animals and treatment : all procedures in this experiment were conducted following protocols approved by the iwate university laboratory animal 
 care and use committee . four primiparous non - lactating , rumen - cannulated healthy holstein cattle , weighing 645  62 ( mean  sd ) kg , were housed in a stanchion 
 barn at the cattle research center of iwate university . during the experimental period , the cattle were fed a sara - inducing diet 2 weeks before and 1 week after 
 first administration of bp .
the sara - inducing diet was composed of a mix of orchard grass and timothy hay with an equivalent amount of flaked barley and corn . 

the ratio of roughage - to - concentrate was 3:7 , and the percentages of total digestible nutrients , crude 
 protein , neutral detergent fiber , non - fiber carbohydrates and starch in the dry matter were adjusted to 75.1 , 12.2 , 37.7 , 42.4 and 37.0% , respectively .
cattle 
 were fed daily at 9:00 and 17:00 and allowed free access to fresh water .
cattle were assigned randomly to a 4  4 latin square experimental design containing 
 three treatments and a control .
, tokyo , japan ) included l. plantarum strain 220 ( 9  10 
 colony - forming units ( cfu)/g ) , e. faecium strain 26 ( 9  10 cfu / g ) and c. butyricum strain miyari ( 9  
 10 cfu / g ) was administered as a daily single dose of 20 , 50 or 100 g per head for 7 consecutive days .
cows fed the sara - inducing diet without bp 
 served as a control . based on the effects of the treatment , the experimental design consisted of 2 weeks of an adaptation period to the respective bp treatment 
 during which cattle were fed only hay .
the bp was stored at 4c , and each dose was mixed with their diet during the morning feeding .
ruminal ph measurements : ruminal ph was measured with a radio transmission ph measurement system ( ycow - s ; dkk - toa yamagata , yamagata , japan ) . 

the ph sensor was calibrated with ph 4 and 7 buffer solutions at the start of each experiment and was placed in the ventral sac of the rumen via the rumen 
 cannula , as the ventral sac of the rumen has more stable ph values than the other ruminal site .
ruminal fluid sampling and vfa , lactic acid and nh3-n assays : ruminal fluids were collected from the same location as the ph sensor 
 through the rumen cannula at 8:00 , 11:00 , 14:00 , 17:00 and 20:00 on 7 days before ( day 7 ) and 7 days after ( day 7 ) the first bp administration ( day 0 ) . these 
 sampling times ( 8:0020:00 )
samples for the vfa , lactic acid and 
 nh3-n measurements were filtered immediately through two layers of cheesecloth .
for the vfa analysis , 10 ml of ruminal fluid was 
 added to 2 ml of 25% metaphosphoric acid in 3 n sulfuric acid .
total vfas and three individual vfas ( acetic acid , propionic acid and butyric 
 acid ) were separated and quantified with gas chromatography ( model 135 , hitachi , tokyo , japan ) using a packed glass column ( 3% thermon-3000 ) on a shimalite tpa 
 6080 support ( shinwa chemical industries ltd . ,
the ruminal fluid was centrifuged immediately at 2,000  
 g for 15 min , and concentrations in the supernatant were determined using a commercially available kit ( f - kit ; d - lactate / l - lactate , j. k. international 
 co. , tokyo , japan ) . to measure nh3-n concentrations , ruminal
fluid was analyzed using the steam distillation method with an nh3-n analyzer 
 ( kjeltec auto sampler system 1035 analyzer , tecator , sweden ) .
blood sampling and plasma metabolite profiles : blood samples were collected from the jugular vein into 10-ml evacuated 
 serum - separating tubes and tubes containing sodium fluoride ( bd vacutainer , franklin lakes , nj , u.s.a . ) .
blood samples were collected at the same time as the 
 ruminal fluid samples ( days 7 and 7 ) , centrifuged ( 1,500  g , 15 min , 4c ) immediately to separate the serum and plasma , and preserved at 80c 
 until the analysis . glucose ( glu ) , non - esterified fatty acids ( nefa ) and -hydroxybutyrate acid ( bhba ) in plasma , as well as total cholesterol ( t - chol ) , 
 triglycerides ( tg ) and blood urea nitrogen ( bun ) in sera were analyzed using an automated biochemistry analyzer ( accute , toshiba ltd . , tokyo , japan ) . statistical analysis : quantitative data are expressed as means  standard errors ( ses ) .
