BACKGROUND
feeding a high-concentrate diet for a longer period of time can cause subacute ruminal acidosis .
it is a digestive disorder with a ph less than  <dig>  lasted for more than 3 h per day  <cit> .
general clinical signs of sara comprise of reduced dry matter intake , decreased ruminal ph and diarrhea  <cit> .
another crucial outcome of sara is the decline of milk quantity and quality  <cit> , and earlier studies also revealed that sara decreased milk protein yield  <cit> .
the persistent consumption of a hc diet by dairy animals enhances the production of organic acids and significantly results in a decline in ph values of the rumen and intestine .
this decrease in ph due to excessive high concentrate diet  <cit>  may affect the alterations of the type of fermentation  <cit>  and the structure of the microbes in the rumen  <cit> .
moreover, the decline in ph also perturbs the balance of microbial population in the rumen causing substantial release of free endotoxin  from gram-negative bacteria  <cit> .
due to possible alterations in permeability and disruption of the gastrointestinal barrier, lps can be translocated into the blood stream  <cit> .
there are also other reports revealed that elevated circulating lps cause a systemic inflammatory response  <cit> .
possibly, being an element of an immune response to lps, sara has been reported to increase the concentration of acute phase proteins in the peripheral blood, such as serum amyloid a  and haptogloblin   <cit> .
the saa has many functions but mainly alters innate immune responses, particularly in the migration of neutrophils and monocytes, while hp has anti-inflammatory effects; lps-binding protein  is activated by microbial infections and facilitates in the neutralization of lps and in the triggering the release of pro-inflammatory cytokines, such as tnf-α, il- <dig> and il-1β .
the innate immune system is the first line of defence against invading pathogens; it involves various types of transmembrane and secreted molecules, recognized as pattern recognition receptors .
toll like receptors, such as tlr <dig>  that join the lps-lbp complex, are located on the outer surfaces of a variety of cells and contribute to the sensing of microbial pathogens in the intracellular endosomes and lysosomes  <cit> .
tlr <dig> recognizes lps with the help of the lbp and cluster of differentiation antigen <dig>   <cit> .
lps activates myeloid differentiating factor  <dig>  after joining with tlr <dig> on the host cell surface  <cit> .
myd <dig> activates tnf receptor-associated factor  <dig> , which stimulates the iκb kinase  complex, and phosphorylated nf-κb is consequently translocated from the cytoplasm to the nucleus  <cit> .
the nf-κb-dependent cascade pathway regulates the production of pro-inflammatory cytokines, including interleukin -1β, tnf-α, il- <dig>  chemokines  and other inflammatory mediators  <cit> .
the principal objectives of this study were to evaluate the inflammatory response in cow uterus and to explore the molecular mechanism triggered by high concentrate-induced sara in mid-lactating dairy cows.
methods
animals, diets and experimental design
twelve healthy multiparous holstein cows, which were purchased from the experimental farm of nanjing agricultural university, with an average body weight of 455 kg were selected for the current study.
the experimental cows were randomly divided into two groups, each group containing six cows: the high-concentrate  group and the low-concentrate  group.
the hc group was provided a high-concentrate diet containing 6: <dig> :  ratio, while lc group was fed low concentrate diet containing 4: <dig>  for the 18-week experimental period.
the details of the nutritional ingredients and their compositions are shown in table  <dig>  the cows were surgically fixed with a ruminal fistula two weeks prior to the experiment.
the cows were kept in individual tie stalls and feed was offered three times per day at  <dig>   <dig> and 2000 h. dmi was ~ <dig>  kg/d/head, and fresh drinking water was available to them during the entire experimental period.
the experiment was started after the animals made a full recovery from the ruminal fistula fixation, and no cows showed any clinical signs of infection during the experiment.table  <dig> ingredient and nutrient composition of diets
premixb
nutritional compositionc
atreatment lc, 40% grain on dm basis; hc, 60% grain on dm basis
ne net energy mega joules, cp crude protein, ee ether extract, ndf neutral detergent fiber, nfc non-forage carbohydrates, adf acid detergent fiber
bthe premix contained va, <dig> ,000 iu/kg; vd,  <dig>  iu/kg; ve,  <dig>  iu/kg; niacin,  <dig>  mg/kg; cu,  <dig>  mg/kg; fe, 525 mg/kg; zn,  <dig>  mg/kg; mn,  <dig>  mg/kg; i, 170 mg/kg; co, 50 mg/kg; se, 27 mg/mg
cthe calculated nutritional composition values
collection of samples and analysis
the rumen fluid was collected at one-hour intervals for 12 h daily for  <dig> continuous days during the 18th week.
blood samples were collected 4 h after feeding on the sampling days  of the 18th week.
