615
J. Anat. (1977), 123, 3, pp. 615-635 With 9 figures Printed in Great Britain
The growth patterns of three hindlimb muscles in the chicken CYRUS HELMI* AND JOEL CRACRAFT
Department of Anatomy, University of Illinois at the Medical Center, Chicago, Illinois 60680
(Accepted 14 May 1976) INTRODUCTION
There is now a large literature on the mechanisms of growth of skeletal muscle (see review by Goldspink, 1972). Nevertheless, very few reported studies have examined the complex interactions of numerous growth parameters for individual muscles and tried to relate patterns of growth to the functional requirements of increasing body size. Two notable exceptions are the studies by Davies (1972) on the deep back muscles of the pig and Rayne & Crawford (1972) on the jaw muscles of the rat. Other workers have mentioned the consequences of increasing body size (Hill, 1950; Schwartz, 1961; Wunder & Bird, 1969; Goldspink, 1970; Stewart, 1972; Bryden, 1973) or have demonstrated the importance of work load (tension) in determining the morphological and physiological properties of growing muscle (e.g. Goldberg, 1967; Rowe & Goldspink, 1969; Gutmann, Schiaffino & Hanzlikova, 1971; Goldspink, 1972; Stewart, 1972; James, 1973; Williams & Goldspink, 1973; Muller, 1974). The purpose of this study was to examine the growth of three hindlimb muscles of the chicken (Gallus gallus), using a number of growth parameters including wet and dry weights, muscle cross sectional area and length, and muscle fasciculus length. In addition to purely descriptive aspects, the investigation compared the growth patterns of certain muscles whose adult architectures differ and whose roles change during postural and locomotor cycles. A major purpose was to determine, if possible, the ways in which architecture (form) and function relate to differences in the growth patterns themselves and to the mechanical demands of increasing body size. Thus, two of the muscles chosen, adductor superficialis and adductor profundus, are postural muscles: the former is parallel fibred whereas the latter is unipennate. The third muscle, biceps femoris, differs in having the non-postural function of flexing the knee joint and in being bipennate. MATERIALS AND METHODS
The chickens used in this study were raised from hatching. A total of 276 individual muscles was obtained from 46 chickens killed in groups 1, 7, 14, 21, 35 and 49 days after hatching. Each age group consisted of 7-9 individuals. The sex was not noted, *
Present address: Department of Anatomy, University of Chicago, Chicago, Illinois 60637.
616
C. HELMI AND J. CRACRAFT
M. femorotibialis
M. semitendinosus M. biceps femorisM. femorotib
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M. adductor superficialis
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Fig. 1. Two views of leg muscles of the chicken in superficial (A) and deep (B) dissection to show muscles examined in this study.
because it seemed irrelevant to the present investigation; we were examining the morphological characteristics of muscles relative to individual body size. Gross morphology of the muscles The adult morphology and functions of the leg muscles examined in this study will be described briefly. The gross morphology and functions of these muscles do not differ significantly among galliform birds (Hudson, Lanzillotti & Edwards, 1959; George & Berger, 1966). M. biceps femoris This is a large two-joint muscle passing posterior to the hip and knee joints (Fig. 1). It arises aponeurotically from the most posterior portion of the anterior iliac crest dorsal to the hip joint and has a fleshy origin as well from the posterior iliac crest. The muscle tapers from its wide origin, and a strong tendon inserts on to the posterior surface of the proximal end of the fibula. The biceps femoris has an asymmetrical bipennate fibre arrangement. The central tendon of insertion is located nearer the posterior margin of the muscle, hence most of the fibres insert on to the anterior part of the tendon. The anterior fibres have a larger angle of pennation (about 200) than the posterior fibres (about 120). The biceps femoris functions to flex the knee joint during terrestrial locomotion; the muscle is probably not active during the weight-bearing propulsive phase of locomotion except perhaps to stabilize the knee joint at the beginning of the stance phase, or perhaps to effect some movement of the pelvis relative to the femur. Cracraft (1971) found that this muscle is composed predominantly of twitch (Fibrillenstruktur) fibres in the domestic pigeon (Columba livia), and this is probably true for most birds. Thus, the long fibres and twitch fibre composition suggest a muscle that during locomotion is developing relatively high velocities, shortening over a
617
Growthl patterns of three hindlimb muscles in chickens
relatively great distance, and acting against comparatively little resistance (in this case, the weight of the lower portion of the leg). M. adductor superficialis This is a flat, nearly trapezoidal, one-joint muscle passing posterior to the hip joint (Fig. 1). It arises by a short aponeurosis from the ventral margin of the ischium. The muscle has a fleshy insertion on to the posteromedial side of the distal half of the femur. The adductor superficialis is composed of long parallel fibres, with the anterior fibres being shorter than those located more posteriorly. This muscle functions to retract the femur. More importantly, it serves a postural function during standing and also resists any forward motion (protraction) of the femur during landing, and at the moment of impact during walking (Cracraft, 1971). In the domestic pigeon this muscle is composed predominantly of tonus (Felderstruktur) fibres, which supports the notion that the muscle is primarily postural, resisting forces over extended periods of time (Cracraft, 1971). M. adductor profundus This is a large, one-joint muscle passing posterior to the hip joint (Fig. 1). It arises from the ventral edge of the ischium by a broad, thin aponeurosis shared with adductor superficialis. The muscle inserts on to the posterior surface of the distal two thirds of the femur. The adductor profundus ,!as a unipennate fibre arrangement, but as the fibres are moderately long and the angle of pennation very small the muscle appears at first glance to have a parallel fibre arrangement. The anterior fibres are shorter than the posterior fibres. In the domestic pigeon this muscle has the same function and fibre-type composition as the adductor superficialis (Cracraft, 1971), and this is undoubtedly true for the chicken also. Fixation and dissection a buffered alcohol-formaldehyde fixative, and care in fixed were muscles The leg in the relaxed position in order to minimize stretching the to leg was taken preserve to be relatively uniform in all muscles. All was assumed muscles. Shrinkage of the a dissecting microscope. binocular out using dissections were carried Measurements The following measurements were taken on each individual: (1) Body weight: recorded to the nearest 0 1 g. (2) Leg weight: the total leg weight distal to the knee joint was recorded to the nearest 0 1 g. (3) Muscle length: the maximum extent of the contractile portion of the muscle (measured along the origin-insertion axis), not including the tendon or aponeurosis, was measured to the nearest 0-1 mm. (4) Fasciculus length: muscle fasciculi were first teased apart under a binocular dissecting microscope. A tracing of a straightened (not stretched) individual fasciculus was obtained using a camera lucida attachment, and the tracings were then measured 39
ANA 123
C. HELMI AND J. CRACRAFT 618 and corrected for magnification. In this manner more accurate measurements were obtained than if the fasciculus had been measured directly. Direct measurement by calipers to the nearest 0-1 mm was made only on the longer fasciculi of the older individuals. Measurements of fasciculi taken from populations of the longest and shortest fasciculi were obtained from each muscle. (5) Muscle cross sectional area: each muscle was transected in the middle of the muscle belly. A tracing of the cross section was then obtained using a binocular dissecting microscope with a camera lucida attachment. From this tracing the area was determined with the aid of a polar planimeter. Each measurement reported below is an average of five determinations. In the muscles studied measurements at the middle of the muscle belly were assumed to be fairly accurate estimations of physiological cross sectional area for purposes of comparison between muscles of different age groups. In all three muscles the fasciculi were long enough to make it reasonably certain that all of them were included in the section. (6) Muscle wet weight: the muscles were lightly blotted and then weighed on an electric microbalance to the nearest 0i001 g. (7) Muscle dry weight: the muscles were dried in an oven to constant weight at 60 'C (about 24 hours). They were than weighed on a microbalance to the nearest 0.001 g. Statistical methods In addition to basic statistics (mean, standard deviation), regression equations were calculated for many variables against body weight. Logarithmic transformations of the raw data were undertaken when it seemed appropriate. In analysing growth patterns the data were fitted by least-squares regression to the well-known allometric growth equation, log Y = log a+b log X,
where Y is a variable changing in value relative to X (usually body weight), log a is the Y-intercept, and b is the slope. RESULTS
Body weight The increase in body weight from hatching to 49 days of age is shown in Figure 2 and data are given in Table 1. The growth curve appears to be approximately logarithmic, although the asymptote has not yet been reached at 49 days. Between the time of hatching and 49 days the mean body weight of the age groups increased about 14-fold (Table 1). Relative to the average final weight (585 g), 2-64 % of that was added in the first week, 8-43 % in the second, and 7-61 0% in the third. During the interval between 21 and 35 days about 32-85 % of the final body weight was added, and in the last 2 weeks 41 36 %o (242 g) of the body weight was added. Church & Johnson (1964) found that after 21 days body weight was sexually dimorphic, with males having a higher gross weight (as mentioned earlier, the goals of this study did not necessitate sex determination).
