BACKGROUND
maintaining good fertility and thereby optimum reproductive performance in dairy cows is of great economic importance for the dairy industry.
knowledge on factors influencing fertility is already being applied to improve or regulate fertility.
for example, the importance of limiting negative energy balance in early lactation cows for proper reproductive performance is well recognized  <cit> .
insight into the hormonal regulation of estrous cycle has found practical application to artificially regulate the cycle in farm animals and to manage or treat fertility related problems.
however, current understanding of genomic factors underlying fertility is limited and this obstructs the development of novel genomic tools and managemental strategies for improving and optimizing reproductive performance, such as biomarkers to monitor the fertility status of cows.
studying the genomic factors underlying fertility may help to optimize nutritional or management systems that improve reproductive performance  <cit>  and also to explain the genetic basis for the decline in several fertility traits of high producing dairy cows.
currently it is known that this decline may be partly attributed to physiological adaptations by the cow to high milk production  <cit> .
among the fertility traits, the expression of estrous behavior , a key fertility trait that marks the fertile period in cows, has decreased both in duration and intensity over generations of cows selected for high milk yield  <cit> .
short heat periods and the absence of clear behavioral signs of heat cause farmers to fail to detect heat or to misjudge the optimum time of insemination of their cows, resulting in financial losses due to prolonged interval from calving to first insemination, reduced conception rates and increased calving intervals.
in an effort to understand the genomic regulation of estrous behavior expression in dairy cows, a microarray experiment was set up to study gene expression levels in  <dig> different brain areas and the anterior pituitary of cows sacrificed at either the start of estrus  or at mid-cycle .
differential gene expression analysis between day <dig> and day <dig> cows for each of these tissues revealed a limited number of significant genes in the anterior pituitary alone and none in the brain areas .
when the trait of interest is quantitative, as in this case , the grouping of individuals into qualitative classes dilutes the available information.
therefore, the association between gene expression and phenotypic trait may be better analyzed using the individual quantitative measurements.
using this approach in an earlier study, we identified a set of a few hundred probes out of approximately  <dig>  probes on a bovine microarray corresponding to genes whose level of expression in the anterior pituitary of experimental cows associated with the degree to which the individual cows expressed estrous behavior  <cit> .
among these probes were genes encoding hormones like fsh and prolactin, whose roles in estrous regulation are well-known.
further, biological processes relevant to estrous behavior were over-represented in this set of genes.
these results give confidence in the association analysis methodology followed, though experimental validation is needed to determine to what extent the associated genes regulate estrous behavior.
in addition to the anterior pituitary, it is likely that a number of areas of the cow brain also have genes whose levels of expression associate with the degree to which cows express estrous behavior.
studies in rodents have revealed that estrogen dependent female reproductive behavior happens via well orchestrated genomic responses in the forebrain with the hypothalamus playing a major role  <cit> .
areas in the limbic region of the forebrain like the amygdala and hippocampus were found to have functions related to sexual behavior and associated emotional responses  <cit> .
as yet, there have been no studies in cows linking gene expression in the brain to estrous behavior.
identifying and studying genes whose level of expression in different brain areas of cows associate with the degree to which these cows express estrous behavior will help improve our understanding of genomic factors underlying fertility.
the objective here is to identify and study those genes that associated with estrous behavior, among genes expressed in four bovine brain areas , either at the start of estrous cycle, or at mid cycle, or regardless of the phase of cycle.
RESULTS
the degree of estrous behavior expression was quantified as a cow's average heat score using heat scores recorded from at least two consecutive cycles .
the data from one of the day <dig> cows was excluded from further analysis because of its high outlier value of  <dig> for average heat score, which we attributed to that cow's several consecutive attempts to mount other cows during one observation period.
the average heat scores for the remaining  <dig> day <dig> cows ranged from  <dig> to  <dig>  with a mean value of  <dig>  , and the average heat scores for the  <dig> day <dig> cows ranged from  <dig> to  <dig>  with a mean value of  <dig>  .
the average heat scores were used with the corresponding gene expression data to run the three analyses per brain area as summarized in table  <dig>
* data from this cow was not used in the analysis due to outlier heat score value
additional file  <dig> lists the associated genes found in each analysis and describes the pattern of association between gene expression and heat score for each of the genes in the list.
the patterns noticed were: linear  or quadratic .
the total number of heat score associated probes found common to all  <dig> gibbs sampling chains per analysis in each brain area is provided in figure  <dig>  additional file  <dig> depicts venn diagrams that show the number of overlapping probes between the different brain areas per analysis.