10 ml of rumen fluid was immediately centrifuged at  <dig>  × g for 45 min, filtered through a disposable  <dig> -μm filter and stored in pyrogen free glass at −20 °c for lps analysis.
the blood samples were collected from the jugular vein into 5-ml vacuum tubes containing sodium heparin as an anticoagulant.
the plasma was separated from the blood samples through centrifugation at 3000 × g at 4 °c for 15 min and stored at −20 °c to determine the concentrations of the pro-inflammatory cytokines il-1β, il- <dig>  and tnfα in the peripheral blood.
at the end of the experiment, the cows were slaughtered after overnight fasting.
the slaughtering was performed according to the law of jiangsu provincial people’s government, china and the study was approved by the animal ethics committee  of nanjing agricultural university, china as described in the end of the manuscript.
after slaughtering, the uterine tissues were removed from all the cows aseptically, as close to the cervix as possible for the analysis.
all collected tissues were immediately frozen in liquid nitrogen and stored at −70 °c within 30 min of slaughtering  <cit> .
determination of ruminal ph
rumen fluids were taken through a rumen fistula at one-hour intervals up to 12 h on days  <dig>   <dig> and  <dig> of the 18th week of the trial.
the ph of rumen was measured each hour immediately following the sample collection by ph-meter .
determination of lps
the concentration of lps in the rumen fluid and plasma of the jugular vein was determined by chromogenic endpoint limulus amebocyte lysate assay kits, ce <dig> and ce <dig> .
briefly, the pre-treated rumen fluid and plasma samples were diluted until their lps concentrations were in the range of  <dig>  to  <dig> endotoxin units /ml relative to the reference endotoxin and assayed as described by gozho et al.
<cit> .
radioimmunoassay
radioimmunoassay was used to calculate the concentrations of il- <dig>  il-1β, and tnf-α in peripheral blood using radioimmunoassay  kits .
rna extraction and real time pcr
total rna was prepared from 100 mg uterus tissue using trizol  via homogenization on ice as described by the manufacturer’s instructions.
the quality of mrna was assessed by both agarose gel  electrophoresis and spectrometry  using the eppendorf bio photometer plus .
only samples with a ratio between  <dig>  and  <dig>  were used in subsequent experiments.
the first-strand cdna was synthesized using 250 ng/μl of the total rna template using prime script rt master mix perfect real time  according to the manufacturer’s instructions.
quantitative real-time pcr  amplification was performed to evaluate the expression of the selected genes.
the primers for genes il-1β, tlr <dig>  il- <dig>  il- <dig>  <cit> , and lbp  <cit>  were used according to previously published work, while myd <dig>  tnf-α, traf <dig>  and nf-κb were designed using the premier primer  <dig>  software.
the primers were assessed for their amplification efficiencies.
the reaction conditions included 2 μl of cdna and  <dig>  μm primers in a total volume of 20 μl of super mix.
thermal cycling parameters consisted of initial denaturation at 95 °c for 15 s, followed by 40 cycles of annealing at 95 °c for 5 s and primer extension at 60 °c for 31 s. all the reactions were run in triplicate.
each cdna sample was amplified using sybr green  in the abi  <dig> fast real-time pcr system .
the data were normalized to the mean of housekeeping gene β-actin to control the unpredictability in expression levels and were analysed using r = 2-ΔΔct method  treatment -  control), as previously depicted  <cit> .
the primer sequences used in this study are presented in table  <dig> table  <dig> the list of primer sequences used for amplification of qrt-pcr
western blotting analysis
the protein was extracted from 100 mg uterus tissue with radio immunoprecipitation assay  buffer using a dounce homogenizer .
after 30 min of incubation on ice, cell extracts were subjected to centrifugation  at 4 °c for 15 min to obtain cell protein.
the supernatant was assayed for protein concentration using the bicinchoninic acid  protein assay kit  and was adjusted to 4 μg/μl.
equal amounts of proteins were isolated using 10% sds-polyacrylamide gel electrophoresis  and shifted to nitrocellulose membrane   at 4 °c.
then, 10% non-fat dry milk was used to block the nitrocellulose membrane.
the membrane was incubated at 4 °c overnight with monoclonal goat antibodies against nf-κb and gapdh , , respectively.
after incubation, the membrane was washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies.
immunoreactive proteins were detected by chemiluminescence using an ecl reagent  and subsequently autoradiographed.
the results were quantified using bio-rad gel doc 2000™ systems  and analysed with bio-rad tds quantity one software .
the relative quantities of proteins were evaluated by a densitometer and expressed as absorbance units .
statistical analyses
the mixed procedure of sas was used to analyze ph with a repeated measures design performed with sas software .
the effects of cows were considered random and the effects of diet and time were considered as fixed factors.
the time within treatments and cows were considered as repeated measurements and the residuals for each variable were used to assess the normality.
the days were averaged within a cow before the analysis for all the data including the ph, radioimmunoassay, ruminal lps, plasma lps and the gene expression data were assessed by independent t-test and expressed as mean ± sem.
differences were concluded to be significant at p <  <dig> .
