Morphofunctional measurements were performed on microphotographs of stained sections, obtained with a light microscope (BX61; Olympus, Tokyo, Japan) connected to a digital camera (DP70; Olympus, Tokyo, Japan) at × 20 magnification. Since GAS muscle has a heterogeneous muscle fiber type distribution and morphometry, three different zones from each muscle (red, intermediate, and white), as previously described by Armstrong and Phelps (1984), were photographed and further analyzed (Armstrong and Phelps, 1984). All the parameters listed below were measured or calculated from transverse cross-section tissues with an area of 5.5 × 105 μm2 using ImageJ software (v. 1.51n; National Institutes of Health, United States).

All muscle fibers were typified according to their metabolic and contractile character (Figures 1B,C) and classified as slow-twitch oxidative (SO), fast-twitch oxidative glycolytic (FOG), fast-twitch glycolytic (FG), or fast-twitch intermediate glycolytic (FIG). SO fibers were unstained for mATPase (pH 10.7) and presented high SDH activity; FOG fibers displayed a dark mATPase and SDH stain; FG fibers presented moderate mATPase stain and remained unstained for SDH assay; and FIG fibers presented moderate to high mATPase and intermediate SDH stain (higher than FG fibers but lower than FOG fibers). Images from eATPase (Figure 1A) were used to measure or calculate the following parameters: fiber cross-sectional area (FCSA), Feret diameter, number of capillaries per 1,000 μm2 of FCSA (CCA = NCF⋅103/FCSA), number of capillaries per fiber (NCF), fiber density (FD), capillary density (CD), and capillary-to-fiber ratio (C/F = CD/FD). The mean total number of fibers per rat included for the analysis was 477 ± 112 (±SD). The same researcher performed fiber typing and identification of the capillaries to guarantee that the same criteria were used in all the analyzed images.

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