Growth patterns of three hindlimb muscles in chickens
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Muscle weight The mean wet and dry weights for each muscle are given for each age group in Table 1 and plotted in Figures 3 and 4. For both wet and dry weight the first 3 weeks is a period of relatively slow growth for all three muscles. During this period both the dry and wet weights of biceps femoris were higher than either of the adductors, and the growth rate of the former was somewhat greater than those of the latter two muscles. The growth of the two adductors was nearly equal during this time. After the third week each muscle rapidly increased in weight, with biceps femoris increasing significantly faster than the two adductors. During this period of rapid growth the two adductors no longer grew at the same rate, and the weights of adductor profundus increased more than those of adductor superficialis. Between hatching and day 49 the mean dry weight for biceps femoris, adductor profundus, and adductor superficialis increased 28-0-, 18-5- and 15-0-fold, respectively, whereas in wet weight they increased 22-7-, 17-2- and 13-5-fold. Table 1 shows that on the first day the total wet and dry weights of the combined adductors were larger than those of the biceps femoris, and that the latter muscle quickly became increasingly larger than the adductor group. 39-2
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Relative growth of muscle weight Log transformed regression statistics for muscle dry and wet weight versus body weight are given in Table 3 and the data are plotted in Figures 5 and 6. The allometric growth coefficient for dry weight of adductor superficialis (slope = 1-003) is not significantly different (P > 005) from unity, indicating that growth of this muscle as expressed by dry weight is proportional to growth of the animal as a whole. The growth coefficients for biceps femoris (slope = 1 228), adductor profundus (slope = 1 089), and the adductors combined (slope = 1-050), on the other hand, are significantly different from isometry, and positive allometry is indicated. It should be noted, however, that the coefficients of adductor profundus and the adductors considered together were barely significant at the 5 % level. The growth coefficient of biceps femoris is also significantly different from those of the adductors considered separately and combined. Regression statistics for log wet weight versus log body weight show similar patterns to those of dry weight. The growth coefficients for biceps femoris (slope = 1'158) and adductor profundus (slope = 1'090) are significantly different from isometry, whereas that of adductor superficialis (slope = 0'959) is not. The combined wet weight of the adductors is also not different from isometry.
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Growth of muscle cross sectional area Raw data for growth of muscle cross sectional area are given in Table 2 and plotted in Figure 7. Cross sectional area of all three muscles increased substantially between 1 and 14 days, but between 14 and 21 days there appears to have been a marked decrease in growth (Fig. 7). After 21 days there was again a marked increase in growth, correlated with a significant increase in the growth of body weight (Fig. 2 and later discussion). Log transformed regression statistics for muscle cross sectional area versus body weight are presented in Table 3 (see Fig. 8). The allometric coefficients for biceps femoris, adductor profundus, and the combined area of both adductors are significantly different from isometry (slope = 067), and all show positive allometry. The slope of 0718 for adductor superficialis is not significantly different from isometry. M. biceps femoris cross sectional area versus leg weight Because biceps femoris functions to lift that portion of the leg distal to the knee joint, we examined the growth pattern of the muscle relative to the leg weight.
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Table 3 presents the regression statistics for this relationship. The allometric coefficient (slope = 0-58) is significantly different at the 5 % level from isometry (slope = 0-67), thus implying a somewhat negatively allometric growth for cross sectional area relative to leg weight.
Growth of leg weight versus body weight In order to assess more fully the growth pattern of biceps femoris, the relationship between growth in leg and body weight was studied (see Table 1 for raw data). Regression statistics indicate that increase in leg weight is slightly positively allometric to body weight (Table 3). The allometric coefficient is 1-08, with a correlation of 0-998, and is significantly different from isometry (slope = 1 -00) at the 5 % level.
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Growth of muscle length Raw data for growth of muscle length are presented in Table 2 and are graphed against time in Figure 7. The lengths of all three muscles increase steadily with time, there being no noticeable change in growth rate at 21 days as there was for cross sectional area. As expected, biceps femoris increases in length more rapidly than do the adductors (Fig. 7), which must lengthen at about the same rate because of their nearly identical position within the leg (Fig. 1). The growth patterns of muscle length relative to body weight were also investigated (Table 3). All three muscles show positive allometric growth, with growth coefficients significantly different from isometry (slope = 033): biceps femoris (050), adductor superficialis (0-46), and adductor profundus (0-47). None of the muscles was significantly different from the others in its growth coefficient. Growth of muscle fasciculus length We examined the relationship between growth in fasciculus length and body size (weight), since a particular rate of growth in muscle length (see above) does not necessarily imply an equal rate of growth in fasciculus length.
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Growth patterns of three hindlimb muscles in chickens
627
Table 3. Results of regression analyses (First line is correlation coefficient, second is slope, and third is y-intercept; asterisk indicates allometric coefficient significantly different from isometry at 5 % level.) Log dry weight versus log body weight
Log wet weight versus log body weight
0994 1-228* -8 255 M. adductor 0-989 1-003 superficialis -8-069 M. adductor 0-983 profundus 1-089* -8-429 M. adductor super0.991 ficialis and M. 1-050* adductor profundus -7 569
0994 1.158* -2-891 0-986 0 959 -2-876 0-985 1 090* -3-083 0 995 1-041 -2-709
0-969 0.832* -2-001 0-960 0-718
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Log X-sect. area versus log muscle wet weight
Log LFL versus log muscle wet weight
Log SFL versus log muscle wet weight
0 947 0-417* 1-643 0-965 0-476* 1-748 0-776 0-270 1*322
0-922 0.412* 1-561 0 911 0.432* 1-432 0-835 0.415* 1-114
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Raw data for fasciculus length are given in Table 2 (see Fig. 9); regression statistics for longest fasciculus length (LFL) and shortest fasciculus length (SFL) against body weight are presented in Table 3. For LFL both biceps femoris (slope = 049) and adductor superficialis (slope = 046) exhibit positive allometric growth (isometry = 033) and are not significantly different from one another. The adductor profundus, on the other hand, shows a much lower growth coefficient (slope = 030), which is not significantly different from isometry. Growth coefficients for muscle length and LFL relative to body weight are not different for biceps femoris and adductor superficialis, but there is a significant difference between the two for adductor profundus. The data presented in Table 3 indicate that all three muscles have coefficients significantly greater (P < 005) than isometry when log SFL is regressed against log body weight. The coefficients of the three muscles are not significantly different from each other.
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Growth patterns of three hindlimb muscles in chickens
629
Growth offasciculus length and muscle length The LFL and SFL were plotted against the muscle length (Table 3). The LFL of adductor superficialis is very nearly equal to the muscle length at any given age. This, of course, was not unexpected since in this parallel fibre muscle the fasciculi essentially run the length of the muscle. In the bipennate muscle, biceps femoris, the fasciculi are also long, and the slope of 0O88 indicates that nearly equal increments are being added to fasciculus length and to muscle length throughout the growth period. In the linear equation, Y = ax+ b, the y-intercept provides a measure for allometric growth (Gould, 1971). The slope merely signifies the constant change, ylx, being added throughout growth. Both adductor superficialis and biceps femoris have y-intercepts that are not significantly different from zero (P > 005), hence no allometry is taking place with respect to fasciculus length versus muscle length. In contrast, adductor profundus exhibits marked negative allometry with respect to fasciculus length. The positive y-intercept of 3 60 indicates that the value of x (here, muscle length) between any two points on the growth equation line is increasing at a greater rate than the y value (here, LFL) of those same two points (if the y-intercept were negative, y values would increase faster than x values); hence negative allometry is observed. For SFL versus muscle length none of the intercepts is significantly different from zero, thus indicating that SFL is increasing isometrically to muscle length. Muscle growth: muscle weight compared to cross sectional area and fasciculus length of muscle Since volume a (and thus weight, assuming a constant specific gravity) is roughly a function of its cross sectional area and its length, it seemed important to assess the relative contributions of cross sectional area and fasciculus length to growth. Table 3 presents the regression statistics for these comparisons. Plotting log cross sectional area against log muscle wet weight it is observed that all of the muscles have slopes that are significantly different from isometry (slope = 0 67). However, we note that the coefficients of all three muscles were barely significant and thus the degree of positive allometry is not great; the larger coefficient of 0757 for adductor profundus suggests that this muscle might be increasing relatively more in cross sectional area than the other muscles, but still the differences among the muscles are not statistically significant. With respect to the regression of log LFL on log muscle wet weight, the growth patterns appear to be somewhat different from those of cross sectional area. In biceps femoris and adductor superficialis the allometric coefficients of LFL are substantially larger than the value of isometry (033), whereas in the comparison of cross sectional area and muscle wet weight allometric coefficients were barely significant. M. adductor profundus, on the other hand, exhibits a coefficient for LFL that is not significantly different from isometry, and thus the coefficient of LFL when regressed against wet weight appears relatively smaller than the coefficient determined in the comparison of cross sectional area and wet weight: cross sectional area exhibits stronger allometry than LFL. When log SFL is regressed against log muscle wet weight, the coefficients for all three muscles exhibit positive allometry (Table 3).