the overlap was highest between the dh and vh and then between the am and hc.
the figure  <dig> and the additional file  <dig> includes the results of the re-analysis on the ap as well, for which the number of associated probes found for the three analyses  were now considerably lower than the numbers  from the association analysis with third order polynomials and one gibbs sampling chain per analysis as done earlier  <cit> .
for the different brain areas, the percentage of associated probes found common to all chains per analysis varied from 50-80%.
for day <dig>  a relatively high number of heat score associated probes were detected for am and ap  whereas for day <dig> this was true for vh .
for dh, the numbers were approximately equal at day <dig> and day <dig> .
figure  <dig> provides the association patterns for three genes whose expression values were found to be associated with heat score at day <dig>  for avp and mchr <dig>  a linear trend was observed in hc and am respectively whereas for oxt, a quadratic trend was observed in dh.
of the total  <dig> unique probes found associated with heat score in all analyses and brain areas,  <dig> had gene annotations.
sets of genes associated with functional categories which we group as: "transcription and regulation of gene expression", "detection of and responses to stimuli" and "signaling pathways" each made up about 15% of the genes that were identified.
in the category "detection of and responses to stimuli" we included the genes involved in neurotransmission through encoding for neurotransmitters/hormones  or neurotransmitter receptors  and metabolizing enzymes .
we could also identify  <dig> more sets of genes, each consisting of between  <dig> to 10% of the total genes.
these included genes associated with: "transport and localization" , "transporter activity" , "metabolism" , "cell cycle"  and "multi-organism processes".
in the last category we included immune system related genes like ctla <dig>  il1rl <dig>  marco, fcrla and il <dig>
a detailed list of all significant enriched go and kegg pathway terms  found in the different brain areas and analyses is provided in additional file  <dig>  for illustration, a summary of only those go biological processes that cleared a fdr cutoff of 20% is provided in table  <dig>  several relevant processes related to behavior, neurotransmission and signaling, and ion regulation were found especially in day <dig>  the presence of enriched processes related to behavior like 'grooming behavior', 'regulation of female receptivity' and 'female mating behavior' within the associated genes found in hc indicates the key role of the genes avp and oxt that contribute to these processes.
terms related to neurotransmission and signaling and the associated ion regulation terms too have biological implications related to estrous behavior as evident from the discussion below on some of the genes that contribute to these processes.
* only terms with p <  <dig>  and fdr < <dig>  are included.
terms typical of other organs  have been removed from this summary table.
where the term is supported by  <dig> or more genes in the gene list, the corresponding brain area is followed by a + sign whereas in cases where the term is supported by only 2- <dig> genes, the sign is absent.
discussion
variation in the behavioral trait to express estrous behavior was found to be associated with variation in the expression of genes in the cow's brain areas: dh, vh, hc and am and also the ap, an endocrine gland pivotal in synchronizing estrous behavior with hormonal and ovarian events preceding ovulation.
the choice of these tissues for this study was based on their reported involvement in regulating female sexual behavior.
although genes differ in their influence on specific traits, it is tempting to consider the am to be of relatively higher importance for regulating estrous behavior as the largest number of associated genes was found here at day <dig> and because am is known for its central role in regulating emotions.
key genes and biological processes as identified from the lists of heat score associated genes are discussed next and linked to estrous behavioral expression, though the links are not always as expected.
genes and biological processes associated with estrous behavior in line with expectations
an association with heat scores was detected for the cga gene, which encodes for the alpha subunit of glycoprotein hormones , with associations being time and brain area specific.
it seems that cows with clear expression of estrous behavior have relatively high expression of this gene in the hypothalamus around mid-cycle and low expression around start of estrus.
the oxt gene was found associated in the hc and dh whereas the avp gene was found associated in the hc and am.
the known functional properties of these genes contributed to several enriched go terms in the david analysis, especially those related to female mating behavior.
oxytocin is released within the brain where it acts on specific oxytocin receptors to elicit effects like female sexual receptivity, grooming behavior and partner bonding  <cit> .
in the presence of estrogen, oxytocin exerts an anxiolytic effect thereby favoring courtship and mating  <cit> .
similar to oxytocin, avp is associated with sexual behavior and bonding  <cit>  and its expression is under control of the steroids progesterone and estrogen  <cit> .
in mice, the absence of estrogen receptors  impairs social recognition similar to the effect of oxt gene deletion  <cit> .
