Histochemical classification of neck and limb muscle fibers in a turtle, Pseudemys scripta: A study using microphotometry and cluster analysis techniques

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Abstract
We have attempted to develop an objective, semiquantitative classification of fiber types in turtle neck and limb muscle using microphotometry and multivariate statistical techniques. We first stained serial sections for myosin adenosine triphosphatase (ATPase) (with acid and alkaline preincubation and without preincubation), NADH‐diaphorase, and two glycolysis‐associated markers, α‐glycerophosphate dehydrogenase (α‐GPDH) and glycogen phosphorylase A (GPA). This allowed us to characterize individual muscle fibers in terms of their contraction speed and metabolic properties. Next we used microphotometry to measure the optical density of the reaction product in each fiber, and we subjected the resulting optical density matrix to cluster and discriminant function analyses in order to assign fibers to groups (fiber types) and to determine which stains contribute most to the distinction between groups. As a control, we processed a well characterized mammalian muscle (rat sternomastoid) simultaneously. Our results suggest that both neck and limb muscle in Pseudemys can best described as falling into three groups: (1) slow oxidative (SO) fibers; (2) fast oxidative glycolytic (FOG) fibers, with relatively high oxidative and glycolytic capacities; and (3) fast glycolytic (Fg) fibers, with low oxidative, low/intermediate α‐GPDH, and high GPA activities. These three fiber types differ from like‐named types in rat muscle both in the pH lability of their myosins and in their metabolic profiles.