Henckel P, Ducro B, Oksbjerg N, Hassing L
Danish Institute of Animal Science, Foulum Research Centre, Tjele, Denmark.
Eur J Histochem. 1998;42(1):49-62.
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.
在马、小牛和猪的肌肉样本中，评估了两种最广泛使用的纤维类型区分方法的客观性，即：1）肌球蛋白ATP酶法（布鲁克和凯泽，1970a，b）；2）联合法，该方法利用肌球蛋白ATP酶反应区分快肌纤维和慢肌纤维，并利用NADH - 四氮唑还原酶活性识别快肌纤维的亚组（阿什莫尔和多尔，1970；彼得等人，1972）。我们还评估了用于观察毛细血管的α - 淀粉酶 - PAS制剂（安德森，1975）在这些物种中的客观性。为了降低肌肉样本组织化学分析的时间成本，我们开发了一个交互式图像分析系统并进行了描述。所有分析均在该系统上进行。与其他几项研究一致，发现两种区分纤维类型的方法之间的差异仅存在于快肌纤维亚组的相对分布中（IIA型和IIB型纤维的相对数量分别为p < 0.02和p < 0.05），而以相对面积计算时，只有IIA型纤维的比例存在显著差异（p < 0.05）。对于相同性状，发现方法与人以及方法与物种之间存在显著的交互作用。当分别分析方法时，联合法在IIA型纤维分布的相对数量和相对面积方面均显示出显著的人员方差成分（分别为p < 0.01和p < 0.001）。ATP酶法的客观性通过以下事实进一步得到证实：对于IIA型纤维相对面积分布的总方差，统计模型解释的比例更高（联合法为34.2%，而ATP酶法为72.1%）。尽管人与人之间测量的重复性通常较高（> 87%），但联合法在进行区分时前提条件（纤维类型之间不同程度的重叠）差异的影响引发了该方法可靠性的问题。显然，对于两种方法区分快肌纤维两个亚组的分布结果进行比较，没有通用规则适用。发现α - 淀粉酶 - PAS法是一种相当客观的方法，可用于识别马、小牛和猪肌肉中的毛细血管。然而，由于结合其他特征描述的毛细血管密度以指示扩散特性时受到人员的显著影响，建议由同一人对一个项目进行所有分析。除了本研究中的方法学结果外，我们还表明，通过应用TEMA图像分析系统，与耗时的传统组织化学制剂评估方法相比，现在可以对大规模实验的样本进行分析。