RESULTS
rumen ph and lps content of rumen fluid and blood plasma
the results of ruminal ph fed a high concentrate diet were published previously by xu et al.
<cit>  and he confirmed the presence of sara by measuring the ruminal ph at different time intervals.
in this study, consuming a high-concentrate diet  for 18 weeks resulted in a significant decline in ruminal ph  than that in the lc group  and lasted for more than three hours  <cit> .
the effect of diet × hours on ph was significant ; however no effect was observed on hours alone.
the lps concentrations in the rumen fluid and the jugular vein plasma were significantly higher in the cows fed a high-concentrate diet compared to cows fed a low-concentrate diet.
the concentration of lps in the rumen fluid of the hc group was  <dig> eu/ml, compared to  <dig> eu/ml in the lc group ; similarly, in the peripheral plasma, the lps level in the hc group was  <dig> eu/ml, whereas it was  <dig> eu/ml in lc group , which indicated that the lps level was significantly increased in the hc group relative to the lc group .table  <dig> lps concentration in rumen and plasma of dairy cows fed low concentrate  and high concentrate
a
hc high concentrate diet, lc low concentrate diet, eu endotoxin unit
b
sem standard error of the mean between the two groups
the lps data were compared using student’s t-test between hc and lc groups
p ≤  <dig>  was considered significant
levels of pro-inflammatory cytokines in the peripheral blood
the results of the measurement of pro-inflammatory cytokines, together with il-1β, il- <dig> and tnf-α are shown in fig.
<dig>  the concentration of il-1β was significantly higher in the hc group compared to the lc group  , the concentration of il- <dig> was significantly increased in the hc group versus lc group   and the concentration of tnf-α was elevated in the hc group relative to the lc group  .
generally, the concentration of pro-inflammatory cytokines in the peripheral blood was significantly higher in the hc group compared to the lc group.fig.
<dig>
a-c the concentration levels of tnf-α, il-1β, and il- <dig> in the peripheral blood.
the data were expressed as the mean ± sem.
; asterisks indicate the differences between the low-concentrate  group and high-concentrate  group.
expressions of genes involved in the inflammation of the uterus
sara significantly increased the expression level of several genes involved in the inflammation of the uterus as shown in fig.
<dig>  the gene expression of tlr <dig>  and myd <dig>  in the hc group was significantly up-regulated compared to the lc group.
similarly, traf- <dig>  and nf-κb  were highly expressed in the hc group relative to the lc group.
the gene expression of tnf-α  and il- <dig>  was significantly increased.
the gene expression of il-1β , il- <dig>  and lbp  was also significantly higher in the hc group compared with the lc group.fig.
<dig> the uterine gene expression profile analysed by qrt-pcr.
the genes involved in transcriptional regulation were measured in the uterine tissues.
the error bars indicate the standard error of the mean and ** indicates significance at p <  <dig>
expression of nf-κb protein in the cow uterus of the hc group and lc group
western blot analysis demonstrated that high concentrate diet-induced inflammation through the increaseduterine concentrations of the nf-κb  proteins in the hc group compared with the lc group .
importantly, the amount of phosphorylated nf-κb  was higher in the uterus of the hc group than in the lc group .
therefore, it was confirmed that nf-κb was activated in hc group in the tlr <dig> signalling pathway.fig.
<dig>
a the western blotting assay of nf-κb  and phosphorylated-p <dig> protein in the uterus.
the nf-κb contents in uterus were evaluated through western blot of the low-concentrate  group and high-concentrate  group.
b the quantities of proteins are measured as arbitrary units relative to gapdh; fold alterations in nf-κb  and phosphorylated nf-κb .
the data were expressed as the mean ± sem., asterisks indicate the differences between the high-concentrate  group compared to the low-concentrate  group
discussion
the present study assessed the uterine inflammation at the molecular level in dairy cows.
the inflammatory response in the uterus due to circulating lps derived from rumen triggered the expression of pro-inflammatory cytokines through tlr <dig> signaling.
to evaluate the inflammatory response in cow uterus and to explore the molecular mechanism triggered by high concentrate diet, sara was confirmed by measuring the ph of the rumen.
xu et al.
