experiments were performed in accordance with the guiding principles for the care and use of experimental 
 animals in the field of physiological sciences ( physiological society of japan ) and were approved by the 
 committee on animal experiments of the school of veterinary medicine , yamaguchi university . in silico mining of the bovine genome database
these genes were predicted by automated computational 
 analysis , best - placed refseq gnomon ver 5.1 , against the bovine genome database ( bioproject number was 
 prjna33843 ) in ddbj / genbanktm / ebi data bank .
table 1table 1.gene numbers in this study ( numbered from the top of the molecular dendrogram ) , gene name from cow 
 sequence , accession number and details of pcr primers#gene nameaccession numberprimernucleotidesequence ( 5to 3)1v1r407xm_002695263.3up2749tggagaacgttgggttttagttadown306326tgaaatccaccggagaacaga2vn1r4lxm_002695264.3up425445cactgagacctgacggcatgt(loc100337475)down684704cgaacgtacgggaaaaatctg3vnt1r4lxm_005195507.1up1634ctggccatgttgccttgtg(loc100847477)down213235tagaggatcctgaatgggaacac4v1r413xm_002695269.2up391413agtgtcttccaggtcatcaccatdown671690agagccactggccaagattg5v1r410xm_002695266.2up1338tatgaagtattggtacgtggagaatgdown288312cagaaccaagatgttacctacgatc6v1r406xm_002695262.2up3352caggtgtccggctgaagagadown218242agtagcaggtcaacaaaaatccaat7vnt1r4lxm_005195506.1up411430actccaggtgggcagagctt(loc101908711)down689710ggactctctgcttgcacctgta8v1r411xm_001789862.3up549571gttgatgtccttccctgatgtgtdown829848gggctgatagttgggaagca9v1r416xm_002695271.1up323ggccagcattgatttgacaatdown283302aggcagatgctaccgatgga10v1r414xm_001254649.3up2644ccaggctcgtggatttgtgdown214235ggtgtgtgctgtatgcatcatg11v1r415xm_001254634.2up90113tatcgtccttcacatcatgggttadown368389ggaacggtgtacttgggagcta12vn1r2xm_002695278.2up4262ggaagcccgagtgaagaacagdown321341gaaaggagggccaagatgttg13v1r418xm_001789884.1up774793gagtccagagccacccaaagdown965990gtacagaaagcagagactggatatga14v1r420xm_001254581.1up6889accttgaatgacaccacagctcdown369386aagaggcaggttgtgcca15v1r421xm_001789887.1up329gtcacaaatttatatgagccattctatdown269386actcaagaggcaggtggtactg16v1r417xm_001789881.3up2746cctttttcctgcgtccagaadown205226agccaactttccttgtgtgctt17v1r419xm_001254591.2up257277tctcaaaagggctccctcagadown537556tgcgccatttgtcagaaaca18vn1r1xm_005219819.1up446464gcgccctctaggcactgatdown726745cccgtggctactctgtggaa19vnt1r4lxm_005215403.1up2444gcgttttgcacaccagttacc(loc100847223)down304323tgtccccttggagacgagaa20v1r427xm_002695464.1up191213tgattattttctcccctgggattdown469490taccctgtggaccagtgactgt21vn1r4lxm_002695460.3up355373acccccaggagagcgaagt(loc100295682)down637654caccctctggcggtgtct22v1r433xm_002695465.1up182202tgatggcagcttttgtgtcaadown459481acttgccttgggcatcagtatag23v1r426xm_002695403.2up118atgtctctaaggggccacdown318338gtgctgagagccatcctctgt24vnt1r1lxm_003585332.1up3756tgcatcaactccctgcagaa(loc100851211)down245256aatgtggcagaaccatctgtgt25v1r425xm_001788382.2up88111gtcaatgtcatccttttcttccaadown366387tctgagcaatgacctcccttct26v1r432xm_002695457.2up359380ctgggagagaagggaggtcactdown589608gaccagaccatgaggccaat27vn1r1lxm_002695462.2up564582gtggtccacctcggatgct(loc100295623)down844863aagggagaaacggtgggaaa28v1r428xm_002695463.2up316336tttcacaaagacgccctgagadown598615ggtgctgcgagccatttt29v1r431xm_002695458.3up329348atggctgtggccaatctcttdown606628tttgagaggaacacggatgtaca30v1r424xm_002695400.2up253276tccagtcttgggtgtaagtttgtgdown534552gcctgtggcagcacctgta31v1r430xm_002695461.3up1738atgccctgagaaccataatccadown273296gccaccctctgtacataaaacaca32v1r437xm_601821.1up6483attgggatcttggccaacacdown345363tgccagacgggagtttctg33v1r403xm_002695191.2up309329gagtgcctaccaagccatcacdown589608agactggaccacgccatgag34v1r404xm_002695192.2up349369aagtgggcgtggcttagatctdown629648ctggactctctggcggtgtt shows the gene names and ncbi reference sequences of the 34 v1r homologs .