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C. HELMI AND J. CRACRAFT
DISCUSSION
Relationship between muscle growth and body size Little attention has been given in the past to the relationship between muscle growth and increasing body size. As the latter increases functional demands on the muscles inevitably change, and consequently modification of structural characteristics of the muscles must ensue if the functional efficiency of the muscle is to be maintained. An important problem facing studies such as this one is determining which parameters will provide the most useful and reliable information about changing functional aspects of growing muscle. We comment on this problem later in the discussion. As body size increases it might be expected that postural muscles will grow allometrically. Body weight is scaling as the cube of the length, muscle cross sectional area (and hence a measure of force development) as the square. Given these geometrical and biomechanical accompaniments of growth, one would predict a change in shape. Examination of Table 3 reveals some complex growth patterns. Only the non-postural biceps femoris shows strong allometry of dry and wet weight, whereas the adductors, which are postural, either exhibit isometric growth (adductor superficialis) or weak allometry (adductor profundus) of these two measures. Furthermore, biceps femoris and adductor profundus show strong allometry of cross sectional area but adductor superficialis exhibits isometric growth of this parameter. One observation of this study that was somewhat unexpected was the marked allometry of biceps femoris in dry and wet weight and in cross sectional area (Table 3): our working hypothesis was that a non-postural muscle should exhibit weaker allometry than the postural muscles. The strong positive allometry of both cross sectional area and muscle length in biceps femoris accounts for the allometric growth of wet and dry weight, but the question remains why cross sectional area exhibits positive allometry. Because of its position within the leg it is reasonable to assume that biceps femoris is not resisting body weight during maintenance of posture. However, the muscle may function to maintain knee joint stability at the time the foot makes contact with the ground upon initiation of the stance phase, much like the hamstring complex does in human locomotion; in this case, the magnitude of body weight may be an important influence on the growth of the muscle. The allometry of biceps femoris may also be related to the pattern of growth of leg weight relative to body weight: cross sectional area of the muscle must scale allometrically in order to compensate for the increase in leg weight (scaling as L3), itself slightly allometric to body weight. This last explanation is less than compelling since cross sectional area of biceps femoris is not allometric to leg weight (on the other hand, it is the area of all the flexors, including semitendinosus and semimembranosus, that should be allometric to leg weight and not biceps femoris alone). Because the adductors are the primary postural muscles acting across the hip joint, we might expect them to exhibit allometric growth relative to body weight. Indeed, cross sectional area and muscle length are both allometric for adductor profundus but only muscle length is allometric for adductor superficialis; most importantly, the combined cross sectional area of the adductors is allometric (Table 3).
Growth patterns of three hindlimb muscles in chickens
631
Table 4. Results of log regression analyses for separate growth periods: 1, 7, 14 days versus 21, 35, 49 days (first line allometric coefficient, second correlation coefficient; asterisk indicates coefficient significantly different from isometry) 1, 7, l4 day period Muscle wet weight (Y) versus body weight (X) (isometry = 1 00) M. biceps femoris M. adductor superficialis M. adductor profundus Muscle dry weight (Y) versus body weight (X) (isometry = 1 -00) M. biceps femoris M. adductor superficialis M. adductor profundus
Muscle length (Y) versus body weight (X) (isometry = 0 33) M. biceps femoris M. adductor superficialis M. adductor profundus
Muscle cross sectional area (Y) versus body weight (X) (isometry = 0 67) M. biceps femoris
period
1-116 0-970 0 919 0 944 0-878 0-893
1-278* 0-992
1-357* 0 977 1-053 0 957 0 947 0 930
1-250* 0-988 1-083 0-978 1-271 * 0-969
0.502* 0-834 0-502* 0-834
0-415* 0-974 0-435* 0 952
0.586*
0.403*
0-886
0 973
1.101*
0-699 0-654 0-680 0-929 0-672 0-826
0 930
M. adductor superficialis M. adductor profundus
21, 35, 49 day
0.999* 0-911 1-126* 0-897
1P091 0-972 1.249* 0-988
The growth patterns of all three muscles are indicated more clearly by comparing an analysis of muscle and body weight during the first three weeks with an analysis made during the last four weeks (Table 4). Neither the wet nor the dry weight of the adductors show allometric growth during the first three weeks: increase in muscle weight (Figs. 3, 4) is exceedingly slow, whereas increase in body weight (Fig. 2) is moderate. During the last 4 weeks muscle weights are allometric to body weight, particularly those of adductor profundus. Nevertheless, this second period of increased growth is not sufficient to overcome the effects of the first three weeks of slow growth, and consequently wet and dry weights of the adductors show isometric growth over the entire period of the study. In contrast, biceps femoris shows allometric growth during both growth periods for dry weight and the second period for wet weight (the coefficient for the first three weeks of wet weight is not quite significantly different from isometry).
632
C. HELMI AND J. CRACRAFT
During the first three weeks muscle length and cross sectional area are allometric to body weight for all three muscles (Table 4). However, the adductors are initially so small relative to body weight that the allometry of these two parameters does not result in a substantial increase in muscle weight. Body weight increases dramatically during the last 4 weeks, and therefore growth coefficients of all three muscles are reduced for muscle length and cross sectional area. At this time cross sectional area of the adductors grows isometrically to body weight (Table 4). Very few other studies have attempted to relate growth patterns of muscle cross sectional area to the problems of scaling during the increase of body size. Davies (1972) compared measurements of transverse sectional area (TSA) of porcine M. longissimus to body weight, and his observed slope of0O69 is not significantly different from isometry. Perhaps this is not surprising since in this complex pennate muscle TSA may not represent a measurement of physiological cross sectional area; if the latter had been measured, it probably would have exhibited allometry. Rayne & Crawford (1972) found that cross sectional area was negatively allometric to body weight in four jaw muscles of the rat. They computed cross sectional area by dividing muscle volume by the origin-insertion length, but again, in complex pennate muscles, such as those of the jaw, this procedure probably will not yield an accurate measure of physiological cross sectional area, and thus the interesting question of allometric scaling could not be investigated.
