ORIGINAL RESEARCH

Reproducibility and Repeatability of Equine MuscleThickness Measurements with UltrasoundArno Lindner, DVM,a Raul Signorini, DVM,b Jorge Vassallo, DVM,b

Fabiana Tomatis, DVM,b Francis M. Flores, DVM,b Mar�ıa E. Gagliano, DVM,b

Jimena Curiotti, DVM,b and Evelina Terragona, DVMb

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ABSTRACT

This study examined the reproducibility and repeat-ability of muscle thickness (MT) measurements withultrasound for the following muscles: extensor carpiradialis, extensor digitorum longus (both flexed andextended), gluteus medius, longissimus lumborum,semitendinosus, and supraspinatus. Three examinersmeasured thickness of these muscles in five Thorough-breds on 3 consecutive days. The day of measurementdid not have any effect on the mean MT value of themuscles examined (P > .05). The left longissimus lum-borummuscle was the only muscle for which the secondmeasurement varied from the first and third (P < .05).The examiners had an effect on the mean coefficient ofvariation (CV) of the thickness of the flexed left extensorcarpi radialis and flexed left extensor digitorum longus.The daily measurements varied more in the case of oneof the examiners. Mean CVs higher or close to 10% werereported for both sides of the flexed extensor digitorumlongus and for the supraspinatus muscles. The lowestCV was calculated for the longissimus lumborum andthe extended semitendinosus (<5%). The largest dis-agreements between the examiners were observed forboth sides of the flexed extensor digitorum longus andthe supraspinatus (>10%–<20%). The best agreementwas measured for the extended extensor carpi radialisand the longissimus lumborum (>3%–<7%). The re-sults showed that for some muscles, it was difficult tolocate the exact anatomical site for taking the MT mea-surements. To reduce CV, it was suggested that onlyone examiner should take all the measurements andthe whole procedure must be such that it is as comfort-able as possible for this particular examiner.

Keywords: Muscle; Thickness; Ultrasound; Horse

rom the Arbeitsgruppe Pferd, Juelich, Germanya; and Facultad de Veterinaria,

niversidad del Litoral, Esperanza, Santa Fe, Argentineb.

eprint requests: Arno Lindner, DVM, Heinrich-Roettgen-Str. 20, D-52428 Juelich,

ermany.

737-0806/$ - see front matter

2010 Elsevier Inc. All rights reserved.

oi:10.1016/j.jevs.2010.10.007

ournal of Equine Veterinary Science � Vol 30, No 11 (2010)

INTRODUCTIONHorses of breeds used in equine sports activities are knownto have a greater muscle mass relative to body mass as com-pared with those that are not used in sports.1 Kearns et al2

found that horses with a tendency to run fast had an overallhigher skeletal muscle mass and a lower percentage of bodyfat. However, the skeletal muscle mass of horses cannot bedetermined routinely,3 and it would be helpful to havea noninvasivemeans to quantify on a regular basis the effectsof training programs on muscles and maybe even to defineparameters of athletic ability. Ultrasound (US) imaging isused to measure thickness and cross-sectional area of mus-cles in human beings, with the purpose of evaluating the ef-fect of training and rehabilitation measures as well as thedevelopment of medical conditions.4-8 US has been provento be sensitive enough to measure changes in body fat inhorses who are fed limited diets9; training for polo competi-tion9; competing in endurance,10 trot, and gallop racing2,3;and taking a repartitioning agent.11 The US measurementof rump fat in horses has been reported to be highly repeat-able, precise, accurate, and easy to perform.9,11,12

US has been used to measure muscle thickness (MT) inhorses to examine effects of Kaatsu,13,14 training,15 scinti-graphic imaging,16 positioning of muscle biopsy needles,17

and to study the relationship with performance.2 However,there are relatively few studies that have examined the repeat-ability and objectivity of the measurement ofMT with US inthe horse. Therefore, in this study, the thickness of severalmuscles in Thoroughbred horses was measured using USto calculate repeatability andobjectivity of themeasurements.

MATERIALS AND METHODS

HorsesFive Thoroughbred horses, three stallions and two geld-ings, ranging in ages from 4 to 6 years were used for thecurrent study. The body condition score of the horsesranged between 3 and 5.5.18 All horses were being usedfor flat racing.