the main effects included the sara challenge and bp 
 treatment , day of the experiment , and hours after administration and feeding .
diurnal measurements of the ruminal ph were analyzed as the 24-hr mean ph from day 
 7 to day 7 .
the ph data collected in 10-min intervals were summarized as a 1-hr mean from 9:00 to 8:00 of the following day to assess circadian changes .
the 
 minimum and maximum ph in one day was determined for days 7 , 0 and 7 .
5.01 ( la jolla , ca , u.s.a . ) was used for the statistical calculations , 
 and two - way repeated - measures analysis of variance ( anova ) followed by the bonferroni post hoc test was used to evaluate the differences between the treatment and 
 control groups .
ruminal ph : sara was successfully induced in cattle fed the sara - inducing diet . according to representative ph data on 3 days after beginning 
 the diet ,
the 1-hr mean ruminal ph decreased rapidly and slowly after the morning and evening feedings , which was indicative of sara ( fig .
1.circadian changes in the 1-hr mean ruminal ph at control day ( n=4 ;  ) and 3 days after beginning the sara - inducing diet ( n=4 ;  ) .
the 1-hr mean ph in cattle fed sara - inducing diet decreased after the morning and evening feedings , which was indicative of 
 successfully induced sara .
data are means  se . * significant difference compared with a control on the same day ( p<0.05 ) . 

considerable disparities in the ruminal ph among the treatment and control groups were observed and continued throughout the treatment period ( fig .
2.changes in the 24-hr mean ruminal ph in cattle administered 20 g ( n=4 ;  ) , 50 g ( n=4 ;  ) or 100 g ( n=4 ;  ) of a bacterial probiotic for 7 consecutive days . 

data are means  se . * significant difference compared with a control on 
 the same day ( p<0.05 ) .
the 24-hr mean ruminal ph in the control was generally lower , whereas that in the bp groups receiving 20 or 50 g was significantly higher from days 2 to 
 7 compared with the control .
the 24-hr mean ph in the 100 g group was also significantly higher from days 2 to 4 compared with the control ; however , it decreased 
 on days 5 to 7 . among the treatment groups , the 20 g bp group maintained a constant ph ( 6.46.5 ) from days 3 to 7 .
circadian changes in the 1-hr mean ruminal ph 
 were almost identical among the treatment groups on days 0 and 7 .
however , the 1-hr mean phs in the treatment groups were slightly higher than those in the 
 control group on day 7 ( fig . 3fig .
3.circadian changes in the 1-hr mean ruminal ph on days 0 and 7 in cattle administered 20 g ( n=4 ;  ) , 50 g ( n=4 ;  ) or 100 g ( n=4 ;  ) of a bacterial 
 probiotic for 7 consecutive days .
additionally , the mean minimum ph in the control on day 7 was < 5.0 , which was significantly lower than in the treatment groups ( fig .
4.box plots of the maximum and minimum ruminal ph on days 7 , 0 and 7 in the treatment groups administered 20 g ( n=4 ; light gray boxes ) , 50 g ( n=4 ; gray 
 boxes ) and 100 g ( n=4 ; dark boxes ) of a bacterial probiotic for 7 consecutive days .
cattle not administered the probiotic served as a control ( n=4 ; white 
 boxes ) . the median and quartiles
the upper and lower bars represent the maximum and minimum values , respectively . * 
 significant difference compared with a control on the same day ( p<0.05 ) .   significant difference compared with 
 the same group on day 7 ( p<0.05 ) . ) .
the mean maximum ph approached > 6.8 in the treatment groups , which did not differ from that in the control .
circadian changes in the 1-hr mean ruminal ph at control day ( n=4 ;  ) and 3 days after beginning the sara - inducing diet ( n=4 ;  ) .
the 1-hr mean ph in cattle fed sara - inducing diet decreased after the morning and evening feedings , which was indicative of 
 successfully induced sara .
data are means  se . * significant difference compared with a control on the same day ( p<0.05 ) . 