drawing a molecular dendrogram : the bovine v1r gene sequences were utilized to draw a 
 molecular dendrogram using the two - parameter model and neighbor - joining method [ 24 , 36 ] with molecular evolutionary genetyx analysis ver.6.0.5 
 ( mega6 ) .
the evolutionary distances were computed using the maximum 
 composite likelihood method . here , the 34 genes are numbered from 
 the top of molecular dendrogram . collecting vos and oe from bulls and steers : we collected vos and oe from healthy , intact 
 japanese black bulls ( n=5 , 46  6 months old , 838  11 kg ) and japanese black steers ( n=5 , 28  1 months old , 
 698  12 kg ) in a local slaughterhouse according to a previous anatomical study .
italian ryegrass hay , rice 
 straw and concentrate were provided according to the japanese feeding standard .
it is generally difficult to obtain sufficient number of bull samples in any 
 country .
the bulls were used for  sumo  wrestling in western japan and were very healthy  they were being 
 sacrificed after losing wrestling rank , not for health reasons .
presence or absence of testis was confirmed in 
 each bull or steer at the slaughterhouse .
vos and oe 
 were obtained within 15 min of slaughter using a disk grinder with a diamond blade , hammer , straight gouge and 
 scissors .
the tissues surrounding the vos and oe were carefully removed and washed with phosphate - buffered 
 saline ( pbs ) . the vos and oe were frozen in liquid nitrogen and preserved at 80c until use for rna 
 extraction . collecting vos and oe from heifers : we collected vos and oe from japanese black heifers in 
 estrus ( n=7 , 30  1 months old , 506  10 kg ) or early luteal phase ( n=6 , 30  1 months old , 505  10 kg ) .
all heifers had received two intramuscular 
 injections of 20 mg dinoprost ( pronalgon f , pfizer , tokyo , japan ) given 11 days apart to synchronize the estrous 
 cycle .
estrous behavior and ovulation were monitored at the farm ; ovarian and uterine morphology were confirmed 
 at the slaughterhouse .
rna extraction , cdna synthesis and semi - quantitative pcr : total rna was extracted from frozen 
 vo and oe samples with rnaiso plus ( takara bio inc . ,
the rna 
 solution was extracted a second time with rnaiso plus , because single extraction yielded a brown product that 
 inhibited rt - pcr that could only be removed by a second extraction ( preliminary study , data not shown ) .
we could 
 not use a column method for rna extraction due to the mucus and brown powder in the vo and oe samples .
total rna 
 samples were digested with dnase i ( promega , madison , wi , u.s.a . ) to remove contaminating genomic dna .
rna 
 concentration and purity were evaluated by using a nanodrop nd-1000 spectrophotometer ( nanodrop technologies , 
 wilmington , de , u.s.a . ) to ensure the a260/a280 nm ratio was in the acceptable range of 1.82.1 .
electrophoresis 
 of total rna followed by staining with ethidium bromide was performed to verify mrna quality ; the 28s:18s ratios 
 were 2:1 .
we synthesized cdna from 2 g rna in 20-l reactions with random 
 hexamer primers and the precise high - capacity cdna reverse transcription kit ( applied biosystems , foster city , 
 ca , u.s.a . ) .
pcr conditions were optimized by conventional pcr with tks gflex dna polymerase ( takara bio ) , 20 
 ng dnase - treated reverse - transcribed rna , a veriti thermal cycler ( applied biosystems ) and 
 primers .
experiments included a no - template control and a no reverse transcription control . in preliminary 
 experiments ,
the optimum cycle number was determined for each target , so that signals were always in the 
 exponential portion of the amplification curve .
