J. Anim Sci.
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J. Anim Sci. 2006. 84:2959-2964. doi:10.2527/jas.2006-345
© 2006 American Society of Animal Science
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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION

Growth- and breed-related changes of muscle bundle structure in cattle1

E. Albrecht, F. Teuscher, K. Ender and J. Wegner2

Research Institute for the Biology of Farm Animals, D-18196 Dummerstorf, Germany


   Abstract
Top
 Abstract
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 IMPLICATIONS
 LITERATURE CITED
 
The objective of this study was to investigate the changes in muscle fiber bundles of cattle of different breeds during growth. Different numbers of muscle fibers are surrounded by connective tissue to form bundles macroscopically visible as meat fibers or meat grain, a common meat quality trait. To determine the influence of breed and age on morphological characteristics of muscle fiber bundles, 4 cattle breeds with different growth impetus and muscularity were reared and slaughtered under experimental conditions. German Angus, a typical beef cattle; Galloway, a smaller beef type; Holstein Friesian, a dairy type; and double-muscled Belgian Blue, an extreme type for muscle growth, were used. Between 5 and 15 bulls of each breed were slaughtered at 2, 4, 6, 12, or 24 mo of age, and slices of semitendinosus muscle were removed. Muscle structure characteristics were determined by computerized image analysis. During growth, the muscle cross-sectional area enlarged (P < 0.001) about 5-fold in double-muscled Belgian Blue bulls and about 4-fold in the other breeds. This was a result of the enlargement (P < 0.001) of primary bundles and muscle fibers. The bundle size was similar (P ≥ 0.15) in bulls of German Angus and Galloway in all age groups and was doubled (P < 0.001) in double-muscled Belgian Blue animals from 4 mo of age on. The Holstein Friesian bulls had the smallest (P < 0.001) muscle fiber bundles at 24 mo of age. The number of muscle fibers per bundle and the number of bundles per muscle remained nearly constant (P > 0.05) during growth. This supports the existing view that the structure of the muscle is already fixed in prenatal life. The double-muscled Belgian Blue bulls showed a more than 2.5-fold greater (P < 0.001) number of muscle fibers per primary bundle compared with the other breeds investigated. The larger muscle fiber bundles led to a smaller amount of connective tissue per muscle area in double-muscled cattle. The coarser grain of meat in double-muscled Belgian Blue bulls and in older animals was not related to greater shear force values.

Key Words: breed • cattle • growth • meat quality • muscle fiber • primary bundle


   INTRODUCTION
Top
 Abstract
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 IMPLICATIONS
 LITERATURE CITED
 
The structural units of muscles are muscle fibers. Different numbers of muscle fibers are surrounded by connective tissue to form bundles, which ensure the contractility of the muscle. Muscle fiber bundles are macroscopically visible as meat fibers or meat grain, a common meat quality trait (Figure 1Go). Consumers prefer fine-grained meat, suggesting that a large grain size is related to less tender meat from older animals. Therefore, the USDA grading scheme for beef also considered grain size for many decades, with top grades requiring fine grain.


Figure 1
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  		Figure 1. Cross sections of semitendinosus muscle demonstrating (A) meat fibers in cooked meat (scale bar = 3 mm), and (B) a cross-section of muscle bundles stained with Aniline Blue and Orange G (scale bar = 1 mm). The white overlays highlight primary bundles containing approximately 100 muscle fibers.

 
As reviewed by Purslow (2005)Go, much of the work on muscle grain and meat texture is more than 40 to 70 yr old. Histological studies about the effect of sex, age, breed, and muscle on grain size by Hammond (1932)Go provided some of the first data in relation to muscle fiber bundles. The size of muscle fiber bundles of 14 muscles in 3 breeds of cattle was studied by Hoshino et al. (1990)Go. Differences were found between the muscles but not between the breeds. However, considering double-muscled cattle compared with normal cattle, there are differences in bundle structure. In double-muscled cattle the connective tissue network is thinner and more open-meshed than in normal cattle. Bundles are larger, and therefore the meat is macroscopically coarser (Boccard, 1981Go).

The objective of the current study was to quantify and compare the muscle fiber bundle structure of the semitendinosus muscle of 4 breeds of cattle during growth from 2 to 24 mo of age by means of computerized image analysis. Additionally, the influence of bundle structure on meat tenderness, as measured by the Warner Bratzler shear force (WBSF) value, was studied.


