Muscle Fiber Typing in Bovine and Porcine Skeletal Muscles Using Immunofluorescence with Monoclonal Antibodies Specific to Myosin Heavy Chain Isoforms

Abstract

The aim of this study was to optimize staining procedures for muscle fiber typing efficiently and rapidly in bovine and porcine skeletal muscles, such as longissimus thoracis, psoas major, semimembranosus, and semitendinosus muscles. The commercially available monoclonal anti-myosin heavy chain (MHC) antibodies and fluorescent dyeconjugated secondary antibodies were applied to immunofluorescence histology. Two different procedures, such as cocktail and serial staining, were adopted to immunofluorescence analysis. In bovine muscles, three pure types (I, IIA, and IIX) and one hybrid type, IIA+IIX, were identified by the cocktail procedure with a combination of BA-F8, SC-71, BF-35, and 6H1 anti-MHC antibodies. Porcine muscle fibers were typed into four pure types (I, IIA, IIX, and IIB) and two hybrid types (IIA+IIX and IIX+IIB) by a serial procedure with a combination of BA-F8, SC-71, BF-35, and BF-F3. Unlike for bovine muscle, the cocktail procedure was not recommended in porcine muscle fiber typing because of the abnormal reactivity of SC-71 antibody under cocktail procedure. Within the four antibodies, combinations of two or more anti-MHC antibodies allowed us to distinguish pure fiber types or all fiber types including hybrid types. Application of other secondary antibodies conjugated with different fluorescent dyes allowed us to get improved image resolution that clearly distinguished hybrid fibers. Muscle fiber characteristics differed depending on species and muscle types.

Pork Loin Chop Quality and Muscle Fiber Characteristics as Affected by the Direction of Cut

“…Our results, regardless of cut groups, corroborated with the previous observations. Additionally, the muscle fiber compositions in M-Vertical and F-Vertical groups were found to be similar to those reported by previous studies that demonstrated that glycolytic fiber (IIX or IIB) content was higher than the oxidative fiber (I or IIA) content in pork loin [19,[24][25][26]. Notably, the F-Parallel group exhibited a nearly double type I and IIA fiber content and nearly half of type IIB fiber content when compared to the M-Vertical and F-Vertical groups.…”

“…Relatively larger muscle fibers were observed on the surface of the F-Parallel group, regardless of muscle fiber types owing to the elongated cylindrical shape of the muscle fibers [ 20 , 21 ]. Among the muscle fiber types, IIB or IIX fibers are generally observed to be larger than types I or IIA, regardless of species and breeds [ 19 , 22 , 23 ]. Our results, regardless of cut groups, corroborated with the previous observations.…”

“…For staining and classification of muscle fiber types, the Song et al [ 19 ] method, with some modifications, was used. Briefly, transverse sections (10-µm thickness) were obtained from muscle cubes using a cryostat microtome (CM1520, Leica Biosystems, Wetzlar, Germany).…”

Muscle Fiber Characteristics on Chop Surface of Pork Loin (M. longissimus thoracis et lumborum) Associated with Muscle Fiber Pennation Angle and Their Relationships with Pork Loin Quality

“…10-μm-thickness sections were obtained by making parallel cuts to the chop surface of M-Vertical group and by making vertical cuts to the muscle fiber orientation of F-Vertical group, respectively, using a cryostat microtome (CM1520, Leica Biosystem, Wtezlar, Germany). The method by Song et al (2020) with some modifications was used to stain the cross-sections and muscle fiber typing. Briefly, each section was incubated with monoclonal anti-myosin heavy chain (MHC) antibodies purchased from DSHB (Iowa, IA, USA), such as BA-F8 (anti-MHC slow/I), SC-71 (anti-MHCs 2a and 2x), BF-35 (anti-MHCs slow/I and 2a), and BF-F3 (anti-MHC 2b).…”

“…The method bySong et al (2020) with some modifications was used to stain the crosssections and muscle fiber typing. Briefly, each section was incubated with monoclonal antimyosin heavy chain (MHC) antibodies purchased from DSHB (Iowa City, IA, USA), such as BA-F8 (anti-MHC slow/I), SC-71 (anti-MHCs 2a and 2x), BF-35 (anti-MHCs slow/I and 2a), and BF-F3 (anti-MHC 2b).…”