studies with rodents and humans  <cit>  demonstrate that oxytocin and vasopressin modulate complex socio-sexual behavior, typically under the influence of reproductive steroid hormones.
the present association of oxt and avp in several brain areas is in line with the above mentioned findings and suggests a major influence of these genes on estrous behavior expression in dairy cows.
the genes cck, pomc, mchr <dig>  gabra <dig>  htr2a and drd <dig>  which associated with heat score in at least one brain area, are known to modulate emotional states like anxiety and satiety  <cit>  or even sexual motivation.
in sheep, dopamine-mediated d <dig> receptor  signaling in the mediobasal hypothalamus affects female sexual motivation and receptivity  <cit> .
interactions between monoamines  and steroid hormones play a major role in the integration of reproductive behavior and gonadal function  <cit> .
the perception and awareness of male-related cues differs with the stage of estrous cycle, with releases of monoamines  and gamma-aminobutyric acid   in the mediobasal hypothalamus being triggered by such cues only when ewes are in estrus  <cit> .
we found serotonin receptor 2a  associated with heat score at day <dig> in vh.
studies in female rats and hamsters have shown the inhibitory and facilitatory effects of serotonin receptor agonists and antagonists on the hypothalamic regulation of sexual receptivity  <cit> , and this regulation is also mediated by gabaergic neurons interacting with serotonin containing neurons  <cit> .
noteworthy, are the heat score associated genes  that regulate prostaglandin functioning.
in the central nervous system, prostaglandins are involved in functions like thermoregulation and influencing neuronal morphology.
prostaglandins are also known to be under the influence of estradiol  <cit>  and are capable of directly affecting neurons that synthesize and secrete gonadotropin-releasing hormone  <cit> .
another heat score associated gene, ttr, a carrier of thyroid hormone, is known to influence anxiety  <cit> , behavioral activity  <cit>  and mental functions  <cit> .
the melanin-concentrating hormone receptor, mchr <dig>  plays a role in metabolic rate and feed intake  <cit> .
changes in anxiety behavior and feeding motivation are likely to facilitate mating.
the present association between heat scores and the expression of ache and several cholinergic receptors  may be explained by the effect of the neurotransmitter, acetylcholine on arousal, plasticity and reward.
the products of the muscarinic cholinergic receptor genes, chrm <dig> and chrm <dig>  are gq-protein coupled receptors whose activation releases intracellular ca2+ via the phospholipase c - inositol  <dig> ,5-trisphosphate signaling pathway  <cit> .
the genes for phospholipase c and inositol triphosphate kinase  and several protein kinases were also found associated to heat score.
these findings may be explained based on the hypothesis put forward by kow and pfaff  <cit>  that the membrane actions of estrogen can modulate the genomic actions of estrogen and that this transcriptional potentiation was mediated via signaling pathways requiring the activation of certain protein kinases and increased intracellular ca2+.
genes and biological processes unexpectedly associated with estrous behavior
the finding of expected processes related to estrus as described in the previous section supports assumed neurophysiological mechanisms underlying female sexual behavior in dairy cows.
here, examples are given of more novel candidate genes and mechanisms.
the tac <dig> gene encodes the protein tachykinin , which in humans has been considered a critical regulator of gonadotropins  via regulating gnrh secretion  <cit> .
the present findings encourage further investigation towards the importance of tachykinin associated mechanisms in dairy cow fertility.
behavioral changes during estrus represent changes in central perception and processing of information, i.e.
cognition, and some of the genes that associated with heat scores have been linked to cognition, e.g.
pebp <dig>  mobp, lta4h and kcnn <dig>  pebp <dig> has been suggested to be involved in chronic stress-induced memory impairment  <cit> , mobp has been linked to mood disorders  <cit> , lta4h to depression in women  <cit> , and kcnn <dig> to anxiety and stress responses  <cit> .
the gene lipn <dig> in mice seems to establish a cross-talk between reproduction and metabolic events  <cit>  and was here associated with heat scores.
also the pou1f <dig> gene, which encodes transcription factors involved in activation of growth hormone and prolactin, was found associated with heat score in the ap on day <dig>  this gene may contribute in part to the generally observed reduction in estrous behavior in high producing cows.
the heat score associated gene garnl <dig> is noted here because it has been linked to egg productivity in laying hens  <cit>  and, thus, may have a fertility related function.
a number of heat score associated genes have been linked to the immune system, e.g.
ctla <dig>  il1rl <dig>  marco, fcrla, il <dig>  ccl <dig> and cxcl <dig>  indicating the importance of cell-cell interactions.