EquipmentA Mindray 6600 Ultrasound machine (Digimed S.A.,Buenos Aires, Argentine) was used to scan the thickness

635

Table 1. Muscles measured and location of the site of measurements

Muscle Site of Measurement

Extensor carpi radialis 30 cm above themedial aspect of the tuberositas radii, holding the probe as much aspossible perpendicular to the latter

Extensor digitorum longus Approximately 30 cm from the tuberositas tibiae, applying the probe as much aspossible perpendicular to the latter. An attempt was made to measure at the sitewhere the muscle appeared to be the thickest

Gluteus medius Midway of the distance between the first coccigeal vertebrae and the tuber coxaeLongissimus lumborum 20 cm cranial to the tuber sacrale of the ilium, 3 cm off the midline of the backSemitendinosus 20 cm below the tuber ischiadicumSupraspinatus In front of the tuber spinae scapulae

636 A Lindner et al � Vol 30, No 11 (2010)

of the muscles. For optimal probe contact and signalpenetration, a US gel was applied directly on the skin aftershaving. The longissimus lumborum, semitendinosus, andgluteus medius muscles were measured with a 2-MHzconvex probe, whereas all the other muscles were measuredwith a 6-MHz convex probe. In comparison with the6-MHz probe, the 2-MHz convex probe increased the clar-ity and resolution of the images taken deep inside the thickermuscles.

Experimental ProcedureHorses were placed in a quiet area of the barn on firmground. Care was taken to prevent them from movingsignificantly; for this, two experienced horse handlerswere introduced, one restrained movement of the horses

Table 2. Repeatability and objectivity results of muscle thickconsecutive days (five horses)

Muscle

Effect

Examiner

Left extensor carpi radialis extended NSLeft extensor carpi radialis flexed NSLeft extensor digitorum longus extended NSLeft extensor digitorum longus flexed NSLeft gluteus medius NSLeft longissimus lumborum NS

Left semitendinosus NSLeft supraspinatus NSRight extensor carpi radialis extended NSRight extensor carpi radialis flexed NSRight extensor digitorum longus extended NSRight extensor digitorum longus flexed NSRight gluteus medius NSRight longissimus lumborum NSRight semitendinosus NSRight supraspinatus NS

NS, nonsignificant.

by holding its head and the other assisted the examiners.Three veterinarians took MT measurements in the horsesonce on 3 consecutive days at the same time.One veterinar-ian was a specialist in US imaging, whereas the other twohad gone through a 3-month training program conductedby the experienced person before engaging in the study.The muscles measured and a description of the anatom-

ical sites where the measurements were taken is shown inTable 1.Each examiner palpated the bony anatomical landmarks

and then using a chalk marked the site of measurement.The extensor carpi radialis and the extensor digitorumlongus muscles were measured in both the extended andflexed position. To measure the extensor carpi radialis inthe flexed position, the leg was lifted till the cannon bone

ness measurements taken by three examiners on 3

Fisher’s TestDay of Measurement

NSNSNSNSNS<0.05 First and second, <0.05;

second and third, <0.01NSNSNSNSNSNSNSNSNSNS

Table 3. Thickness of muscles of horses measured withultrasound (five horses � three examiners � threemeasurements per examiner)

Muscle Thickness (mm)a

Left extensor carpi radialisextended

57.5 � 10.19 [1.32]

Left extensor carpi radialisflexed

58.89 � 8.38 [1.25]

Left extensor digitorumlongus extended

29.71 � 3.72 [0.55]

Left extensor digitorumlongus flexed

24.62 � 6.20 [0.92]

Left gluteus medius 62.00 � 6.94 [1.03]Left longissimus lumborum 103.72 � 5.34 [0.80]Left semitendinosus 104.64 � 9.43 [1.41]Left supraspinatus 34.96 � 6.44 [0.96]Right extensor carpi radialis

extended55.67 � 9.73 [1.45]

Right extensor carpi radialisflexed

56.95 � 9.36 [1.39]

Right extensor digitorumlongus extended

30.35 � 4.48 [0.67]

Right extensor digitorumlongus flexed

23.69 � 5.01 [0.75]

Right gluteus medius 62.52 � 8.07 [1.20]Right longissimus

lumborum103.69 � 5.85 [0.87]

Right semitendinosus 106.02 � 9.73 [1.45]Right supraspinatus 33.98 � 5.75 [0.86]aMean � standard deviation [standard error].

A Lindner et al � Vol 30, No 11 (2010) 637

was parallel to the ground and the hoof was held up. Tomeasure the extensor digitorum longus in the flexed posi-tion, the leg was lifted to a position that was comfortablefor the individual who was holding the hoof and hisarms, legs, and back were maintained fully extended. Thisindividual assisted all the examiners. After the measure-ments were taken, the sites were cleaned and the nextexaminer took images of the muscles. Scanning was per-formed with the probe applied perpendicular to the tissueinterface. Minimal pressure was applied with the probe soas to avoid alterations of the thickness measurements.MT was determined using digital calipers to calculate the

distance between the superficial muscle fascia and the bone.

