this retrospective study included 40 dogs that were diagnosed with biliary sludge and 
 gallbladder mucoceles , and had their gallbladder contents collected during surgery or 
 autopsy at the university of miyazaki veterinary teaching hospital , miyazaki , japan , between 
 may 2012 and july 2016 .
component analysis was performed using 43 samples of gallbladder 
 contents from 40 dogs diagnosed with biliary sludge or gallbladder mucoceles  for three 
 samples , the same animal was re - examined over the course of follow - up .
samples that were 
 diagnosed by autopsy were from dogs that either died of natural causes or were euthanized 
 for reasons other than gallbladder diseases .
euthanization was conducted as below : first , 
 dogs were administered with pentobarbital sodium ( 50 mg / kg , iv ; somnopentyl , kyoritsu 
 seiyaku corp . ,
five min later , potassium chloride ( 2 meq / kg , iv ; kcl 
 corrective injection 1 meq / ml , otsuka pharmaceutical factory , inc .
biliary sludge and gallbladder mucoceles were diagnosed by gross examination during surgery 
 or autopsy .
biliary sludge was diagnosed when the gallbladder contents presented a 
 sludge - like appearance .
gallbladder mucoceles were diagnosed when the gallbladder appeared 
 to be completely filled with a jelly - like substance ( figs .
biliary 
 sludge presents as hyperechogenic accumulated matter in the direction of gravity on 
 ultrasonography images mild * ( a ) , moderate * ( b ) and severe * ( c ) .
gallbladder contents 
 in moderate biliary sludge ( d ) have a black sand - like appearance , while those in 
 severe biliary sludge ( e ) have a black sludge - like appearance .
biliary sludge was classified 
 according to its rate of retention within the gallbladder as mild ( 25% ) , moderate 
 ( 2575% ) or severe ( 75% ) . and 2fig .
. however , gallbladder contents in gallbladder mucoceles present 
 varied appearances ; in the present study , while gallbladder contents in a few 
 mucoceles presented a black and jelly - like appearance ( c ) , those in other mucoceles 
 presented as an indurated , jelly - like material radiating to the margin ( d ) . ) .
biliary sludge was classified according to severity based on the content retention 
 rate relative to size of the gallbladder determined by ultrasonography , as follows : mild 
 ( 25% retention ) , moderate ( 2575% retention ) or severe ( 75% retention ; fig .
biliary 
 sludge presents as hyperechogenic accumulated matter in the direction of gravity on 
 ultrasonography images mild * ( a ) , moderate * ( b ) and severe * ( c ) .
gallbladder contents 
 in moderate biliary sludge ( d ) have a black sand - like appearance , while those in 
 severe biliary sludge ( e ) have a black sludge - like appearance . *
biliary sludge was classified 
 according to its rate of retention within the gallbladder as mild ( 25% ) , moderate 
 ( 2575% ) or severe ( 75% ) . ultrasonography images and gallbladder content findings of gallbladder mucoceles . 