<cit>  also confirmed the presence of sara by measuring the ruminal ph at different time intervals.
in our study, the low rumen ph  lasted for 223 min and high concentrations of lps were observed in the rumen fluid and peripheral blood.
the recent reports also indicated the successful induction of sara, if theduration of low rumen ph was more than 180 min/day  <cit> .
however, there is little literature revealing that sara not only depends on ph, but also on the alteration in the microbial population that develops secondarily to the feed type  <cit> .
as a matter of fact, low ruminal ph values led to the lysis of gram-negative bacteria which released lps into the blood circulation  <cit> .
consistent with previous studies in dairy cows and feedlot, our results presented higher concentrations of lps in the peripheral plasma  and ruminal fluid  of cows fed a high concentrate die trelative to the peripheral plasma of the lc group  <cit> .
moreover, we also found that high concentrate diet feeding increased the plasma concentrations of pro-inflammatory cytokines .
similar results have been reported by other researchers  <cit> .
the increment in pro-inflammatory cytokines concentration in the plasma may provide confirmation for lps translocation into the blood circulation and eliciting of inflammatory responses.
this implies that inflammation in the hc group was due to the high level of lps in the bloodstream, which in turn triggers the pro-inflammatory gene to cope with stress conditions  <cit> .
the rt-qpcr-based findings revealed the presence of inflammation-associated markers in the uterus of sara-induced cows and confirmed that the dairy cows suffered from uterine inflammation at the molecular level.
according to our results, the increased expression of tlr <dig> indicated that tlr <dig> recognized the lps and triggered the inflammatory pathway  <cit> .
identification of inflammatory-stimuli by the innate immune system is organized by ppr that identify external stimuli for example pathogen associated molecular patterns   <cit> .
furthermore, our research demonstrated the increased expression of lbp, myd <dig>  and traf <dig> in the uterus; which provides more clues that the tlr <dig> signaling pathway was stimulated by lps  <cit> .
previous studies demonstrated that as a main transcription factor, nf-κb may bind to the promoter regions of the many inflammatory genes to regulate their transcription activities  <cit> .
similarly, in the current study, nf-κb expression was increased in the hc group, which also confirmed the activation of tlr <dig> signalling pathway.
our results of increased expression of pro-inflammatory cytokines  and chemokine  in the hc group due to sara-induced lps were consistent with the previous studies .
the western blot results further confirmed that the expression levels of nf-κb  and phosphorylated p <dig> proteins were distinctly increased in the hc group compared to the lc group.
taken together, the results suggested that lps triggered the activation of tlr <dig> signalling, which resulted in an inflammatory response in the uterus of cows suffering from sara.
conclusively, lps activates tlr4-nf-κb signalling.
together with the existing study and the previous reports  <cit> , the protein expressions of nf-κb  and p-p <dig> were magnified in the hc group.
CONCLUSIONS
according to our results, long-term feeding of high concentrate diet causes uterine inflammation in mid-lactating dairy cows.
the inflammatory response in the uterus induced by increased level of circulating lps triggers the expression of pro-inflammatory cytokines in the uterus through the tlr4-nf-κb signalling pathway.
