animals : the left hind limbs of 10 ( male , n=8 ; female , n=2 ) clinically 
 healthy beagle dogs ( age , 19 years ; weight , 8.414.2 kg ; and body condition scores were 3 
 out of 5 ) were assessed . the institute of experimental animal sciences of tokyo university of 
 agriculture and technology approved the study protocol ( permit number ; 2669 ) , and all 
 animals were maintained in strict accordance with the recommendations of the guide for the 
 care and use of laboratory animals of the national institutes of health . study protocol : ultrasonography and mri were conducted on different days , 
 but within 3 days . images were taken in 5 different sections , dividing the femur equally 
 into 6 parts between the greater trochanter of the femur and the proximal point of the 
 patella . 1.slice number and scan location for measurement of muscle cross - sectional area and 
 thickness by mri ( left image ) and ultrasonography ( right picture ) . scans were taken 
 from 5 different sections dividing the limb equally into 6 parts between the greater 
 trochanter of the femur ( indicated by a yellow x ) and the proximal point of the 
 patella ( indicated by a red x ) . slice numbers were designated as 15 from proximal to 
 distal . ) . in these slices , qf , bf , st and sm muscles were evaluated for muscle csa and 
 thickness by mri and for muscle thickness by ultrasound . slice number and scan location for measurement of muscle cross - sectional area and 
 thickness by mri ( left image ) and ultrasonography ( right picture ) . scans were taken 
 from 5 different sections dividing the limb equally into 6 parts between the greater 
 trochanter of the femur ( indicated by a yellow x ) and the proximal point of the 
 patella ( indicated by a red x ) . all dogs were 
 subcutaneously injected with 40 g / kg atropine sulfate ( atropine sulfate 
 injection 0.5 mg ; mitsubishi tanabe pharma , osaka , japan ) . anesthesia was induced by 
 intravenous bolus administration of 6 mg / kg propofol ( rapinovet ; intervet , tokyo , japan ) , 
 and a tracheal tube was inserted . anesthesia was maintained by isoflurane inhalation 
 ( forane ; abbott japan , tokyo , japan ) mixed with oxygen during mri . transverse images of the left femoral muscle were obtained by mri using a knee coil 
 ( airis- comfort 0.3 t ; hitachi medical , tokyo , japan ) . all images were scanned in a supine 
 position in the coil with both knees extended to 135. we imagined a femur and tibia and 
 made a template in 135 by cardboard . mri markers ( sunflower oil packaged food ; kobayashi pharmaceutical , osaka , japan ) 
 were set on the body surface at the left greater trochanter of the femur and the proximal 
 point of the patella . the left femoral muscle was imaged in the sagittal plane with a 
 t1-weighted image sequence ( spin - echo sequence : repetition time ( tr ) , 400 ms ; echo time 
 ( te ) , 25 ms ; flip angle , 90 ; number or excitations ( nex ) , 4 ; slice thickness , 2.0 mm ; 
 interslice gap , 6.0 mm ; number of slices , 10 ; field of view ( fov ) , 200 mm ; and matrix , 320  
 224 ) as a positioning imaging ( fig . transverse 
 images were taken from 5 different sections dividing the length between the 2 markers into 6 
 equal parts , with a t1-weighted image sequence ( spin - echo sequence : tr , 400 ms ; te , 25 ms ; 
 flip angle , 90 ; nex , 4 ; slice thickness , 4 mm and 5 mm ; interslice gap , 15.516.5 mm ; fov , 
 200 mm ; and matrix , 320  224 ) and a contrast - enhanced t1-weighted image sequence ( spin - echo 
 sequence : tr , 400 ms ; te , 25 ms ; flip angle , 90 ; nex , 4 ; slice thickness , 4 mm ; interslice 
 gap , 15.516.5 mm ; fov , 200 mm ; and matrix , 320  224 ) . gadodiamide was used as the contrast 
 medium with intravenous administration of 0.2 mg / kg meglumine gadopentetate ( magnevist ; 
 bayer , osaka , japan ) without a pressure injector . ultrasound imaging : muscle thickness was determined using a b - mode 
 ultrasound system ( prosound f75 premier ; hitachi - aloka medical , tokyo , japan ) with a 10-mhz 
 linear transducer ( ust-5415 ; hitachi - aloka medical ) . scans were taken in conscious dogs , but 
 sedation with a subcutaneous injection of 0.1 mg / kg butorphanol tartrate ( vetorphale ; meiji 
 seika pharma , tokyo , japan ) was applied if necessary . each dog was placed in the right 
 lateral recumbent position with the left knee extended to 135. we confirmed the angle of 
 the knee using the same template for mri . to 