 pattern within the gallbladder on abdominal ultrasonography 
 images ( a and b ) . however , gallbladder contents in gallbladder mucoceles present 
 varied appearances ; in the present study , while gallbladder contents in a few 
 mucoceles presented a black and jelly - like appearance ( c ) , those in other mucoceles 
 presented as an indurated , jelly - like material radiating to the margin ( d ) . of the 40 dogs included in the present study , 18 ( 21 samples ) exhibited biliary sludge , and 
 22 ( 22 samples ) exhibited gallbladder mucoceles .
dogs included in this study were of the 
 following breeds : miniature dachshund , chihuahua and toy poodle ( n=6 , each ) ; miniature 
 schnauzer ( n=4 ) ; pug and mixed breed ( n=3 , each ) ; american cocker spaniel , shetland 
 sheepdog , yorkshire terrier , beagle , french bulldog and doberman ( n=2 , each ) and pomeranian , 
 shiba inu and papillon ( n=1 , each ) .
the median age of the 40 dogs ( 43 samples ) included in 
 the present study ( male , 5 ; castrated male , 11 ; female , 7 ; and spayed female , 20 ) was 9 
 years ( range , 115 years , including one dog of unknown age ) , and the median weight was 5.2 
 kg ( range , 1.929.0 kg ) .
all dogs were examined with the owners consent having been 
 obtained prior to surgical treatment ( cholecystectomy , 25 dogs and gallbladder irrigation , 
 15 dogs ) or autopsy ( 3 dogs ) .
gallbladder irrigation was performed by making a small 
 incision on the gallbladder wall , followed by aspiration of contents in the lumen of the 
 gallbladder and irrigation and cleaning up of the inside of the gallbladder with saline . for component analysis ,
43 canine gallbladder content samples with bile ( biliary sludge , 21 
 and gallbladder mucoceles , 22 ) were first dried well and then prepared as potassium bromide 
 ( kbr ) pellets for evaluation by ir spectroscopy ( ir spectroscopy ft / ir-410 , jasco corp .
, 
 tokyo , japan . ) . in a previous study   on the ir 
 spectroscopy of ovary - derived mucins
therefore , mucins derived from swine stomach ( swine mucin ; purity 80% , wako pure 
 chemical industries , ltd . , tokyo , japan . ) were used to establish the reference ir spectrum 
 for mucins in this study .
the ir spectra of biliary sludge and gallbladder mucoceles 
 contents were compared with that of swine mucin in order to determine the composition of the 
 former .
of the 43 canine gallbladder content samples , 41 ( biliary sludge , 20 and gallbladder 
 mucoceles , 21 ) were evaluated by bacterial culture .
the animals were administered 
 antimicrobials after collection of gallbladder contents , but not within 12 hr prior to 
 collection .
bacterial cultures of gallbladder contents were established by aseptically 
 swabbing the inside of the gallbladder with a sterile cotton swab and inoculating the 
 samples onto bacterial media for aerobic and anaerobic culture , followed by bacterial 
 identification .
as determined by ultrasonographic findings , there was no mild case , moderate was 10 
 cases , with all samples exhibiting biliary sludge retention rates 50% , and severe was 11 
 cases .
the ir spectrum of swine mucin corresponded to proteins , with amide bands near 1,650 and 
 1,550 cm , and polysaccharides , with spectral characteristics of sugar chains 
 ( glycosylation bands ) near 1,070 and 1,050 cm(fig .
swine mucin exhibits characteristic absorption bands of 
 proteins ( amide bands near 1,650 and 1,550 cm ; indicated by arrowheads ) 
 and sugar chains ( glycosylation bands near 1,070 and 1,050 cm ; 
 indicated by arrows ) . ) .
this ir spectrum was used as the 
 reference for identification of mucins in gallbladder contents .
swine mucin exhibits characteristic absorption bands of 
 proteins ( amide bands near 1,650 and 1,550 cm ; indicated by arrowheads ) 
 and sugar chains ( glycosylation bands near 1,070 and 1,050 cm ; 
 indicated by arrows ) .
of the 21 samples of gallbladder contents of dogs with biliary sludge , 20 ( 95.2%)ten 
 samples each of moderate and severe biliary sludge  exhibited ir spectra coinciding with 
 amide and glycosylation bands similar to those observed with swine mucin and were thus 
 identified as being composed of mucins ( fig . 

ir spectra of swine mucin ( a ; dotted 
 line ) and severe ( b ; solid line ) and moderate ( c ; solid line ) biliary sludge*. ir 
 spectra of severe biliary sludge that was identified as proteins ( d ; solid line ) . 

moderate ( b ) and severe ( c ) biliary sludge both exhibited amide bands ( near 1,650 
 and 1,550 cm ; indicated by arrowheads ) and glycosylation bands ( near 
 1,070 and 1,050 cm ; indicated by arrows ) , with the exception of one 
 sample ( d ) of severe biliary sludge , where only amide bands were observed .
* ir 
 spectra of only representative samples are presented in b and c ; b ( n=9 ) and c 
 ( n=10 ) exhibited similar spectra . ) .
the ir spectrum of the one remaining sample ( 4.8% ) , which was from a dog with 
 severe biliary sludge , did not exhibit clear glycosylation bands and instead presented 
 only amide bands ; this sample was , therefore , identified as being composed of proteins 
 ( fig .
ir spectra of swine mucin ( a ; dotted 
 line ) and severe ( b ; solid line ) and moderate ( c ; solid line ) biliary sludge*. ir 
 spectra of severe biliary sludge that was identified as proteins ( d ; solid line ) . 

