Immunohistochemistry. Fibre typing
The functional properties of muscles that qualify them for locomotion, postural maintenance or respiration, among others, can be elucidated from their fibre type composition. To the authors' knowledge, the FD muscle has not been previously characterised in cattle. An interesting result of the present study was the abundance of slow-twitch type I fibres and the vestigial expression of the MyHC IIX isoform in the FD, resulting in the absence of IIX pure fibres and a very low proportion of hybrid IIAX fibres (Table 2). Muscles composed mainly of type I could play a major role in maintaining posture by stabilizing the extended joints, while large muscles generating the strong power needed for propulsive force contained a high proportion of the IIX type , as did the PM. The fact that the IIX pure type was not found in FD indicated the specialisation of this muscle towards a greater endurance, corresponding to a muscle continuously used throughout the day for walking, standing, etc.
Similar to the results shown by Picard et al.  in the bovine species, the muscles studied here showed a small proportion of hybrid fibres in comparison to other mammalian species such as horses [13, 18], pigs  or dogs . The role of the hybrid fibres is not fully understood. Some authors  assert that they indicate the dynamic transition from one pure phenotype to the other, whereas some others  claim that they are stable populations that can behave differently depending on external stimuli. In this context, we speculate that a low percentage of hybrid fibres could not significantly change the behaviour of the muscle in response to a external stimuli, whereas a large population of hybrid fibres could. So, species with a low proportion of hybrid fibres (cattle) would support the "dynamic transition" hypothesis, and species having a large percentage of them (dog) the "stable population" one. As the MyHC isoform composition of a single fibre can be used as a "physiological marker", then the extent of hybridism may reveal the diversity of activity that a given muscle or species requires.
The very fast MyHC IIB was not detected in our study, in agreement with Tanabe et al., , Maccatrozzo et al., , and Toniolo et al., . Although large mammalian species were initially supposed not to have the very fast MyHC IIB isoform, it has been found in some of them, such as pig Longissimus muscle,  and llama Semitendinosus and Vastus lateralis muscles [24, 34]. This MyHC isoform was functionally and morphologically compatible with the MyHC IIB gene, commonly reported in eutherian species of mammals . Nevertheless, this third fast MyHC isoform, compatible with the IIB isoform of small rodents, is not expressed in trunk and limb skeletal muscles of humans, carnivores, ruminants or horses [5, 36–39].
The fibre type composition of muscles in meat producing breeds influences their meat quality features. A positive relationship between the percentage of type I fibres and the intramuscular fat (IMF) has been previously described for bovine skeletal muscles . It is noticeable that in the present study a larger proportion of type I fibres was found in the muscle which had a lower IMF content . In relation to this finding, some results in human muscles pointed out the absence of a relationship between the expression of MHY7 (the gene coding for MyHC I) and the expression of genes involved in adipogenesis such as PPARα and PPARδ . The predominance of type II fibres has been associated with a faster post-mortem ageing rate and, therefore, to a rapid rate of tenderisation . In our case, the PM showed a larger proportion of both IIA and IIX types and was perceived as a more tender muscle than the FD . Both IMF and tenderness are relevant traits in the cattle production context.
In a different study, we have performed a microarray experiment to assess the differential gene expression between PM and FD muscles in male Avileña-Negra Ibérica calves  A bovine fat and muscle cDNA microarray  was used and 20 microarray slides were hybridised following a loop design that directly compared both muscles within and between individuals. MYH7 gene (coding for type I isoform) was more expressed in FD, which is in agreement with the higher proportion of type I fibres found in this muscle (see Table 2). Furthermore, the MYH1 gene (coding for type IIX isoform) was more expressed in PM, in agreement with the larger proportion of IIX fibres found in this muscle. However, the MYH2 gene (coding for type IIA isoform) did not show a significant difference in expression in the FD muscle, which had a higher proportion of type IIA fibres in the current experiment. This last observation has also been described in swine [14, 44]. The most common explanation is that mRNA coming from different MyHC genes could hybridise together in the same spots because of their sequence similarity. If this was the case, results would show random patterns in both muscles, but what we have in reality is a particular pattern for MYH7, MYH1 and MYH2 genes specific to the different muscle types. A larger amount of the MYH2 transcript was systematically observed in the PM muscle, which contains less IIA fibres. Different post-transcriptional mechanisms of gene expression, such as siRNA, antisense RNA, RNA interaction with silencing factors, etc., could mediate the relation between MYH2 transcripts and IIA fibres.
Myofibrillar ATPase activity
Muscle studies in cattle have traditionally relied on this method to classify fibre types [15, 30, 45, 46], although in this study the staining for the acid or alkaline stabilities of the mATPase activity was use to determine fibre properties. The immunohistochemistry overcame one of the limitations of the mATPase technique, which could not photometrically distinguish hybrid types with dominance of one isoform from their respective pure phenotypes .
Different mATPase profiles have been reported in bovine skeletal muscles. Our results, in agreement with Totland et al. , showed that the acid stability of mATPase activity at pH 4.42 was lower for IIA than for IIX fibres (Figures 1E and 3B), whereas the reverse was true for the alkaline stability after preincubation at pH 10.35 (Figures 1F and 3C). However, Picard et al., , and Gotoh, , found that IIA and IIX (named IIB in their studies) fibres had the same acid stability after preincubation at pH 4.2 and the mATPase stability after alkaline preincubations at pH 10.48 or 10.5 was opposite to ours. This discrepancy was probably related to the different mATPase histochemical methods applied in the different studies, or to slightly different technical procedures. We found significant correlations between mATPase activities and MyHC isoform (Figure 3B–C), which agreed with similar results in a number of other mammalian species .