abbreviations
ampantimicrobial peptides
appacute phase proteins
cd14cluster of differentiation antigen14
eecsendometrial epithelial cells
gitgastrointestinal tract
hchigh concentrate
il- <dig> α− <dig> β, − <dig>  interleukin- <dig> α,  <dig> β, −6
iκkiκb kinase
lbplps-binding protein
lclow concentrate
lpslipopolysaccharide
myd88myeloid differentiating factor 88
nf-κbnuclear factor kappa-light-chain-enhancer of activated b cells
pagepolyacrylamide gel electrophoresis
pampspathogen-associated molecular patterns
prrpattern recognition receptors
qrt-pcrquantitative real-time pcr
sarasubacute ruminal acidosis
tlrstoll-like receptors
traf6tnf receptor-associated factor 6

 feeding a high-concentrate diet for a longer period of time can cause subacute ruminal acidosis. it is a digestive disorder with a ph less than lasted for more than 3 h per day. general clinical signs of sara comprise of reduced dry matter intake, decreased ruminal ph and diarrhea. another crucial outcome of sara is the decline of milk quantity and quality, and earlier studies also revealed that sara decreased milk protein yield. the persistent consumption of a hc diet by dairy animals enhances the production of organic acids and significantly results in a decline in ph values of the rumen and intestine. this decrease in ph due to excessive high concentrate diet may affect the alterations of the type of fermentation and the structure of the microbes in the rumen. moreover, the decline in ph also perturbs the balance of microbial population in the rumen causing substantial release of free endotoxin from gram-negative bacteria. due to possible alterations in permeability and disruption of the gastrointestinal barrier, lps can be translocated into the blood stream. there are also other reports revealed that elevated circulating lps cause a systemic inflammatory response. possibly, being an element of an immune response to lps, sara has been reported to increase the concentration of acute phase proteins in the peripheral blood, such as serum amyloid a and haptogloblin. the saa has many functions but mainly alters innate immune responses, particularly in the migration of neutrophils and monocytes, while hp has anti-inflammatory effects; lps-binding protein is activated by microbial infections and facilitates in the neutralization of lps and in the triggering the release of pro-inflammatory cytokines, such as tnf-α, il- and il-1β. the innate immune system is the first line of defence against invading pathogens; it involves various types of transmembrane and secreted molecules, recognized as pattern recognition receptors. toll like receptors, such as tlr that join the lps-lbp complex, are located on the outer surfaces of a variety of cells and contribute to the sensing of microbial pathogens in the intracellular endosomes and lysosomes. tlr recognizes lps with the help of the lbp and cluster of differentiation antigen. lps activates myeloid differentiating factor after joining with tlr on the host cell surface. myd activates tnf receptor-associated factor, which stimulates the iκb kinase complex, and phosphorylated nf-κb is consequently translocated from the cytoplasm to the nucleus. the nf-κb-dependent cascade pathway regulates the production of pro-inflammatory cytokines, including interleukin -1β, tnf-α, il- chemokines and other inflammatory mediators. the principal objectives of this study were to evaluate the inflammatory response in cow uterus and to explore the molecular mechanism triggered by high concentrate-induced sara in mid-lactating dairy cows. methods animals, diets and experimental design twelve healthy multiparous holstein cows, which were purchased from the experimental farm of nanjing agricultural university, with an average body weight of 455 kg were selected for the current study. the experimental cows were randomly divided into two groups, each group containing six cows: the high-concentrate group and the low-concentrate group. the hc group was provided a high-concentrate diet containing 6:: ratio, while lc group was fed low concentrate diet containing 4: for the 18-week experimental period. the details of the nutritional ingredients and their compositions are shown in table  the cows were surgically fixed with a ruminal fistula two weeks prior to the experiment. the cows were kept in individual tie stalls and feed was offered three times per day at and 2000 h. dmi was ~  kg/d/head, and fresh drinking water was available to them during the entire experimental period. the experiment was started after the animals made a full recovery from the ruminal fistula fixation, and no cows showed any clinical signs of infection during the experiment.table ingredient and nutrient composition of diets premixb nutritional compositionc atreatment lc, 40% grain on dm basis; hc, 60% grain on dm basis ne net energy mega joules, cp crude protein, ee ether extract, ndf neutral detergent fiber, nfc non-forage carbohydrates, adf acid detergent fiber bthe premix contained va,,000 iu/kg; vd,  iu/kg; ve,  iu/kg; niacin,  mg/kg; cu,  mg/kg; fe, 525 mg/kg; zn,  mg/kg; mn,  mg/kg; i, 170 mg/kg; co, 50 mg/kg; se, 27 mg/mg cthe calculated nutritional composition values collection of samples and analysis the rumen fluid was collected at one-hour intervals for 12 h daily for continuous days during the 18th week. blood samples were collected 4 h after feeding on the sampling days of the 18th week. 10 ml of rumen fluid was immediately centrifuged at  × g for 45 min, filtered through a disposable -μm filter and stored in pyrogen free glass at −20 °c for lps analysis. the blood samples were collected from