Comparison of collagen fibre architecture between slow-twitch cranial and fast-twitch caudal parts of broiler M. latissimus dorsi

1. Collagen fibre architectures of perimysium and endomysium in the slow-twitch cranial and fast-twitch caudal parts of broiler M. latissimus dorsi were compared. 2. Type I and III collagens were distributed in both perimysium and endomysium as indicated by their positive immunohistochemical reactions to polyclonal antibodies. 3. Cells invested by endomysium with no myofibres were larger in the cranial part because of the presence of larger slow-twitch myofibres. The honeycomb structure of endomysium was divided into several parts by thick perimysium. 4. The thick perimysial collagen fibres with parallel fibrils, which were interconnected by the loose reticular fibrils and thin fibres, were more numerous and thicker in the cranial part than the caudal. 5. Thick endomysial sidewall of cells in the cranial part was composed of a rougher reticulum of slightly thicker collagen fibrils compared with the thin sidewall in the caudal part. 6. These results indicated that both perimysial constitutions of collagen fibres and endomysial collagen fibrils had attained much larger growth in the slow-twitch cranial part than the fast-twitch caudal in broiler latissimus dorsi muscle.     

Comparison of collagen content, distribution and architecture among the pectoralis, iliotibialis lateralis and puboischiofemoralis muscles with different myofiber composition in Silky cocks

The total amount of collagen, the relative distributions of types I and III collagens in perimysium and endomysium, and the collagen fiber architecture were compared among the pectoralis (PT), iliotibialis lateralis (ITL) and puboischiofemoralis (PIF) muscles in Silky cocks. All of the myofibers in the PT muscle were type IIB, the myofibers in the ITL muscle were divided into type IIA, 41.7% and IIB, 58.3%, and the PIF muscle was composed of type I, 24.6%; IIA, 64.6%; and transitional, 10.8%. The total amount of collagen differed significantly among the PT (2.92 mg/g), PIF (4.20 mg/g) and ITL (8.06 mg/g) material, where only the PIF was a whole muscle with epimysium. On the image analysis of the immunohistochemical preparations, the percentage area of perimysial collagen to the total area in each type differed significantly among the PIF, PT and ITL muscles, where it was 26.8, 50.0 and 74.4% for the type I collagen and 27.4, 32.9 and 61.7% for the type III collagen, respectively. In the scanning electron micrography of the perimysium in macerated preparations, thick bundles of collagen fibers were observed in the ITL muscle, thinner but broad platelets in the PT muscle, and a coarse tissue of thinner collagen fibers in the PIF muscle. However, the endomysial fabric of collagen fibrils was similar among the muscles. Small, transverse collagen fibers, which branched off from the thicker perimysia, occupied narrow interendomysial spaces and separated the primary myofiber fasciculi. The results indicate that the ITL muscle, localized in the distorted and overextended part of the leg and subject to strong external forces, had highly developed perimysial collagen fiber bundles, but the ITL endomysial collagen architecture was similar to that of the PT and PIF muscles.     

Three-dimensional reconstruction of intramuscular collagen networks of bovine muscle: A demonstration by an immunohistochemical/confocal laser-scanning microscopic method

We undertook a three‐dimensional reconstruction of intramuscular collagen networks of bovine muscle using an immunohistochemical/confocal laser‐scanning microscopic method. By immunohistochemical staining, type I and III collagens were observed mainly in the perimysium, while type IV collagen was observed in the endomysium. On the other hand, type V and VI collagens were observed in both the perimysium and endomysium. By confocal laser‐scanning microscopy, the collagen observed in the perimysium was three‐dimensionally reconstructed as plate‐shaped layers whereas the collagen observed in the endomysium surrounded myofibers. The three‐dimensionally reconstructed observations using immunohistochemical/confocal laser‐scanning microscopic method is useful for investigating collagen networks in muscle.     