moderate ( b ) and severe ( c ) biliary sludge both exhibited amide bands ( near 1,650 
 and 1,550 cm ; indicated by arrowheads ) and glycosylation bands ( near 
 1,070 and 1,050 cm ; indicated by arrows ) , with the exception of one 
 sample ( d ) of severe biliary sludge , where only amide bands were observed .
* ir 
 spectra of only representative samples are presented in b and c ; b ( n=9 ) and c 
 ( n=10 ) exhibited similar spectra .
all 22 samples of gallbladder contents of dogs with gallbladder mucoceles exhibited ir 
 spectra coinciding with amide and glycosylation bands similar to those observed with swine 
 mucin and were thus identified as mucins ( fig . 
 5fig .
ir spectra of swine mucin ( a ; 
 dotted line ) and gallbladder mucoceles * ( b ; solid line ) .
gallbladder mucoceles ( b ) 
 exhibited amide bands ( near 1,650 and 1,550 cm ; indicated by 
 arrowheads ) and glycosylation bands ( near 1,070 and 1,050 cm ; indicated 
 by arrows ) .
* ir spectrum of only a representative sample is presented in b. the 
 gallbladder contents of all gallbladder mucoceles ( n=21 ) exhibited similar 
 spectra . ) .
the ir spectra of biliary sludge and gallbladder mucoceles contents were found to 
 be similar .
ir spectra of swine mucin ( a ; 
 dotted line ) and gallbladder mucoceles * ( b ; solid line ) .
gallbladder mucoceles ( b ) 
 exhibited amide bands ( near 1,650 and 1,550 cm ; indicated by 
 arrowheads ) and glycosylation bands ( near 1,070 and 1,050 cm ; indicated 
 by arrows ) .
* ir spectrum of only a representative sample is presented in b. the 
 gallbladder contents of all gallbladder mucoceles ( n=21 ) exhibited similar 
 spectra .
three of the dogs with biliary sludge received gallbladder irrigation , followed by 12 
 years of follow - up , at the end of which , progressively accumulated gallbladder contents 
 were again collected by gallbladder irrigation .
of the three dogs , one underwent 
 cholecystectomy for extrahepatic bile duct obstruction due to re - accumulation of 
 gallbladder contents about 1 year and 8 months after the second gallbladder irrigation . 