typical cycling conditions were as follows : 94c for 1 min , 
 followed by 35 cycles of 98c for 10 sec , 60c for 15 sec and 68c for 30 sec .
primers were designed with primer express software v3.0 ( applied biosystems ) and reference sequences .
amplicons for most targets were 300 bp 
 with the following exceptions : gene 2 ( 280 bp ) , gene 3 ( 220 bp ) , gene 6 ( 210 bp ) , gene 10 ( 210 bp ) , gene 13 ( 217 
 bp ) , gene 14 ( 320 bp ) , gene 15 ( 383 bp ) , gene 16 ( 200 bp ) , gene 23 ( 338 bp ) , gene 24 ( 220 bp ) and gene 26 ( 250 
 bp ) .
the presence of a single product was confirmed by 2% ( w / v ) agarose gel electrophoresis ( sigma - aldrich , 
 saint louis , mo , u.s.a . ) .
molecular weight markers ranging from 0.1 to 20 kbp ( gene ladder wide 1 ; nippon gene , 
 tokyo , japan ) were used to identify the bands .
pcr products were visualized with a highly sensitive fluorescent 
 stain ( gelstar , lonza , allendale , nj , u.s.a . ) and a charge - coupled device ( ccd ) imaging system ( geldoc ; bio - rad , 
 hercules , ca , u.s.a . ) , and the sensitivity of this system against double - strand dna was 20 pg .
pcr product concentrations were calculated by comparing the band strength of the unknown samples to the 
 molecular weight markers using alphaview software ( proteinsimple , santa clara , ca , u.s.a . ) .
v1r gene expression 
 was normalized to the geometric means of two housekeeping genes , chromosome 2 open reading frame 29 ( c2orf29 ; 
 xm_002691150.2 ) and suppressor of zeste 12 ( suz12 ; nm_001205587.1 ) ; values for each gene were divided by the 
 geometric mean of c2orf29 and suz12 in each sample .
all pcr - amplified products were purified with the nucleospin gel and pcr clean - up kit ( takara bio ) and 
 exosap - it ( affymetrix , santa clara , ca , u.s.a . ) , and then sequenced the dye terminator v3.1 cycle sequencing kit 
 on an abi3130 ( applied biosystems ) .
the obtained sequences were used as query terms for homology searches in the 
 ddbj / genbanktm / ebi data bank using the basic nucleotide local alignment search tool ( blast ) optimized for highly 
 similar sequences ( available on the ncbi website ) . statistical analysis : data were analyzed using statview version 5.0 for windows ( sas 
 institute , inc . ,
two - factor analysis of variance ( anova ) was used to evaluate the effect of 
 organ ( vo or oe ) , group ( bulls , steers , estrous heifers or early luteal - phase heifers ) and interaction between 
 the effects of organ and group on v1r gene expression followed by post - hoc comparisons with fisher s protected 
 least significant difference ( plsd ) test .
the statistical significance of differences among groups was assessed 
 by one - factor anovas followed by post - hoc comparisons with fisher s plsd test using a model consisting of 
 variance from the effect of group and the residual .
1.the evolutionary history of the 34 bovine homologs of v1r was inferred using the neighbor - joining method . 

the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is 
 shown next to the branches .
the tree is drawn to scale , with branch lengths in the same units as those of 
 the evolutionary distances used to infer the phylogenetic tree .
the evolutionary distances were computed 
 using the maximum composite likelihood method , and units represent the number of base substitutions per 
 site . shows the molecular dendrogram of 34 bovine v1r homologs .
there were four clusters : gene 1 to gene 8 ; gene 9 to gene 11 ; gene 13 to gene 17 ; and gene 20 to gene 
 31 .
there was six independent branches for genes 12 , 18 , 19 , 32 , 33 and 34 .
the evolutionary history of the 34 bovine homologs of v1r was inferred using the neighbor - joining method . 

the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is 
 shown next to the branches .
the tree is drawn to scale , with branch lengths in the same units as those of 
 the evolutionary distances used to infer the phylogenetic tree .
the evolutionary distances were computed 
 using the maximum composite likelihood method , and units represent the number of base substitutions per 
 site .
general information of gene expression of bovine v1r homologs : housekeeping genes c2orf29 and 
 suz12 were amplified from all cdna samples .
no amplified products were obtained from the no - template controls 
 and no reverse transcription controls .
no amplified products were obtained from primers specific for genes 3 , 6 , 16 and 23 .
it was impossible to 
 design better primers for these genes , which were thus excluded from further analysis .
