- Email: [email protected]
Reproducibility and Repeatability of Equine Muscle Thickness Measurements with Ultrasound
ORIGINAL RESEARCH Reproducibility and Repeatability of Equine Muscle Thickness Measurements with Ultrasound Arno Lindner, DVM,a Raul Signorini, DVM,b Jorge Vassallo, DVM,b Fabiana Tomatis, DVM,b Francis M. Flores, DVM,b Marıa E. Gagliano, DVM,b Jimena Curiotti, DVM,b and Evelina Terragona, DVMb
ABSTRACT This study examined the reproducibility and repeatability of muscle thickness (MT) measurements with ultrasound for the following muscles: extensor carpi radialis, extensor digitorum longus (both flexed and extended), gluteus medius, longissimus lumborum, semitendinosus, and supraspinatus. Three examiners measured thickness of these muscles in five Thoroughbreds on 3 consecutive days. The day of measurement did not have any effect on the mean MT value of the muscles examined (P > .05). The left longissimus lumborum muscle was the only muscle for which the second measurement varied from the first and third (P < .05). The examiners had an effect on the mean coefficient of variation (CV) of the thickness of the flexed left extensor carpi radialis and flexed left extensor digitorum longus. The daily measurements varied more in the case of one of the examiners. Mean CVs higher or close to 10% were reported for both sides of the flexed extensor digitorum longus and for the supraspinatus muscles. The lowest CV was calculated for the longissimus lumborum and the extended semitendinosus (<5%). The largest disagreements between the examiners were observed for both sides of the flexed extensor digitorum longus and the supraspinatus (>10%–<20%). The best agreement was measured for the extended extensor carpi radialis and the longissimus lumborum (>3%–<7%). The results showed that for some muscles, it was difficult to locate the exact anatomical site for taking the MT measurements. To reduce CV, it was suggested that only one examiner should take all the measurements and the whole procedure must be such that it is as comfortable as possible for this particular examiner. Keywords: Muscle; Thickness; Ultrasound; Horse
From the Arbeitsgruppe Pferd, Juelich, Germanya; and Facultad de Veterinaria, Universidad del Litoral, Esperanza, Santa Fe, Argentineb. Reprint requests: Arno Lindner, DVM, Heinrich-Roettgen-Str. 20, D-52428 Juelich, Germany. 0737-0806/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2010.10.007
Journal of Equine Veterinary Science Vol 30, No 11 (2010)
INTRODUCTION Horses of breeds used in equine sports activities are known to have a greater muscle mass relative to body mass as compared with those that are not used in sports.1 Kearns et al2 found that horses with a tendency to run fast had an overall higher skeletal muscle mass and a lower percentage of body fat. However, the skeletal muscle mass of horses cannot be determined routinely,3 and it would be helpful to have a noninvasive means to quantify on a regular basis the effects of training programs on muscles and maybe even to define parameters of athletic ability. Ultrasound (US) imaging is used to measure thickness and cross-sectional area of muscles in human beings, with the purpose of evaluating the effect of training and rehabilitation measures as well as the development of medical conditions.4-8 US has been proven to be sensitive enough to measure changes in body fat in horses who are fed limited diets9; training for polo competition9; competing in endurance,10 trot, and gallop racing2,3; and taking a repartitioning agent.11 The US measurement of rump fat in horses has been reported to be highly repeatable, precise, accurate, and easy to perform.9,11,12 US has been used to measure muscle thickness (MT) in horses to examine effects of Kaatsu,13,14 training,15 scintigraphic 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 repeatability and objectivity of the measurement of MT with US in the horse. Therefore, in this study, the thickness of several muscles in Thoroughbred horses was measured using US to calculate repeatability and objectivity of the measurements.