Structural weakening of intramuscular connective tissue during postmortem aging of pork

We studied structural changes in the endomysium and perimysium during postmortem aging of pork using the cell‐maceration/scanning electron microscope method. Immediately post mortem, endomysia sheaths that house individual muscle fibers displayed a honeycomb‐like structure. The sheaths of the endomysium consisted of tightly arranged collagen fibrils in a random network. The perimysium comprised several layers of wavy sheets made up of tightly bundled collagen fibers. While the structure of the intramuscular connective tissues remained almost unchanged up to five days post mortem, the endomysium had resolved into individual collagen fibrils, and the thick sheets of the perimysium had separated into collagen fibers and fibrils at 8 days post mortem. These results provide direct evidence for structural weakening of the endomysium and perimysium during postmortem aging of pork. The shear‐force value of raw pork decreased rapidly within six days post mortem and then decreased slowly until 14 days post mortem. Since the rapid increase in tenderness is mainly due to structural weakening of myofibrils, we conclude that the disintegration of the endomysium and perimysium contributes to tenderization of pork during extended postmortem aging.     

Effects of nutritional level on muscle development, histochemical properties of myofibre and collagen architecture in the pectoralis muscle of male broilers

1. The effects of nutritional level on muscle development, histochemical properties of myofibre and collagen architecture in the pectoralis muscle were evaluated using male broilers of Red Cornish x New Hampshire stock, reared on diets of high nutritional value for up to 80 d (H80d) and low nutritional value for up to 80 d (L80d, same age as H80d) or 95 d (L95d, same body weight as H80d). 2. The total live weight and the weight of pectoralis muscle were lower in L80d than in both H80d and L95d. The muscle weight as a percentage of live weight was 8.7% in L80d, 10.7% in H80d and 11.5% in L95d. 3. Pectoralis muscle was composed only of type IIB myofibres and showed no differences in myofibre type composition among the chicken groups. The largest diameter of type IIB myofibres was observed in L95d, followed by H80d and the smallest in L80d. 4. The total amount of intramuscular collagen did not differ among the chicken groups (1.92 to 1.99 mg/g). Types I and III collagens were immunohistochemically detected in both the perimysia and endomysia. The thin perimysia around the primary myofibre fascicles showed larger width in H80d than L80d and L95d, and also the thick perimysia around the secondary fascicles in H80d than L80d. 5. The collagen structure of the perimysium was most developed in H80d, followed by L95d and on the least in L80d. The development of perimysial collagen fibres could be enhanced by a rapid growth rate of the muscle induced by high nutritional level and depressed by a slow growth rate with low nutritional foods. 6. The endomysial collagen architecture was observed as a felt-like tissue of the fibril bundles with many slits. The thinnest endomysial wall was observed in L80d, followed by H80d and the thickest in L95d. 7. From these results, it was indicated that foods of high nutritional value could enhance growth of the pectoralis muscle of broilers, and this is accompanied by hypertrophy of the type IIB myofibres and development of the perimysial collagen architecture.     

Growth changes of the collagen content and architecture in the pectoralis and iliotibialis lateralis muscles of cockerels

1. Growth changes of the collagen content and architecture in the pectoralis (PT) and iliotibialis lateralis (ITL) muscles were examined using cockerels from 1 to 14 weeks of age. 2. Total collagen content in PT muscle showed little change, but in ITL muscle reached a maximum at 5 weeks and thereafter decreased slightly until 14 weeks. The collagen content was markedly larger in ITL muscle after 5 weeks. Pyridinoline content of collagen increased abruptly from 5 to 14 weeks in both muscles, but no difference between muscle types was detected. 3. The cell size of the endomysial honeycombs increased with the development of myofibres, and the mesh size of the perimysium around the honeycombs enlarged. 4. In both muscles endomysia were an incomplete network of collagen fibrils with many foramina at one week, became a very thin membrane of felt-like fabric in 2 to 5 weeks and thereafter increased in thickness until 11 to 14 weeks. 5. Perimysial width around the secondary fasciculus differed between the muscle types after 5 weeks. In the wider perimysium of ITL muscle, the collagen fibres increased in number and size to make a stack of collagen bands around the fasciculus. In the narrower perimysium of PT muscle, a few platelets of collagen fibres also developed. 6. The perimysial collagen fibre at 1 to 2 weeks had a smooth surface and appeared to be composed of fine collagen fibrils. The fibre at 11 to 14 weeks showed a rugged surface and was composed of coarser collagen bundles that combined with each other into a net-like configuration with very slim meshes. 7. Our results showed that the collagenous components of chicken intramuscular connective tissue changed markedly during the early period of muscle growth in distribution, architecture and quality but with little difference in quantity.     