it has been shown that immunoglobulin superfamily proteins may play important roles in brain developmental processes and the functioning of neuronal networks in adults because they provide the ideal structure for protein-protein interactions and, thus, cell-cell interactions  <cit> .
remodeling of synaptic networks, which occurs during estrogen promoted female sexual behavior  <cit> , may also be facilitated by immunoglobulins.
together with the many associated genes that are known to regulate cell fate, activity and morphology, this seems to underline the importance of neural tissue plasticity in the appropriate expression of estrous behavior.
the gene product sodium/potassium-transporting atpase subunit alpha- <dig> , which was found to be associated to heat score at estrus in ap in our earlier study was also found in vh.
atp1a <dig> has been implicated in rapid-onset dystonia parkinsonism , characterized by sudden onset of neurological symptoms over hours to a few days  <cit> , suggesting a role in the sudden onset of behavioral changes like during estrus.
estrous behavior associated genes expressed at estrus and mid-cycle
including data from both day <dig> and day <dig> cows in a combined analysis not only revealed genes that were associated with estrous behavior regardless of phase of cycle but also resulted in greater statistical power and helped reveal some associated genes which could not be found in the separate analysis.
for example, drd <dig>  gene has been found associated in dh.
however, care needs to be taken to interpret the results of the combined analysis where an associated gene was also found in one or both of the separate analyses.
there could be cases where the day0+day <dig> combined analysis found an associated gene because of the effect of a strong association found in one of the separate analyses.
an example of this case is htr2a which did not associate with heat scores on day <dig>  but did on day <dig>  and also weakly  in the day0+day <dig> combined analysis, probably as a carryover effect.
interesting observations were also made by investigating associated genes that appeared in several brain areas and analyses.
for example, the gene slc17a <dig> found in vh at day <dig> and in the combined analysis and also in the ap, is known to mediate the uptake of glutamate into synaptic vesicles at presynaptic nerve terminals of excitatory neural cells and may also mediate the transport of inorganic phosphate  <cit> .
the gene could be a contributor to neurotransmission associated with estrous behavior expression and was one of the genes contributing to several ion transport related go terms found enriched in the david analysis.
the gene nkd <dig>  found in several brain areas across all analyses, was seen to contribute to several go terms related to binding and signal transduction.
it has been reported to have a role as an antagonist of wnt signaling pathway which may influence the development of neurons in dorsal midbrain  <cit> , suggesting again a link between neuronal plasticity and estrous behavior.
the gene ano <dig>  also found associated in several brain areas and analyses, is a member of the anoctamin family which has been implicated in calcium ion-activated chloride channels that perform several important functions including neuronal excitability.
to summarize, several genes were found here whose expression levels in the brain areas were associated with the degree to which cows express estrous behavior.
for some of these genes, there is a known function relating them to processes regulating estrous behavior, while for others, the current association suggests such a relation.
we propose that the genes oxt and avp play major roles in regulating estrous behavior along with genes affecting neurotransmission and neuronal plasticity.
genes, whose expression in mid-cycle associated with estrous behavior, may contribute to preparing the cow for the next estrus.
figure  <dig> depicts the key findings.
this study may assist in the search for biomarkers for estrus detection or in screening for the most likely genes within qtls associated with fertility.
further research with more animals sampled at multiple time points in the estrous cycle may improve our understanding of the dynamic regulation of estrous behavior over the cycle.
nevertheless, this first study provides an understanding of some of the genes and processes related to expression of estrous behavior in dairy cows.
CONCLUSIONS
the study predicted estrous behavior associated genes in the  <dig> brain areas at two time points of the estrous cycle.
these included genes expected to be related to estrous behavior as they influence states like socio-sexual behavior, anxiety, stress and feeding motivation , but also genes whose association with estrous behavior is novel and warrants further investigation.
studying these genes and the processes they control improves our understanding of the genomic regulation of estrous behavior expression, ultimately leading to better management strategies or tools to improve or monitor reproductive performance.