MATERIALS AND METHODS Horses Five Thoroughbred horses, three stallions and two geldings, ranging in ages from 4 to 6 years were used for the current study. The body condition score of the horses ranged between 3 and 5.5.18 All horses were being used for flat racing. Equipment A Mindray 6600 Ultrasound machine (Digimed S.A., Buenos Aires, Argentine) was used to scan the thickness
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Table 1. Muscles measured and location of the site of measurements Muscle
Site of Measurement
Extensor carpi radialis
30 cm above the medial aspect of the tuberositas radii, holding the probe as much as possible perpendicular to the latter Approximately 30 cm from the tuberositas tibiae, applying the probe as much as possible perpendicular to the latter. An attempt was made to measure at the site where the muscle appeared to be the thickest Midway of the distance between the first coccigeal vertebrae and the tuber coxae 20 cm cranial to the tuber sacrale of the ilium, 3 cm off the midline of the back 20 cm below the tuber ischiadicum In front of the tuber spinae scapulae
Extensor digitorum longus
Gluteus medius Longissimus lumborum Semitendinosus Supraspinatus
of the muscles. For optimal probe contact and signal penetration, a US gel was applied directly on the skin after shaving. The longissimus lumborum, semitendinosus, and gluteus medius muscles were measured with a 2-MHz convex probe, whereas all the other muscles were measured with a 6-MHz convex probe. In comparison with the 6-MHz probe, the 2-MHz convex probe increased the clarity and resolution of the images taken deep inside the thicker muscles. Experimental Procedure Horses were placed in a quiet area of the barn on firm ground. Care was taken to prevent them from moving significantly; for this, two experienced horse handlers were introduced, one restrained movement of the horses
by holding its head and the other assisted the examiners. Three veterinarians took MT measurements in the horses once on 3 consecutive days at the same time. One veterinarian was a specialist in US imaging, whereas the other two had gone through a 3-month training program conducted by the experienced person before engaging in the study. The muscles measured and a description of the anatomical sites where the measurements were taken is shown in Table 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 digitorum longus muscles were measured in both the extended and flexed position. To measure the extensor carpi radialis in the flexed position, the leg was lifted till the cannon bone
Table 2. Repeatability and objectivity results of muscle thickness measurements taken by three examiners on 3 consecutive days (five horses) Effect Muscle
Examiner
Day of Measurement
Left extensor carpi radialis extended Left extensor carpi radialis flexed Left extensor digitorum longus extended Left extensor digitorum longus flexed Left gluteus medius Left longissimus lumborum
NS NS NS NS NS NS
NS NS NS NS NS <0.05
Left semitendinosus Left supraspinatus Right extensor carpi radialis extended Right extensor carpi radialis flexed Right extensor digitorum longus extended Right extensor digitorum longus flexed Right gluteus medius Right longissimus lumborum Right semitendinosus Right supraspinatus
NS NS NS NS NS NS NS NS NS NS
NS NS NS NS NS NS NS NS NS NS
NS, nonsignificant.
Fisher’s Test
First and second, <0.05; second and third, <0.01
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Table 3. Thickness of muscles of horses measured with ultrasound (five horses three examiners three measurements per examiner) Muscle Left extensor carpi radialis extended Left extensor carpi radialis flexed Left extensor digitorum longus extended Left extensor digitorum longus flexed Left gluteus medius Left longissimus lumborum Left semitendinosus Left supraspinatus Right extensor carpi radialis extended Right extensor carpi radialis flexed Right extensor digitorum longus extended Right extensor digitorum longus flexed Right gluteus medius Right longissimus lumborum Right semitendinosus Right supraspinatus a
Thickness (mm)a
637
calculated. One-way analysis of variance was used to determine whether there was an effect of the examiner on the CV. Once more, Fisher’s test was applied as a post hoc test when significant effects were found. P < .05 was defined as the level that denoted a significant difference.
57.5 10.19 [1.32] 58.89 8.38 [1.25] 29.71 3.72 [0.55] 24.62 6.20 [0.92] 62.00 6.94 [1.03] 103.72 5.34 [0.80] 104.64 9.43 [1.41] 34.96 6.44 [0.96] 55.67 9.73 [1.45] 56.95 9.36 [1.39] 30.35 4.48 [0.67] 23.69 5.01 [0.75] 62.52 8.07 [1.20] 103.69 5.85 [0.87] 106.02 9.73 [1.45] 33.98 5.75 [0.86]
Mean standard deviation [standard error].