unlike the other two dogs , the gallbladder contents of this dog at the time of the 
 cholecystectomy had clearly transitioned from sludge - like to jelly - like appearance . 
 therefore
, the dog was diagnosed with a gallbladder mucocele rather than biliary sludge 
 based on the findings of analysis of gallbladder contents from the third collection ( fig .
ultrasonography findings at 1 year and 7 months after first collection ( a ) 
 and 10 days after second collection ( b ) indicated biliary sludge .
gallbladder 
 contents of both first and second collections were similar and determined to be 
 biliary sludge ( c ) .
ultrasonography findings at 1 year and 8 months after second 
 collection when extrahepatic duct obstruction occurred ( d ) .
gallbladder content findings at third 
 collection indicated a gallbladder mucocele ( f ) . for treatment of biliary sludge , 
 the inside of the gallbladder
was cleaned with saline , and all of the sludge - like 
 content ( c ) was collected ( b ) .
however , biliary sludge recurred and accumulated 
 progressively ( d ) . during the interval of 1 year and 7 months between the initial 
 collection of biliary sludge and second collection of gallbladder contents , the dog 
 exhibited no clinical symptoms of extrahepatic bile duct obstruction , and its 
 recovery was uneventful .
however , upon recurrence of biliary sludge , the patient 
 received additional follow - up for 1 year and 8 months . to resolve the extrahepatic 
 bile duct obstruction observed during follow - up ( d ) ,
the contents of the excised gallbladder had changed in 
 appearance from sludge ( c ) to jelly - like consistency ( f ) , leading to the diagnosis 
 of gallbladder mucocele . ) .
the gallbladder contents of this dog exhibited nearly similar ir spectra at all 
 three collections and were thus identified each time as being composed of mucins ( fig . 7fig .
( b ) collections , both 
 of which were determined to be biliary sludge .
ir spectra of gallbladder contents 
 from the third collection ( c ) , which was determined to be from a gallbladder 
 mucocele .
gallbladder contents from the first , second and third collections 
 exhibited similar ir spectra ; both amide bands ( near 1,650 and 1,550 
 cm ; indicated by arrowheads ) and glycosylation bands ( near 1,070 and 
 1,050 cm ; indicated by arrows ) were observed , and the contents were 
 identified as mucins . ) .
the gallbladder contents of the remaining two dogs presented a sludge - like 
 appearance at the first and second collections , based on which the dogs were diagnosed 
 with biliary sludge .
the gallbladder contents were identified as being composed of mucins 
 based on their ir spectroscopic findings . ultrasonography and gallbladder content
ultrasonography findings at 1 year and 7 months after first collection ( a ) 
 and 10 days after second collection ( b ) indicated biliary sludge .
gallbladder 
 contents of both first and second collections were similar and determined to be 
 biliary sludge ( c ) .
ultrasonography findings at 1 year and 8 months after second 
 collection when extrahepatic duct obstruction occurred ( d ) .
gallbladder content findings at third 
 collection indicated a gallbladder mucocele ( f ) . for treatment of biliary sludge , 
 the inside of the gallbladder
was cleaned with saline , and all of the sludge - like 
 content ( c ) was collected ( b ) . however , biliary sludge recurred and accumulated 
 progressively ( d ) . during the interval of 1 year and 7 months between the initial 
 collection of biliary sludge and second collection of gallbladder contents
, the dog 
 exhibited no clinical symptoms of extrahepatic bile duct obstruction , and its 
 recovery was uneventful .
however , upon recurrence of biliary sludge , the patient 
 received additional follow - up for 1 year and 8 months . to resolve the extrahepatic 
 bile duct obstruction observed during follow - up ( d ) , the patient underwent 
 cholecystectomy ( e ) .
the contents of the excised gallbladder had changed in 
 appearance from sludge ( c ) to jelly - like consistency ( f ) , leading to the diagnosis 
 of gallbladder mucocele .
second ( b ) collections , both 
 of which were determined to be biliary sludge .
ir spectra of gallbladder contents 
 from the third collection ( c ) , which was determined to be from a gallbladder 
 mucocele .
gallbladder contents from the first , second and third collections 
 exhibited similar ir spectra ; both amide bands ( near 1,650 and 1,550 
 cm ; indicated by arrowheads ) and glycosylation bands ( near 1,070 and 
 1,050 cm ; indicated by arrows ) were observed , and the contents were 
 identified as mucins .
the infective 
 bacterial species were identified to be escherichia coli and 
 serratia marcescens in one sample each .
of the 21 gallbladder mucocele 
 samples , 3 ( 14.3% ) exhibited bacterial infection .
the infective bacterial species were 
 identified to be escherichia coli in two samples and enterococcus 
 spp . in one sample .
as determined by ultrasonographic findings , there was no mild case , moderate was 10 
 cases , with all samples exhibiting biliary sludge retention rates 50% , and severe was 11 
 cases .
the ir spectrum of swine mucin corresponded to proteins , with amide bands near 1,650 and 
 1,550 cm , and polysaccharides , with spectral characteristics of sugar chains 
 ( glycosylation bands ) near 1,070 and 1,050 cm(fig .
swine mucin exhibits characteristic absorption bands of 
 proteins ( amide bands near 1,650 and 1,550 cm ; indicated by arrowheads ) 
 and sugar chains ( glycosylation bands near 1,070 and 1,050 cm ; 
 indicated by arrows ) . ) .
this ir spectrum was used as the 
 reference for identification of mucins in gallbladder contents .
swine mucin exhibits characteristic absorption bands of 
 proteins ( amide bands near 1,650 and 1,550 cm ; indicated by arrowheads ) 
 and sugar chains ( glycosylation bands near 1,070 and 1,050 cm ; 
 indicated by arrows ) .
of the 21 samples of gallbladder contents of dogs with biliary sludge , 20 ( 95.2%)ten 
 samples each of moderate and severe biliary sludge  exhibited ir spectra coinciding with 
 amide and glycosylation bands similar to those observed with swine mucin and were thus 
 identified as being composed of mucins ( fig . 

ir spectra of swine mucin ( a ; dotted 
 line ) and severe ( b ; solid line ) and moderate ( c ; solid line ) biliary sludge*. ir 
 spectra of severe biliary sludge that was identified as proteins ( d ; solid line ) . 

moderate ( b ) and severe ( c ) biliary sludge both exhibited amide bands ( near 1,650 
 and 1,550 cm ; indicated by arrowheads ) and glycosylation bands ( near 
 1,070 and 1,050 cm ; indicated by arrows ) , with the exception of one 
 sample ( d ) of severe biliary sludge , where only amide bands were observed .
* ir 
 spectra of only representative samples are presented in b and c ; b ( n=9 ) and c 
 ( n=10 ) exhibited similar spectra . ) .
the ir spectrum of the one remaining sample ( 4.8% ) , which was from a dog with 
 severe biliary sludge , did not exhibit clear glycosylation bands and instead presented 
 only amide bands ; this sample was , therefore , identified as being composed of proteins 
 ( fig .
ir spectra of swine mucin ( a ; dotted 
 line ) and severe ( b ; solid line ) and moderate ( c ; solid line ) biliary sludge*. ir 
 spectra of severe biliary sludge that was identified as proteins ( d ; solid line ) . 

