animals and diet : all animal procedures conformed to the guidelines of the 
 local ethics committee ( animal experimentation department of the local inc . administrative 
 agency research institute of environment , agriculture and fisheries , osaka prefecture ) . four 
 holstein cows of mean (  standard deviation ) weight 748 kg (  31 kg ) and mean 332 days (  
 60 ) in milk were assigned to the test in a crossover trial . each trial lasted for two days , and the trial interval was set for 
 seven days . animals were fed hay and concentrate separately in four installments at 06:30 , 
 08:30 , 10:30 and 14:30 daily ( table 1table 1.dietary ingredients and chemical composition% dry matteringredientstimothy hay35.0oats hay15.0alfalfa hay10.0beet pulp10.0commercial concentrate30.0chemical compositioncrude protein12.8estimated tdn68.6a ) commercial concentrate containing the following ingredients : heated corn grain , 
 wheat flour , toasted soybean flour , defatted soymeal , rapeseed oil cake , sesame - seed 
 oil cake , corn gluten , wheat bran , sugar beet molasses , calcium carbonate , dehydrated 
 alfalfa meal , calcium phosphate , sodium chloride and supplement ( vitamin a , vitamin 
 d3 , vitamin e , ferric sulfate , copper sulfate , zinc sulfate , manganese sulfate , cobalt 
 sulfate , magnesium carbonate , calcium iodate , magnesium oxide and methionine 
 compound ) . a ) commercial concentrate containing the following ingredients : heated corn grain , 
 wheat flour , toasted soybean flour , defatted soymeal , rapeseed oil cake , sesame - seed 
 oil cake , corn gluten , wheat bran , sugar beet molasses , calcium carbonate , dehydrated 
 alfalfa meal , calcium phosphate , sodium chloride and supplement ( vitamin a , vitamin 
 d3 , vitamin e , ferric sulfate , copper sulfate , zinc sulfate , manganese sulfate , cobalt 
 sulfate , magnesium carbonate , calcium iodate , magnesium oxide and methionine 
 compound ) . capsule preparation : capsules containing thiamine hydrochloride were 
 manufactured by morishita jintan co. , ltd . ( osaka , japan ) with a diameter of 6 mm , specific 
 gravity of 1.3 , approximately 160 mg per capsule itself and a seamless three - layer structure 
 according to the following procedure [ 1 , 2 , 9 ] ( fig . 1.schematic of ( a ) the three - layered capsule and ( b ) method of manufacture . ( a ) core : a 
 mixture of hydrogenated palm oil and water - soluble vitamins , including titanium 
 dioxide as a density - adjusting agent . middle layer : composed of hydrogenated oils and 
 titanium dioxide . ( b ) triple concentric nozzle : core , middle and 
 outer liquid suspension were obtained from respective nozzle . liquid suspension : three 
 kinds of liquid suspension of core , middle layer and outer layer . core liquid included 
 substances intended to be escaped rumen . flowing oil : carrier of spherical 
 droplets . ) : the core liquid suspension consisted of 25% w / w of thiamine hydrochloride and 25% 
 w / w of titanium dioxide to regulate specific gravity , in liquid palm kernel oil having a 
 melting point of 38c . the middle layer was designed to avoid degradation by stomach acid in 
 the abomasum . it was produced from a liquid suspension consisting of 30% w / w titanium 
 dioxide in hardened palm kernel oil having a melting point of 56.5c . the outer layer was 
 intended to protect the middle layer from physical damage caused by jostling during 
 transport . the composition of the outer aqueous suspension was as follows : 3.0% w / w 
 kappa - carrageenan , 11.3% w / w cassava starch , 4.7% w / w sorbitol , 0.02% w / w locust bean gum , 
 1.4% w / w low - methoxy pectin , 0.23% w / w potassium chloride , 0.01% w / w calcium chloride , 0.09% 
 w / w potassium hydrogen phosphate , 0.28% w / w potassium dihydrogen phosphate and 2.3% w / w 
 titanium dioxide . these three types of suspension were applied via an encapsulation machine 
 equipped a triple concentric nozzle . the core , middle and outer layers of the liquid 
 suspension were simultaneously ejected ( via the innermost , intermediate and outermost 
 nozzles , respectively ) to a flowing - oil chamber . the suspension formed a spherical capsule 
 with three layers via its own surface tension in oil . the obtained wet capsules were dried 
 at 20c and 22.6% relative humidity on a rotating fluidized bed , thereby solidifying to form 
 three - layer capsules . the polysaccharide ( kappa - carrageenan , cassava starch , locust bean gum 
 and low - methoxy pectin ) , sugar alcohol ( sorbitol ) and salts ( potassium chloride , calcium 
 chloride , potassium hydrogen phosphate and potassium dihydrogen phosphate ) comprised in the 
 outer thin layer of the capsule are commonly used for food additive and authorized feeding 
 to cattle by inc . administrative agency food and agricultural materials inspection center in 
 japan . the palm kernel oil comprised in the middle layer and core are used for feed of 
 cattle , and also , authorized . titanium dioxide to regulate specific gravity comprised in the 
 all layers of the capsule is used for food additive in japan . wang et al . 