was parallel to the ground and the hoof was held up. To measure the extensor digitorum longus in the flexed position, the leg was lifted to a position that was comfortable for the individual who was holding the hoof and his arms, legs, and back were maintained fully extended. This individual assisted all the examiners. After the measurements were taken, the sites were cleaned and the next examiner took images of the muscles. Scanning was performed with the probe applied perpendicular to the tissue interface. Minimal pressure was applied with the probe so as 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 Analyses Data are shown as mean standard deviation. Analysis of variance for repeated measurements was applied to determine the effect of the day of measurement and the examiner. Fisher’s test was applied as a post hoc test when significant effects were found. The coefficient of variation (CV) for the three measurements taken by each examiner for every muscle was
RESULTS For all the examiners, the day of measurement did not have any effect on the mean value of MT (P > .05; Table 2). The left longissimus lumborum was the only muscle for which the second measurement varied from the first and the third. For both comparisons, values were higher on 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 the mean CV of the thickness measurements of the left extensor carpi radialis and the left extensor digitorum longus in the flexed position (Table 4). The daily measurements of examiner 1 varied more than those of the other two examiners. Overall, mean CVs higher or close to 10% were found for both sides of the extensor digitorum longus scanned in the flexed position and the supraspinatus muscles. The lowest mean CVs were calculated for the longissimus lumborum and the muscle semitendinosus, which were kept in the extended position during the examination (Table 4). To determine the objectivity of the measurements, the CV of the first measurement of each muscle for the three examiners was calculated. The largest CV was observed for the extensor digitorum longus flexed and the supraspinatus on both sides of the horses (Table 5). The best agreement was measured for the extensor carpi radialis held in an extended position and the longissimus lumborum muscles (Table 5).
DISCUSSION US measurements of MT can be made quickly, repeatedly, and in most horses safely without disrupting the horse’s training or conditioning program. This could allow for an objective means to show effects of training on MT,15 and assess the relationship between muscle mass and performance.2 However, the repeatability of the measurements needs to be as low as possible to be able to measure minor training effects and performance differences. In the current study, the MT measurements of most muscles did not differ significantly between the examiners or between their daily measurements. However, the CV varied widely for the different muscles measured by an examiner and also to quite a remarkable extent between the different examiners. Erichsen et al16 described a CV range of 0% to 10% for three examiners who measured the thickness of the gluteus medius muscle at two different sites on each side of the horse (mean of 1.77% and 2.04% for the left and right
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Table 4. Mean CV for the daily ultrasound measurements (three in total) on muscle thickness taken by each examiner on five horses CV of Each Examiner Muscle
Effect of Examiner
1
2
3
Left extensor carpi radialis extended Left extensor carpi radialis flexed Left extensor digitorum longus extended Left extensor digitorum longus flexed Left gluteus medius Left longissimus lumborum Left semitendinosus Left supraspinatus Right extensor carpi radialis extended Right extensor carpi radialis flexed Right extensor digitorum longus extended Right extensor digitorum longus flexed Right gluteus medius Right longissimus lumborum Right semitendinosus Right supraspinatus
NS <0.01 NS <0.05 NS NS NS NS NS NS NS NS NS NS NS NS
5.57 7.70 6.57 19.65 9.78 2.95 6.81 11.14 7.08 8.60 8.98 18.28 13.75 3.68 7.84 9.69
14.46 4.90 6.73 10.36 8.00 4.43 2.46 10.75 6.50 5.50 8.19 11.15 6.88 2.89 3.34 8.52
4.40 2.56 8.71 10.04 3.66 2.54 2.89 6.98 6.87 3.86 5.77 14.35 8.33 3.05 2.97 8.86
CV, coefficient of variation; NS, nonsignificant.