   MATERIALS AND METHODS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 IMPLICATIONS
 LITERATURE CITED
 
Animals

All animals were cared for and killed according to the German rules and regulations for animal care. The experiment was approved by the institutional authorities and by the responsible office of the County of Mecklenburg-Vorpommern, Germany. Bulls of 4 cattle breeds with different muscle growth potential were used. The breeds represented German Angus, a typical beef breed; Galloway, a smaller, environmentally resistant beef breed; Holstein Friesian, a dairy type; and double-muscled Belgian Blue, an extreme type for muscle growth.

Bulls were raised using a tethering system with individual feeding. Calves received a milk replacer diet up to 4 mo of age. After weaning, they were fed twice daily with a common diet consisting of wilted grass silage (1.8 kg of DM), corn silage (0.5 kg of DM), and concentrates (5.1 kg of DM) based on barley grain (92% OM, 15% CP, and 25% crude fiber). The proportion of concentrate in the diet was 70% (DM basis). The level of energy was 1.6-to 1.7-fold greater than the maintenance requirement of 530 kJ/kg of metabolic BW (BW0.75). The maintenance requirement for Galloway cattle was calculated as 500 kJ/kg of metabolic BW. Animals were restricted-fed for concentrates, depending on the BW, and ad libitum for silage. Between 5 and 15 bulls of each breed were slaughtered at 2, 4, 6, 12, or 24 mo of age.

Tissue Collection

After slaughter, chilling at 6°C for 24 h, and dressing, the semitendinosus muscle was removed from the left side of the carcass, trimmed of any external fat, and weighed. Muscle cross-sectional areas were measured on 1-cm thick muscle slices, from the middle region of the muscle (muscle length/2). For histology, muscle samples (1 x 1 x 0.5 cm) were taken at the same location in the superficial, pale region of semitendinosus muscle, according to the studies of Totland and Kryvi (1991)Go. For the measurement of the larger muscle fiber bundles at 24 mo of age, larger muscle samples of 4 x 4 x 1 cm were also taken.

Histology

Samples were immediately frozen in liquid nitrogen and stored at –70°C. Transverse sections, 10-µm thick, were cut using a Cryostat 2800 Frigocut (Reichert-Jung, Leica, Bensheim, Germany) and stained with hematoxylin and eosin. Transverse sections of the larger muscle samples were 20-µm thick. Additionally, sections were stained with Aniline Blue and Orange G to verify the bundle boundaries. Muscle fibers appeared orange, and connective tissue was blue (Figures 1Go and 2Go).


Figure 2
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  		Figure 2. Light micrograph of primary bundles in semitendinosus muscle of (A) a German Angus and (B) a double-muscled Belgian Blue bull, stained with Aniline Blue and Orange G. Scale bar = 100 µm.

 
Computerized Image Analysis

For muscle fiber size and number per cm2, a minimum of 300 muscle fibers per animal were measured by computerized image analysis (Quantimet 570, Cambridge Instruments, Leica, Bensheim, Germany) in randomly selected, muscle fiber primary bundles. The Quantimet 570 device was equipped with a color video camera (DXC-930P, Sony, Japan) and a transmission light microscope (Jenaval, Zeiss Jena, Germany). After calibration, image setup, and acquisition, a delineation was made for enhancing the transition between muscle fibers and connective tissue. The fibers were detected and measured. Adjacent fibers were separated by a binary segmentation operation. The result had to be corrected interactively; therefore, fibers still touching were separated by drawing a line. Artifacts were deleted, and holes were closed. The resulting traits were muscle fiber cross-sectional area and muscle fiber number per cm2.

The measurement of primary bundles was similar, but an additional procedure was necessarily used because of the different magnifications. After detection of muscle fibers, the boundaries of fiber bundles were interactively corrected, incomplete bundles were erased, holes were closed, if necessary boundaries were corrected again, and bundle areas were measured. We defined primary bundles as the smallest units that were covered by connective tissue and consisting of different numbers of muscle fibers. The apparent total muscle fiber number of a primary bundle was calculated from the bundle area and muscle fiber density (fiber number per cm2).

Meat Quality Traits

Standard procedures of our laboratory for determination of meat quality traits were used. For the current study only tenderness at 24 h postmortem was relevant. The WBSF data for the same animals described here were already published by Wegner et al. (2000)Go and were reevaluated together with the bundle structure results.

Statistics

The data were analyzed by ANOVA using the GLM procedure (Version 8, 1999; SAS Inst. Inc., Cary, NC). The factors considered were age and breed as well as the age x breed interaction. The test of significant differences was based on a significance level of P = 0.05. The model used was


Formula

where Yijk = the independent variable; µ = the overall mean; Bi = the effect of breed i (i = 1 to 4); Aj = the effect of slaughter age j (j = 1 to 5); BAij = the effect of interaction; and eijk = residual error. The least squares means were compared by use of the PDIFF statement.