  reported that the titanium was accumulated in 
 the spleen and brain when female mice were one time administrated 5 g / kg body weight of 
 fine - sized titanium dioxide suspensions , but showed no acute toxicity . in the present study , orally administrated titanium dioxide was approximately 0.08 mg / kg 
 body weight of cattle . in addition , we confirmed that specific gravity of the capsule can be adjusted by 
 using talc ( which is authorized feeding to cattle ) in exchange for titanium dioxide . schematic of ( a ) the three - layered capsule and ( b ) method of manufacture . ( a ) core : a 
 mixture of hydrogenated palm oil and water - soluble vitamins , including titanium 
 dioxide as a density - adjusting agent . ( b ) triple concentric nozzle : core , middle and 
 outer liquid suspension were obtained from respective nozzle . liquid suspension : three 
 kinds of liquid suspension of core , middle layer and outer layer . core liquid included 
 substances intended to be escaped rumen . flowing oil : carrier of spherical 
 droplets . in vitro incubation of capsules and preliminary administration : twenty 
 particles of manufactured capsules per flask were anaerobically incubated by shaking in 100 
 ml rumen liquor at 39c for 24 hr in triplicate . rumen liquor was 
 obtained from lactating cows via rumen catheter and was immediately used for in 
 vitro incubation of the capsules . visual inspection established whether or not the 
 capsules had undergone lysis in the rumen liquor . before the main test , the rumen insoluble 
 capsules were preliminarily administered to cows in order to examine the effect of the 
 breaking strength of the capsules on their solubility in the lower digestive tract 
 subsequent to the rumen in vivo . two cows were orally administered the 
 capsule with five kinds of breaking strength ( 0.9 , 2.5 , 4.1 , 5.4 and 8.1 n ) every other 
 week . all feces were picked up from the floor after administration for 24 hr , and the status 
 of excreted capsules was checked visually . a rheometer ( cr-500dx , sun scientific co. , ltd . , 
 tokyo , japan ) was used to measure the breaking strength of the capsule , and the breaking 
 strength was defined as the breaking load when the capsule broke due to constriction . the 
 measurement was carried out using a pressure sensitive disc - shaped shaft with a diameter of 
 10 mm , moving at 20 mm / min at 39c . administration of capsules containing water - soluble vitamins : thiamine 
 hydrochloride was used to determine whether the capsules successfully escaped the rumen . the 
 capsules contained a core of thiamine hydrochloride ( wako pure chemical industries , ltd . the test group was administered encapsulated thiamine hydrochloride , whereas 
 the control group was administered the same amount of thiamine hydrochloride in an aqueous 
 solution . both the test and control groups were orally administered 30 g of thiamine 
 hydrochloride ( approximately 1,500 capsules for test group ) per head . a polyvinyl chloride 
 resin installer rumenfibe ( meiwa sangyo co. , ltd . , kyoto , japan ) was used for oral 
 administration . administration of both the capsules and solution was forced at 10:00 prior 
 to feeding . blood samples were collected from the jugular vein every 3 hr , from immediately 
 before administration until 24 hr after administration . all urine was collected by urinary 
 catheterization for 24 hr after administration and weighed after each excretion . the thiamine concentration was measured using the modifications by fukuwatari et 
 al .   to the high - performance liquid 
 chromatography ( hplc ) specifically , for blood samples , 300 l of whole 
 blood was taken using sodium heparin as an anticoagulant , to which 600 l 
 of 5% w / v trichloroacetic acid solution was added . following column separation using 0.2 m sodium dihydrogen 
 phosphate/0.3% v / v acetonitrile as the mobile phase , it was mixed with 0.1% w / v potassium 
 ferricyanide and 15% w / v naoh before measurement using a fluorescence detector . the columns 
 were reversed - phase hplc columns ( cosmosil 5c18-ms - ii , 4.6  250 mm , nacalai tesque , inc . , kyoto , japan ) . statistical analysis : for samples collected concurrently , student s 
 t - test was used to examine inter - group differences in blood thiamine 
