FIGURE 3.

A productive model system to study regulation of muscle fiber size as whole-animal energetics change for birds is that of the pectoralis of mourning doves. (A) After fixing the pectoralis muscle in 4% paraformaldehyde, we placed fixed muscle tissue in 25% sucrose for 24 h to cryo-protect the samples. Tissues were then flash frozen in isopentane cooled in liquid nitrogen, mounted at resting length in Optimal Cutting Temperature (O.C.T.) compound and allowed to equilibrate to −19°C in a Leica 1800 cryocut microtome before sectioning. Sections were cut at 30 μm, picked up on slides, air-dried at room temperature, stained with a 250 μg/ml solution of wheat germ agglutinin (WGA) labeled with Alexa Fluor 488 (in green), and 4′,6-diamidino-2-phenylindole (DAPI; in blue), for 30 min, and rinsed in avian ringer’s for 60 min. WGA is a lectin that binds to glycoproteins on the basement membrane of the fiber sarcolemma, and effectively outlines the fiber periphery to allow measurements of fiber size, whereas DAPI irreversibly binds to nuclei. Stained slides were examined with an Olympus Fluoview 1000 laser filter confocal microscope, and pictures were taken at a magnification of ×20. Mourning dove pectoralis muscle contain a population of small muscle fibers with a myonuclear domain (MND) surrounded by a population of large muscle fibers. (B,C) Using data from Jimenez and De Jesus (2021b), we isolated the number of nuclei per fiber and MND of N = 4 mourning doves (N = 135 small fibers and N = 63 large fibers). Using a one-way ANOVA, the small fibers demonstrated a significantly fewer nuclei per mm of fiber (F = 108.83, p < 0.0001; Panel (B), and a significantly smaller MND (F = 27.48, p < 0.001; Panel (C).