Patterns of muscle growth A muscle increases in size by longitudinal growth of existing fibres (or perhaps, more rarely, by the longitudinal addition of new fibres in series), by the addition of new fibres to increase girth, and/or by the growth in diameter of existing fibres. We have examined aspects of longitudinal growth of fasciculi and increase in muscle girth, but we have not concerned ourselves with the mechanisms by which this growth is obtained at the cellular level. Our purpose has been to examine growth patterns with respect to problems of allometric scaling and the concomitant changes in functional demands brought on by increase in body size. The results of this study confirm that increase in muscle size (e.g. weight) may take place quite differently in different muscles. We compared the growth curves of cross sectional area versus muscle wet weight with those of fasciculus length (LFL, SFL) versus wet weight (Table 3). In biceps femoris and adductor superficialis cross sectional area grows only slightly allometrically to wet weight whereas LFL and SFL exhibit stronger allometry. This suggests that growth in fasciculus length may be contributing relatively more to the increase of muscle weight than is growth of cross sectional area. Both muscles exhibit characteristics consistent with this pattern of growth. M. biceps femoris arises from a broad area of attachment, but narrows distally. Growth of biceps femoris would be limited posteriorly by the presence of semimembranosus and semitendinosus, laterally by iliotibialis, medially by piriformis, semimembranosus, and semitendinosus, and anteriorly by the lack of an area of origin. Growth in length will be limited only by the final adult leg size; functionally, as the animal grows, the fibres of biceps femoris must lengthen substantially in order that the muscle can continue to shorten (or stretch) over the increasingly greater absolute distances demanded by larger body size and a bone-muscle arrangement like this
Growth patterns of three hindlimb muscles in chickens 633 knee joint flexor. Similarly, adductor superficialis is a very thin, straplike muscle, closely bounded on both the medial and lateral sides by other muscles, and growth therefore would be accomplished more easily by increase in length than in girth. Nevertheless, in contrast to the bone-muscle arrangement of biceps femoris, that of the adductors (i.e. one-joint muscles whose origins and insertions are close to the centre of rotation) allows for less increase in length with increasing body size (more elements contribute to absolute length increase in biceps femoris than the adductors), and indeed adductor superficialis has a slower rate of growth than does biceps femoris (Table 3). The growth pattern of adductor profundus is somewhat different from that of the above two muscles. Although cross sectional area and SFL grow allometrically to muscle wet weight, LFL does not (see also below). In this muscle increase in cross sectional area apparently contributes more to weight increase than does growth in fasciculus length. The geometry of the muscle in the leg may play a role. Unlike the other two muscles, adductor profundus can grow in a medial direction; there are no muscles covering the medial surface, except distally at the insertion, and ultimate limits to growth probably include the amount of area available for origin and insertion of fibres. A comparison of growth of LFL and SFL in the three muscles contributes some information about differential growth rates of fasciculi within the same muscle. In biceps femoris and adductor superficialis the shortest (SFL) and longest (LFL) fasciculi appear to be growing at relatively the same rates (Table 3). In adductor profundus the SFL appears to have growth rates significantly higher than the LFL. Very little information is available on this aspect of muscle growth. In a study of M. tibialis anterior of the rabbit Crawford (1954) gathered data which suggest fairly uniform growth rates of fasciculi throughout the muscle, but in this study he was examining growth rates within the middle portion of the muscle belly rather than changes in the lengths of fasciculi as measured from their ends. Mackay & Harrop (1969), employing techniques similar to those of Crawford, also found uniform growth in M. sternomastoideus and M. anterior gracilis of the rat; these authors also measured growth within the middle portion of the muscle belly. It is not possible at this time to suggest an explanation for apparent differences in fasciculus growth rate in adductor profundus. Very probably these differences are associated with subtle changes in fasciculus orientation within the muscle as it grows: because the absolute amount of growth in these fasciculi is small, changes in architecture are difficult to detect. Functional analysis of growing muscle One conclusion from this study is that an analysis of muscle growth based only on one or two parameters, especially muscle weight, may not provide sufficient information to make functional interpretations about the growth patterns. Many workers have studied the relative growth of muscle wet or dry weight (see review of Stewart, 1972). It is clear from the literature that growth rates of muscle weight do often change during ontogeny and that these differences may reflect changes in function at certain periods of the life cycle (see, for example, the interpretations of Bryden (1973) for over 70 muscles of the elephant seal, Mirounga leonina). However, muscle 40
ANA 123
634 C. HELMI AND J. CRACRAFT weight itself may not be an accurate index of physiological properties such as relative force development, speed of contraction, or length of stretching and shortening (Gans & Bock, 1965; Bock, 1974), and thus the use of muscle weight alone will not provide an adequate descriptive picture of functional changes during growth. For example, wet and dry weights of biceps femoris have growth patterns (Figs. 3, 4) that correlate well with the increase in body size at 21 days (Fig. 2), but this relationship is less precise for the weights of the adductors. On the other hand, cross sectional area is the parameter that is functionally relevant for comparison with body size, and cross sectional area of adductor profundus exhibits a growth pattern that very closely mirrors the increase of body weight (Fig. 7). In conclusion, we can expect to learn much more about the functional aspects of muscle growth if future workers will examine a number of parameters rather than just one or two. Furthermore, events of muscle growth need to be related to changes in behaviour of living animals. In this connexion one of us (J. C.) is examining the details of ontogenetic changes in locomotor behaviour of chickens using high speed cinematography, and one of the expectations is that the patterns of muscle growth described above can be correlated with locomotion more closely than is presently possible. Virtually all students of muscle growth have neglected to correlate their findings with changes in behaviour, so that this remains an important area of research for functional morphologists. SUMMARY
This study was designed to investigate the growth patterns of three hindlimb muscles of the chicken relative to the functional-biomechanical demands of increasing body size. The biceps femoris, a bipennate non-postural muscle, grew relatively faster in terms of wet and dry weight than did the parallel-fibred adductor superficialis or the unipennate adductor profundus, both postural muscles. All three muscles exhibited positive allometry (relative to body weight) in muscle length but only biceps femoris and adductor profundus showed positive allometry in cross sectional area, adductor superficialis having isometric growth in this parameter. In biceps femoris and adductor superficialis the lengths of the longest and shortest fasciculi grew at equal rates, whereas in adductor profundus the shortest fasciculi grew faster than the longest. We conclude that muscle weight alone is an insufficient indicator of changing function in growing muscle. Hence, growth studies should include other functionally relevant parameters such as cross sectional area, which is proportional to the forceproducing capabilities of the muscle, or fibre (fasciculus) length, which is indicative of the absolute amount of stretching or shortening that is possible and of the contraction velocity. We wish to thank the following colleagues for their help and comments on the manuscript: Drs E. F. Allin, H. R. Barghusen, S. M. Herring, and R. P. Scapino. We are also grateful to Ms Kathryn Sisson for her excellent illustrations. This research was supported by a National Science Foundation grant (GB-41089) to J. C. and by funds from the Department of Anatomy, University of Illinois, to C. H.
Growth patterns of three hindlimb muscles in chickens
635
REFERENCES
BOCK, W. J. (1974). The avian skeletomuscular system. In Avian Biology, vol. 4 (ed. D. S. Farner and J. R. King), pp. 119-257. New York: Academic Press. BRYDEN, M. M. (1973). Growth patterns of individual muscles of the elephant seal, Mirounga leonina. Journal of Anatomy 116, 121-133. CHURCH, L. E. & JOHNSON, L. C. (1964). Growth of long bones in the chicken, rates of growth in length and diameter of the humerus, tibia, and metatarsus. American Journal of Anatomy 114, 521-538. CRACRAFT, J. (1971). The functional morphology of the hind limb of the domestic pigeon, Columba livia. Bulletin of the American Museum of Natural History 144, 173-268. CRAWFORD, G. N. C. (1954). An experimental study of muscle growth in the rabbit. Journal of Bone and Joint Surgery 36, 294-303. DAVIES, A. S. (1972). Postnatal changes in the histochemical fibre types of porcine skeletal muscle. Journal of Anatomy 113, 213-240. GANS, C. & BOCK, W. J. (1965). The functional analysis of muscle architecture: a theoretical analysis. Ergebnisse der Anatomie und Entwicklungsgeschichte 38, 115-142. GEORGE, J. C. & BERGER, A. J. (1966). Avian Myology. New York: Academic Press. GOLDBERG, A. L. (1967). Work-induced growth of skeletal muscle in normal and hypophysectomized rats. American Journal of Physiology 213, 1193-1198. GOLDSPINK, G. (1970). Morphological adaptations due to growth and activity. In The Physiology and Biochemistry of Muscle as a Food, vol. 2 (ed. E. J. Brisky, R. G. Cassens and B. B. Marsh), pp. 521-536. Madison: University of Wisconsin Press. GOLDSPINK, G. (1972). Postembryonic growth and differentiation of striated muscle. In The Structure and Function of Muscle (ed. G. H. Bourne), pp. 179-236. New York: Academic Press. GOULD, S. J. (1971). Muscular mechanics and the ontogeny of swimming in scallops. Palaeontology 14, 61-94. GUTMANN, E., SCHIAFFINO, S. & HANZLIKOVA, V. (1971). Mechanism of compensatory hypertrophy in skeletal muscle of the rat. Experimental Neurology 31, 451-464. HILL, A. V. (1950). The dimensions of animals and their muscular dynamics. Science Progress 38, 209-230. HUDSON, G. E., LANZILLOTHI, P. J. & EDWARDS, G. D. (1959). Muscles of the pelvic limb in galliform birds. American Midland Naturalist 61, 1-67. JAMES, N. T. (1973). Compensatory hypertrophy in the extensor digitorum longus muscle of the rat. Journal of Anatomy 116, 57-65. MACKAY, B. & HARROP, T. J. (1969). An experimental study of the longitudinal growth of skeletal muscle in the rat. Acta anatomica 72, 38-49. MUJLLER, W. (1974). Temporal progress of muscle adaptation to endurance training in hind limb muscles of young rats. A histochemical and morphometrical study. Cell Tissue Research 156, 61-87. RAYNE, J. & CRAWFORD, G. N. C. (1972). The growth of muscles of mastication in the rat. Journal of Anatomy 113, 391-408. ROWE, R. W. D. & GOLDSPINK, G. (1969). Muscle fibre growth in five different muscles in both sexes of mice. I. Normal mice. Journal of Anatomy 104, 519-530. SCHWARTZ, N. B. (1961). Changing size, composition, and contraction strength of gastrocnemius muscle. American Journal of Plhysiology 201, 164-170. STEWART, D. M. (1972). The role of tension in muscle growth. In Regulation of Organ and Tissue Growth (ed. R. J. Goss), pp. 77-100. New York: Academic Press. WILLIAMS, P. E. & GOLDSPINK, G. (1973). The effects of immobilization on the longitudinal growth of striated muscle fibres. Journal of Anatomy 116, 45-55. WUNDER, C. C. & BIRD, J. W. C. (1969). Growth of the white-mouse gastrocnemius muscle. I. Terrestrial gravity. Proceedings of the Iowa Academy of Sciences 76, 368-375.
