DISTRIBUTION HETEROGENEITY OF MUSCLE FIBER TYPES IN THE RABBIT LONGISSIMUS MUSCLE 1
Institut National de la Recbercbe Agronomique, 34060 Montpellier-Cedex France University of California, Davis 95616
SUMMARY
The Longissimus muscle of the rabbit was examined at four cross-sectional levels from anterior to posterior, and the total number of muscle fibers as well as the percentage of ~R, c~R and ~W fiber types were estimated at each level. Significant differences were found between section levels for percentages of ~ R and 13R fiber types, but not for the ~W fiber type. The total number of fibers increased fourfold from anterior to posterior. The percentage of ~R fibers decreased from approximately 10% at the anterior end of the muscle to 3% at the posterior end, while the percentage of ~R fibers increased from 34% to 41%. Enzymatic analysis of isocitrate dehydrogenase and aldolase showed no significant differences between three selected areas of the muscle which were observed to have significant differences in distribution of ~R and/JR fiber types. (Key Words: Muscle, Muscle Fiber, Rabbit.) INTRODUCTION
In 1873, Ranvier reported the existence of two fiber types in skeletal muscle. A more recent study of Romanul (1964) describes as many as eight muscle fiber types. Although Guth and YeUin (1971) question the validity of any nomenclature founded on simple and static muscle characteristics, we believe it is necessary to study and describe muscle fibers of animals for comparative purposes. There is no consensus on a system of classification for typing muscle fibers. In one system, (Ashmore and Doerr, 1971) three main types of fibers are differentiated by cytochemical analyses:
1Supported in part by a grant from the l~l~gation g~n~rale ~I la Recherche seientifique et technique. M~ TAAP 594 bis. 2Station de Physiologie Animale, a Department of Animal Science.
a) slowly contracting fibers with aerobic metabolism (13R) b) rapidly contracting fibers with aerobic metabolism (aR) c) rapidly contracting fibers with anaerobic metabolism (aW) However, it is emphasized that the energy metabolism of a fibers is highly adaptable and may tend to become more aerobic, or more anaerobic depending upon functional demand. Generally in muscles, fibers of different types are arranged in a check-board manner and are not randomly distributed (James, 1971). This characteristic has been seldom studied and one objective of the present work is to contribute to the understanding of this phenomena. We use a histochemical approach as well as a quantitative assessment of enzymatic activity in the longissimus muscle of the rabbit. MATERIALS AND METHODS
Five New Zealand male rabbits were sacrificed at 900 g live weight. The left longissimus muscles were removed and divided into four sections at levels between the fifth and sixth ribs, between the 10th and l l t h ribs, between the first and second lumbar vertebrae and between the fourth and fifth lumbar vertebrae. The cross-sectional area of each section was measured at its anterior end by planimetry and the area was reduced by 10%. This corrected value takes into account the extra-cellular space (Nouguds, 1973). Transverse serial sections 32 /am in thickness were cut through the middle of each of the four pieces of muscle with a cryostat microtome. The sections were air dryed at 4 C and reacted for myofibrillar ATPase after alkali preincubation (Padykula and Herman, 1955) and after acid preincubation (Brooke and Kaiser, 1969). Succinic dehydrogenase (SDH) was visualized according to Nachlas et al. (1957). With these techniques we
985 JOURNAL OF ANIMAL SCIENCE, Vol. 43, No. 5 (1976)
Downloaded from https://academic.oup.com/jas/article-abstract/43/5/985/4697231 by Iowa State University user on 23 January 2019
P. Vigneron 2, F. Bacou 2 and C. R. Ashmore 3
986
VIGNERON, BACOU AND ASHMORE
Downloaded from https://academic.oup.com/jas/article-abstract/43/5/985/4697231 by Iowa State University user on 23 January 2019