Relationship between development of intramuscular connective tissue and toughness of pork during growth of pigs

We investigated changes in structures and properties of the endomysium and perimysium during development of semitendinosus muscle in relation to the increase in toughness of pork using samples from neonates to 55-mo-old pigs. The shear force value of pork increased linearly until 6 mo of age, and the rate of increase slowed down thereafter. The secondary perimysium thickened owing to an increase in the number and thickness of perimysial sheets consisting of collagen fibers, which became thicker and wavy with the growth of the pigs. This increase in thickness of the secondary perimysium was correlated significantly with the increase in the shear force value (r = .98). The endomysial sheaths became thicker and denser in the muscle of 6-mo-old pigs. Maturation of the endomysium was accompanied by hypertrophy of muscle fibers. The amount of heat-soluble collagen decreased almost linearly, indicating that nonreducible cross-links between collagen molecules were formed throughout chronological aging. We conclude that thickening of the perimysium is closely related to an increase in the toughness of pork during growth of pigs.     

Oral ingestion of collagen peptide causes change in width of the perimysium of the chicken iliotibialis lateralis muscle

Skeletal muscle is mainly composed of myofibers and intramuscular connective tissue. Bundles composed of many myofibers, with each myofiber sheathed in connective tissue called the endomysium, are packed in the perimysium, which occupies the vast bulk of the intramuscular connective tissue. The perimysium is a major determination factor for muscle texture. Some studies have reported that collagen peptide (Col-Pep) ingestion improves the connective tissue architecture, such as the tendon and dermis. The present study evaluated the effects of Col-Pep ingestion on the chicken iliotibialis lateralis (ITL) muscle. Chicks were allocated to three groups: the 0.15% or 0.3% Col-Pep groups and a control group. Col-Pep was administered by mixing in with commercial food. On day 49, the ITL muscles were analyzed by morphological observation and the textural property test. The width of the perimysium in the 0.3% Col-Pep group was significantly larger than other two groups. Although scanning electron microscopic observations did not reveal any differences in the architecture of the endomysium, elastic improvement of the ITL muscle was observed as suggested by an increase of the width of perimysium and improved rheological properties. Our results indicate that ingestion of Col-Pep improves the textural property of ITL muscle of chickens by changing structure of the perimysium.     

Structural changes in intramuscular connective tissue during the fattening of Japanese black cattle: effect of marbling on beef tenderization

We investigated changes in structures and mechanical properties of the intramuscular connective tissue during the fattening of Japanese Black steers, using the cell maceration method for scanning electron microscopy. During the early fattening period, from 9 to 20 mo of age, collagen fibrils of the endomysium in longissimus muscle associated more closely with each other, and collagen fibers in the perimysium increased in thickness and their wavy pattern became more regular. These changes were closely related to the increase in mechanical strength of the intramuscular connective tissue and resulted in a toughening of the beef during the period. The shear force value of longissimus muscle decreased after 20 mo of age, concomitantly with the rapid increase in the crude fat content. Scanning electron micrographs of the longissimus muscle dissected from 32-mo-old steers clearly showed that the adipose tissues were formed between muscle fiber bundles, that the honeycomb structure of endomysia was partially broken, and that the perimysium separated into thinner collagen fibers. In semitendinosus muscle, in which the crude fat content was lower (P<.05) than that in longissimus muscle, the structure of the intramuscular connective tissue remained rigid at 32 mo of age. The shear force value of the muscle increased even in the late fattening period, from 20 to 32 mo of age. Thus, the development of adipose tissues in longissimus muscle appears to disorganize the structure of the intramuscular connective tissue and contributes to tenderization of highly marbled beef from Japanese Black cattle during the late fattening period.     