Relations among bundle structure traits and between muscle bundle structure and WBSF values were analyzed by the CORR procedure of SAS. The simple Pearson’s correlation coefficient describing the relationship among all bulls was determined.


   RESULTS AND DISCUSSION
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
IMPLICATIONS
 LITERATURE CITED
 
In the current study primary bundles were defined according to Judge et al. (1989)Go as the smallest units in a muscle consisting of muscle fibers surrounded by connective tissue (Figure 1Go). The number of muscle fibers per primary bundle was given by Judge et al. (1989)Go as 20 to 40 and by Wegner et al. (1993)Go as about 100. The smallest muscle fiber bundles were also defined as primary bundles by Lepetit and Culioli (1992)Go, and these were summarized to secondary, tertiary, and main bundles. In contrast, Totland et al. (1988)Go and Hoshino et al. (1990)Go named the largest bundles primary bundles, which are subdivided in secondary bundles. Therefore, a clear definition of primary bundles is necessary when comparing results of bundle size or number of muscle fibers per bundle.

Muscle cross-sectional area, primary bundle area, and muscle fiber area of semitendinosus muscle were measured at 5 ages to assess changes in muscle structure during growth (Table 1Go). From these measured traits the apparent number of muscle fibers per primary bundle and the apparent number of primary bundles per muscle were calculated (Table 2Go).


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  		Table 1. Cross-sectional areas of muscle, primary bundle, and muscle fibers in semitendinosus muscle of different cattle breeds during growth
 

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  		Table 2. Number of primary bundles per muscle and number of muscle fibers per bundle in semitendinosus muscle of different cattle breeds during growth
 
The analysis of variance demonstrated an influence of breed, age, and their interaction on the cross-sectional areas of muscle, of primary bundles, and of muscle fibers (P ≤ 0.006). The apparent number of muscle fibers per primary bundle and the apparent number of primary bundles per muscle were also influenced by breed (P < 0.001) and age (P ≤ 0.009). The interaction between breed and age had no influence (P = 0.44) on the apparent number of primary bundles per muscle but had an influence (P = 0.018) on the apparent number of muscle fibers per primary bundle.

The cross-sectional areas of muscle, of primary bundles, and of muscle fibers increased (P < 0.05) in bulls of all 4 breeds during growth. The Pearson’s correlation coefficients showed close relations (r = 0.9) among these traits within all investigated breeds. The muscle cross-sectional area enlarged about 5-fold in double-muscled Belgian Blue bulls and about 4-fold in the other breeds. Comparing the breeds, differences appeared in the development of the muscle area in contrast to the development of the muscle fiber area. Whereas the muscle fibers had similar size in all breeds up to 12 mo of age, the muscle cross-sectional area differed already at 4 mo of age. Double-muscled Belgian Blue bulls had a greater muscle area than bulls of Galloway (P = 0.001) and Holstein Friesian (P < 0.001) at 4 mo of age and the greatest muscle area of all breeds from 6 mo of age onward (P < 0.01). The muscle fiber cross-sectional area at 24 mo of age was similar (P = 0.182) in German Angus and Galloway bulls but greater (P ≤ 0.04) compared with Holstein Friesian and double-muscled Belgian Blue bulls. Muscle fiber bundles increased (P < 0.05) during growth about 3.5-fold in Holstein Friesian and about 4.5-fold in the other investigated breeds. This enlargement of muscle fiber bundles leads to the visible coarser grain of meat in older animals.

Differences in muscle fiber bundle areas among the 4 breeds were more evident. The primary bundle area in double-muscled Belgian Blue bulls was already approximately double that in other breeds at 2 mo of age. Differences among the 4 studied breeds increased with age. Holstein Friesian bulls had smaller primary bundle areas than German Angus (P = 0.021) and double-muscled Belgian Blue bulls (P < 0.001) at 12 mo of age and the smallest bundles (P < 0.001) of all 4 breeds at 24 mo of age. German Angus and Galloway cattle showed no differences in muscle fiber bundle size (P ≥ 0.156).

The shape of muscle fiber bundles is influenced by the region within the muscle as shown by Totland et al. (1988)Go. Therefore, the shape of muscle fiber bundles was additionally measured. In our study the samples were always taken at the same region. There were no changes during growth and no differences among breeds in this trait (data not shown).