the jugular vein into 5-ml vacuum tubes containing sodium heparin as an anticoagulant. the plasma was separated from the blood samples through centrifugation at 3000 × g at 4 °c for 15 min and stored at −20 °c to determine the concentrations of the pro-inflammatory cytokines il-1β, il- and tnfα in the peripheral blood. at the end of the experiment, the cows were slaughtered after overnight fasting. the slaughtering was performed according to the law of jiangsu provincial people’s government, china and the study was approved by the animal ethics committee of nanjing agricultural university, china as described in the end of the manuscript. after slaughtering, the uterine tissues were removed from all the cows aseptically, as close to the cervix as possible for the analysis. all collected tissues were immediately frozen in liquid nitrogen and stored at −70 °c within 30 min of slaughtering. determination of ruminal ph rumen fluids were taken through a rumen fistula at one-hour intervals up to 12 h on days and of the 18th week of the trial. the ph of rumen was measured each hour immediately following the sample collection by ph-meter. determination of lps the concentration of lps in the rumen fluid and plasma of the jugular vein was determined by chromogenic endpoint limulus amebocyte lysate assay kits, ce and ce. briefly, the pre-treated rumen fluid and plasma samples were diluted until their lps concentrations were in the range of to endotoxin units /ml relative to the reference endotoxin and assayed as described by gozho et al.. radioimmunoassay radioimmunoassay was used to calculate the concentrations of il- il-1β, and tnf-α in peripheral blood using radioimmunoassay kits. rna extraction and real time pcr total rna was prepared from 100 mg uterus tissue using trizol via homogenization on ice as described by the manufacturer’s instructions. the quality of mrna was assessed by both agarose gel electrophoresis and spectrometry using the eppendorf bio photometer plus. only samples with a ratio between and were used in subsequent experiments. the first-strand cdna was synthesized using 250 ng/μl of the total rna template using prime script rt master mix perfect real time according to the manufacturer’s instructions. quantitative real-time pcr amplification was performed to evaluate the expression of the selected genes. the primers for genes il-1β, tlr il- il-, and lbp were used according to previously published work, while myd tnf-α, traf and nf-κb were designed using the premier primer software. the primers were assessed for their amplification efficiencies. the reaction conditions included 2 μl of cdna and  μm primers in a total volume of 20 μl of super mix. thermal cycling parameters consisted of initial denaturation at 95 °c for 15 s, followed by 40 cycles of annealing at 95 °c for 5 s and primer extension at 60 °c for 31 s. all the reactions were run in triplicate. each cdna sample was amplified using sybr green in the abi fast real-time pcr system. the data were normalized to the mean of housekeeping gene β-actin to control the unpredictability in expression levels and were analysed using r = 2-ΔΔct method treatment - control), as previously depicted. the primer sequences used in this study are presented in table  table the list of primer sequences used for amplification of qrt-pcr western blotting analysis the protein was extracted from 100 mg uterus tissue with radio immunoprecipitation assay buffer using a dounce homogenizer. after 30 min of incubation on ice, cell extracts were subjected to centrifugation at 4 °c for 15 min to obtain cell protein. the supernatant was assayed for protein concentration using the bicinchoninic acid protein assay kit and was adjusted to 4 μg/μl. equal amounts of proteins were isolated using 10% sds-polyacrylamide gel electrophoresis and shifted to nitrocellulose membrane at 4 °c. then, 10% non-fat dry milk was used to block the nitrocellulose membrane. the membrane was incubated at 4 °c overnight with monoclonal goat antibodies against nf-κb and gapdh,, respectively. after incubation, the membrane was washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies. immunoreactive proteins were detected by chemiluminescence using an ecl reagent and subsequently autoradiographed. the results were quantified using bio-rad gel doc 2000™ systems and analysed with bio-rad tds quantity one software. the relative quantities of proteins were evaluated by a densitometer and expressed as absorbance units. statistical analyses the mixed procedure of sas was used to analyze ph with a repeated measures design performed with sas software. the effects of cows were considered random and the effects of diet and time were considered as fixed factors. the time within treatments and cows were considered as repeated measurements and the residuals for each variable were used to assess the normality. the days were averaged within a cow before the analysis for all the data including the ph, radioimmunoassay, ruminal lps, plasma lps and the gene expression data were assessed by independent t-test and expressed as mean ± sem. differences were concluded to be significant at p < .  