Relationships between physical and structural properties of intramuscular connective tissue and toughness of raw pork

We studied the relationships between the shear-force value and physical and structural properties of the intramuscular connective tissue (IMCT) in six classes of porcine skeletal muscle to elucidate the contribution of IMCT to toughness of raw pork. The shear-force value of raw pork correlated significantly with that of the IMCT model prepared from each class of skeletal muscle ( P  < 0.05). The correlation suggested that the variable toughness of pork was caused by the mechanical strength of the endomysium and perimysium. The thickness of the secondary perimysium correlated significantly with the shear-force value of raw pork ( P  < 0.05) and with that of the IMCT model ( P  < 0.05). The shear-force value of raw pork correlated significantly with the total amount of collagen ( P  < 0.05) but not with the heat-solubility of collagen. We concluded therefore that the thickness of the secondary perimysium determines the mechanical strength of IMCT and contributes to toughness in raw pork.     

Comparative observations on the growth changes of the histochemical property and collagen architecture of the Musculus pectoralis from Silky, layer-type and meat-type cockerels

Growth‐related changes in the histochemical properties and collagen architecture of the Musculus pectoralis were compared among Silky, layer‐type and meat‐type cockerels. Histochemical and immunohistochemical methods were used and collagen architecture was studied using scanning electron microscopy. The total amount of collagen present was also measured. The diameter of type IIB myofibers was similar or rather larger in the layer‐type birds compared with the meat‐type. The collagen content was generally low for 5–10 weeks across the breeds and then increased in the other breeds except for Silky. In the perimysium, the collagen bundles gradually increased in size and the density of the fibrils also increased during growth. At 30 weeks of age, the layer‐type birds showed compact collagen bundles while the meat‐type had loose bundles. The endomysial collagen network appeared relatively denser in the meat‐type chicks compared to the others at week 1. At 30 weeks of age, compact and felt‐like structure of endomysium was shown by Silky and layer‐type chickens, while the meat‐type showed a relatively loose arrangement of tissue in the endomysial collagen. From these results, it appears that the meat‐type chicken can produce a large M. pectoralis with many, relatively thinner myofibers and a relatively undeveloped form of intramuscular collagen structure.     

Muscular architecture in the omasal laminae of cattle and sheep

The muscular architecture of the bovine omasal laminae was examined. The omasal laminae had three thin smooth muscle layers which consisted of an intermediate layer and two lateral layers. The muscle bundles of the intermediate layers ran radially in the laminae and those of the two lateral layers ran parallel to the free border of the laminae. At the free border, three types of the muscular architecture were observed. In a histological study, the muscle bundles of the intermediate layer penetrated into the connective tissue around the smooth muscle fasciculi of the inner layer of the tunica muscularis of the omasal wall and attached to the bundles of both the inner layer and of the outer layer of the tunica muscularis. These results indicate that a re-evaluation of the muscular architecture in the omasal laminae is required to facilitate better understanding of the omasal morphology and physiology.     

Comparative observations of the growth changes of the histochemical properties and collagen architecture of the iliotibialis lateralis muscle from Silky, layer and meat type cockerels

Growth‐related changes in the histochemical property and collagen architecture of the iliotibialis lateralis muscle were compared among Silky, layer and meat cockerels. Histochemical and immunohistochemical methods were employed to observe the collagen architecture. The total intramuscular collagen was also determined. The muscle consisted of type IIA, IIB and IIC myofibers, of which type IIB occurred at the highest frequency. The diameter of type IIB myofibers in each week was largest in the layer, followed by the meat, and was smallest in the Silky. The total amount of collagen reached 3.38 mg/g in the meat bird, 3.03 mg/g in the layer and 2.71 mg/g in the Silky by 30 weeks of age, respectively. In the perimysium, the collagen bundles increased in size and density of fibrils with growth. At 30 weeks of age the layer had compact collagen platelets while the Silky had loose collagen bundles. In the meat bird, the collagen bundles were moderately compact. The endomysial collagen network had a large mesh size at 1 week and thereafter accumulated many collagen fibrils to form a felt‐like fabric of fibrils at 30 weeks of age. From these results it appears that growth‐related changes in the iliotibialis lateralis muscle are not necessarily causally affected by the different growth rates of chicken breeds.     

