microarray data from this study are available at [ provide url below ] : http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse75240 . lactating holstein - friesian cows ( n  =  10 ) were supplemented with a daily oral dose ( 50ml ) of the commercial probiotics fasttrak microbial pack ( conklin company , kansas city , mo , usa ) for 60  days . peripheral blood samples were collected aseptically from probiotics - treated animals into vaccutainer tubes containing anticoagulant acd . the in vitro effect of lipopolysaccharide ( lps ) , endotoxin treatment was evaluated using blood samples collected from probiotics - treated animals at ( day 60 ) . whole blood ( 5  ml ) samples with 1    10  viable  cells / ml were pretreated with 100  ng / ml of lipopolysaccharide   ( lps , sigma - a ) and untreated sample served as control for 30  min using incubation conditions previously described by . after treatment , samples were centrifuged at 10,000  rpm for 15  min and blood cell pellets were used for rna isolation ( fig . 1 ) . total rna from blood cell pellet was isolated using the zr whole blood rna miniprep kit ( zymo research , irvine , ca ) following the manufacturer 's recommendations . the rna procedure included differential lysis of red and white blood cells , and an on - column dnase digestion . total rna was quantified using a nanodrop-1000 spectrophotometer ( thermo fisher scientific , waltham , ma ) , and rna integrity number ( rin ) and quality was determined with the agilent 2100 bioanalyzer ( agilent technologies , santa clara , ca ) . samples with high rna integrity ( rin  >  7 ) were stored at -80  c until used for microarray analysis . a pooled rna sample was generated by taking an equal concentration of rna from experimental animals in each group ( lps vs control ) . total rna ( 500  ng , rin  >  7 ) was used to prepare cyanine-3 ( cy3 ) labeled crna using the one - color low rna input linear amplification plus kit ( agilent technologies , santa clara , ca , usa ) according to the manufacturer 's instructions . sample purification was done with the rnaeasy column purification ( qiagen , valencia , ca , usa ) . dye incorporation and crna yield ( table 1 ) were checked with the nanodrop nd-1000 spectrophotometer ( thermo - scientific , waltham , ma ) . hybridization was done on the agilent bovine v2 4x44k gene expression microarray slide ( agilent technologies , santa clara ca , usa ) for 17  h. slides were scanned immediately after washing on the agilent dna microarray scanner ( g2505b ) using one color scan default settings for 4x44k array slides . signal intensity data were extracted with the agilent feature extraction software 10.10.1.1 ( agilent ) using default parameters ( ge1-v5_95_feb07 ) according to the manufacturer 's protocol . signal intensity data were extracted with the agilent feature extraction software ( v 9.5.1 ) . data normalization and statistical analysis were performed using genespring software 13.0 ( agilent technologies , santa clara ca , usa ) . boxwhisker , matrix , and scatter plots displaying the log ratio distribution of microarray data after normalization is shown in fig . the raw data are available from the ncbi geo repository , under the accession number gse75240 . in conclusion probiotics supplementation had an effect on the response to lps exposure in bovine whole blood . treatment with lps resulted in 13,658 differentially expressed genes ( fold change cutoff   2 , p  <  0.05 ) , 3816 upregulated genes and 9842 downregulated genes . exposure to lps had specific effects on toll - like receptors genes expression including tlr4 , tlr2 and tlr7 . lactating holstein - friesian cows ( n  =  10 ) were supplemented with a daily oral dose ( 50ml ) of the commercial probiotics fasttrak microbial pack ( conklin company , kansas city , mo , usa ) for 60  days . peripheral blood samples were collected aseptically from probiotics - treated animals into vaccutainer tubes containing anticoagulant acd . the in vitro effect of lipopolysaccharide ( lps ) , endotoxin treatment was evaluated using blood samples collected from probiotics - treated animals at ( day 60 ) . whole blood ( 5  ml ) samples with 1    10  viable  cells / ml were pretreated with 100  ng / ml of lipopolysaccharide   ( lps , sigma - a ) and untreated sample