 avoid muscle compression , a generous amount of gel was used under the transducer . the 
 cross - sectional planes of each muscle were scanned in the same lines as for mri ( fig . , 2 sonographers measured 
 the muscle thickness of the measuring sites ( 20 sites ) of 1 dog to confirm that muscle 
 thickness could be measured precisely using ultrasound . measurement and calculation by mri and ultrasound : from each transverse 
 image of the left femoral muscle by mri , an outline of each muscle was traced , and the csa 
 was calculated . among the t1-weighted or contrast - enhanced t1-weighted images , we selected 
 images in which it was easy to distinguish the outline of muscles . from each transverse 
 image by mri and each cross - sectional image by ultrasound , muscle thickness was measured at 
 the maximal vertical thickness in each muscle . qf muscle thickness was measured passing 
 through the center of the rectus femoris muscle . the trace and thickness were digitized 
 using software ( osirix ; newton graphics , sapporo , japan ) . statistics : the results are expressed as mean  standard deviation ( sd ) . the correlation between muscle csa and muscle thickness as measured by mri was analyzed 
 using pearson s product - moment correlation coefficient in each slice . the correlation 
 between muscle thickness as measured by mri and muscle thickness as measured by 
 ultrasonography was then analyzed in those slices showing a correlation between muscle csa 
 and muscle thickness as measured by mri . we compared the differences of both measurement 
 procedures with muscle thickness using the bland - altman comparison . finally , the correlation 
 between muscle csa as calculated by mri and muscle thickness as measured by ultrasonography 
 was analyzed in those slices showing a correlation in the second step . all data were 
 statistically analyzed using graphpad prism version 5.0 ( graphpad software , la jolla , ca , 
 u.s.a . ) . the intraclass 
 correlation coefficient for muscle thickness as measured using ultrasound was statistically 
 analyzed using ibm spss statistics 20.0 ( ibm spss statistics server ) . the intraclass correlation coefficient ( icc ( 2.1 ) ) was 0.948 based on the measurements of 2 
 sonographers for the muscle thickness of 20 sites of 1 dog . values of muscle csa as measured by mri , as well as muscle thickness as measured by mri and 
 ultrasound , are shown in table 1table 1.muscle cross - sectional areas and thickness as measured by mri and 
 ultrasonographyslice 1slice 2slice 3slice 4slice 5qfcsa ( cm)10.5  2.011.9  2.110.8  1.87.5  1.83.6  1.2mtmr ( mm)29.2  3.327.2  4.123.2  4.316.7  3.611.0  2.2mtus ( mm)34.0  3.531.3  3.328.0  2.719.9  4.112.4  2.0bfcsa ( cm)5.4  1.26.4  1.37.4  1.57.9  1.55.8  1.7mtmr ( mm)14.3  2.015.8  2.517.8  3.518.2  2.912.6  3.4mtus ( mm)12.6  2.014.6  2.217.6  3.116.6  2.513.0  1.4stcsa ( cm)3.7  1.24.7  0.94.5  0.74.5  0.93.8  1.0mtmr ( mm)15.5 2.015.5  1.116.1  1.717.2  2.116.5  3.6mtus ( mm)14.2  1.614.4  2.014.0  2.515.0  1.814.5  2.3smcsa ( cm)1.8  0.56.0  0.86.5  0.94.9  1.13.2  1.0mtmr ( mm)11.3  1.222.0  2.724.3  2.821.8  4.116.9  2.1mtus ( mm)15.0  1.919.1  2.421.2  2.119.7  1.417.7  1.8data are expressed as mean  sd . csa , muscle cross - sectional area ; mtmr , 
 muscle thickness as measured by mri ; mtus , muscle thickness as measured by 
 ultrasonography ; qf , quadriceps femoris muscle ; bf , biceps femoris muscle ; st , 
 semitendinosus muscle ; sm , semimembranosus muscle .. t1-weighted images with a slice thickness of 4 mm were most suitable for 
 measurement , because the muscle outlines were more clearly depicted ( fig . 2fig . 2.transverse images of femoral muscles to measure muscle cross - sectional area by mri . green lines show the outlines of each femoral muscle : quadriceps femoris ( qf ) muscle ; 
 biceps femoris ( bf ) muscle ; semitendinosus ( st ) muscle ; and semimembranosus ( sm ) 
 muscle . t1-weighted imaging with a slice thickness of 4 mm was most suitable for 
 measurement , because the muscle outlines were clearly depicted . ) . however , identification of the vastus medialis , vastus intermedius and vastus 
 lateralis muscles was difficult in the qf muscle group . in the sm muscle , identification of 
 muscle outlines was also difficult , because little connective tissue was present among the 
 st and adductor muscles in slices 24 . muscle contrast was increased in the t1-weighted 
 images with a slice thickness of 5 mm . in the contrast - enhanced t1-weighted images , the 