moderate ( b ) and severe ( c ) biliary sludge both exhibited amide bands ( near 1,650 
 and 1,550 cm ; indicated by arrowheads ) and glycosylation bands ( near 
 1,070 and 1,050 cm ; indicated by arrows ) , with the exception of one 
 sample ( d ) of severe biliary sludge , where only amide bands were observed .
* ir 
 spectra of only representative samples are presented in b and c ; b ( n=9 ) and c 
 ( n=10 ) exhibited similar spectra .
all 22 samples of gallbladder contents of dogs with gallbladder mucoceles exhibited ir 
 spectra coinciding with amide and glycosylation bands similar to those observed with swine 
 mucin and were thus identified as mucins ( fig . 

ir spectra of swine mucin ( a ; 
 dotted line ) and gallbladder mucoceles * ( b ; solid line ) .
gallbladder mucoceles ( b ) 
 exhibited amide bands ( near 1,650 and 1,550 cm ; indicated by 
 arrowheads ) and glycosylation bands ( near 1,070 and 1,050 cm ; indicated 
 by arrows ) .
* ir spectrum of only a representative sample is presented in b. the 
 gallbladder contents of all gallbladder mucoceles ( n=21 ) exhibited similar 
 spectra . ) .
the ir spectra of biliary sludge and gallbladder mucoceles contents were found to 
 be similar .
ir spectra of swine mucin ( a ; 
 dotted line ) and gallbladder mucoceles * ( b ; solid line ) .
gallbladder mucoceles ( b ) 
 exhibited amide bands ( near 1,650 and 1,550 cm ; indicated by 
 arrowheads ) and glycosylation bands ( near 1,070 and 1,050 cm ; indicated 
 by arrows ) .
* ir spectrum of only a representative sample is presented in b. the 
 gallbladder contents of all gallbladder mucoceles ( n=21 ) exhibited similar 
 spectra .
three of the dogs with biliary sludge received gallbladder irrigation , followed by 12 
 years of follow - up , at the end of which , progressively accumulated gallbladder contents 
 were again collected by gallbladder irrigation .
of the three dogs , one underwent 
 cholecystectomy for extrahepatic bile duct obstruction due to re - accumulation of 
 gallbladder contents about 1 year and 8 months after the second gallbladder irrigation . 

unlike the other two dogs , the gallbladder contents of this dog at the time of the 
 cholecystectomy had clearly transitioned from sludge - like to jelly - like appearance . 