During growth there was no change in the number of muscle fibers per bundle (P ≥ 0.65, P ≥ 0.06) or in the number of primary bundles per muscle (P ≥ 0.15, P ≥ 0.19) in German Angus and Galloway bulls, respectively (Table 2Go). In Holstein Friesian bulls, the number of bundles per muscle increased slightly (P < 0.05), whereas the number of muscle fibers per bundle did not change during growth (P ≥ 0.188). In double-muscled Belgian Blue bulls, the apparent muscle fiber number per bundle decreased during growth (P < 0.05). A technical reason for this result may be that large bundles in the cross sections were often incomplete and so only smaller bundles were measured. The relatively constant number of muscle fibers per bundle and number of bundles per muscle agree with Wegner et al. (2000)Go for the constant apparent total muscle fiber number in semitendinosus muscle from birth to 24 mo of age. In general, this study shows that there is no change in the number of muscle fibers per bundle or in the number of primary bundles per muscle during growth. This supports the hypothesis that the muscle bundles are determined in prenatal life like muscle fibers, and they cannot divide during postnatal growth because of the contractile function of muscle tissue. In contrast, the amounts and composition of i.m. connective tissue can be manipulated by nutrition and exercise (Purslow, 2005Go).

Primary bundles contained a similar number of muscle fibers (Table 2Go) in German Angus, Galloway, and Holstein Friesian bulls (P ≥ 0.14) despite the differences in muscle fiber size (Table 1Go) between beef and dairy breeds. In contrast a 2.5- to 3-fold greater number of muscle fibers (P < 0.001) was recorded for double-muscled Belgian Blue bulls. In Figure 2Go, primary bundles of a double-muscled Belgian Blue and a German Angus bull are shown, demonstrating the greater number of muscle fibers in primary bundles of double-muscled Belgian Blue cattle. The condition of double muscling observed in Belgian Blue is associated with mutations in the myostatin gene (Grobet et al., 1997Go). This mutation results in excessive muscle fiber formation (hyperplasia) during prenatal life, whereas the muscle fiber size is not affected (Picard and Cassar-Malek, 1998Go). The excessive muscle fiber formation is not accompanied by additional i.m. connective tissue formation. In prenatal life, the i.m. connective tissue seems to be developed earlier than muscle fibers. Previously, Boccard (1981)Go observed an open-meshed i.m. connective tissue framework in culard (double-muscled) animals. A consequence of the greater number of muscle fibers in primary bundles of double-muscled cattle is the lower content of connective tissue per cm2 of muscle area in double-muscled Belgian Blue bulls. This should contribute to a greater degree of tenderness in double-muscled cattle, despite the fact that i.m. fat, a second contributor for sensory perception of tenderness, is very low in double-muscled Belgian Blue bulls (Albrecht et al., 2006Go).

In addition to growth and breed related changes of bundle structure, its relation to WBSF value 24 h postmortem (published in Wegner et al., 2000Go) was studied. There was no relationship (P ≥ 0.099) between bundle structure and WBSF value 24 h postmortem. The coarser grain of meat by larger muscle fiber bundles in older animals was not related to greater WBSF values. Furthermore, the coarser grain of meat in double-muscled Belgian Blue bulls was not related to greater WBSF values because of the smaller amount of i.m. connective tissue per area by larger muscle fiber bundles. This supports the results of Oliván et al. (2004)Go regarding the reduced content of collagen in muscles of double-muscled Belgian Blue bulls. In sensory panel scores of meat texture, fibrousness is influenced by bundle structure. In the study of Otremba et al. (1999)Go there were no significant correlations between fibrousness and WBSF. Nishimura et al. (1999)Go showed by electron microscopy the disorganization of the perimysium by developing fat cells. Therefore, i.m. connective tissue and marbling are opponents in their influence on tenderness.


   IMPLICATIONS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 IMPLICATIONS
LITERATURE CITED
 
In postnatal life of cattle, growth of muscle cross-sectional area, of primary bundle area, and of muscle fiber area are closely related. No muscle fibers are newly developed nor primary bundles divided by additional growth of connective tissue. The number of muscle fibers per bundle and the number of bundles per muscle remain constant. Quantitative changes are limited to the size of structural units, leading to a coarser structure in older animals. Comparing meat of different breeds at the same age, meat can be fine granulated (e.g., Holstein Friesian) or very coarse granulated (e.g., double-muscled Belgian Blue). Relations between bundle structure and meat quality traits were not found.


   Footnotes
 
1 The authors thank Karola Marquardt for excellent technical assistance. Back

2 Corresponding author: wegner{at}fbn-dummerstorf.de

Received for publication May 30, 2006. Accepted for publication July 15, 2006.