 concentrations . tukey s multiple comparison analysis was used to determine differences in 
 the concentrations of blood thiamine within each group . all analyses were performed using r 
 statistical software ( version 3.1.0 ; www.r-project.org ) . in vitro incubation of capsules and preliminary administration : for 
 in vitro incubation , there were no lysed capsules in the rumen liquor for 
 24 hr . when the capsules with a breaking strength of 4.7 n were orally administrated to 
 cattle , the capsules were excreted having maintained their spherical shape in feces ( table 2table 2.status of capsules after in vitro incubation and preliminary 
 administrationbreaking strength of a capsule ( n)0.92.54.15.48.1status of capsules after in vitro incubationspherical shapespherical shapespherical shapespherical shapespherical shapestatus of capsules in fecesfragmentfragmentfragmentspherical shapespherical shape ) . on the other hand , only fragments of the capsule were obtained from the 
 feces when capsules with breaking strength of 4.1 n or less were administrated . administration of capsules : the results of thiamine hydrochloride level in 
 the blood are shown in fig . 2.fig . blood thiamine concentrations 
 were measured before administration and every 3 hr after administration .  : 
 encapsulated thiamine hydrochloride - administered group ,  : aqueous thiamine 
 hydrochloride - administered group . * : denotes significant differences between the 
 groups at the given time ( * * , p<0.01 ; * * * , 
 p<0.001 ) . : significant differences 
 between time periods are noted with different letters 
 ( p<0.05 ) . no change in blood thiamine concentrations was found in the control group that was 
 administered an aqueous solution of thiamine hydrochloride . in contrast , in the test group 
 administered thiamine hydrochloride via the three - layer capsules , a marked rise in blood 
 thiamine concentration was found 3 hr following administration . the concentration 
 significantly increased from 12.4  1.03 ng / ml before 
 administration to 54.8  2.21 ng / ml at 6 hr after 
 administration . the elevated blood thiamine concentration was maintained for 9 hr after 
 administration before gradually decreasing . blood concentrations were 4.1-fold higher in the 
 test group than in the control group 6 hr after administration . 3.accumulated thiamine hydrochloride excreted via urine , calculated from the 
 concentration and volume of each urine excretion . each plot shows average accumulated 
 thiamine hydrochloride in urine for the time elapsed since administration . : 
 encapsulated thiamine hydrochloride -administered group ,  : aqueous thiamine 
 hydrochloride -administered group . shows accumulated thiamine hydrochloride excreted in urine , calculated from urine 
 thiamine hydrochloride level and urine volume after administration of capsules . there was 
 considerable individual variability in the thiamine value , and no difference was found 
 between the test group ( 801.7  348.2 g ) and the control group ( 674.6  
 184.1 g , p=0.7993 ) at 24 hr after administration . blood thiamine concentrations 
 were measured before administration and every 3 hr after administration .  : 
 encapsulated thiamine hydrochloride - administered group ,  : aqueous thiamine 
 hydrochloride - administered group . * : denotes significant differences between the 
 groups at the given time ( * * , p<0.01 ; * * * , 
 p<0.001 ) . : significant differences 
 between time periods are noted with different letters 
 ( p<0.05 ) . accumulated thiamine hydrochloride excreted via urine , calculated from the 
 concentration and volume of each urine excretion . each plot shows average accumulated 
 thiamine hydrochloride in urine for the time elapsed since administration .  : 
 encapsulated thiamine hydrochloride -administered group ,  : aqueous thiamine 
 hydrochloride -administered group . there were no lysed capsules in in vitro incubation , and hence , it was 
 concluded that capsules placed into the rumen retained their shape until flowing out to the 
 omasum . our previous study in dairy cattle showed that the proportion of ruminated 
 indigestible plastic balls ( which are similar to the capsules used in this study in shape , 
 size and specific gravity ) was 2% or less . 