sides, respectively). These data indicate that it would be best to have only one individual to take all the measurements. For the flexed left extensor carpi radialis and flexed extensor digitorum longus, the difference between the examiners was significant: Examiner 1 had markedly higher CVs in comparison with the others. This finding suggests that the more pronounced differences between the examiners are for muscles on the left side of the horses. This may be because all the examiners were right-handed, thus perhaps making it more difficult to position adequately the scanning head of the probe with the right hand on the 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. Possible causes for this result, as discussed between the examiners, were that examiner 1 felt much more confident in the use of the US and simultaneously was less comfortable with horses as compared with the other examiners. Both factors could have reduced the scanning accuracy of examiner 1. Overall, the supraspinatus and the flexed extensor digitorum longus muscles had the highest CV with values of >10%. Therefore, it is possible that the exact location of the scanning site of the supraspinatus muscle in front of the tuber spinae scapulae was not as clear as assumed, whereas the difficulties in scanning the flexed extensor digitorum longus 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 addition to the longissimus dorsi, examiner 2 reported a CV of <5% for the semitendinosus muscle. Examiner 3
achieved a CV of <5% for the aforementioned two muscles, as well as the flexed extensor carpi radialis muscle and the left gluteus medius muscle. In previous studies, the CV of MT measurements with US for the extensor digitorum communis 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 measures to reduce the CV of measurements. All the examiners in this study were veterinary surgeons; two were primarily in equine practice and one (examiner 1) in academia. The examiners were selected to conduct the study because of their interest in the subject and willingness to be trained in US scanning (examiners 2 and 3). It is quite possible that the examiners in previous studies2,13,16 were much more skilled than those working in this study. However, the results of this study clearly indicate that a very careful approach is required to obtain repeatable results. Other than the examiner, a minimum of two more individuals need to be present during the study, one to hold the head of the horse to keep it as still as possible, and the other to assist the examiner in all means so that the examiner can concentrate 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 thickness measurements of the extensor digitorum longus, the longissimus lumborum, the semitendinosus, and the supraspinatus muscles of horses. The thickness of the extensor carpi radialis measured in the current study is similar to the values described by Kearns et al2 for Standardbreds,
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Table 5. Mean CV of the measurements taken by three examiners of the thickness of different muscles of five horses
Muscle Left extensor carpi radialis extended Left extensor carpi radialis flexed Left extensor digitorum longus extended Left extensor digitorum longus flexed Left gluteus medius Left longissimus lumborum Left semitendinosus Left supraspinatus Right extensor carpi radialis extended Right extensor carpi radialis flexed Right extensor digitorum longus extended Right extensor digitorum longus flexed Right gluteus medius Right longissimus lumborum Right semitendinosus Right supraspinatus
CV for the First Measurement by the Three Examiners Together 3.32 9.19 8.65 10.81 7.75 4.43 5.93 13.43 5.54
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were made in the dorsal or ventral compartment of the muscle. Age also affects the depth of the gluteus medius muscle: horses aged >3 years had a slightly slimmer MT of the gluteus medius.17 In conclusion, care has to be taken to maintain a low CV of MT measurements done by US. Keeping the repeatability of MT at <5% seems to be easiest for the longissimus lumborum and the semitendinosus, whereas the CVs of the extensor carpi radialis, the extensor digitorum longus, and the gluteus medius are most likely to be higher. The thickness measurements of the supraspinatus and the flexed extensor carpi radialis as well as the flexed extensor digitorum longus muscles would quite likely vary by a lot to be of practical benefit. ACKNOWLEDGMENTS The authors thank Holly M. Greene for revising the manuscript. REFERENCES 1. Gunn HM. Muscle, bone and fat proportions and the muscle distri-
8.57
bution of Thoroughbreds and other horses. In: Gillespie JR, Robinson NE, eds. Equine exercise physiology 2: Proceedings of
5.04
the 2nd International Conference of the Equine Exercise Physiology.
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2. Kearns CF, McKeever KH, Kumagai K, Abe T. Fat-free mass is related to one-mile race performance in elite standardbred horses.
7.83 3.62
3. Kearns CF, McKeever KH, Abe T. Overview of horse body composi-
Davis, CA: ICEEP Publications; 1987:253–264.
Vet J 2002;163:260–266. tion and muscle architecture: implications for performance. Vet J
6.43 11.24
CV, coefficient of variation.
but much lower than that for Thoroughbreds3 (70 mm). The competitive level of the Thoroughbreds in both studies may provide an explanation for the reported difference. The Thoroughbreds measured by Kearns et al3 were elite Thoroughbreds, whereas those in this study were not. The gluteus medius muscle measurements obtained in Swedish riding horses used for dressage, eventing, and show jumping16 were approximately thicker by 2 cm as compared with those obtained from the horses used in this study, whereas the MT in French Standardbreds was similar.17 Valette et al17 showed that the thickness of the gluteus medius muscle was dependent not only on the location of the scanning head but also on the inclination of the hip. In Standardbreds with a straighter hip, the ventral compartment of the gluteus medius muscle was on an average 3 cm deeper than in horses with a wide hip. They found that the MT of male Standardbreds was thicker as compared with females, regardless of whether measurements
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. Reeves ND, Maganaris CN, Narici MV. 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 studies and ultrasonography. Clin Neurophysiol 2007;118:2172–2175. 8. Uremovi M, Bosnjak PM, Seric V, Vargek Solter V, Budic R, Bosnjak B, et al. Ultrasound measurement 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.