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J. Anat. (1977), 123, 3, pp. 615-635 With 9 figures Printed in Great Britain
The growth patterns of three hindlimb muscles in the chicken CYRUS HELMI* AND JOEL CRACRAFT
Department of Anatomy, University of Illinois at the Medical Center, Chicago, Illinois 60680
(Accepted 14 May 1976) INTRODUCTION
There is now a large literature on the mechanisms of growth of skeletal muscle (see review by Goldspink, 1972). Nevertheless, very few reported studies have examined the complex interactions of numerous growth parameters for individual muscles and tried to relate patterns of growth to the functional requirements of increasing body size. Two notable exceptions are the studies by Davies (1972) on the deep back muscles of the pig and Rayne & Crawford (1972) on the jaw muscles of the rat. Other workers have mentioned the consequences of increasing body size (Hill, 1950; Schwartz, 1961; Wunder & Bird, 1969; Goldspink, 1970; Stewart, 1972; Bryden, 1973) or have demonstrated the importance of work load (tension) in determining the morphological and physiological properties of growing muscle (e.g. Goldberg, 1967; Rowe & Goldspink, 1969; Gutmann, Schiaffino & Hanzlikova, 1971; Goldspink, 1972; Stewart, 1972; James, 1973; Williams & Goldspink, 1973; Muller, 1974). The purpose of this study was to examine the growth of three hindlimb muscles of the chicken (Gallus gallus), using a number of growth parameters including wet and dry weights, muscle cross sectional area and length, and muscle fasciculus length. In addition to purely descriptive aspects, the investigation compared the growth patterns of certain muscles whose adult architectures differ and whose roles change during postural and locomotor cycles. A major purpose was to determine, if possible, the ways in which architecture (form) and function relate to differences in the growth patterns themselves and to the mechanical demands of increasing body size. Thus, two of the muscles chosen, adductor superficialis and adductor profundus, are postural muscles: the former is parallel fibred whereas the latter is unipennate. The third muscle, biceps femoris, differs in having the non-postural function of flexing the knee joint and in being bipennate. MATERIALS AND METHODS
The chickens used in this study were raised from hatching. A total of 276 individual muscles was obtained from 46 chickens killed in groups 1, 7, 14, 21, 35 and 49 days after hatching. Each age group consisted of 7-9 individuals. The sex was not noted, *
Present address: Department of Anatomy, University of Chicago, Chicago, Illinois 60637.
616
C. HELMI AND J. CRACRAFT
M. femorotibialis
M. semitendinosus M. biceps femorisM. femorotib
S
bic psM. adductor profundus
M. adductor superficialis
A
B
Fig. 1. Two views of leg muscles of the chicken in superficial (A) and deep (B) dissection to show muscles examined in this study.
because it seemed irrelevant to the present investigation; we were examining the morphological characteristics of muscles relative to individual body size. Gross morphology of the muscles The adult morphology and functions of the leg muscles examined in this study will be described briefly. The gross morphology and functions of these muscles do not differ significantly among galliform birds (Hudson, Lanzillotti & Edwards, 1959; George & Berger, 1966). M. biceps femoris This is a large two-joint muscle passing posterior to the hip and knee joints (Fig. 1). It arises aponeurotically from the most posterior portion of the anterior iliac crest dorsal to the hip joint and has a fleshy origin as well from the posterior iliac crest. The muscle tapers from its wide origin, and a strong tendon inserts on to the posterior surface of the proximal end of the fibula. The biceps femoris has an asymmetrical bipennate fibre arrangement. The central tendon of insertion is located nearer the posterior margin of the muscle, hence most of the fibres insert on to the anterior part of the tendon. The anterior fibres have a larger angle of pennation (about 200) than the posterior fibres (about 120). The biceps femoris functions to flex the knee joint during terrestrial locomotion; the muscle is probably not active during the weight-bearing propulsive phase of locomotion except perhaps to stabilize the knee joint at the beginning of the stance phase, or perhaps to effect some movement of the pelvis relative to the femur. Cracraft (1971) found that this muscle is composed predominantly of twitch (Fibrillenstruktur) fibres in the domestic pigeon (Columba livia), and this is probably true for most birds. Thus, the long fibres and twitch fibre composition suggest a muscle that during locomotion is developing relatively high velocities, shortening over a
617
Growthl patterns of three hindlimb muscles in chickens
relatively great distance, and acting against comparatively little resistance (in this case, the weight of the lower portion of the leg). M. adductor superficialis This is a flat, nearly trapezoidal, one-joint muscle passing posterior to the hip joint (Fig. 1). It arises by a short aponeurosis from the ventral margin of the ischium. The muscle has a fleshy insertion on to the posteromedial side of the distal half of the femur. The adductor superficialis is composed of long parallel fibres, with the anterior fibres being shorter than those located more posteriorly. This muscle functions to retract the femur. More importantly, it serves a postural function during standing and also resists any forward motion (protraction) of the femur during landing, and at the moment of impact during walking (Cracraft, 1971). In the domestic pigeon this muscle is composed predominantly of tonus (Felderstruktur) fibres, which supports the notion that the muscle is primarily postural, resisting forces over extended periods of time (Cracraft, 1971). M. adductor profundus This is a large, one-joint muscle passing posterior to the hip joint (Fig. 1). It arises from the ventral edge of the ischium by a broad, thin aponeurosis shared with adductor superficialis. The muscle inserts on to the posterior surface of the distal two thirds of the femur. The adductor profundus ,!as a unipennate fibre arrangement, but as the fibres are moderately long and the angle of pennation very small the muscle appears at first glance to have a parallel fibre arrangement. The anterior fibres are shorter than the posterior fibres. In the domestic pigeon this muscle has the same function and fibre-type composition as the adductor superficialis (Cracraft, 1971), and this is undoubtedly true for the chicken also. Fixation and dissection a buffered alcohol-formaldehyde fixative, and care in fixed were muscles The leg in the relaxed position in order to minimize stretching the to leg was taken preserve to be relatively uniform in all muscles. All was assumed muscles. Shrinkage of the a dissecting microscope. binocular out using dissections were carried Measurements The following measurements were taken on each individual: (1) Body weight: recorded to the nearest 0 1 g. (2) Leg weight: the total leg weight distal to the knee joint was recorded to the nearest 0 1 g. (3) Muscle length: the maximum extent of the contractile portion of the muscle (measured along the origin-insertion axis), not including the tendon or aponeurosis, was measured to the nearest 0-1 mm. (4) Fasciculus length: muscle fasciculi were first teased apart under a binocular dissecting microscope. A tracing of a straightened (not stretched) individual fasciculus was obtained using a camera lucida attachment, and the tracings were then measured 39
ANA 123
C. HELMI AND J. CRACRAFT 618 and corrected for magnification. In this manner more accurate measurements were obtained than if the fasciculus had been measured directly. Direct measurement by calipers to the nearest 0-1 mm was made only on the longer fasciculi of the older individuals. Measurements of fasciculi taken from populations of the longest and shortest fasciculi were obtained from each muscle. (5) Muscle cross sectional area: each muscle was transected in the middle of the muscle belly. A tracing of the cross section was then obtained using a binocular dissecting microscope with a camera lucida attachment. From this tracing the area was determined with the aid of a polar planimeter. Each measurement reported below is an average of five determinations. In the muscles studied measurements at the middle of the muscle belly were assumed to be fairly accurate estimations of physiological cross sectional area for purposes of comparison between muscles of different age groups. In all three muscles the fasciculi were long enough to make it reasonably certain that all of them were included in the section. (6) Muscle wet weight: the muscles were lightly blotted and then weighed on an electric microbalance to the nearest 0i001 g. (7) Muscle dry weight: the muscles were dried in an oven to constant weight at 60 'C (about 24 hours). They were than weighed on a microbalance to the nearest 0.001 g. Statistical methods In addition to basic statistics (mean, standard deviation), regression equations were calculated for many variables against body weight. Logarithmic transformations of the raw data were undertaken when it seemed appropriate. In analysing growth patterns the data were fitted by least-squares regression to the well-known allometric growth equation, log Y = log a+b log X,
where Y is a variable changing in value relative to X (usually body weight), log a is the Y-intercept, and b is the slope. RESULTS
Body weight The increase in body weight from hatching to 49 days of age is shown in Figure 2 and data are given in Table 1. The growth curve appears to be approximately logarithmic, although the asymptote has not yet been reached at 49 days. Between the time of hatching and 49 days the mean body weight of the age groups increased about 14-fold (Table 1). Relative to the average final weight (585 g), 2-64 % of that was added in the first week, 8-43 % in the second, and 7-61 0% in the third. During the interval between 21 and 35 days about 32-85 % of the final body weight was added, and in the last 2 weeks 41 36 %o (242 g) of the body weight was added. Church & Johnson (1964) found that after 21 days body weight was sexually dimorphic, with males having a higher gross weight (as mentioned earlier, the goals of this study did not necessitate sex determination).