 maintaining good fertility and thereby optimum reproductive performance in dairy cows is of great economic importance for the dairy industry. knowledge on factors influencing fertility is already being applied to improve or regulate fertility. for example, the importance of limiting negative energy balance in early lactation cows for proper reproductive performance is well recognized. insight into the hormonal regulation of estrous cycle has found practical application to artificially regulate the cycle in farm animals and to manage or treat fertility related problems. however, current understanding of genomic factors underlying fertility is limited and this obstructs the development of novel genomic tools and managemental strategies for improving and optimizing reproductive performance, such as biomarkers to monitor the fertility status of cows. studying the genomic factors underlying fertility may help to optimize nutritional or management systems that improve reproductive performance and also to explain the genetic basis for the decline in several fertility traits of high producing dairy cows. currently it is known that this decline may be partly attributed to physiological adaptations by the cow to high milk production. among the fertility traits, the expression of estrous behavior, a key fertility trait that marks the fertile period in cows, has decreased both in duration and intensity over generations of cows selected for high milk yield. short heat periods and the absence of clear behavioral signs of heat cause farmers to fail to detect heat or to misjudge the optimum time of insemination of their cows, resulting in financial losses due to prolonged interval from calving to first insemination, reduced conception rates and increased calving intervals. in an effort to understand the genomic regulation of estrous behavior expression in dairy cows, a microarray experiment was set up to study gene expression levels in different brain areas and the anterior pituitary of cows sacrificed at either the start of estrus or at mid-cycle. differential gene expression analysis between day and day cows for each of these tissues revealed a limited number of significant genes in the anterior pituitary alone and none in the brain areas. when the trait of interest is quantitative, as in this case, the grouping of individuals into qualitative classes dilutes the available information. therefore, the association between gene expression and phenotypic trait may be better analyzed using the individual quantitative measurements. using this approach in an earlier study, we identified a set of a few hundred probes out of approximately probes on a bovine microarray corresponding to genes whose level of expression in the anterior pituitary of experimental cows associated with the degree to which the individual cows expressed estrous behavior. among these probes were genes encoding hormones like fsh and prolactin, whose roles in estrous regulation are well-known. further, biological processes relevant to estrous behavior were over-represented in this set of genes. these results give confidence in the association analysis methodology followed, though experimental validation is needed to determine to what extent the associated genes regulate estrous behavior. in addition to the anterior pituitary, it is likely that a number of areas of the cow brain also have genes whose levels of expression associate with the degree to which cows express estrous behavior. studies in rodents have revealed that estrogen dependent female reproductive behavior happens via well orchestrated genomic responses in the forebrain with the hypothalamus playing a major role. areas in the limbic region of the forebrain like the amygdala and hippocampus were found to have functions related to sexual behavior and associated emotional responses. as yet, there have been no studies in cows linking gene expression in the brain to estrous behavior. identifying and studying genes whose level of expression in different brain areas of cows associate with the degree to which these cows express estrous behavior will help improve our understanding of genomic factors underlying fertility. the objective here is to identify and study those genes that associated with estrous behavior, among genes expressed in four bovine brain areas, either at the start of estrous cycle, or at mid cycle, or regardless of the phase of cycle.  the degree of estrous behavior expression was quantified as a cow's average heat score using heat scores recorded from at least two consecutive cycles. the data from one of the day cows was excluded from further analysis because of its high outlier value of for average heat score, which we attributed to that cow's several consecutive attempts to mount other cows during one observation period. the average heat scores for the remaining day cows ranged from to with a mean value of, and the average heat scores for the day cows ranged from to with a mean value of. the average heat scores were used with the corresponding gene expression data to run the three analyses per brain area as summarized in table * data from this cow was not used in the analysis due to outlier heat score value additional file lists the associated genes found in each analysis and describes the pattern of association between gene expression and heat score for each of the genes in the list. the patterns noticed were: linear or quadratic. the total number of heat score associated probes found common to all gibbs sampling chains per analysis in each brain area is provided in figure additional file depicts venn diagrams that show the number of overlapping probes between the different brain areas per analysis. the overlap was highest between the dh and vh and then between the am and hc. the figure and the additional file includes the results of the re-analysis on the ap as well, for which the number of associated probes found for the three analyses were now considerably lower than the numbers from the association analysis with third order polynomials and one gibbs sampling chain per analysis as done earlier. for the different brain areas, the percentage of associated probes found common