changes in the 24-hr mean ruminal ph in cattle administered 20 g ( n=4 ;  ) , 50 g ( n=4 ;  ) or 100 g ( n=4 ;  ) of a bacterial probiotic for 7 consecutive days . 

* significant difference compared with a control on 
 the same day ( p<0.05 ) .
circadian changes in the 1-hr mean ruminal ph on days 0 and 7 in cattle administered 20 g ( n=4 ;  ) , 50 g ( n=4 ;  ) or 100 g ( n=4 ;  ) of a bacterial 
 probiotic for 7 consecutive days .
box plots of the maximum and minimum ruminal ph on days 7 , 0 and 7 in the treatment groups administered 20 g ( n=4 ; light gray boxes ) , 50 g ( n=4 ; gray 
 boxes ) and 100 g ( n=4 ; dark boxes ) of a bacterial probiotic for 7 consecutive days .
cattle not administered the probiotic served as a control ( n=4 ; white 
 boxes ) . the median and quartiles
the upper and lower bars represent the maximum and minimum values , respectively . * 
 significant difference compared with a control on the same day ( p<0.05 ) .   significant difference compared with 
 the same group on day 7 ( p<0.05 ) .
ruminal vfa , lactic acid and nh3-n concentrations : total vfa increased from 8:00 to 20:00 in the treatment and control groups on days 
 0 and 7 ( fig . 5fig .
5.circadian changes in the ruminal vfa , a : p ratio , lactic acid and nh3-n concentrations on days 0 and 7 in cattle administered 20 g ( n=4 ;  ) , 50 g 
 ( n=4 ;  ) or 100 g ( n=4 ;  ) of a bacterial probiotic for 7 consecutive days .
* significant difference compared with a control on the same day ( p<0.05 ) . )
. however , no difference was observed in the total or individual vfa concentrations between the treatment and control groups . the acetic acid - to - propionic 
 acid ratio ( a : p ) was almost identical among the treatment groups .
lactic acid concentrations remained stable in the treatment groups , and the concentrations at 
 20:00 on day 7 were significantly lower than that in the control . no difference was observed in nh3-n concentrations among the treatment groups , 
 although the nh3-n concentrations in the 50 g and 100 g groups at 8:00 on day 7 were significantly higher than that in the control .
circadian changes in the ruminal vfa , a : p ratio , lactic acid and nh3-n concentrations on days 0 and 7 in cattle administered 20 g ( n=4 ;  ) , 50 g 
 ( n=4 ;  ) or 100 g ( n=4 ;  ) of a bacterial probiotic for 7 consecutive days .
data are 
 means  se . * significant difference compared with a control on the same day ( p<0.05 ) .
blood metabolite profiles : glu concentrations decreased from 8:00 to 20:00 , but were almost the same among the treatment and control groups 
 ( fig .
6.circadian changes in the blood glu , nefa , bhba and bun concentrations on days 0 and 7 in cattle administered 20 g ( n=4 ;  ) , 50 g ( n=4 ;  ) or 100 g ( n=4 ;  ) 
 of a bacterial probiotic for 7 consecutive days .
data are means  se . * 
 significant difference compared with a control on the same day ( p<0.05 ) . ) .
nefa levels were affected by feeding time and were significantly lower at 20:00 compared with at 8:00 in the treatment groups on days 0 and 7 . 