Growth patterns of three hindlimb muscles in chickens
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Muscle weight The mean wet and dry weights for each muscle are given for each age group in Table 1 and plotted in Figures 3 and 4. For both wet and dry weight the first 3 weeks is a period of relatively slow growth for all three muscles. During this period both the dry and wet weights of biceps femoris were higher than either of the adductors, and the growth rate of the former was somewhat greater than those of the latter two muscles. The growth of the two adductors was nearly equal during this time. After the third week each muscle rapidly increased in weight, with biceps femoris increasing significantly faster than the two adductors. During this period of rapid growth the two adductors no longer grew at the same rate, and the weights of adductor profundus increased more than those of adductor superficialis. Between hatching and day 49 the mean dry weight for biceps femoris, adductor profundus, and adductor superficialis increased 28-0-, 18-5- and 15-0-fold, respectively, whereas in wet weight they increased 22-7-, 17-2- and 13-5-fold. Table 1 shows that on the first day the total wet and dry weights of the combined adductors were larger than those of the biceps femoris, and that the latter muscle quickly became increasingly larger than the adductor group. 39-2
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Relative growth of muscle weight Log transformed regression statistics for muscle dry and wet weight versus body weight are given in Table 3 and the data are plotted in Figures 5 and 6. The allometric growth coefficient for dry weight of adductor superficialis (slope = 1-003) is not significantly different (P > 005) from unity, indicating that growth of this muscle as expressed by dry weight is proportional to growth of the animal as a whole. The growth coefficients for biceps femoris (slope = 1 228), adductor profundus (slope = 1 089), and the adductors combined (slope = 1-050), on the other hand, are significantly different from isometry, and positive allometry is indicated. It should be noted, however, that the coefficients of adductor profundus and the adductors considered together were barely significant at the 5 % level. The growth coefficient of biceps femoris is also significantly different from those of the adductors considered separately and combined. Regression statistics for log wet weight versus log body weight show similar patterns to those of dry weight. The growth coefficients for biceps femoris (slope = 1'158) and adductor profundus (slope = 1'090) are significantly different from isometry, whereas that of adductor superficialis (slope = 0'959) is not. The combined wet weight of the adductors is also not different from isometry.
622
C. HELMI AND J. CRACRAFT 075
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0-55
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21 28 35 42 49 Age (days) Fig. 4. Relationship between muscle dry weight and age for adductor superficialis, adductor profundus, and biceps femoris. Note the marked increase in growth after 21 days, especially in biceps femoris. 1
7
Growth of muscle cross sectional area Raw data for growth of muscle cross sectional area are given in Table 2 and plotted in Figure 7. Cross sectional area of all three muscles increased substantially between 1 and 14 days, but between 14 and 21 days there appears to have been a marked decrease in growth (Fig. 7). After 21 days there was again a marked increase in growth, correlated with a significant increase in the growth of body weight (Fig. 2 and later discussion). Log transformed regression statistics for muscle cross sectional area versus body weight are presented in Table 3 (see Fig. 8). The allometric coefficients for biceps femoris, adductor profundus, and the combined area of both adductors are significantly different from isometry (slope = 067), and all show positive allometry. The slope of 0718 for adductor superficialis is not significantly different from isometry. M. biceps femoris cross sectional area versus leg weight Because biceps femoris functions to lift that portion of the leg distal to the knee joint, we examined the growth pattern of the muscle relative to the leg weight.
623
Growth patterns of three hindlimb muscles in chickens 3 -00 12 *00
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Fig. 5. Allometric relationship between muscle wet weight and body weight for adductor superficialis, adductor profundus, and biceps femoris. Log-log scales.
Table 3 presents the regression statistics for this relationship. The allometric coefficient (slope = 0-58) is significantly different at the 5 % level from isometry (slope = 0-67), thus implying a somewhat negatively allometric growth for cross sectional area relative to leg weight.
Growth of leg weight versus body weight In order to assess more fully the growth pattern of biceps femoris, the relationship between growth in leg and body weight was studied (see Table 1 for raw data). Regression statistics indicate that increase in leg weight is slightly positively allometric to body weight (Table 3). The allometric coefficient is 1-08, with a correlation of 0-998, and is significantly different from isometry (slope = 1 -00) at the 5 % level.
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C. HELMI AND J. CRACRAFT
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Fig. 6. Allometric relationship between muscle dry weight and body weight for adductor superficialis, adductor profundus and biceps femoris. Log-log scales.
Growth of muscle length Raw data for growth of muscle length are presented in Table 2 and are graphed against time in Figure 7. The lengths of all three muscles increase steadily with time, there being no noticeable change in growth rate at 21 days as there was for cross sectional area. As expected, biceps femoris increases in length more rapidly than do the adductors (Fig. 7), which must lengthen at about the same rate because of their nearly identical position within the leg (Fig. 1). The growth patterns of muscle length relative to body weight were also investigated (Table 3). All three muscles show positive allometric growth, with growth coefficients significantly different from isometry (slope = 033): biceps femoris (050), adductor superficialis (0-46), and adductor profundus (0-47). None of the muscles was significantly different from the others in its growth coefficient. Growth of muscle fasciculus length We examined the relationship between growth in fasciculus length and body size (weight), since a particular rate of growth in muscle length (see above) does not necessarily imply an equal rate of growth in fasciculus length.
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Growth patterns of three hindlimb muscles in chickens
627
Table 3. Results of regression analyses (First line is correlation coefficient, second is slope, and third is y-intercept; asterisk indicates allometric coefficient significantly different from isometry at 5 % level.) Log dry weight versus log body weight
Log wet weight versus log body weight
0994 1-228* -8 255 M. adductor 0-989 1-003 superficialis -8-069 M. adductor 0-983 profundus 1-089* -8-429 M. adductor super0.991 ficialis and M. 1-050* adductor profundus -7 569
0994 1.158* -2-891 0-986 0 959 -2-876 0-985 1 090* -3-083 0 995 1-041 -2-709
0-969 0.832* -2-001 0-960 0-718
0-974 0.579* 2 529 -
0965 0.497* 1-040 0965
-1P910
-
0 899 0-969
-
0-465* 0-884
0956 0-491* 0-971 0-966 0.464* 0-839 0-768 0-295 1-123
LFL versus
SFL versus muscle length
Log X-sect. area versus log muscle wet weight
Log LFL versus log muscle wet weight
Log SFL versus log muscle wet weight
0 947 0-417* 1-643 0-965 0-476* 1-748 0-776 0-270 1*322
0-922 0.412* 1-561 0 911 0.432* 1-432 0-835 0.415* 1-114
M. biceps femoris
Log SFL versus log body weight M. biceps femoris
M. adductor
superficialis M. adductor profundus
0-933 0-486* 0-351 0-920 0-425* 0-167 0-855 0.470* -0-203
muscle length
0'991 0-884 0 574 0-980 0-992 -0 404 0-827 0 400 3 603
Log X-sect. Log muscle area X-sect. area versus versus log body log leg weight weight
0.457*
0957
0.839* -2-142 0-971 0-788* -1 353
0-981 0-717 1-138 0 959 0-465 1-072 0-874 0 304 -0 704
Log muscle length versus log body weight
0-978 0-721* 2-786 0-967
0.744* 3-036 0-956 0-757*
3.300
Log LFL versus log body weight
Raw data for fasciculus length are given in Table 2 (see Fig. 9); regression statistics for longest fasciculus length (LFL) and shortest fasciculus length (SFL) against body weight are presented in Table 3. For LFL both biceps femoris (slope = 049) and adductor superficialis (slope = 046) exhibit positive allometric growth (isometry = 033) and are not significantly different from one another. The adductor profundus, on the other hand, shows a much lower growth coefficient (slope = 030), which is not significantly different from isometry. Growth coefficients for muscle length and LFL relative to body weight are not different for biceps femoris and adductor superficialis, but there is a significant difference between the two for adductor profundus. The data presented in Table 3 indicate that all three muscles have coefficients significantly greater (P < 005) than isometry when log SFL is regressed against log body weight. The coefficients of the three muscles are not significantly different from each other.