 muscle signal became higher , and the outlines of the muscle became clearer . therefore , when 
 we could not identify the outlines of the sm or st muscles , we used t1-weighted images with 
 a slice thickness of 5 mm or contrast - enhanced t1-weighted images ( fig . 3fig . 3.transverse images of semimembranosus ( sm ) muscles in slice 4 by mri . in the 
 t1-weighted images ( t1wi ) with a slice thickness of 4 mm , identification of the muscle 
 outline of sm from the semitendinosus ( st ) muscle and adductor ( ad ) muscle was 
 difficult ( yellow arrowhead ) . in the t1-weighted images with a slice thickness of 5 
 mm , muscle contrast was increased ( red arrowhead ) . in the contrast - enhanced 
 t1-weighted images ( t1wi - c ) , the mean amount of time required for mri was 29.3 min ( range , 2247 min ) . csa , muscle cross - sectional area ; mtmr , 
 muscle thickness as measured by mri ; mtus , muscle thickness as measured by 
 ultrasonography ; qf , quadriceps femoris muscle ; bf , biceps femoris muscle ; st , 
 semitendinosus muscle ; sm , semimembranosus muscle . transverse images of femoral muscles to measure muscle cross - sectional area by mri . green lines show the outlines of each femoral muscle : quadriceps femoris ( qf ) muscle ; 
 biceps femoris ( bf ) muscle ; semitendinosus ( st ) muscle ; and semimembranosus ( sm ) 
 muscle . t1-weighted imaging with a slice thickness of 4 mm was most suitable for 
 measurement , because the muscle outlines were clearly depicted . transverse images of semimembranosus ( sm ) muscles in slice 4 by mri . in the 
 t1-weighted images ( t1wi ) with a slice thickness of 4 mm , identification of the muscle 
 outline of sm from the semitendinosus ( st ) muscle and adductor ( ad ) muscle was 
 difficult ( yellow arrowhead ) . in the t1-weighted images with a slice thickness of 5 
 mm , muscle contrast was increased ( red arrowhead ) . in the contrast - enhanced 
 t1-weighted images ( t1wi - c ) , the muscle signal became higher ( red arrowhead ) . during ultrasonography , 3 dogs that could not maintain a right lateral recumbent position 
 required sedation . qf muscle thickness was measured passing through the 
 center of the rectus femoris ( rf ) muscle from the muscle surface to the femoral bone 
 ( fb ) . horizontal stripes in slices 4 and 5 represent artifacts due to failure to 
 maintain contact between the body and transducer . vl , vastus lateralis muscle ; vm , 
 vastus medialis muscle ; vi , vastus intermedius muscle . muscle fibers appeared hypoechoic , and perimysia and fascia appeared hyperechoic . the 
 qf and bf muscles were easy to scan in all slices , because they were closest to the skin . the st and sm muscles in the proximal slices of the femur were difficult to scan , because 
 the interface between the body and transducer was not flat . the sm muscle in the distal 
 slices was also difficult to scan , because the muscle finished medial to the tibia and the 
 echo was attenuated . the st muscle was 
 imaged caudal to the sm in slices 1 and 2 and between the bf and sm muscles at a deep site 
 in slices 35 . the mean amount of time required for ultrasonography was 16 min ( range , 1130 
 min ) . qf muscle thickness was measured passing through the 
 center of the rectus femoris ( rf ) muscle from the muscle surface to the femoral bone 
 ( fb ) . horizontal stripes in slices 4 and 5 represent artifacts due to failure to 
 maintain contact between the body and transducer . vl , vastus lateralis muscle ; vm , 
 vastus medialis muscle ; vi , vastus intermedius muscle . correlation coefficients between muscle csa and muscle thickness as measured by mri are 
 shown in table 2table 2.correlation coefficient between muscle cross - sectional area and diameter as 
 measured by mrislice 1slice 2slice 3slice 4slice 5qf0.7570.7800.6670.7740.521bf0.7630.6900.7760.8840.405st0.8340.6320.6440.8250.887sm0.6360.5710.6630.6300.860a ) p<0.05 , b ) p<0.01 , c ) 
 p<0.001 . qf , quadriceps femoris muscle ; bf , biceps femoris muscle ; 
 st , semitendinosus muscle ; sm , semimembranosus muscle .. correlations were obtained in slices 14 in the qf and bf muscles , slices 15 
 in the st muscle and slices 1 , 3 and 5 in the sm muscle . correlation coefficients between 
 muscle thickness as measured by mri and ultrasound in the slices for which a correlation was 
 obtained between muscle csa and muscle thickness as measured by mri are shown in table 3table 3.correlation coefficient between muscle thickness as measured by mri and muscle 
