Rapid High-Throughput Analysis of Bovine Skeletal Muscle Fiber Morphology via Automated Fluorescent Microscopy and MuscleBos software

Skeletal muscle tissue is comprised of many individual muscle cells (myofibers) that can be classified as different types based on their morphology, histochemistry, enzymatic reactivity, and biochemical characteristics. One of the most common methods of classification of muscle fiber type relies on the local expression of specific myosin heavy chain (MyHC) isoforms. Adult mammalian muscle fibers are generally categorized into four major types including I, IIA, IIX, and IIB. However, the distribution of these muscle fiber types varies across both different species and muscle groups within species, influencing muscle function and physiological responses. In bovine species, skeletal muscle plays a critical role in determining in-vivo metabolic physiological processes and impacting post-harvest meat quality traits. Immunostaining methods using isoform-specific MyHC antibodies have been widely adopted to characterize muscle fiber morphology. However, manual capture and analysis of immunofluorescent images of muscle fiber type staining is time consuming, labor-intensive, and potentially susceptible to investigator bias. To address these limitations, we established and validated a high-throughput method for the analysis of bovine muscle fiber morphology that combines automated fluorescent microscopy with high-content image analysis using a customized version of the MuscleJ plugin for FIJI/ImageJ that we named MuscleBos. This refined method enables rapid quantitative characterization of muscle fiber type profile and fiber type-specific myofiber cross-sectional area in bovine skeletal muscle tissue cross-sections. This methodology should enable valuable deeper insights into future studies of muscle composition in bovine species and its impact on in vivo animal physiology and meat science.