therefore , the dog was diagnosed with a gallbladder mucocele rather than biliary sludge 
 based on the findings of analysis of gallbladder contents from the third collection ( fig .
ultrasonography findings at 1 year and 7 months after first collection ( a ) 
 and 10 days after second collection ( b ) indicated biliary sludge .
gallbladder 
 contents of both first and second collections were similar and determined to be 
 biliary sludge ( c ) .
ultrasonography findings at 1 year and 8 months after second 
 collection when extrahepatic duct obstruction occurred ( d ) .
gallbladder content findings at third 
 collection indicated a gallbladder mucocele ( f ) . for treatment of biliary sludge , 
 the inside of the gallbladder
was cleaned with saline , and all of the sludge - like 
 content ( c ) was collected ( b ) . however , biliary sludge recurred and accumulated 
 progressively ( d ) . during the interval of 1 year and 7 months between the initial 
 collection of biliary sludge and second collection of gallbladder contents
, the dog 
 exhibited no clinical symptoms of extrahepatic bile duct obstruction , and its 
 recovery was uneventful .
however , upon recurrence of biliary sludge , the patient 
 received additional follow - up for 1 year and 8 months . to resolve the extrahepatic 
 bile duct obstruction observed during follow - up ( d ) ,
the contents of the excised gallbladder had changed in 
 appearance from sludge ( c ) to jelly - like consistency ( f ) , leading to the diagnosis 
 of gallbladder mucocele . ) .
the gallbladder contents of this dog exhibited nearly similar ir spectra at all 
 three collections and were thus identified each time as being composed of mucins ( fig . 7fig .
( b ) collections , both 
 of which were determined to be biliary sludge .
ir spectra of gallbladder contents 
 from the third collection ( c ) , which was determined to be from a gallbladder 
 mucocele .
gallbladder contents from the first , second and third collections 
 exhibited similar ir spectra ; both amide bands ( near 1,650 and 1,550 
 cm ; indicated by arrowheads ) and glycosylation bands ( near 1,070 and 
 1,050 cm ; indicated by arrows ) were observed , and the contents were 
 identified as mucins . ) .
the gallbladder contents of the remaining two dogs presented a sludge - like 
 appearance at the first and second collections , based on which the dogs were diagnosed 
 with biliary sludge .
the gallbladder contents were identified as being composed of mucins 
 based on their ir spectroscopic findings .
ultrasonography findings at 1 year and 7 months after first collection ( a ) 
 and 10 days after second collection ( b ) indicated biliary sludge .
gallbladder 
 contents of both first and second collections were similar and determined to be 
 biliary sludge ( c ) .
ultrasonography findings at 1 year and 8 months after second 
 collection when extrahepatic duct obstruction occurred ( d ) .
gallbladder content findings at third 
 collection indicated a gallbladder mucocele ( f ) . for treatment of biliary sludge , 
 the inside of the gallbladder
was cleaned with saline , and all of the sludge - like 
 content ( c ) was collected ( b ) . however , biliary sludge recurred and accumulated 
 progressively ( d ) . during the interval of 1 year and 7 months between the initial 
 collection of biliary sludge and second collection of gallbladder contents
, the dog 
 exhibited no clinical symptoms of extrahepatic bile duct obstruction , and its 
 recovery was uneventful .
however , upon recurrence of biliary sludge , the patient 
 received additional follow - up for 1 year and 8 months . to resolve the extrahepatic 
 bile duct obstruction observed during follow - up ( d ) ,
the contents of the excised gallbladder had changed in 
 appearance from sludge ( c ) to jelly - like consistency ( f ) , leading to the diagnosis 
 of gallbladder mucocele .
ir 
 spectra of gallbladder contents from the first ( a ) and second ( b ) collections , both 
 of which were determined to be biliary sludge .
ir spectra of gallbladder contents 
 from the third collection ( c ) , which was determined to be from a gallbladder 
 mucocele .
gallbladder contents from the first , second and third collections 
 exhibited similar ir spectra ; both amide bands ( near 1,650 and 1,550 
 cm ; indicated by arrowheads ) and glycosylation bands ( near 1,070 and 
 1,050 cm ; indicated by arrows ) were observed , and the contents were 
 identified as mucins .
the infective 
 bacterial species were identified to be escherichia coli and 
 serratia marcescens in one sample each .
of the 21 gallbladder mucocele 
 samples , 3 ( 14.3% ) exhibited bacterial infection .
the infective bacterial species were 
 identified to be escherichia coli in two samples and enterococcus 
 spp . in one sample .
canine gallbladder diseases  especially gallbladder mucoceles  are caused by excessive mucin 
 secretion .
however , to our knowledge , there are no 
 reports regarding component analysis of actual contents from gallbladder mucoceles .
a 
 previous study performed ir spectroscopic analysis of human ovary - derived mucins with the ir 
 spectrum of bovine submaxillary gland - derived mucins as reference . therefore , in the present study , the ir spectrum of swine mucin was 
 employed as the reference for comparative evaluation of ir spectra of biliary sludge and 
 gallbladder mucocele samples .
nearly all of the gallbladder content samples of dogs with 
 biliary sludge ( 95.2% ) exhibited similar ir spectra as swine mucin , irrespective of the 
 severity of biliary sludge , which ranged from moderate ( 5075% biliary sludge ) to severe 
 ( fig .
4 ) . in case of gallbladder mucocele 
 contents , all samples exhibited similar ir spectra as swine mucin ( fig . 5 ) .
these findings indicated that the principal components of 
 gallbladder contents in both biliary sludge and gallbladder mucoceles were mucins , which 
 suggested the possibility that mucins were involved in the pathogenesis of not only 
 gallbladder mucoceles but also biliary sludge .
7 , it was observed that there were some changes 
 between biliary sludge ( a and b ) and gallbladder mucocele ( c ) . in particular ,
these changes 
 were observed near 1,460 , 1,375 and 1,055 cm in fig . 7 ( c ) . these were characteristic ir spectra showing cholesterol 
 .
it was suggested that these ir spectra were 
 possible to have been affected by cholesterol because cholesterol was contained in the bile . 