2.the expression of bovine v1r homologs genes 1 to 11 in the vo and oe of bulls ( b ; black bar ) , steers ( s ; 
 dark gray bars ) , estrous heifers ( e ; dotted bars ) and early luteal - phase heifers ( l ; white bars ) . v1r
gene 
 expressions were normalized to the geometric means of two housekeeping genes , c2orf29 and 
 suz12 ; values for each gene were divided by the geometric mean of 
 c2orf29 and suz12 in each sample .
the header in the upper right corner 
 of each graph represents the results of two - factor anova followed by fisher s plsd test , including the 
 effect of organ ( vo or oe ) , effect of group ( bulls , steers , estrous heifers or anestrous heifers ) and 
 interaction between the effects of organ and group .
letters ( a , b or c ) above the left - side bars indicate 
 significant between - group differences in expression in vos ; greek letters (  or  ) above the right - side 
 bars indicate significant between - group differences in expression in oes ( one - factor anova followed by 
 fisher s plsd test).fig .
3.the expression of bovine v1r homologs genes 12 to 21 in the vo and oe of bulls ( b ; black bar ) , steers ( s ; 
 dark gray bars ) , estrous heifers ( e ; dotted bars ) and early luteal - phase heifers ( l ; white bars ) .
v1r gene 
 expressions were normalized to the geometric means of two housekeeping genes , c2orf29 and 
 suz12 ; values for each gene were divided by the geometric mean of 
 c2orf29 and suz12 in each sample .
the header in the upper right corner 
 of each graph represents the results of two - factor anova followed by fisher s plsd test , including the 
 effect of organ ( vo or oe ) , effect of group ( bulls , steers , estrous heifers or anestrous heifers ) and 
 interaction between the effects of organ and group .
letters ( a , b or c ) above the left - side bars indicate 
 significant between - group differences in expression in vos ; greek letters (  or  ) above the right - side 
 bars indicate significant between - group differences in expression in oes ( one - factor anova followed by 
 fisher s plsd test).fig .
4.the expression of bovine v1r homologs genes 22 to 34 in the vo and oe of bulls ( b ; black bar ) , steers ( s ; 
 dark gray bars ) , estrous heifers ( e ; dotted bars ) and early luteal - phase heifers ( l ; white bars ) .
v1r gene 
 expressions were normalized to the geometric means of two housekeeping genes , c2orf29 and 
 suz12 ; values for each gene were divided by the geometric mean of 
 c2orf29 and suz12 in each sample .
the header in the upper right corner 
 of each graph represents the results of two - factor anova followed by fisher s plsd test , including the 
 effect of organ ( vo or oe ) , effect of group ( bulls , steers , estrous heifers or anestrous heifers ) and 
 interaction between the effects of organ and group .
letters ( a , b or c ) above the left - side bars indicate 
 significant between - group differences in expression in vos ; greek letters (  or  ) above the right - side 
 bars indicate significant between - group differences in expression in oes ( one - factor anova followed by 
 fisher s plsd test ) .
show v1r mrna expression in the oe and vo of bulls , steers , estrous heifers and early luteal - phase 
 heifers , and outcomes of anova .
the organ effect ( oe or vo ) was significant for 22 genes ( genes 2 , 4 , 5 , 7 , 8 , 
 9 , 10 , 12 , 13 , 14 , 15 , 17 , 18 , 20 , 21 , 25 , 26 , 28 , 30 , 31 , 33 and 34 ) , all of which were more highly expressed 
 in vos than in the oe .
the effect of group ( bulls , steers , estrous heifers or early luteal - phase heifers ) was 
 significant for ten genes ( genes 2 , 12 , 15 , 17 , 18 , 22 , 24 , 26 , 28 and 34 ) .
the interaction between the effects 
 of organ and group was significant for five genes ( genes 2 , 12 , 15 , 17 and 20 ) .