could classify muscle fibers as 3R, 0~R, or ~W. longitudinally divided to obtain one sample Using the sections reacted for myofibrillar devoid of~/R fibers, as well as one sample which ATPase, cross-sectional diameters of between was richer in ~R fibers. (see level IV, figure 1). 3,500 and 4,000 fibers were measured at each The plane of section for producing these latter of the four levels. By calculating the mean fiber two samples was predetermined by cytochemiarea and dividing that value into the corrected cal analyses of the sample area for fiber type cross-sectional area of the muscle, the total distribution within the muscle. number of fibers at each level was estimated. These samples were carefully minced and For determination of the accuracy of this homogenized with an Ultra Turrax homogemethod, the total number of muscle fibers was nizer followed by homogenization with a counted directly at each level in the muscle of Thomas homogenizer in .25 M sucrose solution. one animal. The homogenate was centrifuged 1 hr at Estimation of percentages of/3R, a R and aW 100,000 g. The supernatant was used to meaat each level was made using the serial sections sure the activity o f two enzymes, aldolase reacted for ATPase and SDH by identifying (E.C.4.1.2.13) and NADP + Isocitrate dehydrofrom 2,500 to 11,OOO fibers in four to 10 genase (ICDH, E.C.1.1.1.42), as representative microscopic fields at each level. All quantitative of anaerobic and oxidative metabolism, respechistology was done using a Visopan Reichert tively. Aldolase activity was assayed by followprojection microscope. ing the change in absorbance of NAD according In order to visualize the distribution o f / J to Rutter et al. (1961). ICDH activity was fibers within the muscle, levels I and II were assayed by following the change in absorbance subdivided into six areas, and levels III and IV of NADP according to Moyle (1956). into nine areas. The percentage of B fibers was estimated for each area at each level. RESULTS For quantitative estimation of enzyme activA good correspondence was found between ities the longissimus muscle of 10 rabbits was removed and sampled in three areas. The first total numbers of fibers as estimated in five sample was taken between the 10th and 14th . animals and the number actually counted in the four-section levels of one animal (table 1). The ribs. A second sample was taken between the mean fiber number, as estimated for five fourth and fifth lumbar vertebrae. It was animals increases from anterior to posterior, until the first lumbar vertebra level is attained. It is similar at the two posterior levels (table 1). Mean fiber diameter (table 1), as calculated, shows no significant variation. Mean percentage of aW fibers (nearly 57%), and of aerobic fibers ( a R +/3R) does not vary much from end to end of the muscle. We did observe, however, a progressive increase from anterior to posterior of the percentage of a R Level I,i = 10.02 Level H,i= 7.26 fibers which is compensated by a decrease in the percentage of/3R fibers (table 1). The mean percentage of aW, a R and /3R fibers varied significantly between themselves at each section level (P
Institut National de la Recbercbe Agronomique, 34060 Montpellier-Cedex France University of California, Davis 95616
SUMMARY
The Longissimus muscle of the rabbit was examined at four cross-sectional levels from anterior to posterior, and the total number of muscle fibers as well as the percentage of ~R, c~R and ~W fiber types were estimated at each level. Significant differences were found between section levels for percentages of ~ R and 13R fiber types, but not for the ~W fiber type. The total number of fibers increased fourfold from anterior to posterior. The percentage of ~R fibers decreased from approximately 10% at the anterior end of the muscle to 3% at the posterior end, while the percentage of ~R fibers increased from 34% to 41%. Enzymatic analysis of isocitrate dehydrogenase and aldolase showed no significant differences between three selected areas of the muscle which were observed to have significant differences in distribution of ~R and/JR fiber types. (Key Words: Muscle, Muscle Fiber, Rabbit.) INTRODUCTION
In 1873, Ranvier reported the existence of two fiber types in skeletal muscle. A more recent study of Romanul (1964) describes as many as eight muscle fiber types. Although Guth and YeUin (1971) question the validity of any nomenclature founded on simple and static muscle characteristics, we believe it is necessary to study and describe muscle fibers of animals for comparative purposes. There is no consensus on a system of classification for typing muscle fibers. In one system, (Ashmore and Doerr, 1971) three main types of fibers are differentiated by cytochemical analyses:
1Supported in part by a grant from the l~l~gation g~n~rale ~I la Recherche seientifique et technique. M~ TAAP 594 bis. 2Station de Physiologie Animale, a Department of Animal Science.