therefore , it was possible that amount of cholesterol in the gallbladder content was 
 increased with varying from biliary sludge to gallbladder mucocele in this case .
although 
 these characteristic ir spectra showing cholesterol were observed in some of gallbladder 
 mucocele cases , not in all gallbladder mucocele cases .
therefore , since all gallbladder 
 mucocele samples in this study showed amide bands and glycosylation bands , it was considered 
 that the principal component of gallbladder mucocele was mucin . in present study ,
the gallbladder contents of one of the dogs with biliary sludge were 
 identified as being composed of proteins ( fig .
the gallbladder in this case had presented extensive bleeding upon histopathological 
 examination , which indicated the possibility that gallbladder bleeding affected the 
 composition of the biliary sludge contents . in order to verify these hypothesis ,
the ir 
 spectrum of a clot of peripheral venous blood from a healthy dog was compared with that of 
 the gallbladder contents in this case .
the ir spectrum of the blood clot exhibited amide 
 bands but no glycosylation bands , and therefore , the blood clot was identified as being 
 composed of proteins .
the ir spectrum of the proteinaceous biliary sludge sample was found 
 to be similar to that of the blood clot ( fig . 

ir 
 spectra of swine mucin ( a ; dotted line ) and a blood clot from a healthy dog ( b ; dashed 
 line ) .
gallbladder contents of biliary sludge determined as being composed of proteins 
 ( c ; solid line ) .
the blood clot exhibited only amide bands in its ir spectrum and was , 
 therefore , determined as being composed of proteins .
the ir spectra of the blood clot 
 and the biliary sludge determined as being composed of proteins were similar . 

arrowheads indicate amide bands ( near 1,650 and 1,550 cm ) ; arrows 
 indicate glycosylation bands ( near 1,070 and 1,050 cm ) . ) , which may be attributable to the mixing of biliary sludge with plasma proteins from 
 blood because of gallbladder bleeding .
these findings suggest that , in cases where 
 histopathological findings reveal gallbladder bleeding , the composition of gallbladder 
 contents might be different from mucin .
ir 
 spectra of swine mucin ( a ; dotted line ) and a blood clot from a healthy dog ( b ; dashed 
 line ) .
gallbladder contents of biliary sludge determined as being composed of proteins 
 ( c ; solid line ) .
the blood clot exhibited only amide bands in its ir spectrum and was , 
 therefore , determined as being composed of proteins .
the ir spectra of the blood clot 
 and the biliary sludge determined as being composed of proteins were similar . 