rumen ph and lps content of rumen fluid and blood plasma the results of ruminal ph fed a high concentrate diet were published previously by xu et al. and he confirmed the presence of sara by measuring the ruminal ph at different time intervals. in this study, consuming a high-concentrate diet for 18 weeks resulted in a significant decline in ruminal ph than that in the lc group and lasted for more than three hours. the effect of diet × hours on ph was significant; however no effect was observed on hours alone. the lps concentrations in the rumen fluid and the jugular vein plasma were significantly higher in the cows fed a high-concentrate diet compared to cows fed a low-concentrate diet. the concentration of lps in the rumen fluid of the hc group was eu/ml, compared to eu/ml in the lc group; similarly, in the peripheral plasma, the lps level in the hc group was eu/ml, whereas it was eu/ml in lc group, which indicated that the lps level was significantly increased in the hc group relative to the lc group.table lps concentration in rumen and plasma of dairy cows fed low concentrate and high concentrate a hc high concentrate diet, lc low concentrate diet, eu endotoxin unit b sem standard error of the mean between the two groups the lps data were compared using student’s t-test between hc and lc groups p ≤  was considered significant levels of pro-inflammatory cytokines in the peripheral blood the results of the measurement of pro-inflammatory cytokines, together with il-1β, il- and tnf-α are shown in fig.  the concentration of il-1β was significantly higher in the hc group compared to the lc group, the concentration of il- was significantly increased in the hc group versus lc group and the concentration of tnf-α was elevated in the hc group relative to the lc group. generally, the concentration of pro-inflammatory cytokines in the peripheral blood was significantly higher in the hc group compared to the lc group.fig. a-c the concentration levels of tnf-α, il-1β, and il- in the peripheral blood. the data were expressed as the mean ± sem.; asterisks indicate the differences between the low-concentrate group and high-concentrate group. expressions of genes involved in the inflammation of the uterus sara significantly increased the expression level of several genes involved in the inflammation of the uterus as shown in fig.  the gene expression of tlr and myd in the hc group was significantly up-regulated compared to the lc group. similarly, traf- and nf-κb were highly expressed in the hc group relative to the lc group. the gene expression of tnf-α and il- was significantly increased. the gene expression of il-1β, il- and lbp was also significantly higher in the hc group compared with the lc group.fig. the uterine gene expression profile analysed by qrt-pcr. the genes involved in transcriptional regulation were measured in the uterine tissues. the error bars indicate the standard error of the mean and ** indicates significance at p <  expression of nf-κb protein in the cow uterus of the hc group and lc group western blot analysis demonstrated that high concentrate diet-induced inflammation through the increaseduterine concentrations of the nf-κb proteins in the hc group compared with the lc group. importantly, the amount of phosphorylated nf-κb was higher in the uterus of the hc group than in the lc group. therefore, it was confirmed that nf-κb was activated in hc group in the tlr signalling pathway.fig. a the western blotting assay of nf-κb and phosphorylated-p protein in the uterus. the nf-κb contents in uterus were evaluated through western blot of the low-concentrate group and high-concentrate group. b the quantities of proteins are measured as arbitrary units relative to gapdh; fold alterations in nf-κb and phosphorylated nf-κb. the data were expressed as the mean ± sem., asterisks indicate the differences between the high-concentrate group compared to the low-concentrate group discussion the present study assessed the uterine inflammation at the molecular level in dairy cows. the inflammatory response in the uterus due to circulating lps derived from rumen triggered the expression of pro-inflammatory cytokines through tlr signaling. to evaluate the inflammatory response in cow uterus and to explore the molecular mechanism triggered by high concentrate diet, sara was confirmed by measuring the ph of the rumen. xu et al. also confirmed the presence of sara by measuring the ruminal ph at different time intervals. in our study, the low rumen ph lasted for 223 min and high concentrations of lps were observed in the rumen fluid and peripheral blood. the recent reports also indicated the successful induction of sara, if theduration of low rumen ph was more than 180 min/day. however, there is little literature revealing that sara not only depends on ph, but also on the alteration in the microbial population that develops secondarily to the feed type. as a matter of fact, low ruminal ph values led to the lysis of gram-negative bacteria which released lps into the blood circulation. consistent with previous studies in dairy cows and feedlot, our results presented higher concentrations of lps in the peripheral plasma and ruminal fluid of cows fed a high concentrate die trelative to the peripheral plasma of the lc group. moreover, we also found that high concentrate diet feeding increased the plasma concentrations of pro-inflammatory cytokines. similar results have been reported by other researchers. the increment in pro-inflammatory cytokines concentration in the plasma may provide confirmation for lps translocation into the blood circulation and eliciting of inflammatory responses. this implies that inflammation in the hc group was due to the high level of lps in the bloodstream, which in turn triggers the pro-inflammatory gene to cope with stress conditions. the rt-qpcr-based findings revealed the presence of inflammation-associated markers in the uterus of sara-induced cows and confirmed that the dairy cows suffered from uterine inflammation at the molecular level. according to our results, the increased expression