the expression of bovine v1r homologs genes 1 to 11 in the vo and oe of bulls ( b ; black bar ) , steers ( s ; 
 dark gray bars ) , estrous heifers ( e ; dotted bars ) and early luteal - phase heifers ( l ; white bars ) . v1r
gene 
 expressions were normalized to the geometric means of two housekeeping genes , c2orf29 and 
 suz12 ; values for each gene were divided by the geometric mean of 
 c2orf29 and suz12 in each sample .
the header in the upper right corner 
 of each graph represents the results of two - factor anova followed by fisher s plsd test , including the 
 effect of organ ( vo or oe ) , effect of group ( bulls , steers , estrous heifers or anestrous heifers ) and 
 interaction between the effects of organ and group .
letters ( a , b or c ) above the left - side bars indicate 
 significant between - group differences in expression in vos ; greek letters (  or  ) above the right - side 
 bars indicate significant between - group differences in expression in oes ( one - factor anova followed by 
 fisher s plsd test ) .
the expression of bovine v1r homologs genes 12 to 21 in the vo and oe of bulls ( b ; black bar ) , steers ( s ; 
 dark gray bars ) , estrous heifers ( e ; dotted bars ) and early luteal - phase heifers ( l ; white bars ) . v1r
gene 
 expressions were normalized to the geometric means of two housekeeping genes , c2orf29 and 
 suz12 ; values for each gene were divided by the geometric mean of 
 c2orf29 and suz12 in each sample .
the header in the upper right corner 
 of each graph represents the results of two - factor anova followed by fisher s plsd test , including the 
 effect of organ ( vo or oe ) , effect of group ( bulls , steers , estrous heifers or anestrous heifers ) and 
 interaction between the effects of organ and group .
letters ( a , b or c ) above the left - side bars indicate 
 significant between - group differences in expression in vos ; greek letters (  or  ) above the right - side 
 bars indicate significant between - group differences in expression in oes ( one - factor anova followed by 
 fisher s plsd test ) .
the expression of bovine v1r homologs genes 22 to 34 in the vo and oe of bulls ( b ; black bar ) , steers ( s ; 
 dark gray bars ) , estrous heifers ( e ; dotted bars ) and early luteal - phase heifers ( l ; white bars ) . v1r
gene 
 expressions were normalized to the geometric means of two housekeeping genes , c2orf29 and 
 suz12 ; values for each gene were divided by the geometric mean of 
 c2orf29 and suz12 in each sample .
the header in the upper right corner 
 of each graph represents the results of two - factor anova followed by fisher s plsd test , including the 
 effect of organ ( vo or oe ) , effect of group ( bulls , steers , estrous heifers or anestrous heifers ) and 
 interaction between the effects of organ and group . letters ( a , b or c ) above the left - side bars indicate 
 significant between - group differences in expression in vos ; greek letters (  or  ) above the right - side 
 bars indicate significant between - group differences in expression in oes ( one - factor anova followed by 
 fisher s plsd test )
. bulls vs. steers : gene 1 expression in the oe was higher in the bull than in the steer .
gene 7 
 expression in vos was higher in the bull than in the steer .
estrous heifers vs. early luteal - phase heifers : gene 9 showed higher expression in the oe of 
 estrous heifers than in the oe of early luteal - phase heifers .
gene 15 showed higher expression in the vos of 
 estrous heifers than in the vos of early luteal - phase heifers .
eight genes ( genes 5 , 12 , 17 , 20 , 22 , 28 , 33 and 
 34 ) showed higher expression in vos of early luteal - phase heifers than in those of estrous heifers .
furthermore , 
 no genes showed higher expression in the oe of early luteal - phase heifers than in the oe of estrous heifers .
bulls vs. estrous or early luteal - phase heifers : the expression of gene 1 in the oes was 
 higher in bulls than in both estrous and early luteal - phase heifers . the expressions of three genes ( 12 , 17 and 
 33 ) were higher in the vos of bulls than of estrous heifers , but not of early luteal - phase heifers .
the 
 expression of gene 15 in the vos was higher in bulls than in early luteal - phase heifers , but not in estrous 
 heifers .
the expression of gene 26 in the vos was higher in estrous heifer than in bulls , but not in early 
 luteal - phase heifers .
further , the expressions of six genes ( 2 , 12 , 18 , 20 , 22 and 28 ) were higher in the vos of 
 early luteal - phase heifers than of bulls .