   LITERATURE CITED
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 IMPLICATIONS
 LITERATURE CITED
 


Albrecht, E., F. Teuscher, K. Ender, and J. Wegner. 2006. Growth-and breed-related changes of marbling characteristics in cattle. J. Anim. Sci. 84:1067–1075.[Abstract/Free Full Text]

Boccard, R. 1981. Facts and reflections on muscular hypertrophy in cattle: Double muscling or culard. Pages 1–27 in Developments in Meat Science-2. R. Lawrie, ed. Elsevier Applied Science, London, UK, and New York, NY.

Grobet, L., L. J. Martin, D. Poncelet, D. Pirottin, B. Brouwers, J. Riquet, A. Schoeberlein, S. Dunner, F. Menisssier, J. Massabanda, R. Fries, R. Hanset, and M. Georges. 1997. A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle. Nat. Genet. 17:71–74.[CrossRef][Medline]

Hammond, J. 1932. Growth and the development of mutton qualities in the sheep. Biological monographs and manuals (Vol. X). Oliver and Boyd, London, UK.

Hoshino, T., A. Suzuki, T. Yamaguchi, S. Ohwada, and M. Ota. 1990. A comparative morphometrical analysis of the amount and distribution of fat within muscles of Japanese Black Cattle, Japanese Shorthorn, and their crossbred (F1) steers. Tohoku J. Agric. Res. 40:57–64.

Judge, M. D., E. D. Aberle, J. C. Forrest, H. B. Hedric, and R. A. Merkel. 1989. Principles of Meat Science. 2. Edition, Kendall, Hunt Publishing Company, Dubuque, IA.

Lepetit, J., and J. Culioli. 1992. Mechanical properties of meat. 38th ICoMST, Clermont - Ferrand, France:135–151.

Nishimura, T., A. Hattori, and K. Takahashi. 1999. Structural changes in intramuscular connective tissue during the fattening of Japanese Black Cattle: Effect of marbling on beef tenderization. J. Anim. Sci. 77:93–104.[Abstract/Free Full Text]

Oliván, M., A. Martinez, K. Osoro, C. Sañudo, B. Panea, J. L. Olleta, M. M. Campo, M. À. Oliver, X. Serra, M. Gil, and J. Piedrafita. 2004. Effect of muscular hypertrophy on physicochemical, biochemical and texture traits of meat from yearling bulls. Meat Sci. 68:567–575.[CrossRef]

Otremba, M. M., M. E. Dikeman, G. A. Milliken, S. L. Stroda, J. A. Unruh, and E. Chambers, IV. 1999. Interrelationships among evaluations of beef longissimus and semitendinosus muscle tenderness by Warner-Bratzler shear force, a descriptive-texture profile sensory panel, and a descriptive attribute sensory panel. J. Anim. Sci. 77:865–873.[Abstract/Free Full Text]

Picard, B., and I. Cassar-Malek. 1998. Differentiation of skeletal muscle and its hormonal control during the fetal stage. Pages 13–24 in Proc. Symposium on Growth in Ruminants. H. I. Blum, T. H. Elsasser, and P. Guilloteau, ed. Univ., Berne, Switzerland.

Purslow, P. P. 2005. Intramuscular connective tissue and its role in meat quality. Meat Sci. 70:435–447.[CrossRef]

SAS Inst. Inc. 1999. Online SAS users guide: Statistics. Version 8.02. ed. SAS Institute, Inc., Cary, NC.

Totland, G. K., and H. Kryvi. 1991. Distribution patterns of muscle fiber types in major muscles of the bull (Bos taurus). Anat. Embryol. 184:441–450.[CrossRef][Medline]

Totland, G. K., H. Kryvi, and E. Slinde. 1988. Composition of muscle fibre types and connective tissue in bovine M. semitendinosus and its relation to tenderness. Meat Sci. 23:303–315.[CrossRef]

Wegner, J., E. Albrecht, I. Fiedler, F. Teuscher, H. J. Papstein, and K. Ender. 2000. Growth- and breed-related changes of muscle fiber characteristics in cattle. J. Anim. Sci. 78:1485–1496.[Abstract/Free Full Text]

Wegner, J., I. Fiedler, C. Rehfeldt, and K. Ender. 1993. Die Mikrostruktur des Muskels und ihre Bedeutung für Wachstum und Fleischqualität. Pages 11–23 in Rinderzucht, Fleischqualität und Extensivierung. H. D. Matthes and L. Panicke, ed. FBN Dummerstorf, Germany.


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