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12. Kane RA, Fisher M, Parrett D, Lawrence LM. Estimating fatness in
bouts over 100 m at near maximal speed on the thickness of Musculus
horses. In: Proceedings of the 10th Equine Nutrition and Physiology
supraspinalis and extensor carpi radialis. In: Proceedings of the
Symposium; Fort Collins, CO; 1987:127–131.
Equine Science Society; Tucson, Arizona; 2005:114–116.
13. Abe T, Kearns CF, Manso Filho HC, Sato Y, Sleeper M,
16. Erichsen C, Eksell P, Widstr€ om C, Berger M, Holm KR, Johnston C.
McKeever KH. Acute vascular occlusion in horses: effects on skeletal
Scintigraphy of the sacroiliac joint region in asymptomatic riding
muscle size and blood flow. Equine Comp Exerc Physiol 2004;1:
horses: scintigraphic appearance and evaluation of method. Vet
239–243. 14. Abe T, Kearns CF, Manso Filho HC, Sato Y, McKeever KH. Muscle,
Radiol Ultrasound 2003;44:699–706. 17. Valette JP, Barrey E, Jouglin M, Courouce A, Auvinet B, Flaux B.
tendon, and somatotropin responses to the restriction of muscle
Standardisation of muscular biopsy of gluteus medius in French trot-
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
ters. Equine Vet J Suppl 1999;30:342–344. 18. Henneke DR, Potter GD, Kreider JL, Yeates BF. Relationship between body condition score, physical measurement, and body fat percentage in mares. Equine Vet J 1983;15:371–372.
ABSTRACT This study examined the reproducibility and repeatability of muscle thickness (MT) measurements with ultrasound for the following muscles: extensor carpi radialis, extensor digitorum longus (both flexed and extended), gluteus medius, longissimus lumborum, semitendinosus, and supraspinatus. Three examiners measured thickness of these muscles in five Thoroughbreds on 3 consecutive days. The day of measurement did not have any effect on the mean MT value of the muscles examined (P > .05). The left longissimus lumborum muscle was the only muscle for which the second measurement varied from the first and third (P < .05). The examiners had an effect on the mean coefficient of variation (CV) of the thickness of the flexed left extensor carpi radialis and flexed left extensor digitorum longus. The daily measurements varied more in the case of one of the examiners. Mean CVs higher or close to 10% were reported for both sides of the flexed extensor digitorum longus and for the supraspinatus muscles. The lowest CV was calculated for the longissimus lumborum and the extended semitendinosus (<5%). The largest disagreements between the examiners were observed for both sides of the flexed extensor digitorum longus and the supraspinatus (>10%–<20%). The best agreement was measured for the extended extensor carpi radialis and the longissimus lumborum (>3%–<7%). The results showed that for some muscles, it was difficult to locate the exact anatomical site for taking the MT measurements. To reduce CV, it was suggested that only one examiner should take all the measurements and the whole procedure must be such that it is as comfortable as possible for this particular examiner. Keywords: Muscle; Thickness; Ultrasound; Horse
From the Arbeitsgruppe Pferd, Juelich, Germanya; and Facultad de Veterinaria, Universidad del Litoral, Esperanza, Santa Fe, Argentineb. Reprint requests: Arno Lindner, DVM, Heinrich-Roettgen-Str. 20, D-52428 Juelich, Germany. 0737-0806/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2010.10.007
Journal of Equine Veterinary Science Vol 30, No 11 (2010)
INTRODUCTION Horses of breeds used in equine sports activities are known to have a greater muscle mass relative to body mass as compared with those that are not used in sports.1 Kearns et al2 found that horses with a tendency to run fast had an overall higher skeletal muscle mass and a lower percentage of body fat. However, the skeletal muscle mass of horses cannot be determined routinely,3 and it would be helpful to have a noninvasive means to quantify on a regular basis the effects of training programs on muscles and maybe even to define parameters of athletic ability. Ultrasound (US) imaging is used to measure thickness and cross-sectional area of muscles in human beings, with the purpose of evaluating the effect of training and rehabilitation measures as well as the development of medical conditions.4-8 US has been proven to be sensitive enough to measure changes in body fat in horses who are fed limited diets9; training for polo competition9; competing in endurance,10 trot, and gallop racing2,3; and taking a repartitioning agent.11 The US measurement of rump fat in horses has been reported to be highly repeatable, precise, accurate, and easy to perform.9,11,12 US has been used to measure muscle thickness (MT) in horses to examine effects of Kaatsu,13,14 training,15 scintigraphic 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 repeatability and objectivity of the measurement of MT with US in the horse. Therefore, in this study, the thickness of several muscles in Thoroughbred horses was measured using US to calculate repeatability and objectivity of the measurements.