this study clarified sexual polymorphisms of v1r gene expression in bovine vos and oe . although intraspecies 
 chemosignals and receptors in this species are poorly understood , the expression polymorphisms discovered herein 
 suggest the presence of multiple intraspecies chemosignals in females and males .
the genomes of mice , bovines 
 and humans contain 530 , 34 and 4 v1r family genes [ 16 , 21 ] ; thus , bovines represent a mid - point in the loss of v1r genes through 
 evolution .
therefore , the results of this study may give an insight into the evolution of such receptor genes in 
 mammals and their involvement in sex - specific behavior .
it is well known that there are sex differences in intraspecies chemosignal detection , although the mechanisms for these sex differences were unknown in mammals prior to this 
 study .
our study 
 results show higher gene expression in bulls than in estrous heifers ( genes 1 , 12 , 17 and 33 ) .
in addition , we 
 also show higher gene expression in estrous heifers than in bulls ( gene 26 ) .
therefore , we believe that these 
 genes may be important causes of sex differences in intraspecies chemosignal detection
. our results also suggest 
 that different receptor clusters may have different roles and effects on the central nervous system . however , we 
 believe that other mechanisms for sex differences in intraspecies chemosignal detection may exist in cattle . 

indeed , a recent study , which was published after we completed our study , showed that progesterone can silence 
 sensory neurons detecting urinary protein pheromones in female mice .
the estrous heifers showed higher expression of gene 9 in oe and gene 15 in vos in comparison to early 
 luteal - phase heifers .
the vos of early luteal - phase heifers showed higher expression of eight genes than those 
 of estrous heifers .
blood estradiol is higher 
 in estrous heifers than in early luteal - phase heifers .
this and the 
 findings of doty and cameron   suggest estradiol may be one 
 controlling factor .
interestingly , mouse vo synthesizes estrogen , and the vo neuronal response to putative urine 
 pheromones is reduced by estradiol .
we found no previous reports on the effect 
 of sex steroids on v1r gene expression ; however , the ability to detect putative human pheromone , androstenone , 
 is more likely to diminish in boys than in girls after puberty [ 10 , 13 , 27 ] .
further studies are required 
 to test the hypothesis that estradiol and testosterone control sex - associated polymorphisms in v1r gene 
 expression .
ohara 
 et al .   reported that all of the 24 identified v1r 
 genes are expressed in the vo and oe of a goat , the genome of which has not been fully sequenced .
however , the 
 authors did not report on the characteristics of the goat ( age , sex and intact or gonadectomized ) , and they used 
 nested pcr ( both first and second pcrs were 35 cycles ) to evaluate gene expression .
therefore , it is impossible 
 to compare our data with theirs . sniffing is the behavioral response for pheromone binding to the oe receptors , 
 and flehmen is the behavioral response for pheromone binding to the vo receptors . in general ,
therefore , the smaller number 
 of ligand - receptor binding sites on the oe may be enough to induce the flehmen response , and a higher number of 
 ligand - receptor binding sites on the vos may be interpreted ( or treated ) by the accessory olfactory bulb and 
 lead to various neurological and behavioral effects in cattle .
sexual pheromones induce male and female effects 
 to activate reproductive functions in the opposite sex in small ruminants [ 11 , 17 , 37 ] .
however , an 
 intraspecies chemosignal was reported in dorsett ewes , which utilize their vo for neonatal offspring recognition 
 .
rats have the ability to communicate affective states , such as 
 stress , alarm , fear , anxiety or sexual interest , by releasing intraspecies chemosignals [ 25 , 26 ] .
therefore , some of the 20 v1rs may have 
 important roles in the detection of intraspecies chemosignals for non - sexual purposes .
we compared the expression of each of 30 v1rs in the vo and oe among the 4 different groups .
individual gene 
 expression patterns did not show uniformity . because all 30 v1rs are orphan receptors , it is impossible to 
 explain the precise characteristics of each receptor and explain the relationship with each physiological change 
 or gene expression requirement .
however , generally , physiological responses and decisions of animals are in 
 response to situations in their living environment and for survival and species conservation .
intraspecies 
 chemosignals have sexual and non - sexual purposes [ 3 , 6 , 18 ] .
rodents can discriminate among conspecific 
 animals based on various sensory cues , including dietary history , 
 illness - associated state   and major histocompatibility complex .
therefore , expression patterns of v1rs may have important roles in 
 complex intraspecific chemical communication .