 thickness as measured by ultrasonographyslice 1slice 2slice 3slice 4slice 5qf0.7010.8570.7510.486bf0.3850.9340.9700.638st0.1400.7900.6530.2770.229sm0.0080.5430.126a ) p<0.05 , b ) p<0.01 , c ) 
 p<0.001 . qf , quadriceps femoris muscle ; bf , biceps femoris muscle ; 
 st , semitendinosus muscle ; sm , semimembranosus muscle .. correlations were obtained in slices 13 in the qf muscle , slices 24 in the 
 bf muscle and slices 2 and 3 in the st muscle . bland - altman plots presenting differences between the 2 measurement procedures and muscle 
 thickness for these slices are given in fig . 5fig . differences in muscle thickness measurements between mri 
 and ultrasonography for : a ) quadriceps femoris ( qf ) at slices 13 ; b ) biceps femoris 
 ( bf ) at slices 24 ; and c ) semitendinosus ( st ) at slices 2 and 3 . solid circles indicate values from 1 dog .. these comparisons indicated that the thickness of the qf muscle measured by 
 ultrasound was overestimated compared with that measured by mri , as well as that the 
 thicknesses of the bf and st muscles measured by ultrasound were underestimated compared 
 with those measured by mri . however , except for 1 measurement in slice 3 of the qf muscle 
 and 1 measurement in slice 4 of the bf muscle , the values remained within the 95% confidence 
 intervals . correlation coefficients between muscle csa as measured by mri and muscle 
 thickness as measured by ultrasonography are shown in table 4table 4.correlation coefficient between muscle cross - sectional area as measured by mri 
 and muscle thickness as measured by ultrasonographyslice 1slice 2slice 3slice 4slice 5qf0.6910.8780.737bf0.6510.7740.671st0.6350.359sma ) p<0.05 , b ) p<0.01 , c ) 
 p<0.001 . qf , quadriceps femoris muscle ; bf , biceps femoris muscle ; 
 st , semitendinosus muscle ; sm , semimembranosus muscle .. correlations were obtained in slices 13 in the qf muscle , slices 24 in the 
 bf muscle and slice 2 in the st muscle . qf , quadriceps femoris muscle ; bf , biceps femoris muscle ; 
 st , semitendinosus muscle ; sm , semimembranosus muscle . qf , quadriceps femoris muscle ; bf , biceps femoris muscle ; 
 st , semitendinosus muscle ; sm , semimembranosus muscle . bland - altman comparisons . differences in muscle thickness measurements between mri 
 and ultrasonography for : a ) quadriceps femoris ( qf ) at slices 13 ; b ) biceps femoris 
 ( bf ) at slices 24 ; and c ) semitendinosus ( st ) at slices 2 and 3 . qf , quadriceps femoris muscle ; bf , biceps femoris muscle ; 
 st , semitendinosus muscle ; sm , semimembranosus muscle . this study was performed to validate the use of ultrasonography as a clinical tool for 
 measuring muscle thickness in dogs . muscle thickness as measured by ultrasound was found to 
 be accurate and correlated well with measurements from mri . furthermore , the thicknesses of 
 the qf , bf and st muscles as measured by ultrasonography at slices 13 ( from the proximal 
 end to the middle of the femur ) , 24 ( middle of the femur ) and 2 ( more proximal than the 
 middle of the femur ) , respectively , correlated with muscle csa as measured by mri . these 
 sites were easy to scan , because the interface between the body and transducer was flat , and 
 measurements at these sites were stable . because these sites represented the belly muscle , 
 where muscle csa and muscle thickness were large , the changes of these parameters might have 
 been smaller than those of other sites . in humans , the csa of the qf muscle has been 
 measured by ultrasonography at a point halfway between the greater trochanter and lateral 
 joint line of the knee   and at 30%50% of the 
 distance from the superior border of the patella to the medial aspect of the anterior 
 superior iliac spine . measurements of the qf 
 muscle thickness by ultrasonography have also been performed at the halfway point between 
 the epicondylus lateralis and trochanter major of the femur [ 24 , 25 ] , mid - thigh , and 45% of the distance between the popliteal crease and the greater 
 trochanter   in humans . the present canine study 
 showed that the measurement of muscle thickness for the qf muscle is suitable at 16.7%50% 
 of the distance from the greater trochanter to the proximal point of the patella , but the 
 results also revealed that sites near the belly muscle are most suitable for 
 ultrasonographic measurements . conversely , muscles in which csa and muscle shape were irregular showed no correlation 
 between muscle csa and thickness according to mri , or between muscle thickness as measured 
