To determine which myosin heavy chain (MHC) isoforms are expressed in canine skeletal muscles, different muscle samples of five mixed-breed dogs were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated MHC isoforms were identified by immunoblotting technique using a set of specific monoclonal antibodies. To compare the results of the electrophoretic and immunoblotting study, the pattern of MHC isoform expression and histochemical profiles of canine fibres were additionally demonstrated on serial muscle sections by immunohistochemistry and myofibrillar adenosine triphosphatase (mATPase) histochemistry. Not more than three MHC isoforms were demonstrated by SDS-PAGE in the analysed canine muscles. By the immunoblotting technique, the fastest migrating MHC band was identified as slow or MHC-I, the intermediate one as MHC-IIx and the slowest migrating band as MHC-IIa isoform. Since none of the three MHC bands and none of the analysed fibres were recognized by the antibody specific to MHC-IIb of rats, we concluded that MHC-IIb is not expressed in large skeletal muscles of dogs. Similarly, only three major fibre types, i.e. I, IIA and IIX, were revealed according to the pattern of MHC immunohistochemistry and mATPase reaction. Type IIA fibres were more alkali- and acid-stable than type IIX fibres after mATPase histochemistry; hence, the latter corresponded to type IIDog fibres. However, beside the three major fibre types, scarce hybrid fibres co-expressing two MHC isoforms (I/IIA and IIA/IIX) were demonstrated by immunohistochemistry.

The aim of this study was to determine the pattern of myosin heavy chain (MHC) isoform expressions within the muscle fibres of functionally diverse trunk and limb dog muscles using monoclonal antibodies that are specific to MHC isoforms. We found that three MHC isoforms are expressed in dog skeletal muscles. The pattern of their expressions determined the existence of ‘pure’ fibres, i.e. I and IIa, both expressing only one MHC isoform, and ‘hybrid’ fibres, i.e. I/IIa and IIa/x, that co-expressed two MHC isoforms. While the MHCI, MHCIIa and MHCI/IIa fibres corresponded to the myofibrillar ATPase type fibres I, IIA and IIC, respectively, the hybrid MHCIIa/x fibres mostly behaved like the IIDog fibre type in myofibrillar ATPase reaction as described by Latorre et al. [J Anat 1993a;182:329–337]. No pure MHCIIx fibres were found. Though MHCIIa/x fibres were quite numerous, their presence varied not only within different muscles but within the same muscle of different animals as well. We suggest that the discrepancies in the classification of fibre types according to their myofibrillar ATPase activity between different studies of dog skeletal muscles are probably a consequence of the variable content of the MHCIIa and MHCIIx isoforms in the MHCIIa/x hybrid fibres. Estimating the histochemical metabolic profile of fibres we found that in all fast fibres oxidative-glycolytic metabolism prevailed, whereas in slow fibres oxidative metabolism was more pronounced.

Testes samples of 52 brown hares (Lepus europaeus L.), sacrificed between July and January, were subjected to immuno histochemical analysis. The terminal deoxynucleotidyl transferase-mediated d'UTP nick end labelling (TUNEL) method was applied to detect apoptosis; and antibodies to proliferating cell nuclear antigen (PCNA) were used to evaluate cell proliferation in the testes. In the seminiferous epithelium, the apoptotic processes were evident from August to early November with maximal values in September. Cell death in germ cells occurs predominantly during the prophase of the first meiotic division. In July, and from mid-November onwards, only the occasional TUNEL-positive cells can be seen. The proliferation of germ cells continues during the testis regression phase. The average number of PCNA-positive cells decreases slightly from September onwards and rises again in mid-November.

To further elucidate the pattern of MHC isoform expression in skeletal muscles of large mammals, in this study the skeletal muscles of brown bear, one of the largest mammalian predators with an extraordinary locomotor capacity, were analyzed. Fiber types in longissimus dorsi, triceps brachii caput longum, and rectus femoris muscles were determined according to the myofibrillar ATPase (mATPase) histochemistry and MHC isoform expression, revealed by a set of antibodies specific to MHC isoforms. The oxidative (SDH) and glycolytic enzyme (alpha-GPDH) capacity of fibers was demonstrated as well. By mATPase histochemistry five fiber types, i.e., I, IIC, IIA, IIAX, IIX were distinguished. Analyzing the MHC isoform expression, we assume that MHC-I, -IIa, and -IIx are expressed in the muscles of adolescent bears. MHC-I isoform was expressed in Type-I fibers and coexpressed with presumably -IIa isoform, in Type-IIC fibers. Surprisingly, two antibodies specific to rat MHC-IIa stained those fast fibers, that were histochemically and immunohistochemically classified as Type IIX. This assumption was additionally confirmed by complete absence of fiber staining with antibody specific to rat MHC-IIb and all fast fiber staining with antibody that according to our experience recognizes MHC-IIa and -IIx of rat. Furthermore, quite high-oxidative capacity of all fast fiber types and their weak glycolytic capacity also imply for MHC-IIa and -IIx isoform expression in fast fibers of bear. However, in adult, full-grown animal, only MHC-I and MHC-IIa isoforms were expressed. The expression of only two fast isoforms in bear, like in many other large mammals (humans, cat, dog, goat, cattle, and horse) obviously meets the weight-bearing and locomotor demands of these mammals.

To gain a better understanding of the normal characteristics of developing canine muscles, myosin heavy chain (MHC) isoform expression was analysed in the axial and limb skeletal muscles of 18 young dogs whose ages ranged from the late prenatal stage to 6 months. We compared the results of immunohistochemistry using ten monoclonal antibodies, specific to different MHC isoforms, and enzyme-histochemical reactions, which demonstrate the activity of myofibrillar ATPase, succinate dehydrogenase (SDH) and α -glycerophosphate dehydrogenase ( α -GPDH). In the skeletal muscles of fetuses and neonatal dogs the developmental isoforms MHC-emb and MHC-neo were prevalent.In all muscles the primary fibres, located centrally in each muscle fascicle, strongly expressed the slow isoform MHC-I.The adult fast isoform MHC-IIa was first noted in some of the secondary fibres on fetal day 55. During the first 10 days after birth, the expression of MHC-emb declined, as did that of MHC-neo during the second and third weeks. Correspondingly, the expression of MHC-IIa, and later, of MHC-I increased in the secondary fibres. Between the sixth week and second month the expression of MHC-IIx became prominent. The slow rhomboideus muscle exhibited an early expression of the slow isoform in the secondary fibres. Our results indicate that the timing of muscle maturation depends on its activity immediately following birth. The fastest developing muscle was the diaphragm, followed by the fast muscles. A pronounced changeover from developmental to adult isoforms was noted at 4 -6 weeks of age, which coincides with the increased physical activity of puppies.