628
C. HELMI AND J. CRACRAFT
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Growth patterns of three hindlimb muscles in chickens
629
Growth offasciculus length and muscle length The LFL and SFL were plotted against the muscle length (Table 3). The LFL of adductor superficialis is very nearly equal to the muscle length at any given age. This, of course, was not unexpected since in this parallel fibre muscle the fasciculi essentially run the length of the muscle. In the bipennate muscle, biceps femoris, the fasciculi are also long, and the slope of 0O88 indicates that nearly equal increments are being added to fasciculus length and to muscle length throughout the growth period. In the linear equation, Y = ax+ b, the y-intercept provides a measure for allometric growth (Gould, 1971). The slope merely signifies the constant change, ylx, being added throughout growth. Both adductor superficialis and biceps femoris have y-intercepts that are not significantly different from zero (P > 005), hence no allometry is taking place with respect to fasciculus length versus muscle length. In contrast, adductor profundus exhibits marked negative allometry with respect to fasciculus length. The positive y-intercept of 3 60 indicates that the value of x (here, muscle length) between any two points on the growth equation line is increasing at a greater rate than the y value (here, LFL) of those same two points (if the y-intercept were negative, y values would increase faster than x values); hence negative allometry is observed. For SFL versus muscle length none of the intercepts is significantly different from zero, thus indicating that SFL is increasing isometrically to muscle length. Muscle growth: muscle weight compared to cross sectional area and fasciculus length of muscle Since volume a (and thus weight, assuming a constant specific gravity) is roughly a function of its cross sectional area and its length, it seemed important to assess the relative contributions of cross sectional area and fasciculus length to growth. Table 3 presents the regression statistics for these comparisons. Plotting log cross sectional area against log muscle wet weight it is observed that all of the muscles have slopes that are significantly different from isometry (slope = 0 67). However, we note that the coefficients of all three muscles were barely significant and thus the degree of positive allometry is not great; the larger coefficient of 0757 for adductor profundus suggests that this muscle might be increasing relatively more in cross sectional area than the other muscles, but still the differences among the muscles are not statistically significant. With respect to the regression of log LFL on log muscle wet weight, the growth patterns appear to be somewhat different from those of cross sectional area. In biceps femoris and adductor superficialis the allometric coefficients of LFL are substantially larger than the value of isometry (033), whereas in the comparison of cross sectional area and muscle wet weight allometric coefficients were barely significant. M. adductor profundus, on the other hand, exhibits a coefficient for LFL that is not significantly different from isometry, and thus the coefficient of LFL when regressed against wet weight appears relatively smaller than the coefficient determined in the comparison of cross sectional area and wet weight: cross sectional area exhibits stronger allometry than LFL. When log SFL is regressed against log muscle wet weight, the coefficients for all three muscles exhibit positive allometry (Table 3).
630
C. HELMI AND J. CRACRAFT
DISCUSSION
Relationship between muscle growth and body size Little attention has been given in the past to the relationship between muscle growth and increasing body size. As the latter increases functional demands on the muscles inevitably change, and consequently modification of structural characteristics of the muscles must ensue if the functional efficiency of the muscle is to be maintained. An important problem facing studies such as this one is determining which parameters will provide the most useful and reliable information about changing functional aspects of growing muscle. We comment on this problem later in the discussion. As body size increases it might be expected that postural muscles will grow allometrically. Body weight is scaling as the cube of the length, muscle cross sectional area (and hence a measure of force development) as the square. Given these geometrical and biomechanical accompaniments of growth, one would predict a change in shape. Examination of Table 3 reveals some complex growth patterns. Only the non-postural biceps femoris shows strong allometry of dry and wet weight, whereas the adductors, which are postural, either exhibit isometric growth (adductor superficialis) or weak allometry (adductor profundus) of these two measures. Furthermore, biceps femoris and adductor profundus show strong allometry of cross sectional area but adductor superficialis exhibits isometric growth of this parameter. One observation of this study that was somewhat unexpected was the marked allometry of biceps femoris in dry and wet weight and in cross sectional area (Table 3): our working hypothesis was that a non-postural muscle should exhibit weaker allometry than the postural muscles. The strong positive allometry of both cross sectional area and muscle length in biceps femoris accounts for the allometric growth of wet and dry weight, but the question remains why cross sectional area exhibits positive allometry. Because of its position within the leg it is reasonable to assume that biceps femoris is not resisting body weight during maintenance of posture. However, the muscle may function to maintain knee joint stability at the time the foot makes contact with the ground upon initiation of the stance phase, much like the hamstring complex does in human locomotion; in this case, the magnitude of body weight may be an important influence on the growth of the muscle. The allometry of biceps femoris may also be related to the pattern of growth of leg weight relative to body weight: cross sectional area of the muscle must scale allometrically in order to compensate for the increase in leg weight (scaling as L3), itself slightly allometric to body weight. This last explanation is less than compelling since cross sectional area of biceps femoris is not allometric to leg weight (on the other hand, it is the area of all the flexors, including semitendinosus and semimembranosus, that should be allometric to leg weight and not biceps femoris alone). Because the adductors are the primary postural muscles acting across the hip joint, we might expect them to exhibit allometric growth relative to body weight. Indeed, cross sectional area and muscle length are both allometric for adductor profundus but only muscle length is allometric for adductor superficialis; most importantly, the combined cross sectional area of the adductors is allometric (Table 3).
Growth patterns of three hindlimb muscles in chickens
631
Table 4. Results of log regression analyses for separate growth periods: 1, 7, 14 days versus 21, 35, 49 days (first line allometric coefficient, second correlation coefficient; asterisk indicates coefficient significantly different from isometry) 1, 7, l4 day period Muscle wet weight (Y) versus body weight (X) (isometry = 1 00) M. biceps femoris M. adductor superficialis M. adductor profundus Muscle dry weight (Y) versus body weight (X) (isometry = 1 -00) M. biceps femoris M. adductor superficialis M. adductor profundus
Muscle length (Y) versus body weight (X) (isometry = 0 33) M. biceps femoris M. adductor superficialis M. adductor profundus
Muscle cross sectional area (Y) versus body weight (X) (isometry = 0 67) M. biceps femoris
period
1-116 0-970 0 919 0 944 0-878 0-893
1-278* 0-992
1-357* 0 977 1-053 0 957 0 947 0 930
1-250* 0-988 1-083 0-978 1-271 * 0-969
0.502* 0-834 0-502* 0-834
0-415* 0-974 0-435* 0 952
0.586*
0.403*
0-886
0 973
1.101*
0-699 0-654 0-680 0-929 0-672 0-826
0 930
M. adductor superficialis M. adductor profundus
21, 35, 49 day
0.999* 0-911 1-126* 0-897
1P091 0-972 1.249* 0-988
The growth patterns of all three muscles are indicated more clearly by comparing an analysis of muscle and body weight during the first three weeks with an analysis made during the last four weeks (Table 4). Neither the wet nor the dry weight of the adductors show allometric growth during the first three weeks: increase in muscle weight (Figs. 3, 4) is exceedingly slow, whereas increase in body weight (Fig. 2) is moderate. During the last 4 weeks muscle weights are allometric to body weight, particularly those of adductor profundus. Nevertheless, this second period of increased growth is not sufficient to overcome the effects of the first three weeks of slow growth, and consequently wet and dry weights of the adductors show isometric growth over the entire period of the study. In contrast, biceps femoris shows allometric growth during both growth periods for dry weight and the second period for wet weight (the coefficient for the first three weeks of wet weight is not quite significantly different from isometry).