SDH and GPDH activities have been determined in several species, such as goats , dogs  and swine , but, to the best of our knowledge, this is the first study in which they were histochemically quantified in bovine skeletal muscle fibre types. The oxidative and glycolitic capacities differed among fibre types, and showed a negative correlation whose value indicates that the MyHC isoform is not the only factor influencing the metabolic profile of the fibres. This was also reflected in the variation of SDH:GPDH ratios across fibres that, rather, showed quite a remarkable stepwise decline from slow to fast types. The SDH:GPDH ratio expresses the capacity of myofibres for synthesizing ATP from oxidative (SDH) and glycolitic (GPDH) pathways, showing the ability of myofibres to produce energy in aerobic or anaerobic form.
Biological systems have acquired effective adaptive strategies to cope with physiological challenges and to maximise biochemical processes under imposed constraints . Contractile and metabolic properties appeared related in our study (Figure 4D and 4E). Although correlations among MyHC types and metabolic properties were different from zero, the magnitude of such correlations indicated that fibre types did not necessarily exhibit a precise metabolic specialisation. The hybrid fibres had intermediate properties between their respective pure types. However, the metabolic pattern, represented by the SDH and GPDH activities, of hybrid fibres was different to the one described for the contractile properties, in this case indirectly measured by the mATPase activities, as previously described in the Results section (Figures 3A and 4A). In order to assess the effect of both, mATPase and metabolic properties, on the position of hybrid fibres in relation to their pure types, Mahalanobis distances among all groups of fibres were calculated removing mATPase information: the Mahalanobis distances (results not shown) indicated a pattern similar to the one observed in Fig 4a. When metabolic attributes dominated the analysis, hybrid fibres tended to have an intermediate position closer to type IIA in both hybrid fibre populations. However, when the metabolic information was removed, a pattern similar to the one shown in Fig 3A was found. Mahalanobis distances among all groups of fibres were also similar to the ones shown in Table 4. Thus, our results suggest that metabolic and contractile properties appear to position hybrid fibres differently, although they are always between their pure types. Striated muscle tissue demonstrates a remarkable malleability and can adjust its metabolic and contractile makeup in response to alterations in functional demands , which could explain the discrepancies between metabolic and contractile patterns.
The fact that the correlation value between the SDH activity and MyHC type was higher than the one between the GPDH and MyHC type indicated that the oxidative specialisation was more preserved among fibre types than the glycolitc one in these muscles and species. Our results, together with previous studies [11, 18, 39] indicate that the magnitude of the correlations between contractile and metabolic properties differs across species, and such differences could be related to differences in the SDH:GPDH ratio within fibre types among them.
The significant differences regarding histological features of the distinct fibre types might have a functional reason, as reported in similar studies .
Although the reasons for the difference in the number of nuclei among fibre types are not fully understood, it has been related to different activity patterns among fibre types [47, 48]. More active muscle fibres usually have higher levels of both protein synthesis and turnover than those scarcely recruited . Fibres in FD are expected to be more active than fibres in PM, as the number of nuclei were significantly higher in FD than in PM (Table 3). The over-expression of genes related to protein synthesis and turnover observed in FD  corroborated this idea.
A small fibre size is an advantage for the diffusion of oxygen and nutrients for oxidative metabolism  and is related to more fatigue resistance as well. The mean CSA of the fibre types decreased in the order IIX>IIAX>IIA>I>I+IIA in PM and IIA>I>I+IIA>IIAX in FD, in agreement with a similar study in goats  but contrary to a study in dogs, in which the CSA was IIX>I>IIA . An inverse relationship between fibre diameter and oxidative capacity of muscle fibres has been reported , which is in accordance with our results.
Capillarisation has been associated with the transport of oxygen and lipids (among other nutrients), and consequently with a large oxidative capacity . In this context, the oxidative capacity of a muscle is related to MyHC isoform distribution, as well as to histological features . Although our study was not designed to compare metabolic activities of the muscles, we observed that all fibre types tended to have a higher oxidative activity in PM than in FD. Therefore, the smaller amount of MyHC I in PM may be compensated for by their large oxidative ability, which could then be more related to capillarisation than to fibre type.
Provided that different motor units are recruited at postural and phasic activities, their constituent muscle fibres might have different sizes and capillary supply [50, 51]. Features such as small size and high capillarisation, typical of I and IIA fibre types, mean these motor units are more frequently activated and have a higher oxidative metabolism than the fast IIX motor units. This relationship is also related to the fatigue resistance of the motor units.
Carbohydrates are imported from the capillary supply lines to the myofibres, where they may be stored as either intramuscular triglycerides or glycogen, for later combustion. Fatty acid metabolism is an aerobic process that takes place in the mitochondria . When compared to FD, PM showed a larger relative capillarisation and a smaller cell size, a high expression of mitochondrial genes  and a larger IMF content . All these features account for the great oxidative ability of PM in cattle, even when compared to a muscle mainly composed of type I fibres, such as FD.