to all chains per analysis varied from 50-80%. for day a relatively high number of heat score associated probes were detected for am and ap whereas for day this was true for vh. for dh, the numbers were approximately equal at day and day. figure provides the association patterns for three genes whose expression values were found to be associated with heat score at day for avp and mchr a linear trend was observed in hc and am respectively whereas for oxt, a quadratic trend was observed in dh. of the total unique probes found associated with heat score in all analyses and brain areas, had gene annotations. sets of genes associated with functional categories which we group as: "transcription and regulation of gene expression", "detection of and responses to stimuli" and "signaling pathways" each made up about 15% of the genes that were identified. in the category "detection of and responses to stimuli" we included the genes involved in neurotransmission through encoding for neurotransmitters/hormones or neurotransmitter receptors and metabolizing enzymes. we could also identify more sets of genes, each consisting of between to 10% of the total genes. these included genes associated with: "transport and localization", "transporter activity", "metabolism", "cell cycle" and "multi-organism processes". in the last category we included immune system related genes like ctla il1rl marco, fcrla and il a detailed list of all significant enriched go and kegg pathway terms found in the different brain areas and analyses is provided in additional file for illustration, a summary of only those go biological processes that cleared a fdr cutoff of 20% is provided in table several relevant processes related to behavior, neurotransmission and signaling, and ion regulation were found especially in day the presence of enriched processes related to behavior like'grooming behavior','regulation of female receptivity' and'female mating behavior' within the associated genes found in hc indicates the key role of the genes avp and oxt that contribute to these processes. terms related to neurotransmission and signaling and the associated ion regulation terms too have biological implications related to estrous behavior as evident from the discussion below on some of the genes that contribute to these processes. * only terms with p < and fdr < are included. terms typical of other organs have been removed from this summary table. where the term is supported by or more genes in the gene list, the corresponding brain area is followed by a + sign whereas in cases where the term is supported by only 2- genes, the sign is absent. discussion variation in the behavioral trait to express estrous behavior was found to be associated with variation in the expression of genes in the cow's brain areas: dh, vh, hc and am and also the ap, an endocrine gland pivotal in synchronizing estrous behavior with hormonal and ovarian events preceding ovulation. the choice of these tissues for this study was based on their reported involvement in regulating female sexual behavior. although genes differ in their influence on specific traits, it is tempting to consider the am to be of relatively higher importance for regulating estrous behavior as the largest number of associated genes was found here at day and because am is known for its central role in regulating emotions. key genes and biological processes as identified from the lists of heat score associated genes are discussed next and linked to estrous behavioral expression, though the links are not always as expected. genes and biological processes associated with estrous behavior in line with expectations an association with heat scores was detected for the cga gene, which encodes for the alpha subunit of glycoprotein hormones, with associations being time and brain area specific. it seems that cows with clear expression of estrous behavior have relatively high expression of this gene in the hypothalamus around mid-cycle and low expression around start of estrus. the oxt gene was found associated in the hc and dh whereas the avp gene was found associated in the hc and am. the known functional properties of these genes contributed to several enriched go terms in the david analysis, especially those related to female mating behavior. oxytocin is released within the brain where it acts on specific oxytocin receptors to elicit effects like female sexual receptivity, grooming behavior and partner bonding. in the presence of estrogen, oxytocin exerts an anxiolytic effect thereby favoring courtship and mating. similar to oxytocin, avp is associated with sexual behavior and bonding and its expression is under control of the steroids progesterone and estrogen. in mice, the absence of estrogen receptors impairs social recognition similar to the effect of oxt gene deletion. studies with rodents and humans demonstrate that oxytocin and vasopressin modulate complex socio-sexual behavior, typically under the influence of reproductive steroid hormones. the present association of oxt and avp in several brain areas is in line with the above mentioned findings and suggests a major influence of these genes on estrous behavior expression in dairy cows. the genes cck, pomc, mchr gabra htr2a and drd which associated with heat score in at least one brain area, are known to modulate emotional states like anxiety and satiety or even sexual motivation. in sheep, dopamine-mediated d receptor signaling in the mediobasal hypothalamus affects female sexual motivation and receptivity. interactions between monoamines and steroid hormones play a major role in the integration of reproductive behavior and gonadal function. the perception and awareness of male-related cues differs with the stage of estrous cycle, with releases of monoamines and gamma-aminobutyric acid in the mediobasal hypothalamus being triggered by such cues only when ewes are in estrus. we found serotonin receptor 2a associated with heat score at day in vh. studies in female rats and hamsters have shown the inhibitory and facilitatory effects of serotonin receptor agonists and antagonists on the hypothalamic regulation of sexual receptivity, and this regulation is