MATERIALS AND METHODS Horses Five Thoroughbred horses, three stallions and two geldings, ranging in ages from 4 to 6 years were used for the current study. The body condition score of the horses ranged between 3 and 5.5.18 All horses were being used for flat racing. Equipment A Mindray 6600 Ultrasound machine (Digimed S.A., Buenos Aires, Argentine) was used to scan the thickness
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Table 1. Muscles measured and location of the site of measurements Muscle
Site of Measurement
Extensor carpi radialis
30 cm above the medial aspect of the tuberositas radii, holding the probe as much as possible perpendicular to the latter Approximately 30 cm from the tuberositas tibiae, applying the probe as much as possible perpendicular to the latter. An attempt was made to measure at the site where the muscle appeared to be the thickest Midway of the distance between the first coccigeal vertebrae and the tuber coxae 20 cm cranial to the tuber sacrale of the ilium, 3 cm off the midline of the back 20 cm below the tuber ischiadicum In front of the tuber spinae scapulae
Extensor digitorum longus
Gluteus medius Longissimus lumborum Semitendinosus Supraspinatus
of the muscles. For optimal probe contact and signal penetration, a US gel was applied directly on the skin after shaving. The longissimus lumborum, semitendinosus, and gluteus medius muscles were measured with a 2-MHz convex probe, whereas all the other muscles were measured with a 6-MHz convex probe. In comparison with the 6-MHz probe, the 2-MHz convex probe increased the clarity and resolution of the images taken deep inside the thicker muscles. Experimental Procedure Horses were placed in a quiet area of the barn on firm ground. Care was taken to prevent them from moving significantly; for this, two experienced horse handlers were introduced, one restrained movement of the horses
by holding its head and the other assisted the examiners. Three veterinarians took MT measurements in the horses once on 3 consecutive days at the same time. One veterinarian was a specialist in US imaging, whereas the other two had gone through a 3-month training program conducted by the experienced person before engaging in the study. The muscles measured and a description of the anatomical sites where the measurements were taken is shown in Table 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 digitorum longus muscles were measured in both the extended and flexed position. To measure the extensor carpi radialis in the flexed position, the leg was lifted till the cannon bone
Table 2. Repeatability and objectivity results of muscle thickness measurements taken by three examiners on 3 consecutive days (five horses) Effect Muscle
Examiner
Day of Measurement
Left extensor carpi radialis extended Left extensor carpi radialis flexed Left extensor digitorum longus extended Left extensor digitorum longus flexed Left gluteus medius Left longissimus lumborum
NS NS NS NS NS NS
NS NS NS NS NS <0.05
Left semitendinosus Left supraspinatus Right extensor carpi radialis extended Right extensor carpi radialis flexed Right extensor digitorum longus extended Right extensor digitorum longus flexed Right gluteus medius Right longissimus lumborum Right semitendinosus Right supraspinatus
NS NS NS NS NS NS NS NS NS NS
NS NS NS NS NS NS NS NS NS NS
NS, nonsignificant.