632
C. HELMI AND J. CRACRAFT
During the first three weeks muscle length and cross sectional area are allometric to body weight for all three muscles (Table 4). However, the adductors are initially so small relative to body weight that the allometry of these two parameters does not result in a substantial increase in muscle weight. Body weight increases dramatically during the last 4 weeks, and therefore growth coefficients of all three muscles are reduced for muscle length and cross sectional area. At this time cross sectional area of the adductors grows isometrically to body weight (Table 4). Very few other studies have attempted to relate growth patterns of muscle cross sectional area to the problems of scaling during the increase of body size. Davies (1972) compared measurements of transverse sectional area (TSA) of porcine M. longissimus to body weight, and his observed slope of0O69 is not significantly different from isometry. Perhaps this is not surprising since in this complex pennate muscle TSA may not represent a measurement of physiological cross sectional area; if the latter had been measured, it probably would have exhibited allometry. Rayne & Crawford (1972) found that cross sectional area was negatively allometric to body weight in four jaw muscles of the rat. They computed cross sectional area by dividing muscle volume by the origin-insertion length, but again, in complex pennate muscles, such as those of the jaw, this procedure probably will not yield an accurate measure of physiological cross sectional area, and thus the interesting question of allometric scaling could not be investigated.
Patterns of muscle growth A muscle increases in size by longitudinal growth of existing fibres (or perhaps, more rarely, by the longitudinal addition of new fibres in series), by the addition of new fibres to increase girth, and/or by the growth in diameter of existing fibres. We have examined aspects of longitudinal growth of fasciculi and increase in muscle girth, but we have not concerned ourselves with the mechanisms by which this growth is obtained at the cellular level. Our purpose has been to examine growth patterns with respect to problems of allometric scaling and the concomitant changes in functional demands brought on by increase in body size. The results of this study confirm that increase in muscle size (e.g. weight) may take place quite differently in different muscles. We compared the growth curves of cross sectional area versus muscle wet weight with those of fasciculus length (LFL, SFL) versus wet weight (Table 3). In biceps femoris and adductor superficialis cross sectional area grows only slightly allometrically to wet weight whereas LFL and SFL exhibit stronger allometry. This suggests that growth in fasciculus length may be contributing relatively more to the increase of muscle weight than is growth of cross sectional area. Both muscles exhibit characteristics consistent with this pattern of growth. M. biceps femoris arises from a broad area of attachment, but narrows distally. Growth of biceps femoris would be limited posteriorly by the presence of semimembranosus and semitendinosus, laterally by iliotibialis, medially by piriformis, semimembranosus, and semitendinosus, and anteriorly by the lack of an area of origin. Growth in length will be limited only by the final adult leg size; functionally, as the animal grows, the fibres of biceps femoris must lengthen substantially in order that the muscle can continue to shorten (or stretch) over the increasingly greater absolute distances demanded by larger body size and a bone-muscle arrangement like this
Growth patterns of three hindlimb muscles in chickens 633 knee joint flexor. Similarly, adductor superficialis is a very thin, straplike muscle, closely bounded on both the medial and lateral sides by other muscles, and growth therefore would be accomplished more easily by increase in length than in girth. Nevertheless, in contrast to the bone-muscle arrangement of biceps femoris, that of the adductors (i.e. one-joint muscles whose origins and insertions are close to the centre of rotation) allows for less increase in length with increasing body size (more elements contribute to absolute length increase in biceps femoris than the adductors), and indeed adductor superficialis has a slower rate of growth than does biceps femoris (Table 3). The growth pattern of adductor profundus is somewhat different from that of the above two muscles. Although cross sectional area and SFL grow allometrically to muscle wet weight, LFL does not (see also below). In this muscle increase in cross sectional area apparently contributes more to weight increase than does growth in fasciculus length. The geometry of the muscle in the leg may play a role. Unlike the other two muscles, adductor profundus can grow in a medial direction; there are no muscles covering the medial surface, except distally at the insertion, and ultimate limits to growth probably include the amount of area available for origin and insertion of fibres. A comparison of growth of LFL and SFL in the three muscles contributes some information about differential growth rates of fasciculi within the same muscle. In biceps femoris and adductor superficialis the shortest (SFL) and longest (LFL) fasciculi appear to be growing at relatively the same rates (Table 3). In adductor profundus the SFL appears to have growth rates significantly higher than the LFL. Very little information is available on this aspect of muscle growth. In a study of M. tibialis anterior of the rabbit Crawford (1954) gathered data which suggest fairly uniform growth rates of fasciculi throughout the muscle, but in this study he was examining growth rates within the middle portion of the muscle belly rather than changes in the lengths of fasciculi as measured from their ends. Mackay & Harrop (1969), employing techniques similar to those of Crawford, also found uniform growth in M. sternomastoideus and M. anterior gracilis of the rat; these authors also measured growth within the middle portion of the muscle belly. It is not possible at this time to suggest an explanation for apparent differences in fasciculus growth rate in adductor profundus. Very probably these differences are associated with subtle changes in fasciculus orientation within the muscle as it grows: because the absolute amount of growth in these fasciculi is small, changes in architecture are difficult to detect. Functional analysis of growing muscle One conclusion from this study is that an analysis of muscle growth based only on one or two parameters, especially muscle weight, may not provide sufficient information to make functional interpretations about the growth patterns. Many workers have studied the relative growth of muscle wet or dry weight (see review of Stewart, 1972). It is clear from the literature that growth rates of muscle weight do often change during ontogeny and that these differences may reflect changes in function at certain periods of the life cycle (see, for example, the interpretations of Bryden (1973) for over 70 muscles of the elephant seal, Mirounga leonina). However, muscle 40
ANA 123
634 C. HELMI AND J. CRACRAFT weight itself may not be an accurate index of physiological properties such as relative force development, speed of contraction, or length of stretching and shortening (Gans & Bock, 1965; Bock, 1974), and thus the use of muscle weight alone will not provide an adequate descriptive picture of functional changes during growth. For example, wet and dry weights of biceps femoris have growth patterns (Figs. 3, 4) that correlate well with the increase in body size at 21 days (Fig. 2), but this relationship is less precise for the weights of the adductors. On the other hand, cross sectional area is the parameter that is functionally relevant for comparison with body size, and cross sectional area of adductor profundus exhibits a growth pattern that very closely mirrors the increase of body weight (Fig. 7). In conclusion, we can expect to learn much more about the functional aspects of muscle growth if future workers will examine a number of parameters rather than just one or two. Furthermore, events of muscle growth need to be related to changes in behaviour of living animals. In this connexion one of us (J. C.) is examining the details of ontogenetic changes in locomotor behaviour of chickens using high speed cinematography, and one of the expectations is that the patterns of muscle growth described above can be correlated with locomotion more closely than is presently possible. Virtually all students of muscle growth have neglected to correlate their findings with changes in behaviour, so that this remains an important area of research for functional morphologists. SUMMARY
This study was designed to investigate the growth patterns of three hindlimb muscles of the chicken relative to the functional-biomechanical demands of increasing body size. The biceps femoris, a bipennate non-postural muscle, grew relatively faster in terms of wet and dry weight than did the parallel-fibred adductor superficialis or the unipennate adductor profundus, both postural muscles. All three muscles exhibited positive allometry (relative to body weight) in muscle length but only biceps femoris and adductor profundus showed positive allometry in cross sectional area, adductor superficialis having isometric growth in this parameter. In biceps femoris and adductor superficialis the lengths of the longest and shortest fasciculi grew at equal rates, whereas in adductor profundus the shortest fasciculi grew faster than the longest. We conclude that muscle weight alone is an insufficient indicator of changing function in growing muscle. Hence, growth studies should include other functionally relevant parameters such as cross sectional area, which is proportional to the forceproducing capabilities of the muscle, or fibre (fasciculus) length, which is indicative of the absolute amount of stretching or shortening that is possible and of the contraction velocity. We wish to thank the following colleagues for their help and comments on the manuscript: Drs E. F. Allin, H. R. Barghusen, S. M. Herring, and R. P. Scapino. We are also grateful to Ms Kathryn Sisson for her excellent illustrations. This research was supported by a National Science Foundation grant (GB-41089) to J. C. and by funds from the Department of Anatomy, University of Illinois, to C. H.
Growth patterns of three hindlimb muscles in chickens
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