a) slowly contracting fibers with aerobic metabolism (13R) b) rapidly contracting fibers with aerobic metabolism (aR) c) rapidly contracting fibers with anaerobic metabolism (aW) However, it is emphasized that the energy metabolism of a fibers is highly adaptable and may tend to become more aerobic, or more anaerobic depending upon functional demand. Generally in muscles, fibers of different types are arranged in a check-board manner and are not randomly distributed (James, 1971). This characteristic has been seldom studied and one objective of the present work is to contribute to the understanding of this phenomena. We use a histochemical approach as well as a quantitative assessment of enzymatic activity in the longissimus muscle of the rabbit. MATERIALS AND METHODS
Five New Zealand male rabbits were sacrificed at 900 g live weight. The left longissimus muscles were removed and divided into four sections at levels between the fifth and sixth ribs, between the 10th and l l t h ribs, between the first and second lumbar vertebrae and between the fourth and fifth lumbar vertebrae. The cross-sectional area of each section was measured at its anterior end by planimetry and the area was reduced by 10%. This corrected value takes into account the extra-cellular space (Nouguds, 1973). Transverse serial sections 32 /am in thickness were cut through the middle of each of the four pieces of muscle with a cryostat microtome. The sections were air dryed at 4 C and reacted for myofibrillar ATPase after alkali preincubation (Padykula and Herman, 1955) and after acid preincubation (Brooke and Kaiser, 1969). Succinic dehydrogenase (SDH) was visualized according to Nachlas et al. (1957). With these techniques we
985 JOURNAL OF ANIMAL SCIENCE, Vol. 43, No. 5 (1976)
Downloaded from https://academic.oup.com/jas/article-abstract/43/5/985/4697231 by Iowa State University user on 23 January 2019
P. Vigneron 2, F. Bacou 2 and C. R. Ashmore 3
986
VIGNERON, BACOU AND ASHMORE
Downloaded from https://academic.oup.com/jas/article-abstract/43/5/985/4697231 by Iowa State University user on 23 January 2019
could classify muscle fibers as 3R, 0~R, or ~W. longitudinally divided to obtain one sample Using the sections reacted for myofibrillar devoid of~/R fibers, as well as one sample which ATPase, cross-sectional diameters of between was richer in ~R fibers. (see level IV, figure 1). 3,500 and 4,000 fibers were measured at each The plane of section for producing these latter of the four levels. By calculating the mean fiber two samples was predetermined by cytochemiarea and dividing that value into the corrected cal analyses of the sample area for fiber type cross-sectional area of the muscle, the total distribution within the muscle. number of fibers at each level was estimated. These samples were carefully minced and For determination of the accuracy of this homogenized with an Ultra Turrax homogemethod, the total number of muscle fibers was nizer followed by homogenization with a counted directly at each level in the muscle of Thomas homogenizer in .25 M sucrose solution. one animal. The homogenate was centrifuged 1 hr at Estimation of percentages of/3R, a R and aW 100,000 g. The supernatant was used to meaat each level was made using the serial sections sure the activity o f two enzymes, aldolase reacted for ATPase and SDH by identifying (E.C.4.1.2.13) and NADP + Isocitrate dehydrofrom 2,500 to 11,OOO fibers in four to 10 genase (ICDH, E.C.1.1.1.42), as representative microscopic fields at each level. All quantitative of anaerobic and oxidative metabolism, respechistology was done using a Visopan Reichert tively. Aldolase activity was assayed by followprojection microscope. ing the change in absorbance of NAD according In order to visualize the distribution o f / J to Rutter et al. (1961). ICDH activity was fibers within the muscle, levels I and II were assayed by following the change in absorbance subdivided into six areas, and levels III and IV of NADP according to Moyle (1956). into nine areas. The percentage of B fibers was estimated for each area at each level. RESULTS For quantitative estimation of enzyme activA good correspondence was found between ities the longissimus muscle of 10 rabbits was removed and sampled in three areas. The first total numbers of fibers as estimated in five sample was taken between the 10th and 14th . animals and the number actually counted in the four-section levels of one animal (table 1). The ribs. A second sample was taken between the mean fiber number, as estimated for five fourth and fifth lumbar vertebrae. It was animals increases from anterior to posterior, until the first lumbar vertebra level is attained. It is similar at the two posterior levels (table 1). Mean fiber diameter (table 1), as calculated, shows no significant variation. Mean percentage of aW fibers (nearly 57%), and of aerobic fibers ( a R +/3R) does not vary much from end to end of the muscle. We did observe, however, a progressive increase from anterior to posterior of the percentage of a R Level I,i = 10.02 Level H,i= 7.26 fibers which is compensated by a decrease in the percentage of/3R fibers (table 1). The mean percentage of aW, a R and /3R fibers varied significantly between themselves at each section level (P