furthermore , nefa level in the 50 g group at 20:00 on day 7 was significantly lower than that in the control .
in contrast , bhba and bun concentrations were higher 
 and lower , respectively , in all groups from 8:00 to 20:00 .
bhba concentrations in the 20 g and 50 g at 17:00 and 20:00 , and bun concentrations in the treatment 
 groups at 8:00 were significantly higher than that in the controls , respectively .
circadian changes in the blood glu , nefa , bhba and bun concentrations on days 0 and 7 in cattle administered 20 g ( n=4 ;  ) , 50 g ( n=4 ;  ) or 100 g ( n=4 ;  ) 
 of a bacterial probiotic for 7 consecutive days .
data are means  se . * 
 significant difference compared with a control on the same day ( p<0.05 ) .
sara occurs when the ruminal ph decreases due to a combination of overproduction of vfa and lactic acid and a decrease in vfa absorption in the rumen [ 1 , 22 ] .
sara is diagnosed when the ph in the ruminal fluid is < 5.6 for at least 3 hr 
 per day [ 5 , 17 , 21 ] . in this study ,
the 
 ruminal ph measured continuously indicated that sara was successfully induced during the experimental period , especially during the first week of the experiment , 
 by feeding a sara - inducing diet .
studies have shown that the main ruminal bacteria affecting the ruminal fermentative capacity are classified as lactic acid - producers and consumers [ 4 , 15 ] , and both of these bacterial groups have been used as bps .
( 2002 )   reported that a bp containing both lactate - consuming 
 ( propionibacterium ) and lactate - producing ( enterococcus ) bacteria activated ruminal fermentation and reduced the risk of 
 acidosis in cattle . in another study , a bp consisting of l. plantarum and e. faecium induced changes in the ruminal ph of cows 
 fed a high - grain diet .
however , the mechanism of the effects of bp on ruminal fermentation is unclear , and the effects 
 of bp on sara in cattle are unknown . in this study , the 24-hr mean ruminal ph was higher in the bp - treated groups compared with in the control group during sara 
 challenge .
this was indicative of increased ruminal fermentation in the cows , as reported previously [ 12 , 27 , 28 ] .
( 2012 )   examined the effects of administering bp in sara - challenged cows and found no effects on ruminal ph when bp was administered as a single strain ; 
 however , ruminal ph increased compared over that in the control group when using a combination of e. faecium and yeast .
we used the same bp in a 
 previous study on weaned calves fed a high - concentrate diet , and the mean ruminal ph was significantly higher in the treatment group compared with in the control 
 group . in this study , different doses of a multi - strain bp were administered to sara - challenged cows , and the 24-hr 
 mean ruminal ph was notably higher on days 3 to 7 , whereas the minimum ph increased on day 7 in the treatment groups .
bp appears to increase the ability of ruminal bacteria to metabolize lactic acid and regulate ruminal ph [ 20 , 21 ] .
it has been hypothesized that the functionality and efficacy of bps can be determined based on their effects on the 
 predominant rumen microbiota [ 7 , 12 ] . in this study , cattle receiving lower bp doses 
 ( 20 g per head ) had a constant mean ph of 6.46.6 .
in contrast , cattle receiving higher bp doses ( 100 g per head ) had higher mean ph on days 2 and 3 , which 
 decreased on days 5 to 7 .
previous reports have indicated that changes in rumen bacterial composition and diversity , increased activities of lactate - consuming 
 bacteria and greater lactate absorption affect ruminal ph [ 13 , 15 ] .
the higher bp 
 dose might increase lab numbers in the rumen , which could be related to increased ruminal fermentation capacity , higher carbohydrate fermentation and increased 
 ruminal ph .
therefore , ruminal lab might have been overly increased due to the higher bp dose , causing the ruminal 
 fermentation and ph to decrease on day 5 .
these results are consistent with previous reports that lower bp doses improve gastrointestinal tract microbiota in 
 calves .
furthermore , weaned calves administered lower bp doses showed a higher mean ruminal ph compared with controls 
 . based on our results , administration of an extremely high dose ( 100 g per head ) of lab could cause decreased 
 ruminal fermentation and ruminal ph .
decreases in ruminal ph are related to vfa production and lactic acid accumulation after feeding a high - grain diet [ 1 , 
 18 ] . in this study , although ruminal vfa concentrations were not affected by bp treatment , they were affected by feeding 
 time .
this is in agreement with previous reports in which ruminal vfas were not affected by probiotics including lab [ 6 , 
 20 ] .
administration of lab probiotics is thought to help rumen microbiota adapt to the presence of lactic acid   and prevent lactate accumulation in the rumen .
