Objectivity of two methods of differentiating fibre types and repeatability of measurements by application of the TEMA image analysis system.

The objectivity of two of the most widely used methods for differentiation of fibre types, i.e. 1) the myosin ATP-ase method (Brooke and Kaiser, 1970a,b) and 2) the combined method, by which the myosin ATP-ase reaction is used to differentiate between fast and slow twitch fibres and NADH-tetrazolium reductase activity is used to identify the subgroups of fast twitch fibres (Ashmore and Doerr, 1970, Peter et al., 1972), was assessed in muscle samples from horses, calves and pigs. We also assessed the objectivity of the alpha-amylase-PAS preparation for the visualisation of capillaries (Andersen, 1975) in these species. For the purpose of reducing the time costs of histochemical analysis of muscle samples, we have developed an interactive image analysis system which is described. All analyses are performed on this system. In accordance with several other investigations, differences between the two methods of differentiating fibre types were found only for the relative distribution of the fast-twitch fibre subgroups (p < 0.02 and p < 0.05 for the relative number of type IIA and IIB, respectively) whereas when calculated in relative area, only the proportion of type IIA fibres differed significantly (p < 0.05). Significant interaction effects between method and person and method and species were found for the same traits. When methods were analysed separately, the combined method displayed a significant variance component of person for the distribution of type IIA fibres both in relative number and in relative area (p < 0.01 and p < 0.001, respectively). The objectivity of the ATP-ase method was further substantiated by the fact that a higher fraction of the total variance for the relative area distribution of type IIA fibres was explained by the statistical model (72.1% versus 34.2% for the combined method). Although the repeatability of measurements between persons was generally high (> 87%), the impact of differences in pre-requisites (varied degrees of overlap between the fibre types) for performing the differentiation by the combined method raises a question of the reliability of this method. Apparently, no general rules for comparison of results of distribution of the two subgroups of fast twitch fibres by the two methods are applicable. The alpha-amylase-PAS method was found to be a fairly objective method to identify capillaries in muscles from horses, calves and pigs. However, as capillarity described in combination with other traits to give an indication of diffusion characteristics is significantly influenced by person, it is recommended that the same person perform all the analysis of a project. In addition to the methodological results in this study, we have shown that by application of the TEMA image analysis system, which is more rapid compared with the time-consuming traditional method for evaluation of histochemical preparations, analysis of samples from large-scale experiments are now possible.