Fisher’s Test
First and second, <0.05; second and third, <0.01
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Table 3. Thickness of muscles of horses measured with ultrasound (five horses three examiners three measurements per examiner) Muscle Left extensor carpi radialis extended Left extensor carpi radialis flexed Left extensor digitorum longus extended Left extensor digitorum longus flexed Left gluteus medius Left longissimus lumborum Left semitendinosus Left supraspinatus Right extensor carpi radialis extended Right extensor carpi radialis flexed Right extensor digitorum longus extended Right extensor digitorum longus flexed Right gluteus medius Right longissimus lumborum Right semitendinosus Right supraspinatus a
Thickness (mm)a
637
calculated. One-way analysis of variance was used to determine whether there was an effect of the examiner on the CV. Once more, Fisher’s test was applied as a post hoc test when significant effects were found. P < .05 was defined as the level that denoted a significant difference.
57.5 10.19 [1.32] 58.89 8.38 [1.25] 29.71 3.72 [0.55] 24.62 6.20 [0.92] 62.00 6.94 [1.03] 103.72 5.34 [0.80] 104.64 9.43 [1.41] 34.96 6.44 [0.96] 55.67 9.73 [1.45] 56.95 9.36 [1.39] 30.35 4.48 [0.67] 23.69 5.01 [0.75] 62.52 8.07 [1.20] 103.69 5.85 [0.87] 106.02 9.73 [1.45] 33.98 5.75 [0.86]
Mean standard deviation [standard error].
was parallel to the ground and the hoof was held up. To measure the extensor digitorum longus in the flexed position, the leg was lifted to a position that was comfortable for the individual who was holding the hoof and his arms, legs, and back were maintained fully extended. This individual assisted all the examiners. After the measurements were taken, the sites were cleaned and the next examiner took images of the muscles. Scanning was performed with the probe applied perpendicular to the tissue interface. Minimal pressure was applied with the probe so as 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 Analyses Data are shown as mean standard deviation. Analysis of variance for repeated measurements was applied to determine the effect of the day of measurement and the examiner. Fisher’s test was applied as a post hoc test when significant effects were found. The coefficient of variation (CV) for the three measurements taken by each examiner for every muscle was
RESULTS For all the examiners, the day of measurement did not have any effect on the mean value of MT (P > .05; Table 2). The left longissimus lumborum was the only muscle for which the second measurement varied from the first and the third. For both comparisons, values were higher on 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 the mean CV of the thickness measurements of the left extensor carpi radialis and the left extensor digitorum longus in the flexed position (Table 4). The daily measurements of examiner 1 varied more than those of the other two examiners. Overall, mean CVs higher or close to 10% were found for both sides of the extensor digitorum longus scanned in the flexed position and the supraspinatus muscles. The lowest mean CVs were calculated for the longissimus lumborum and the muscle semitendinosus, which were kept in the extended position during the examination (Table 4). To determine the objectivity of the measurements, the CV of the first measurement of each muscle for the three examiners was calculated. The largest CV was observed for the extensor digitorum longus flexed and the supraspinatus on both sides of the horses (Table 5). The best agreement was measured for the extensor carpi radialis held in an extended position and the longissimus lumborum muscles (Table 5).
DISCUSSION US measurements of MT can be made quickly, repeatedly, and in most horses safely without disrupting the horse’s training or conditioning program. This could allow for an objective means to show effects of training on MT,15 and assess the relationship between muscle mass and performance.2 However, the repeatability of the measurements needs to be as low as possible to be able to measure minor training effects and performance differences. In the current study, the MT measurements of most muscles did not differ significantly between the examiners or between their daily measurements. However, the CV varied widely for the different muscles measured by an examiner and also to quite a remarkable extent between the different examiners. Erichsen et al16 described a CV range of 0% to 10% for three examiners who measured the thickness of the gluteus medius muscle at two different sites on each side of the horse (mean of 1.77% and 2.04% for the left and right
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Table 4. Mean CV for the daily ultrasound measurements (three in total) on muscle thickness taken by each examiner on five horses CV of Each Examiner Muscle
Effect of Examiner
1
2
3
Left extensor carpi radialis extended Left extensor carpi radialis flexed Left extensor digitorum longus extended Left extensor digitorum longus flexed Left gluteus medius Left longissimus lumborum Left semitendinosus Left supraspinatus Right extensor carpi radialis extended Right extensor carpi radialis flexed Right extensor digitorum longus extended Right extensor digitorum longus flexed Right gluteus medius Right longissimus lumborum Right semitendinosus Right supraspinatus
NS <0.01 NS <0.05 NS NS NS NS NS NS NS NS NS NS NS NS
5.57 7.70 6.57 19.65 9.78 2.95 6.81 11.14 7.08 8.60 8.98 18.28 13.75 3.68 7.84 9.69
14.46 4.90 6.73 10.36 8.00 4.43 2.46 10.75 6.50 5.50 8.19 11.15 6.88 2.89 3.34 8.52
4.40 2.56 8.71 10.04 3.66 2.54 2.89 6.98 6.87 3.86 5.77 14.35 8.33 3.05 2.97 8.86
CV, coefficient of variation; NS, nonsignificant.