also mediated by gabaergic neurons interacting with serotonin containing neurons. noteworthy, are the heat score associated genes that regulate prostaglandin functioning. in the central nervous system, prostaglandins are involved in functions like thermoregulation and influencing neuronal morphology. prostaglandins are also known to be under the influence of estradiol and are capable of directly affecting neurons that synthesize and secrete gonadotropin-releasing hormone. another heat score associated gene, ttr, a carrier of thyroid hormone, is known to influence anxiety, behavioral activity and mental functions. the melanin-concentrating hormone receptor, mchr plays a role in metabolic rate and feed intake. changes in anxiety behavior and feeding motivation are likely to facilitate mating. the present association between heat scores and the expression of ache and several cholinergic receptors may be explained by the effect of the neurotransmitter, acetylcholine on arousal, plasticity and reward. the products of the muscarinic cholinergic receptor genes, chrm and chrm are gq-protein coupled receptors whose activation releases intracellular ca2+ via the phospholipase c - inositol,5-trisphosphate signaling pathway. the genes for phospholipase c and inositol triphosphate kinase and several protein kinases were also found associated to heat score. these findings may be explained based on the hypothesis put forward by kow and pfaff that the membrane actions of estrogen can modulate the genomic actions of estrogen and that this transcriptional potentiation was mediated via signaling pathways requiring the activation of certain protein kinases and increased intracellular ca2+. genes and biological processes unexpectedly associated with estrous behavior the finding of expected processes related to estrus as described in the previous section supports assumed neurophysiological mechanisms underlying female sexual behavior in dairy cows. here, examples are given of more novel candidate genes and mechanisms. the tac gene encodes the protein tachykinin, which in humans has been considered a critical regulator of gonadotropins via regulating gnrh secretion. the present findings encourage further investigation towards the importance of tachykinin associated mechanisms in dairy cow fertility. behavioral changes during estrus represent changes in central perception and processing of information, i.e. cognition, and some of the genes that associated with heat scores have been linked to cognition, e.g. pebp mobp, lta4h and kcnn pebp has been suggested to be involved in chronic stress-induced memory impairment, mobp has been linked to mood disorders, lta4h to depression in women, and kcnn to anxiety and stress responses. the gene lipn in mice seems to establish a cross-talk between reproduction and metabolic events and was here associated with heat scores. also the pou1f gene, which encodes transcription factors involved in activation of growth hormone and prolactin, was found associated with heat score in the ap on day this gene may contribute in part to the generally observed reduction in estrous behavior in high producing cows. the heat score associated gene garnl is noted here because it has been linked to egg productivity in laying hens and, thus, may have a fertility related function. a number of heat score associated genes have been linked to the immune system, e.g. ctla il1rl marco, fcrla, il ccl and cxcl indicating the importance of cell-cell interactions. it has been shown that immunoglobulin superfamily proteins may play important roles in brain developmental processes and the functioning of neuronal networks in adults because they provide the ideal structure for protein-protein interactions and, thus, cell-cell interactions. remodeling of synaptic networks, which occurs during estrogen promoted female sexual behavior, may also be facilitated by immunoglobulins. together with the many associated genes that are known to regulate cell fate, activity and morphology, this seems to underline the importance of neural tissue plasticity in the appropriate expression of estrous behavior. the gene product sodium/potassium-transporting atpase subunit alpha-, which was found to be associated to heat score at estrus in ap in our earlier study was also found in vh. atp1a has been implicated in rapid-onset dystonia parkinsonism, characterized by sudden onset of neurological symptoms over hours to a few days, suggesting a role in the sudden onset of behavioral changes like during estrus. estrous behavior associated genes expressed at estrus and mid-cycle including data from both day and day cows in a combined analysis not only revealed genes that were associated with estrous behavior regardless of phase of cycle but also resulted in greater statistical power and helped reveal some associated genes which could not be found in the separate analysis. for example, drd gene has been found associated in dh. however, care needs to be taken to interpret the results of the combined analysis where an associated gene was also found in one or both of the separate analyses. there could be cases where the day0+day combined analysis found an associated gene because of the effect of a strong association found in one of the separate analyses. an example of this case is htr2a which did not associate with heat scores on day but did on day and also weakly in the day0+day combined analysis, probably as a carryover effect. interesting observations were also made by investigating associated genes that appeared in several brain areas and analyses. for example, the gene slc17a found in vh at day and in the combined analysis and also in the ap, is known to mediate the uptake of glutamate into synaptic vesicles at presynaptic nerve terminals of excitatory neural cells and may also mediate the transport of inorganic phosphate. the gene could be a contributor to neurotransmission associated with estrous behavior expression and was one of the genes contributing to several ion transport related go terms found enriched in the david analysis. the gene nkd found in several brain areas across all analyses, was seen to contribute to several go terms related to