( 1992 )   reported that lactate - consuming bacteria increased only when lactic acid accumulated and ruminal ph decreased . 

based on these results , lab probiotics might affect ruminal ph by increasing the activity of lactate - consuming bacteria . 
 in our previous study , significantly lower lactic acid concentrations were observed in the ruminal fluid of weaned calves receiving bp compared with the control 
 . in this study ,
lactic acid concentrations were stable in the treatment groups ; however , lactic acid was higher and 
 correlated with a lower mean ruminal ph in the control group . gradually increasing ruminal
ph might be due to time after feeding and the absorption capacity of 
 the rumen .
bps may prevent a decline in ruminal ph by increasing the lactic acid consumption by some microbes [ 3 , 6 ] .
a combination of certain probiotic bacteria that synthesize lactic acid may sustain 
 a tonic level of lactic acid in the rumen , stimulating rumen microbial communities that consume lactic acid and reducing acidity , causing the ruminal ph to remain 
 constant and stable .
conversely , the nh3-n concentrations in the ruminal fluid of the bp groups remained stable in this study , although significant difference was observed 
 at 8:00 on day 7 between the bp - treated and control groups .
bp has no effect on the nh3-n concentrations in the rumen [ 7 , 12 ] .
decreases in nh3-n levels after morning feeding in this study were in agreement with the 
 previous study , which found that nh3-n concentrations decreased with decreasing ruminal ph [ 7 , 13 ] .
it has been reported that nefa levels are 
 lower in steers with sara compared with in the control ; therefore , the decrease in nefa levels indicates a more 
 efficient use of dietary energy and greater dry matter intake in the bp - treated groups . in conclusion ,
repeated administration of a bp comprised of l. plantarum , e. faecium and c. butyricum improved 
 the 24-hr mean ruminal ph in cattle with experimentally induced sara at doses of 20 or 50 g per head .
diurnal patterns of the 1-hr mean ruminal ph were identical 
 among the treatment and control groups .
ruminal vfa was not affected by bp treatment ; however , lactic acid was lower in the treatment groups than in the control 
 group . based on these results
, bp might affect ruminal ph by increasing lactate consumption and decreasing lactic acid concentrations , resulting in a consistently 
 higher ruminal ph in sara cattle .
these results suggest that repeated administration of a multi - strain bp might reduce the risk of sara in cattle and that 
 consecutive treatment with 20 or 50 g of a bp containing lab during high - concentrate feeding might reduce the incidence of sara in dairy cattle .
 effects of a bacterial probiotic ( bp ) on ruminal fermentation and plasma metabolites were evaluated in four holstein cattle ( body weight , 645  62 kg ; mean  
 sd ) with induced subacute ruminal acidosis ( sara ) . 
 sara was induced by feeding a sara - inducing diet , and thereafter , 20 , 50 or 100 g per head of a commercial bp 
 was administered for 7 consecutive days during the morning feeding . 
 cattle without bp served as the control . 
 the 24-hr mean ruminal ph in the control was lower , 
 whereas those in the bp groups administered 20 or 50 g were significantly higher compared to the control from days 2 to 7 . 
 circadian patterns of the 1-hr mean 
 ruminal ph were identical ( 6.46.8 ) among all cattle receiving bp . although the mean minimum ph in the control on day 7 and day 0 was < 5.8 , the ph in the 
 treatment groups on day 7 was > 5.8 and significantly higher than that of the control group ( > 5.2 ) . 
 ruminal volatile fatty acid ( vfa ) concentrations were 
 not affected by bp treatment ; however , the bp groups had lower lactic acid levels compared with the control group at 20:00 on day 7 . 
 additionally , 
 non - esterified fatty acid levels decreased from 8:00 to 20:00 in all bp groups on day 7 . 
 these results suggest that administration of 20 to 50 g of a 
 multi - strain bp for 7 days might improve the low ph and high lactic acid level of the ruminal fluid in sara cattle . 