sides, respectively). These data indicate that it would be best to have only one individual to take all the measurements. For the flexed left extensor carpi radialis and flexed extensor digitorum longus, the difference between the examiners was significant: Examiner 1 had markedly higher CVs in comparison with the others. This finding suggests that the more pronounced differences between the examiners are for muscles on the left side of the horses. This may be because all the examiners were right-handed, thus perhaps making it more difficult to position adequately the scanning head of the probe with the right hand on the 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. Possible causes for this result, as discussed between the examiners, were that examiner 1 felt much more confident in the use of the US and simultaneously was less comfortable with horses as compared with the other examiners. Both factors could have reduced the scanning accuracy of examiner 1. Overall, the supraspinatus and the flexed extensor digitorum longus muscles had the highest CV with values of >10%. Therefore, it is possible that the exact location of the scanning site of the supraspinatus muscle in front of the tuber spinae scapulae was not as clear as assumed, whereas the difficulties in scanning the flexed extensor digitorum longus 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 addition to the longissimus dorsi, examiner 2 reported a CV of <5% for the semitendinosus muscle. Examiner 3
achieved a CV of <5% for the aforementioned two muscles, as well as the flexed extensor carpi radialis muscle and the left gluteus medius muscle. In previous studies, the CV of MT measurements with US for the extensor digitorum communis 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 measures to reduce the CV of measurements. All the examiners in this study were veterinary surgeons; two were primarily in equine practice and one (examiner 1) in academia. The examiners were selected to conduct the study because of their interest in the subject and willingness to be trained in US scanning (examiners 2 and 3). It is quite possible that the examiners in previous studies2,13,16 were much more skilled than those working in this study. However, the results of this study clearly indicate that a very careful approach is required to obtain repeatable results. Other than the examiner, a minimum of two more individuals need to be present during the study, one to hold the head of the horse to keep it as still as possible, and the other to assist the examiner in all means so that the examiner can concentrate 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 thickness measurements of the extensor digitorum longus, the longissimus lumborum, the semitendinosus, and the supraspinatus muscles of horses. The thickness of the extensor carpi radialis measured in the current study is similar to the values described by Kearns et al2 for Standardbreds,
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Table 5. Mean CV of the measurements taken by three examiners of the thickness of different muscles of five horses
Muscle Left extensor carpi radialis extended Left extensor carpi radialis flexed Left extensor digitorum longus extended Left extensor digitorum longus flexed Left gluteus medius Left longissimus lumborum Left semitendinosus Left supraspinatus Right extensor carpi radialis extended Right extensor carpi radialis flexed Right extensor digitorum longus extended Right extensor digitorum longus flexed Right gluteus medius Right longissimus lumborum Right semitendinosus Right supraspinatus
CV for the First Measurement by the Three Examiners Together 3.32 9.19 8.65 10.81 7.75 4.43 5.93 13.43 5.54
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were made in the dorsal or ventral compartment of the muscle. Age also affects the depth of the gluteus medius muscle: horses aged >3 years had a slightly slimmer MT of the gluteus medius.17 In conclusion, care has to be taken to maintain a low CV of MT measurements done by US. Keeping the repeatability of MT at <5% seems to be easiest for the longissimus lumborum and the semitendinosus, whereas the CVs of the extensor carpi radialis, the extensor digitorum longus, and the gluteus medius are most likely to be higher. The thickness measurements of the supraspinatus and the flexed extensor carpi radialis as well as the flexed extensor digitorum longus muscles would quite likely vary by a lot to be of practical benefit. ACKNOWLEDGMENTS The authors thank Holly M. Greene for revising the manuscript. REFERENCES 1. Gunn HM. Muscle, bone and fat proportions and the muscle distri-
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