 13.3 � 4.6Treatment 20 929 � 615 1398 � 736 6.5 � 4 1.76 � 0.8 10.67 � 0.64P value <0.05 <0.05 NS <0.05 NS

Values are expressed as round mean � standard deviation.Abbreviations: B, butanone; C, coumarin; Conc, concentration of ejaculate; Et, time to ejaculation; N, number of bulls; n, number of ejaculates; Nb spz,number of spermatozoa in the ejaculate; NS, not significant; OA, oleic acid; Rt, reaction time; SQ þ DCE, squaleneþ1,2-dichloroethylene; Vol, volume of theejaculate.

a Rt and Et are expressed in seconds.b Volume is expressed in mL.c Concentration is expressed in billions of spermatozoa/mL.d Number of spermatozoa per ejaculate is expressed in billions.

C. Le Danvic et al. / Theriogenology 83 (2015) 1381–1388 1387

communication). Oleic acid (9-octadecenoic acid) was foundto act as a sex pheromone in buffaloes, leading to penileerection and mounting behavior after detection [17]. Squa-lene is described as a putative male pheromone in the giantpanda, Ailuropodamelanoleuca [18]. Presence of coumarin (or2H-1-benzopyran-2-one) in cow urine could be attributed tofeeding as this natural substance is found in many plants.However, during individual collection sessions, cows werefed in the same way. In addition, after ingestion, this com-pound is degraded in the liver, and only its degradationproducts are excreted in theurine [19], excluding the variablepresence of coumarin in cow urine as due to diet. Conse-quently, we assume the presence of coumarin in cowurine toconstitute a chemical cue. The characterization of DCE in cowurine is intriguing as organohalogen compounds are rarelyfound in biological fluids. Contamination by solvents couldexplain the presence of thismolecule in our samples (even if,the risk was minimized using high quality solvents). But, inour study, 1,2-dicholorethylene was only found duringpreestrus or/and estrus suggesting that this molecule is apotential chemical cue in estrus cows. This statement is re-enforced by the characterization of another organohalogen,the 1-chloro-octane, in buffalo during estrus [17].

Inhalation of those estrous-specific compounds led to asignificant or slightly significant (except for the coumarin)

decrease in reaction and ejaculation times in bulls (inde-pendently of the libido group). In all trials, the “bull” effectwas always significant, indicating that behavior after treat-ment is specific to each animal. Nobull treatment interactionhas emerged as significant, including for the libido parame-ters, Rt and Et, when the positive effect of the treatmentwasnoted. Beneficial effects of treatments on observed param-eters (libido and/or production) could therefore entirely beattributed to the tested molecules. The results support theidea that themoleculeshave aneffectonmost bulls althoughthemagnitude of the response depends on the animals and/or to their receptivity to the treatment. An average gain from10 minutes to 2 hours on the time spent by herdsmen persemen session collection (based on collection of 20 bulls perday) is observed after inhalation of 2-butanone and OA.Interestingly, an increase of concentration in spermatozoaperejaculate couldbeobserved forbulls presenting anormallibido with coumarin and SQ þ DCE treatments. A similareffect is observed on weak libido bulls after inhalation of 2-butanone. No negative effects on semen production havebeen observed whatever the treatment.

All bulls were naïve; their reaction to estrous-specificmolecules was not the result of previous training orlearning. The identified molecules are recognized by thebull and could be considered as chemical signals implicated

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in the sex communication between cows and bulls in areproductive context.

4.1. Conclusions

The present study has led to the identification of severalchemical compounds both present and varying duringestrus cycle stages in cows: coumarin, SQ, DCE, 6-amino-undecane, OA, and 2-butanone. Two of these molecules,2-butanone and oleic acid, enhanced libido in bulls(reduction of Rt and Et) after inhalation.

This work has led to the development of a nasal spraycontaining molecules enhancing bull sexual behavior andcapacity (PheroBull, IMV Technologies) with a significantdecrease in the mounting and ejaculation time and no effecton sperm quality, increasing sperm quantity by 9% in somebulls. This spray could be used to enhance bull sexualbehavior in semen collection centers to facilitate handlingandproductivity, especially for bulls presentingaweak libido.

Acknowledgments

The authors thank Catherine Joly, Cyril Gonzalez, PascalSalvetti, Nadine Jeanguyot, andMarie-Christine Deloche fortheir help in collecting samples and in the following ofestrous cycle. Chrystelle Le Danvic also thanks Yves Leroyfor his helpful participation in gas chromatography andmass spectrometry analyses. This work was supported by aCIFRE grant from the French ANRT (Association NationaleRecherche Technologie) to Chrystelle Le Danvic. They thankINRA (Institut National de la Recherche Agronomique) andUNCEIA (Union Nationale des Coopératives d’Elevage etd’Inséminations Animales) for funding.

Author contributions: Chrystelle Le Danvic, Olivier Ger-ard, Eli Sellem, Claire Ponsart, Philippe Chemineau, PatriceHumblot, and Patricia Nagnan-Le Meillour contributed tothe design of the study. Chrystelle Le Danvic collected theurinary samples. Chrystelle Le Danvic and Patricia Nagnan-Le Meillour were responsible for the chemical analysis andthe preparation of sprays. Olivier Gerard and Eli Sellemwere responsible for bull stud trial organization and sta-tistical analysis of the data. Chrystelle Le Danvic, OlivierGerard, Eli Sellem, Philippe Chemineau, Patrice Humblot,and Patricia Nagnan-Le Meillour contributed to the writingof the article.

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