Statistical AnalysesData are shown as mean � standard deviation. Analysis ofvariance for repeated measurements was applied to deter-mine the effect of the day of measurement and the exam-iner. Fisher’s test was applied as a post hoc test whensignificant effects were found.The coefficient of variation (CV) for the three measure-

ments taken by each examiner for every muscle was

calculated. One-way analysis of variance was used to deter-mine whether there was an effect of the examiner on theCV. Once more, Fisher’s test was applied as a post hoctest when significant effects were found. P < .05 wasdefined as the level that denoted a significant difference.

RESULTSFor all the examiners, the day of measurement did nothave any effect on the mean value of MT (P > .05; Table2). The left longissimus lumborum was the only musclefor which the second measurement varied from the firstand the third. For both comparisons, values were higheron the second day (P < .05, Table 2).The average values of the thickness measurements of all

muscles are shown in Table 3.The examiners had an effect on themeanCV of the thick-

ness measurements of the left extensor carpi radialis andthe left extensor digitorum longus in the flexed position(Table 4). The daily measurements of examiner 1 variedmore than those of the other two examiners. Overall,mean CVs higher or close to 10% were found for both sidesof the extensor digitorum longus scanned in the flexedposition and the supraspinatus muscles. The lowest meanCVs were calculated for the longissimus lumborum andthe muscle semitendinosus, which were kept in the ex-tended position during the examination (Table 4).To determine the objectivity of the measurements, the

CV of the first measurement of each muscle for the threeexaminers was calculated. The largest CV was observedfor the extensor digitorum longus flexed and the supraspi-natus on both sides of the horses (Table 5). The best agree-ment was measured for the extensor carpi radialis held in anextended position and the longissimus lumborum muscles(Table 5).

DISCUSSIONUS measurements of MT can be made quickly, repeatedly,and in most horses safely without disrupting the horse’straining or conditioning program. This could allow for anobjective means to show effects of training on MT,15 andassess the relationship between muscle mass and perfor-mance.2 However, the repeatability of the measurementsneeds to be as low as possible to be able to measure minortraining effects and performance differences.In the current study, the MTmeasurements of most mus-

cles did not differ significantly between the examiners or be-tween their daily measurements. However, the CV variedwidely for the different muscles measured by an examinerand also to quite a remarkable extent between the differentexaminers. Erichsen et al16 described a CV range of 0% to10% for three examiners who measured the thickness of thegluteus medius muscle at two different sites on each side ofthe horse (mean of 1.77% and 2.04% for the left and right

Table 4. Mean CV for the daily ultrasound measurements (three in total) on muscle thickness taken by each examineron five horses

Muscle Effect of Examiner

CV of Each Examiner

1 2 3

Left extensor carpi radialis extended NS 5.57 14.46 4.40Left extensor carpi radialis flexed <0.01 7.70 4.90 2.56Left extensor digitorum longus extended NS 6.57 6.73 8.71Left extensor digitorum longus flexed <0.05 19.65 10.36 10.04Left gluteus medius NS 9.78 8.00 3.66Left longissimus lumborum NS 2.95 4.43 2.54Left semitendinosus NS 6.81 2.46 2.89Left supraspinatus NS 11.14 10.75 6.98Right extensor carpi radialis extended NS 7.08 6.50 6.87Right extensor carpi radialis flexed NS 8.60 5.50 3.86Right extensor digitorum longus extended NS 8.98 8.19 5.77Right extensor digitorum longus flexed NS 18.28 11.15 14.35Right gluteus medius NS 13.75 6.88 8.33Right longissimus lumborum NS 3.68 2.89 3.05Right semitendinosus NS 7.84 3.34 2.97Right supraspinatus NS 9.69 8.52 8.86

CV, coefficient of variation; NS, nonsignificant.

