the role of pheromones in reproduction has been well documented in several species of mammals [ 14 ] . the pheromonal cues excreted through faeces , urine , vaginal secretions , saliva , and specialised scent glands play a significant role in sexual and social behaviour . in cattle , it is known that bulls can detect pheromone odours and differentiate between oestrus and nonoestrus urine or vaginal fluid [ 68 ] . the physical and chemical properties of bovine vaginal mucus have been of long standing interest to reproductive biologists because the mucus enhances conception by facilitating viability and transport of sperm . the oestrus female attracts the male and informs its receptive state by means of chemical signals deriving from vaginal discharge   and urine . there is considerable evidence that olfactory components produced from vaginal fluid influence the male sexual behaviour in buffaloes , hamsters , sheep , and bovines [ 15 , 16 ] . in addition , it has reported that vaginal secretions of heifers at oestrus stimulated sexual activity and mounting behaviour . the individual animal may produce several volatile compounds from a single source but the influence of pheromone activity may be one compound or a mixture of compounds . hence , it is necessary to analyse the bioactivity of individual volatile compounds identified in the bovine vaginal fluid . however , identification of the volatile compounds in bovine vaginal fluid and knowledge of how their biological activity may influence sexual behaviour remained to date . the present investigations were designed to analyse the chemistry of vaginal fluid and assess their bioactivity . vaginal fluid was collected from twelve healthy cows , bos taurus at the exotic cattle breeding centre , tanjore , india . they were artificially bred , and the females were approximately 20 to 30 months old , and the males were 30 to 36 months old . the animals were fed a standard diet in which the presence of the chemicals has entirely differed from the identified compounds from the vaginal fluid . the same diet was provided throughout the study . examination per rectum of each heifer was performed regularly at one- or two - week intervals to verify the normal morphological changes in the internal organ of uterus . since the detection of vaginal fluid pheromones by bulls is more important for the success of oestrus detection under natural conditions , the samples were collected on the basis of bull behaviours such as licking , sniffing , flehmen , and mounting during oestrus and nonoestrus phases under natural conditions . the technique involved rectal massage of the reproductive tract or an infusion tube placed within the vagina prior to a.i . the samples were screened through cheese cloth or nylon mesh ( 60120  m ) at the time of collection . immediately after screening , the samples were stored frozen at 20c and analyzed by gas chromatography mass spectrometry ( gc - ms ) . the samples collected from the particular stage as per the experimental protocol were pooled to minimise the effect of individual variation . in a preliminary study , nine organic solvents , namely , n - hexane , acetone , methanol , ethanol , petroleum ether , diethyl ether , chloroform , dichloromethane , and benzene were used to extract the compounds from the vaginal fluid samples . among the different solvents used , the bull exhibited maximum response ( i.e. , flehmen ) when exposed to the sample dissolved in diethyl ether . hence , this solvent was used throughout the study . triplicate 15  ml of samples were taken from the pooled samples and separately mixed with 15  ml of diethyl ether . the supernatant was filtered through a silica - gel column ( 60120 mesh ) 30  min at room temperature . the filtered extract was reduced to 1/5 of its original volume by cooling with liquid nitrogen to condense it . the sample was fractionated , and chemical compounds were identified by gc - ms ( qp-5050 , shimadzu ) . two microlitres of extract were injected into the gc - ms system on a 30  m glass capillary column with a film thickness of 0.25  m ( 30  0.2  mm i.d . coated with ucon hb 2000 ) using the following temperature program : initial oven temperature of 40c for 4  min , increasing to 250c at 15c / min , and then held at 250c for 10  min . the vaginal fluids were analyzed repeatedly six times and subjected to cross - checking and confirmation . these data were already stored in a compact library of chemical substance ( nist 6221b ) . the synthetic compounds were procured and individually applied manually onto the genital region of nonoestrus animals ( dummy cows ) . the bulls were allowed to sniff the genital region of experimental cows for a period of 30  min . the diethyl ether was used for gc - ms analysis as well as dissolving the synthetic compounds in various concentrations for example , 0.5% , 1.0% , 2.0% , 5.0% . since 1% concentration of oestrus - specific compounds were found to be effective in eliciting the flehmen and other behaviours of penile erection and mounting , the same concentration was taken in the ratio of 1  :  1  :  1 and used throughout the experiment to assess the bioactivity . the duration of flehmen behaviour exhibited by the bulls in response to dummy cows that received synthetic ( oestrus and nonoestrus ) compounds was recorded . subsequently , other behaviour responses like penile erection , mounting , and act of copulation were also recorded . the data were compiled using spss statistical software 10th version and subjected to analysis of variance ( anova ) with post hoc comparison ( one - way ) using duncan 's multiple range test ( dmrt ) . the gc - ms profiles shown in figures 1 and 2 are the representative of vaginal fluid obtained in the oestrus and