 by mri and ultrasonography . moreover , the proximal and distal sites of the femur correspond 
 approximately to the muscle - tendon junctions , which could have influenced measurements . in 
 humans , muscle csa in the lower limbs varies with changes in the distribution of tissue 
 fluid in the supine and standing positions . positional differences between mri and ultrasonography could thus be related to differences 
 in the measured values . according to the bland - altman plots , the qf muscle diameter at 
 slices 13 was overestimated by ultrasonography to a greater extent than by mri . we scanned 
 the qf muscle at these slices from the lateral side of the femur . when we scanned 
 immediately above the rectus femoris muscle , the measurement was complicated by the deep 
 location of the femur . the angle between the direction of the ultrasonic scan and the 
 measurement line was increased , causing measurement error . in addition , ultrasonography 
 underestimated st muscle diameter at slices 2 and 3 compared with mri . the scan site at 
 these slices was located at a transition from the lateral side to the caudal femur . the 
 interface between the body and transducer was not flat at this point , and the muscle might 
 therefore have been compressed . a compound scanning technique is used when the entire muscle cross - section can not be 
 encompassed [ 21 , 24 ] . however , this technique is relatively complicated . weiss criticized the 
 assessment of muscle csa by compound scanning , because distortions of the muscle tissue are 
 inevitable . if muscle function can be estimated 
 from muscle thickness as measured by ultrasonography in dogs , this technique would thus be 
 useful in the clinical field . the amount of time required for the assessment of muscle thickness by ultrasonography was 
 relatively long when measuring all 20 sites examined in the present study . however , we have 
 identified measuring sites where evaluation by ultrasonography is convenient . therefore , the 
 thicknesses of the sites and muscles indicated by the present study can be measured 
 relatively quickly . it is important to confirm the precision and reliability of muscle thickness measurements 
 using ultrasound . the intraclass correlation coefficient ( icc ( 2.1 ) ) 
 was 0.948 , indicating high reliability . this study revealed that ultrasonography offers a useful tool for measuring muscle 
 thickness in the femoral region , particularly that of the qf , bf and st muscles . however , we 
 must confirm the applicability of this assessment method to dogs of various breeds , sizes , 
 ages and muscle pathologies . further studies are also needed to determine whether 
 ultrasonography can be applied to assess muscle function by measuring muscle thickness .
assessment of muscle mass is important for evaluating muscle function and rehabilitation 
 outcomes . 
 ultrasound has recently been successfully used to estimate muscle mass in humans 
 by measuring muscle thickness . 
 this study attempted to standardize procedures for 
 measuring femoral muscle thickness ultrasonographically , as well as quantify the 
 reliability and validity of ultrasound evaluations of muscle thickness compared to 
 measurements made by magnetic resonance imaging ( mri ) in dogs . 
 we evaluated the quadriceps 
 femoris ( qf ) , biceps femoris ( bf ) , semitendinosus ( st ) and semimembranosus ( sm ) muscles of 
 10 clinically healthy beagle dogs . 
 scans were taken in 5 different sections divided 
 equally between the greater trochanter and proximal patella . 
 mri was performed , followed 
 by t1-weighted and contrast - enhanced t1-weighted imaging . 
 muscle cross - sectional area 
 ( csa ) was measured with mri , and muscle thickness was measured with mri and 
 ultrasonography . 
 the thickness of the qf , bf and st muscles as measured by ultrasound at 
 slices 13 ( from the proximal end to the middle of the femur ) , 24 ( middle of the femur ) 
 and 2 ( more proximal than the middle of the femur ) , respectively , was correlated with 
 muscle thickness and csa as measured by mri . 
 these sites showed a flat interface between 
 muscle and transducer and were situated over belly muscle . 
 no correlation between 
 measurement types was seen in sm muscle . 
 we must confirm this assessment method for 
 various breeds , sizes , ages and muscle pathologies in dogs , thereby confirming that muscle 
 thickness as measured ultrasonographically can reflect muscle function .