binding and signal transduction. it has been reported to have a role as an antagonist of wnt signaling pathway which may influence the development of neurons in dorsal midbrain, suggesting again a link between neuronal plasticity and estrous behavior. the gene ano also found associated in several brain areas and analyses, is a member of the anoctamin family which has been implicated in calcium ion-activated chloride channels that perform several important functions including neuronal excitability. to summarize, several genes were found here whose expression levels in the brain areas were associated with the degree to which cows express estrous behavior. for some of these genes, there is a known function relating them to processes regulating estrous behavior, while for others, the current association suggests such a relation. we propose that the genes oxt and avp play major roles in regulating estrous behavior along with genes affecting neurotransmission and neuronal plasticity. genes, whose expression in mid-cycle associated with estrous behavior, may contribute to preparing the cow for the next estrus. figure depicts the key findings. this study may assist in the search for biomarkers for estrus detection or in screening for the most likely genes within qtls associated with fertility. further research with more animals sampled at multiple time points in the estrous cycle may improve our understanding of the dynamic regulation of estrous behavior over the cycle. nevertheless, this first study provides an understanding of some of the genes and processes related to expression of estrous behavior in dairy cows.  the study predicted estrous behavior associated genes in the brain areas at two time points of the estrous cycle. these included genes expected to be related to estrous behavior as they influence states like socio-sexual behavior, anxiety, stress and feeding motivation, but also genes whose association with estrous behavior is novel and warrants further investigation. studying these genes and the processes they control improves our understanding of the genomic regulation of estrous behavior expression, ultimately leading to better management strategies or tools to improve or monitor reproductive performance. 
these included genes expected to be related to estrous behavior as they influence states like socio-sexual behavior, anxiety, stress and feeding motivation, but also genes whose association with estrous behavior is novel and warrants further investigation. here, we aimed to identify and study those genes that associated with a key fertility trait namely estrous behavior, among genes expressed in four bovine brain areas, either at the start of estrous cycle, or at mid cycle, or regardless of the phase of cycle. an average heat score was calculated for each of primiparous cows in which estrous behavior was recorded for at least two consecutive estrous cycles starting from days post-partum. several genes were identified whose expression levels in the bovine brain associated with the level of expression of estrous behavior. the genes oxt and avp play major roles in regulating estrous behavior in dairy cows. genes related to neurotransmission and neuronal plasticity are also involved in estrous regulation, with several genes and processes expressed in mid-cycle probably contributing to proper expression of estrous behavior in the next estrus. studying these genes and the processes they control improves our understanding of the genomic regulation of estrous behavior expression. the decline noticed in several fertility traits of dairy cattle over the past few decades is of major concern. for each brain area, gene expression was modeled as a function of the orthogonally transformed average heat score values using a bayesian hierarchical mixed model. gene expression was then measured in brain tissue samples collected from these cows, of which were sacrificed at the start of estrus and around mid cycle. understanding of the genomic factors underlying fertility, which could have potential applications to improve fertility, is very limited. genes whose expression patterns showed significant linear or quadratic relationships with heat scores were identified.
 the decline noticed in several fertility traits of dairy cattle over the past few decades is of major concern. understanding of the genomic factors underlying fertility, which could have potential applications to improve fertility, is very limited. here, we aimed to identify and study those genes that associated with a key fertility trait namely estrous behavior, among genes expressed in four bovine brain areas, either at the start of estrous cycle, or at mid cycle, or regardless of the phase of cycle.  an average heat score was calculated for each of primiparous cows in which estrous behavior was recorded for at least two consecutive estrous cycles starting from days post-partum. gene expression was then measured in brain tissue samples collected from these cows, of which were sacrificed at the start of estrus and around mid cycle. for each brain area, gene expression was modeled as a function of the orthogonally transformed average heat score values using a bayesian hierarchical mixed model. genes whose expression patterns showed significant linear or quadratic relationships with heat scores were identified. these included genes expected to be related to estrous behavior as they influence states like socio-sexual behavior, anxiety, stress and feeding motivation, but also genes whose association with estrous behavior is novel and warrants further investigation.  several genes were identified whose expression levels in the bovine brain associated with the level of expression of estrous behavior. the genes oxt and avp play major roles in regulating estrous behavior in dairy cows. genes related to neurotransmission and neuronal plasticity are also involved in estrous regulation, with several genes and processes expressed in mid-cycle probably contributing to proper expression of estrous behavior in the next estrus. studying these genes and the processes they control improves our understanding of the genomic regulation of estrous behavior expression. 