638 A Lindner et al � Vol 30, No 11 (2010)

sides, respectively). These data indicate that it would be bestto have only one individual to take all the measurements.For the flexed left extensor carpi radialis and flexed exten-

sor digitorum longus, the difference between the exam-iners was significant: Examiner 1 had markedly higherCVs in comparison with the others. This finding suggeststhat the more pronounced differences between the exam-iners are for muscles on the left side of the horses. Thismay be because all the examiners were right-handed, thusperhaps making it more difficult to position adequatelythe scanning head of the probe with the right hand onthe left side of the horse.It should also be noted that the examiner who was best

trained in US was the one with the highest CVs. Possiblecauses for this result, as discussed between the examiners,were that examiner 1 felt much more confident in the useof the US and simultaneously was less comfortable withhorses as compared with the other examiners. Both factorscould have reduced the scanning accuracy of examiner 1.Overall, the supraspinatus and the flexed extensor digito-

rum longus muscles had the highest CV with values of>10%. Therefore, it is possible that the exact location ofthe scanning site of the supraspinatus muscle in front of thetuber spinae scapulae was not as clear as assumed, whereasthe difficulties in scanning the flexed extensor digitorumlongus from below were obvious.Examiner 1 achieved a CV of <5% for only one muscle,

the longissimus dorsi on both sides of the horse. In addi-tion to the longissimus dorsi, examiner 2 reported a CVof <5% for the semitendinosus muscle. Examiner 3

achieved a CV of<5% for the aforementioned twomuscles,as well as the flexed extensor carpi radialis muscle and theleft gluteus medius muscle. In previous studies, the CVof MT measurements with US for the extensor digitorumcommunis has been calculated to be 1%,13 between 0%and 10% for gluteus medius16 (mean, 0%–4.3%), and 2%for the vastus lateralis and the extensor carpi radialis.2

However, none of those studies described any special mea-sures to reduce the CV of measurements. All the examinersin this study were veterinary surgeons; two were primarilyin equine practice and one (examiner 1) in academia. Theexaminers were selected to conduct the study because oftheir interest in the subject and willingness to be trainedin US scanning (examiners 2 and 3). It is quite possiblethat the examiners in previous studies2,13,16 were muchmore skilled than those working in this study. However,the results of this study clearly indicate that a very carefulapproach is required to obtain repeatable results. Otherthan the examiner, a minimum of two more individualsneed to be present during the study, one to hold thehead of the horse to keep it as still as possible, and the otherto assist the examiner in all means so that the examiner canconcentrate entirely on the positioning of the probe.On the basis of review of previously published data, it was

found that this is the first reported study on the thicknessmeasurements of the extensor digitorum longus, the long-issimus lumborum, the semitendinosus, and the supraspi-natus muscles of horses. The thickness of the extensorcarpi radialis measured in the current study is similar tothe values described by Kearns et al2 for Standardbreds,

Table 5. Mean CV of the measurements taken by threeexaminers of the thickness of different muscles of fivehorses

Muscle

CV for the FirstMeasurement by theThree ExaminersTogether

Left extensor carpi radialisextended

3.32

Left extensor carpi radialisflexed

9.19

Left extensor digitorumlongus extended

8.65

Left extensor digitorumlongus flexed

10.81

Left gluteus medius 7.75Left longissimus lumborum 4.43Left semitendinosus 5.93Left supraspinatus 13.43Right extensor carpi radialis

extended5.54

Right extensor carpi radialisflexed

8.57

Right extensor digitorumlongus extended

5.04

Right extensor digitorumlongus flexed

19.68

Right gluteus medius 7.83Right longissimus

lumborum3.62

Right semitendinosus 6.43Right supraspinatus 11.24

CV, coefficient of variation.

A Lindner et al � Vol 30, No 11 (2010) 639

but much lower than that for Thoroughbreds3 (70 mm).The competitive level of the Thoroughbreds in both stud-ies may provide an explanation for the reported difference.The Thoroughbreds measured by Kearns et al3 were eliteThoroughbreds, whereas those in this study were not.The gluteus medius muscle measurements obtained in

Swedish riding horses used for dressage, eventing, andshow jumping16 were approximately thicker by 2 cm ascompared with those obtained from the horses used inthis study, whereas the MT in French Standardbreds wassimilar.17 Valette et al17 showed that the thickness of thegluteus medius muscle was dependent not only on the lo-cation of the scanning head but also on the inclination ofthe hip. In Standardbreds with a straighter hip, the ventralcompartment of the gluteus medius muscle was on an aver-age 3 cm deeper than in horses with a wide hip. They foundthat the MT of male Standardbreds was thicker as com-pared with females, regardless of whether measurements

were made in the dorsal or ventral compartment of themuscle. Age also affects the depth of the gluteus mediusmuscle: horses aged >3 years had a slightly slimmer MTof the gluteus medius.17

In conclusion, care has to be taken to maintain a low CVof MTmeasurements done by US. Keeping the repeatabil-ity of MT at <5% seems to be easiest for the longissimuslumborum and the semitendinosus, whereas the CVs ofthe extensor carpi radialis, the extensor digitorum longus,and the gluteus medius are most likely to be higher. Thethickness measurements of the supraspinatus and the flexedextensor carpi radialis as well as the flexed extensor digito-rum longus muscles would quite likely vary by a lot to be ofpractical benefit.