tks gflex dna polymerase possesses high fidelity and excellent extension activity , because it contains a 
 specific priming accelerator that suppresses non - specific binding , takara bio s original strong elongation 
 factor and reaction buffer elements that sequester pcr inhibitors .
thus , the polymerase can amplify templates 
 from over a wide range of nucleic acid concentrations , even from targets containing gc- or at - rich sequences . in 
 our preliminary trials ,
pcr amplification failed , likely because of inhibiting contaminants , and we subsequently 
 evaluated several cdna reverse transcription kits and enzymes and concluded that the high - capacity cdna reverse 
 transcription kit and tks gflex provided the best pcr results .
ibarra - soria et al .   used rnaseq to quantify whole 
 transcriptomes of the mouse vo and oe ; however , they measured only one vir gene among the 530 annotated v1r 
 genes by using real - time pcr with a commercial primer and probe set .
the amplicon size was 59 bp for the 
 commercial primer and probe set ; however , the sequences of primers and probe in the set were not specified . 

thus , it was impossible to compare the homology of the 59-bp amplified products with that of the other 529 
 genes . in this study , we did not include any real - time pcr analysis data , because first , we did not obtain a 
 product by using tks gflex , which could be because of the high risk of failure to amplify due to the presence of 
 inhibiting contaminants .
second , it is difficult to ensure specificity , because of the high level of sequence 
 similarity within the v1r family .
third , for precise real - time pcr 
 analysis results , the amplicon size must be less than 150 bp ( and more than 50 bp ) .
however , such small amplicon size is insufficient to obtain sequence data for blast 
 search to identify the gene .
fourth , the manufacturer of the dye terminator cycle sequencing kit , namely , 
 applied biosystems , advised us that the excitation and emission wavelengths of the fluorescent dyes used in 
 real - time pcr kits are almost similar to those of the fluorescent dyes used in the dye terminator cycle 
 sequencing kit .
therefore , the manufacturer was unable to guarantee the quality of the sequence data of the 
 amplified products obtained by real - time pcr .
therefore , we used semi - quantitative rt - pcr followed by sequencing 
 for identification of the expressed gene in the present study ; however , we must be cautious , because 
 quantification by using semi - quantitative rt - pcrs may not always be accurate .
in addition , the number of receptors 
 on the cell surface is not solely controlled by their mrna expressions .
therefore , specific antibodies to 
 extra - cellular regions of each receptor must be developed to clarify the role and importance of each v1r . in conclusion , in this study , we discovered sexual polymorphisms of v1r expressions among bulls , steers , and 
 estrous and early luteal - phase heifers .
this is the first report of sexual polymorphisms of v1r expression in 
 ruminant species and the first reported estrous stage - dependent difference of v1r expression in any mammalian 
 species .
 vomeronasal 1 receptors ( v1r ) are a family of receptors for intraspecies chemosignals , including pheromones , 
 and are expressed in the olfactory epithelium ( oe ) and vomeronasal organ ( vo ) . even in the well - studied 
 rodents , 
 it is unclear which members of the v1r family cause sexual polymorphisms , as there are numerous genes 
 and it is difficult to quantify their expressions individually . 
 bovine species carry only 34 v1r homologs , and 
 the oe and vos are large enough to sample . here 
 , v1r expression was quantified in the oe and vos of individual 
 bovines . 
 based on the 34 gene sequences , we obtained a molecular dendrogram consisting of four clusters and 
 six independent branches . 
 semi - quantitative rt - pcr was used to obtain gene expression profiles in the vos and 
 oe of 5 japanese black bulls , 5 steers , 7 estrous heifers and 6 early luteal - phase heifers . 
 ten genes showed 
 significant between - group differences , and 22 showed high expression in vos than in oe . 
 the bulls showed 
 higher expression of one gene more in oe and another in vos ( both p<0.05 ) than did steers ; 
 both genes belonged to the first cluster . 
 no genes were expressed more abundantly in steers than in bulls . 
 the 
 estrous heifers showed higher expression of a gene of the second cluster in oe , and a gene of the third 
 cluster in vos ( both p<0.05 ) than did early luteal - phase heifers . 
 these results suggest 
 v1r expression exhibits sexual polymorphisms in cattle . 
MATERIALS AND METHODS
RESULTS
DISCUSSION