Age-related changes in the intramuscular distribution of melanocytes in the Silky fowl

1. The characteristics of melanocyte distribution in skeletal muscles in the Silky fowl were investigated in association with growth. 2. Pectoralis (PT) and iliotibialis lateralis (ITL) muscles from 1-, 3-, 5-, 10-, 20- and 30-week-old Silky males were weighed and collagen type I was detected in frozen sections immunohistochemically. 3. Melanocytes were observed in the collagen type I-immunopositive endomysium and perimysium in both muscles. 4. Image analysis indicated that the total area occupied by melanocytes in histological sections sharply decreased from 0.61% to 0.16% in PT muscle and from 1.67% to 0.33% in ITL muscle at 1 to 3 weeks, and then gradually decreased. The melanocyte area was larger in ITL muscle than in PT muscle until 10 weeks of age. 5. We concluded that the proportion of intramuscular melanocytes in the Silky fowl differs between types of muscles in the early stages of development, and it decreases with growth.     

Identification and characterization of proteoglycans in bovine neonatal skeletal muscle

In order to provide background for understanding biological roles of proteoglycans (PG) in developing skeletal muscle, we have isolated and characterized PG in bovine neonatal skeletal muscle. Two types of PG were isolated from skeletal muscle by density gradient ultracentrifugation and ion‐exchange chromatography. One was a small PG (PG‐S) with a molecular size of 100–130 kDa, another was a large PG (PG‐L) with a molecular size of 300–500 kDa. The glycosaminoglycan chains of PG‐S and PG‐L were dermatan sulfate and chondroitin sulfate, respectively, judged by cellulose acetate membrane electrophoresis. Immunoblot assays revealed that both PG bound to type I, II, III and IV collagen, laminin and fibronectin. Unlike PG‐S, PG‐L bound to type V collagen and hyaluronic acid. Small proteoglycans had a core protein of 45 kDa, which reacted with the antibody against the decorin core protein. The N‐terminal amino acid sequence of the PG‐S core protein was consistent with that of decorin from bovine bone and tendon. Thus, PG‐S from neonatal skeletal muscle was identified as decorin in bovines. Immunohistochemical analysis with antibodies against PG‐L and PG‐S demonstrated that PG‐L was located both in the perimysium and endomysium, but PG‐S was localized exclusively in the perimysium. These findings suggest that the characterized PG may have distinct roles in the ECM construction of developing skeletal muscle.     

Immunohistochemical and scanning electron microscopic comparison of the collagen network constructions between pig, goat and chicken livers

The distribution and three-dimensional architecture of collagen fibers were compared between pig, goat and chicken livers. Immunohistochemical staining revealed that collagen type I was identified in the interlobular connective tissue region and intralobular areas in pigs and goats. Type III collagen was also identified in the interlobular connective tissue region and intralobular sinusoidal walls. In the chicken liver, only the circumference region of the vessels was immunostained with collagen type I and III antibodies and the interlobular connective tissue wall could not be distinguished clearly. In the intralobular region, collagen type I antibody immunoreacted around the hepatic cells but collagen type III antibody immunoreacted weakly. In the NaOH macerated specimen, well-developed collagen bundles formed the prominent interlobular walls in pigs. In contrast, the wall in the goat liver comprised a thin layer of the bundles. In the chicken liver, there were no notable collagen septa between lobules. The intralobular collagen construction was quite different between the animals, indicating a fragile collagen fibril networks in pigs, a robust framework in goats and dense fabric-like septa in chickens. These results indicate that the distinct collagen frameworks may contribute to the histological strength of the livers in each of the animal species.     

Histopathological study of feline eosinophilic dermatoses

A retrospective study was conducted on skin specimens from 24 cats with eosinophilic granuloma complex. The specimens were stained with haematoxylin and eosin and Gallego's trichrome stain. In all specimens, flame figures and/or large foci of so-called "collagen degeneration" were detected and histopathological features were not predictive of the clinical picture. Use of the term eosinophilic dermatosis was advocated in diagnostic dermatopathology. On trichrome-stained sections, normally stained collagen fibres were identified in the middle of both flame figures and large foci of "collagen degeneration" and the debris surrounding collagen bundles showed the same tinctorial properties as eosinophil granules. Eosinophil degranulation around collagen bundles seemed to represent the major pathogenetic event in these lesions, analogous with human flame figures. The term flame figures might therefore be more accurately used to designate those foci of eosinophilic to partly basophilic debris commonly referred to as "collagen degeneration".     