ACKNOWLEDGMENTS

The authors thank Holly M. Greene for revising the manu-script.

REFERENCES

1. Gunn HM. Muscle, bone and fat proportions and the muscle distri-

bution of Thoroughbreds and other horses. In: Gillespie JR,

Robinson NE, eds. Equine exercise physiology 2: Proceedings of

the 2nd International Conference of the Equine Exercise Physiology.

Davis, CA: ICEEP Publications; 1987:253–264.

2. Kearns CF, McKeever KH, Kumagai K, Abe T. Fat-free mass is re-

lated to one-mile race performance in elite standardbred horses.

Vet J 2002;163:260–266.

3. Kearns CF, McKeever KH, Abe T. Overview of horse body composi-

tion and muscle architecture: implications for performance. Vet J

2002;164:224–234.

4. Bemben MG. Use of diagnostic ultrasound for assessing muscle size.

J Strength Cond Res 2002;16:103–108.

5. Perkin HM, Bond EA, Thompson J, Woods EC, Smith C. Realtime

ultrasound: an objective measure of skeletal muscle. Phys Ther Rev

2003;8:99–108.

6. ReevesND,Maganaris CN,NariciMV.Ultrasonographic assessment

of human skeletal muscle size. Eur J Appl Physiol 2004;91:116–118.

7. Severinsen K, Andersen H. Evaluation of atrophy of foot muscles in

diabetic neuropathy—a comparative study of nerve conduction stud-

ies and ultrasonography. Clin Neurophysiol 2007;118:2172–2175.

8. Uremovi M, Bosnjak PM, Seric V, Vargek Solter V, Budic R,

Bosnjak B, et al. Ultrasoundmeasurement of the volume of musculus

quadriceps after knee joint injury. Coll Antropol 2004;28(Suppl 2):

227–233.

9. Westervelt RG, Stouffer RG, Hintz HF, Schryver HF. Estimating

fatness in horses and ponies. J Anim Sci 1976;43:781–785.

10. Lawrence LM, Jackson S, Kline K, Moser L, Biel M. Observations on

body weight and condition of horses in a 150-mile endurance ride.

J Equine Vet Sci 1992;12:320–324.

11. Kearns CF, McKeever KH, Malinowski K, Struck MB, Abe T.

Chronic administration of therapeutic levels of clenbuterol acts as

a repartitioning agent. J Appl Physiol 2001;91:2064–2070.

640 A Lindner et al � Vol 30, No 11 (2010)

12. Kane RA, Fisher M, Parrett D, Lawrence LM. Estimating fatness in

horses. In: Proceedings of the 10th Equine Nutrition and Physiology

Symposium; Fort Collins, CO; 1987:127–131.

13. Abe T, Kearns CF, Manso Filho HC, Sato Y, Sleeper M,

McKeever KH. Acute vascular occlusion in horses: effects on skeletal

muscle size and blood flow. Equine Comp Exerc Physiol 2004;1:

239–243.

14. Abe T, Kearns CF, Manso Filho HC, Sato Y, McKeever KH. Muscle,

tendon, and somatotropin responses to the restriction of muscle

blood flow induced by KAATSU-walk training. Equine Vet J Suppl

2006;36:345–348.

15. Lindner A, Signorini R, Brero L, Arn E,Mancini R, Enrique A. Effect

of conditioning horses one, two or three times a week with 2 exercise

bouts over 100m at nearmaximal speed on the thickness ofMusculus

supraspinalis and extensor carpi radialis. In: Proceedings of the

Equine Science Society; Tucson, Arizona; 2005:114–116.

16. Erichsen C, Eksell P, Widstr€omC, BergerM, Holm KR, Johnston C.

Scintigraphy of the sacroiliac joint region in asymptomatic riding

horses: scintigraphic appearance and evaluation of method. Vet

Radiol Ultrasound 2003;44:699–706.

17. Valette JP, Barrey E, Jouglin M, Courouce A, Auvinet B, Flaux B.

Standardisation of muscular biopsy of gluteus medius in French trot-

ters. Equine Vet J Suppl 1999;30:342–344.

18. Henneke DR, Potter GD, Kreider JL, Yeates BF. Relationship be-

tween body condition score, physical measurement, and body fat

percentage in mares. Equine Vet J 1983;15:371–372.