nonoestrus periods . eight different peaks were noted in the vaginal fluid of two different phases ( figure 1 ) . a single volatile , that is , 3-hexanol , was found in both the phases whereas other two volatiles , cyclohexane 3,3,5 trimethyl and phosphonic acid , were found only in nonoestrus vaginal fluid ( table 1 ) . of these , trimethylamine , acetic acid , and propionic acid were unique in the oestrus phase but were absent in the nonoestrus . the peaks between 5 and 6 and after the peak 7 in nonoestrus are referred as contaminants and not matching with any related compounds . the volatile compounds identified in the vaginal fluid have the molecular weight range between 59 and 174 dalton ( table 1 ) . the statistical analyses showed that the flehmen behaviour of bulls was greatly influenced by the mixture of three synthetic compounds ( p < 0.001 ) than that of individual compounds and control sample ( table 3 ) . the number of mounting activity and flehmen ( table 3 ) was higher in response to mixture of the three compounds than that of individual and in combinations . the results of table 2 summarises the flehmen and mounting behaviour performed by the male responder on exposure to synthetic compounds . among the individual synthetic compounds tested the combination of acetic and propionic acid showed higher sexual behaviour than that of individual and combination of acetic acid and trimethylamine ; propionic acid and trimethylamine . the present results revealed that the acetic acid , propionic acid , and trimethylamine appeared during oestrus phase but were not found in the nonoestrus phase . among the compounds identified in oestrus vaginal fluid , the acetic and propionic acids belong to fatty acids , and the identification of volatile fatty acids in the bovine vaginal fluid is consistent with the report of the predominant presence of short chain aliphatic acids , acetic acid , prop- , isotonic , in vaginal secretion of rhesus monkey . such volatile aliphatics have also been demonstrated in the vaginal secretion of an inside range of primates , including human female . furthermore ,   reported that acetic , propionic , and isobutyric acid can act as pheromone in chimpanzee .   in the present study the bulls exhibited high frequency of flehmen response when exposed to oestrus sample as well as mixture of synthetic compounds tested . it clearly indicates that these three oestrus - specific compounds probably act as sex attractants ( table 1 ) . it has been demonstrated that the olfactory chemical signals produced from oestrus females are mediated through the vomeronasal organ ( vno ) facilitating the matting behaviour through expression of flehmen response in many species [ 20 , 21 ] . the present findings further confirm that the identified compounds in oestrus and to activate the bull to mount are the sex attractants .   it is reported that in some mammals , pheromones are not single compounds but a mixture . reference   reported that three oestrus - specific compounds , namely , acetic acid , propionic acid , and 1-iodo undecane of cattle ( bos taurus ) were involved in the attraction of the opposite sex . oestrus - specific bovine urinary signal , 1-iodo undecane , is identified successfully in our laboratory by gas chromatography linked with mass spectrometry . similarly , in the present study the mixture of acetic , propionic , and trimethylamine revealed high incidence of precopulatory behaviour . the oestrus indication may have been initiated through urine followed by vaginal secretion , and accordingly the bull responded . it is , therefore , possible that the chemical signals produced from urine and vaginal fluid may act together for initiation of the precopulatory behaviours and successful coitus . chemical signals are less likely to be based on the presence of unique chemical compounds than on the relative ratios of concentrations in a complex mixture . this concept is commonly termed as  bouquet effect  [ 25 , 26 ] , also proposed that volatile compounds in specific ratios and in the presence of unique components might confer pheromonal effects . consistent with this suggestion , the present study also reveals that estrus vaginal fluid contains three specific compounds , which may be the basis of estrus odours . it is noteworthy that the present study on behaviour responses showed similar results using the mixture of synthetic compounds to that of oestrus vaginal fluid . we conclude that the specific volatile fatty acids present in bovine vaginal fluid during oestrus appear to be involved in sexual communication .
the chemical 
 profiles of vaginal fluid collected from cows in 
 oestrus and nonoestrus were analysed by gas 
 chromatography - mass spectrometry ( gc - ms ) 
 to establish any qualitative differences that 
 might have potential value in bovine 
 biocommunication . 
 eight different organic 
 compounds were detected using the two 
 chromatograms . 
 the chemical profiles of oestrus 
 vaginal fluid were distinguished significantly by 
 the presence of three specific substances , namely , 
 trimethylamine , acetic acid , and propionic acid 
 that were not present in nonoestrus phase . 
 the 
 oestrus specific synthetic compounds were rubbed 
 onto the genital region of nonoestrus animals 
 ( dummy cows ) , and the bulls were allowed to 
 sniff the genital region and observed sexual 
 behaviours . 
 the statistical significance was 
 higher ( p < 0.001 ) in bulls exhibiting repeated flehmen and mounting 
 behaviours towards the mixture of acetic acid , propionic acid , and 
 trimethylamine as compared to test these compounds separately . 
 it was concluded that the volatile substances present in the bovine vaginal fluid during oestrus may act as chemical communicators .