of tlr indicated that tlr recognized the lps and triggered the inflammatory pathway. identification of inflammatory-stimuli by the innate immune system is organized by ppr that identify external stimuli for example pathogen associated molecular patterns. furthermore, our research demonstrated the increased expression of lbp, myd and traf in the uterus; which provides more clues that the tlr signaling pathway was stimulated by lps. previous studies demonstrated that as a main transcription factor, nf-κb may bind to the promoter regions of the many inflammatory genes to regulate their transcription activities. similarly, in the current study, nf-κb expression was increased in the hc group, which also confirmed the activation of tlr signalling pathway. our results of increased expression of pro-inflammatory cytokines and chemokine in the hc group due to sara-induced lps were consistent with the previous studies. the western blot results further confirmed that the expression levels of nf-κb and phosphorylated p proteins were distinctly increased in the hc group compared to the lc group. taken together, the results suggested that lps triggered the activation of tlr signalling, which resulted in an inflammatory response in the uterus of cows suffering from sara. conclusively, lps activates tlr4-nf-κb signalling. together with the existing study and the previous reports, the protein expressions of nf-κb and p-p were magnified in the hc group.  according to our results, long-term feeding of high concentrate diet causes uterine inflammation in mid-lactating dairy cows. the inflammatory response in the uterus induced by increased level of circulating lps triggers the expression of pro-inflammatory cytokines in the uterus through the tlr4-nf-κb signalling pathway. abbreviations ampantimicrobial peptides appacute phase proteins cd14cluster of differentiation antigen14 eecsendometrial epithelial cells gitgastrointestinal tract hchigh concentrate il- α− β, − interleukin- α, β, −6 iκkiκb kinase lbplps-binding protein lclow concentrate lpslipopolysaccharide myd88myeloid differentiating factor 88 nf-κbnuclear factor kappa-light-chain-enhancer of activated b cells pagepolyacrylamide gel electrophoresis pampspathogen-associated molecular patterns prrpattern recognition receptors qrt-pcrquantitative real-time pcr sarasubacute ruminal acidosis tlrstoll-like receptors traf6tnf receptor-associated factor 6 
the lps concentrations in the rumen fluid and the peripheral plasma were found significantly increased in the hc group compared to the lc group. therefore, the results indicated that lps derived from the rumen triggered the genes associated with inflammation in the uterus of mid-lactating dairy cows fed a high-concentrate diet, causing endometritis. the aims of the current study were to evaluate the inflammatory response in cow uterus and to explore the molecular mechanism triggered by high concentrate-induced subacute ruminal acidosis in mid-lactating dairy cows. the hc group showed lower ruminal ph and higher lipopolysaccharide concentrations in both the rumen and peripheral plasma compared to the lc group. the concentrations of il-1β, tnf-α and il- were significantly higher in the hc group compared to the lc group. similarly, the inflammatory genes tlr lbp, myd traf- nf-κb, il- il- tnf-α and il-1β were significantly upregulated in the uterus of the hc versus the lc group. the occurrence of uterine inflammation was further validated by the increased protein expression of nf-κb-p and its active phosphorylated variant in the uterus of sara cows. the uterus of sara cows revealed elevated mrna concentrations of nuclear transcription factors and pro-inflammatory cytokines, which confirmed the presence of inflammation.
 the aims of the current study were to evaluate the inflammatory response in cow uterus and to explore the molecular mechanism triggered by high concentrate-induced subacute ruminal acidosis in mid-lactating dairy cows. twelve mid-lactating holstein cows with an average weight of 455 kg were allocated into two groups subjected to two diets for 18-weeks either a low-concentrate group containing 4: and a high-concentrate group containing 6:: ratio based on dry matter.  the hc group showed lower ruminal ph and higher lipopolysaccharide concentrations in both the rumen and peripheral plasma compared to the lc group. the lps concentrations in the rumen fluid and the peripheral plasma were found significantly increased in the hc group compared to the lc group. the concentrations of il-1β, tnf-α and il- were significantly higher in the hc group compared to the lc group. the uterus of sara cows revealed elevated mrna concentrations of nuclear transcription factors and pro-inflammatory cytokines, which confirmed the presence of inflammation. the occurrence of uterine inflammation was further validated by the increased protein expression of nf-κb-p and its active phosphorylated variant in the uterus of sara cows. similarly, the inflammatory genes tlr lbp, myd traf- nf-κb, il- il- tnf-α and il-1β were significantly upregulated in the uterus of the hc versus the lc group.  therefore, the results indicated that lps derived from the rumen triggered the genes associated with inflammation in the uterus of mid-lactating dairy cows fed a high-concentrate diet, causing endometritis. keywords lipopolysaccharidesub-acute ruminal acidosiscowlbpimmune gene expressionthe national natural science foundation of china31172371shen xiangzhen the priority academic programme development of jiangsu higher education institutionspapdshen xiangzhen issue-copyright-statement© the author 2016 