arrowheads indicate amide bands ( near 1,650 and 1,550 cm ) ; arrows 
 indicate glycosylation bands ( near 1,070 and 1,050 cm ) .
both biliary sludge and gallbladder mucocele samples exhibited a low incidence of bacterial 
 infection ( 10.0 and 14.3% , respectively ) .
a previous study reported a 13.5% rate of 
 bacterial infection in gallbladder mucoceles , 
 which corresponds with the findings of the present study .
several bacterial species 
 identified in gallbladder infections are among those that constitute normal intestinal flora 
 . therefore , it is possible that the cases of 
 gallbladder infection observed in the present study were due to ascending infection from the 
 duodenum .
the findings of component analysis of gallbladder contents from canine biliary sludge and 
 gallbladder mucoceles in the present study have demonstrated the previously unreported fact 
 that mucins are the principle components of gallbladder content in both pathophysiologies . 
 there were no differences in gallbladder composition among biliary sludge samples of various 
 severities .
additionally , the low rates of bacterial infection in both biliary sludge and 
 gallbladder mucocele samples indicated that these two disease differ in pathophysiology from 
 gallstones . from the results of this study
, it was considered that the pathophysiology of canine 
 biliary sludge may be occurred by progressive accumulation of mucin in the gallbladder . in 
 one of the dogs
included in the present study , gallbladder disease progressed from biliary 
 sludge to gallbladder mucocele ( fig .
these 
 findings suggest not only that biliary sludge and gallbladder mucoceles share a 
 pathophysiology involving the progressive accumulation of mucins , but also that biliary 
 sludge possibly represents the stage preceding gallbladder mucoceles . however , the factors 
 responsible for the transition of gallbladder contents from sludge to jelly - like appearance 
 in the present study are not clear . as a criterion for differentiating between the sludge 
 and jelly - like appearances of gallbladder contents , in the present study
, a resected 
 gallbladder with contents that were semisolid , low in liquid content and appeared molded to 
 the gallbladder was diagnosed as a gallbladder mucocele .
the water content of gallbladder 
 contents might be one of the factors responsible for the transition of gallbladder contents 
 from sludge to jelly - like appearance .
all of gallbladder contents in this study were dried 
 with bile and analyzed by ir spectroscopy .
generally , the components contained in bile are 
 bile acid , cholesterol , lecithin , bilirubin and more , and do not contain volatile components 
 .
therefore , these analyses by ir spectroscopy 
 were carried out in gallbladder contents which contained liquid components , and it was 
 considered that only the water content of gallbladder contents was changed by drying . 
 moisture in bile is removed by the actions of the na  k and 
 cl 
hco3 pumps of gallbladder epithelial cells [ 19 , 21 ] 
 and / or  based on reports in mice 
aquaporins , which are channels responsible for transporting 
 water into the body [ 18 , 24 ] .
these transport channels have been reported to possess the ability 
 to concentrate bile or manipulate the composition of gallbladder bile .
therefore , abnormal 
 functioning of bile moisture absorption mechanisms might be involved in the pathophysiology 
 of canine biliary sludge and gallbladder mucoceles . in this study , since the ir spectroscopy was a qualitative analysis method , it is necessary 
 to quantitatively analyze components of gallbladder content , such as amount of mucin and/or 
 cholesterol , in the future study .
it is not known whether biliary sludge is similar to 
 gallbladder mucoceles in terms of involvement of excessive mucin secretion from the 
 gallbladder mucosal epithelium . therefore
, future studies on histopathological evaluation of 
 gallbladder mucosal epithelium have been planned to investigate whether excessive mucin 
 secretion is involved in the pathophysiology of biliary sludge .
additionally , in the case of 
 gallbladder content transition with histological findings of severe gallbladder bleeding 
 observed in the present study , it is necessary to histopathologically investigate the 
 gallbladder and its principal components in detail . to conclude , the present findings have demonstrated that the principle components of both 
 biliary sludge and gallbladder mucoceles are mucins .
these findings suggest the possibility 
 that the two diseases are not independent of each other , but in fact , represent one 
 continuous disease .
further studies involving extensive data collection of gallbladder 
 contents from biliary sludge and gallbladder mucocele , and histopathological evaluation of 
 these gallbladder will be conducted to elucidate the pathophysiologies of gallbladder 
 mucoceles and biliary sludge .
 the pathophysiology of canine gallbladder diseases , including biliary sludge , gallbladder 
 mucoceles and gallstones , is poorly understood . 
 this study aimed to evaluate the component 
 of gallbladder contents and bacterial infection of the gallbladder in order to elucidate 
 the pathophysiology of biliary sludge and gallbladder mucoceles . 
 a total of 43 samples of 
 canine gallbladder contents ( biliary sludge , 21 and gallbladder mucoceles , 22 ) were 
 subjected to component analysis by infrared spectroscopy , and the resultant infrared 
 spectra were compared with that of swine mucin . of the 43 samples , 41 were also evaluated 
 by aerobic and anaerobic bacterial culture . 
 the contents of 20 ( 95.2% ) biliary sludge and 
 22 ( 100% ) gallbladder mucocele samples exhibited similar infrared spectra as swine mucin . 
 
 although biliary sludge and gallbladder mucocele contents exhibited similar infrared 
 spectra , one sample of biliary sludge ( 4.8% ) was determined to be composed of proteins . 
 
 the rate of bacterial infection of the gallbladder was 10.0% for biliary sludge and 14.3% 
 for gallbladder mucoceles . 
 almost all of the identified bacterial species were intestinal 
 flora . 
 these results indicate that the principal components of gallbladder contents in 
 both gallbladder mucoceles and biliary sludge are mucins and that both pathophysiologies 
 exhibit low rates of bacterial infection of the gallbladder . 
 therefore , it is possible 
 that gallbladder mucoceles and biliary sludge have the same pathophysiology , and , rather 
 than being independent diseases , they could possibly represent a continuous disease . 
 thus , 
 biliary sludge could be considered as the stage preceding the appearance of gallbladder 
 mucoceles . 
MATERIALS AND METHODS
RESULTS
Severity classification of canine biliary sludge
IR spectrum of swine mucin
IR spectra and component analysis of gallbladder contents
IR spectra of gallbladder contents of the same dog at multiple time points
Bacterial infection of the gallbladder
DISCUSSION