Collagen fiber arrangement in canine hepatic venules

Cell-maceration/scanning electron microscopy, serial sections and scanning electron microscopy of vascular resin casts were employed to demonstrate the arrangement of collagen fibers in the terminal hepatic venules, involving the central, intercalated and collecting veins in dog liver. In cell-maceration specimens, each collagen fiber was observed to run in various directions, forming a sheath with a compact meshwork of collagen fibers. The collagenous meshwork in the hepatic venules was looser than those of the terminal portal venules and hepatic arterioles. Some collagen fibers formed bundles with an elongated spiral arrangement encircling the wall of the terminal hepatic venules. In resin casts, these venules were observed as a twisted configuration caused by spiral collagen bundles. A helical modification of such connective tissue bundles might provide a mechanically stable vascular structure and permit reversible changes in linear and circumferential vascular dimensions at the terminal tributaries of veins. Round or oval pores with diameters of approximately 9 microns were also observed in the sheath of collagen fibers. These pores, together with the relatively loose collagenous meshwork in the hepatic venules, might play a role in lymphocyte migration from these venules into the surrounding tissue and provide high permeability to the venule walls. No such helical configuration and pores were observed in either the portal venules or the hepatic arterioles.     

3-D ultrastructural distribution of collagen in human placental villi at term in relation to vascular tree

In order to understand the 3-D distribution of collagen in relation to vascularization, chorionic villi of human placentae, belonging to normal pregnancies at term, were studied by scanning electron microscopy (SEM) after alkali maceration techniques, and by transmission electron microscopy (TEM). The villous tree appeared made of an uninterrupted structure of collagen fibres. The collagen fibres connected the chorionic villi axis with their basal plates and organised differently according to the various levels of villous branching. The collagen of stem villi showed copious fibres. The external fibres (facing the villous surface) were arranged mainly longitudinally. The central core of the villi (inner fibres) were arranged concentrically around the wall of the fetal vessels. Both external and internal fibres formed stratified lamellae or small parallel bundles. The inner core of stem villi showed small holes housing capillary spaces. Mature intermediate and terminal villi showed a scarce amount of collagen arranged in thin concentric layer within the villous core, surrounding numerous dilated capillary and sinusoid spaces.These observations demonstrated that the extracellular matrix of human chorionic villi is highly compartmentalised and shows a variable structural 3-D distribution depending on the branching level of the villous tree, such a distribution ensures the most favourable microenvironment for feto-maternal exchanges and it is likely able to provide a modulated support to the developing chorionic fetal vessels and trophoblastic layer as well.     

Increased collagen III in culled chicken meat after feeding dietary wood charcoal and vinegar contributes to palatability and tenderness

We comprehensively evaluated meat quality in chickens fed a diet consisting of wood charcoal and vinegar (WCV) using food scientific and histological approaches. In culled hens, lipid and fatty acid in Musculus semimembranosus, cooking loss and sensory tests of whole thigh meat, and meat texture of breast meat were observed. In male broilers, cross section of M. semimembranosus was used for observations on muscle area, perimysium, non‐collagen total protein and total collagen content, and anti‐collagen I and III reactions. In frozen male broilers, conventional morphology of M. semimembranosus as well as chicken anti‐collagen III reaction to selected muscles of thigh meat and breast meat were compared between the control and WCV‐fed birds. Increased lipid and fatty acids, decreased cooking loss, high score in total evaluation for sensory test of thigh meat, and decreased meat texture values were observed for culled hens fed WCV. The higher values of muscle area, total collagen and collagen III were observed for broilers fed WCV. No perimysium collapse for M. semitendinosus or increased collagen III reactions of M. tensor fasciae latae, the flexor muscle group and M. pectoralis superficialis were observed for frozen muscles in the WCV group. These total results suggest that WCV produces palatable and tender meat by increasing collagen III.