 according to that finding , almost all the capsules possibly passed through the rumen without 
 being degraded by microorganisms in rumen and/or physical destruction by rumination . in the 
 present study , the capsules were found to be insoluble in rumen liquor in 
 vitro . at the same time , no intact capsules were found in feces after the 
 preliminary administration when the breaking strength of capsule was set for less than 4.1 
 n. in addition , we have reported in our previous study that oral administration of the same 
 three - layered capsules containing live lactic acid bacteria had altered the intestinal 
 bacterial flora of cattle . these results 
 strongly suggest that the capsules used in the main administration tests in this study 
 escaped from being broken in the rumen , but dissolved in lower digestive tract . the elevated blood thiamine concentration in the test group suggests that the capsule 
 reached the intestinal tract after passing quickly through the anterior stomach . it has been 
 suggested that solids with a high specific gravity , such as the capsules used in this study , 
 quickly enter the ruminal ventral sacs rather than being limited to the mat formed in the 
 rumen . additionally , it is known that once solids 
 have entered the ventral sac , they flow through the omasum without reentering the dorsal sac 
 . hoeller et al .   reported that the rumen wall of sheep has an extremely 
 low permeability to thiamine and that the rumen may not be a site of significant absorption 
 of thiamin . [ 12 , 13 ] reported that absorption of 
 thiamine occurs mainly in the small intestine . in the results of our study , the blood 
 thiamine levels started to increase 3 hr after administration in the test group , even though 
 thiamine half - life in blood is very short , reported as 4.7 and 7.1 min in fitting two 
 exponential functions . furthermore , plasma 
 thiamine concentration peaked between 6 and 9 hr after administration before subsequently 
 starting to decline . these results suggest that the capsules moved rapidly to the duodenum 
 without stopping in the rumen ; and that almost all the capsules had dissolved and released 
 their contents in a rapid manner , since no capsules lysed in rumen liquor in in 
 vitro incubation , as indicated above . the rise of blood thiamine level for the 
 test group seems to be relatively low with respect to the amount of administered thiamine . it is thought to be due to that thiamine is quickly metabolized in blood and its half - life 
 is very short . in contrast , the ruminal retention time of the aqueous solutions , such as those used in the 
 control group , is estimated to be about 10 hr in dairy cows . assuming that as much as 52% administered thiamine hydrochloride reached the 
 lower digestive tract intact , a rise in blood 
 concentrations would be expected to occur 10 hr following administration . however , in 
 practice , this was not observed . this is considered to be due to metabolic action of ruminal 
 microorganisms and/or quick hydrolysis or tissue uptake that somewhat absorbed thiamin , as 
 mentioned above . with regard to urine , there was no difference in accumulated thiamine 
 hydrochloride between control and test groups . nakajima et al .   reported that in cattle , thiamine is absorbed from 
 blood by some tissues , such as the brain , heart , liver and kidney . for this reason , it was 
 concluded that tissue uptake of migrated thiamine from capsules occurred in a matter of 
 hours and that thiamine was not excreted to urine . the above results show that encasing water - soluble vitamins within a multi - layer capsule 
 structure allows for efficient absorption by cows . the three - layered capsules developed in 
 this study for cows were shown to function as a rumen escapable tool . additionally , it was 
 demonstrated that the capsules rapidly moved to the duodenum following administration , since 
 peak blood thiamine concentration occurred between 6 and 9 hr after oral capsule 
 administration . thiamine is effective for prevention and treatment of bovine ketosis , but has been 
 administered intravenously because it is degraded in the rumen . our present work suggests 
 that the three - layered capsule presented here could enable oral administration of thiamine 
 to cattle , which is easier than intravenous injection on - site . furthermore , capsules could 
 be used in cases where the bypass oil method is not available , such as for medical products , 
 probiotics or transmucosally administered vaccines that have little resistance to heat or 
 acid . a further study is required for more detailed evaluation of the capacity of 
 three - layered capsules , such as the escape efficiency of capsules and quantification of the 
 rumen escape level of contents for clinical veterinary use .
a new rumen escapable tool is presented for cattle in prospect of developing medical 
 treatment or supplementing trace elements for disease prevention . 
 this tool consists of a 
 three - layered capsule that dissolves in the lower digestive tract , but not in the rumen . 
 the capsule was manufactured by capsule - forming techniques through the use of liquid 
 surface tension . 
 this method does not involve high - temperature treatment , so the capsule 
 can contain not only lipophilic substances but also hydrophilic or heat - sensitive 
 substances . furthermore , the capsule has a specific gravity of 1.3 and diameter of 6.0 mm , 
 which were previously shown to be appropriate to avoid rumination . 
 the objective of this 
 study was to confirm the effectiveness of the capsule pertinent to rumen escaping . in 
 order to validate rumen escape , capsules containing 30 g of water - soluble vitamin 
 ( thiamine hydrochloride ) per head 
 were administered to four lactating cows assigned in a 
 crossover trial . in the group 
 administered encapsulated thiamine hydrochloride , blood 
 thiamine levels increased from 12.4  1.03 ng / ml before 
 administration to 54.8  2.21 ng / ml at 6 hr following 
 administration , whereas the level remained at 13.3  2.05 
 ng / ml in the control group administered via aqueous 
 solution . 
 this indicates that the three - layered capsules passed through the rumen and 
 dissolved in the lower digestive tract , thus functioning as a rumen escapable tool .