served as control for 30  min using incubation conditions previously described by . after treatment , samples were centrifuged at 10,000  rpm for 15  min and blood cell pellets were used for rna isolation ( fig . total rna from blood cell pellet was isolated using the zr whole blood rna miniprep kit ( zymo research , irvine , ca ) following the manufacturer 's recommendations . the rna procedure included differential lysis of red and white blood cells , and an on - column dnase digestion . total rna was quantified using a nanodrop-1000 spectrophotometer ( thermo fisher scientific , waltham , ma ) , and rna integrity number ( rin ) and quality was determined with the agilent 2100 bioanalyzer ( agilent technologies , santa clara , ca ) . samples with high rna integrity ( rin  >  7 ) were stored at -80  c until used for microarray analysis . a pooled rna sample was generated by taking an equal concentration of rna from experimental animals in each group ( lps vs control ) . total rna ( 500  ng , rin  >  7 ) was used to prepare cyanine-3 ( cy3 ) labeled crna using the one - color low rna input linear amplification plus kit ( agilent technologies , santa clara , ca , usa ) according to the manufacturer 's instructions . sample purification was done with the rnaeasy column purification ( qiagen , valencia , ca , usa ) . dye incorporation and crna yield ( table 1 ) were checked with the nanodrop nd-1000 spectrophotometer ( thermo - scientific , waltham , ma ) . hybridization was done on the agilent bovine v2 4x44k gene expression microarray slide ( agilent technologies , santa clara ca , usa ) for 17  h. slides were scanned immediately after washing on the agilent dna microarray scanner ( g2505b ) using one color scan default settings for 4x44k array slides . signal intensity data were extracted with the agilent feature extraction software 10.10.1.1 ( agilent ) using default parameters ( ge1-v5_95_feb07 ) according to the manufacturer 's protocol . signal intensity data were extracted with the agilent feature extraction software ( v 9.5.1 ) . data normalization and statistical analysis were performed using genespring software 13.0 ( agilent technologies , santa clara ca , usa ) . boxwhisker , matrix , and scatter plots displaying the log ratio distribution of microarray data after normalization is shown in fig . the raw data are available from the ncbi geo repository , under the accession number gse75240 . in conclusion probiotics supplementation had an effect on the response to lps exposure in bovine whole blood . treatment with lps resulted in 13,658 differentially expressed genes ( fold change cutoff   2 , p  <  0.05 ) , 3816 upregulated genes and 9842 downregulated genes . exposure to lps had specific effects on toll - like receptors genes expression including tlr4 , tlr2 and tlr7 .
probiotic supplements are beneficial for animal health and rumen function ; and lipopolysaccharides ( lps ) from gram negative bacteria have been associated with inflammatory diseases . in this study the transcriptional profile in whole blood collected from probiotics - treated cows was investigated in response to stimulation with lipopolysaccharides ( lps ) in vitro . 
 microarray experiment was performed between lps - treated and control samples using the agilent one - color bovine v2 bovine ( v2 ) 4x44k array slides . 
 global gene expression analysis identified 13,658 differentially expressed genes ( fold change cutoff    2 , p  <  0.05 ) , 3816 upregulated genes and 9842 downregulated genes in blood in response to lps . 
 treatment with lps resulted in increased expression of tlr4 ( fold change ( fc )  =  3.16 ) and transcription factor nfkb ( fc  =  5.4 ) and decreased the expression of genes including tlr1 ( fc  =    2.54 ) , tlr3 ( fc  =    2.43 ) , tlr10 ( fc  =    3.88 ) , nod2 ( fc  =    2.4 ) , nod1 ( fc  =    2.45 ) and pro - inflammatory cytokine il1b (   3.27 ) . 
 the regulation of the genes involved in inflammation signaling pathway suggests that probiotics may stimulate the innate immune response of animal against parasitic and bacterial infections . we have provided a detailed description of the experimental design , microarray experiment and normalization and analysis of data which have been deposited into ncbi gene expression omnibus ( geo ) : gse75240 .