Standardization of metachromatic staining method of myofibrillar ATPase activity of myosin to skeletal striated muscle of mules and donkeys

Abstract

Pesq. Vet. Bras. 34(9):917-922, setembro 2014 917 RESUMO.-[Padronização do método de coloração metacromático da atividade da miosina ATPase miofibrilar para músculo estriado esquelético de muares e asininos.] O presente estudo objetivou padronizar o pH e a temperatura da pré-incubação e incubação do método de coloração metacromática de myofibrillar atividade ATPase da miosina (mATPase) utilizada para asininos e muares.Vinte e quatro jumentos e 10 muares, sete machos e três fêmeas, foram usados no estudo. De cada animal, fragmentos do músculo Gluteus medius foram coletados e biópsia muscular percutânea foi realizada por meio de uma agulha Bergström tipo de 6,0 mm. Em adição ao método de coloração metacromática de mATPase, a técnica de nicotinamida adenina dinucleótido tetrazólio redutase (NADH-TR) foi também realizada para confirmar os dados histoquímicos. O resultado histoquímico da mATPase por pré-incubação ácida (pH=4,50) e incubação alcalina (pH=10,50), a uma temperatura de 37 o C, foi o que proporcionou a melhor diferenciação das fibras coradas com azul de toluidina. As fibras musculares foram identificadas de acordo com as seguintes cores: tipo I (oxidativa, azul claro), tipo IIA (oxidativo-glico- This study aims at standardizing the pre-incubation and incubation pH and temperature used in the metachromatic staining method of myofibrillar ATPase activity of myosin (mATPase) used for asses and mules. Twenty four donkeys and 10 mules, seven females and three males, were used in the study. From each animal, fragments from the Gluteus medius muscle were collected and percutaneous muscle biopsy was performed using a 6.0-mm Bergström-type needle. In addition to the metachromatic staining method of mATPase, the technique of nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) was also performed to confirm the histochemical data. The histochemical result of mATPase for acidic pre-incubation (pH=4.50) and alkaline incubation (pH=10.50), at a temperature of 37 o C, yielded the best differentiation of fibers stained with toluidine blue. Muscle fibers were identified according to the following colors: type I (oxidative, light blue), type IIA (oxidative-glycolytic, intermediate blue) and type IIX (glycolytic, dark blue). There are no reports in the literature regarding the characterization and distribution of different types of muscle fibers used by donkeys and mules when performing traction work, cargo transportation, endurance sports (horseback riding) and marching competitions. Therefore, this study is the first report on the standardization of the mATPase technique for donkeys and mules.INDEX TERMS: Standardization, metachromatic staining method, myofibrillar ATPase activity of myosin, skeletal striated muscle, mules, donkeys, histochemistry.1 Received on October 10, 2013.Accepted for publication on July 27, 2014. lítico, azul intermediário) e tipo IIX (glycolytic, azul escuro). Na literatura, não foram encontradas publicações pertinentes à caracterização e distribuição dos diferentes tipos de fibr...

Donkey Internal Medicine—Part II: Cardiovascular, Respiratory, Neurologic, Urinary, Ophthalmic, Dermatology, and Musculoskeletal Disorders

“…Muscle atrophy secondary to starvation or gastrointestinal disorders as well as degenerative muscular lesions and myonecrosis due to selenium deficiency were also seen in this study [157]. Biopsy sampling and histochemical techniques useful for myopathies are described in donkeys and mules [158]. Exertional rhabdomyolysis, polysaccharide storage myopathy, grass sickness, and atypical myopathy have been described in donkeys or mules [157,159e162].…”

Skeletal Muscle Fiber Composition of Untrained Mangalarga Marchador Fillies

“…Muscle samples were sectioned serially (12-mm thickness) in a cryostat (Mícron gmbH, H1599 OM, 69,190, walldorf, Germany) at À20 C. Histochemical analysis was used to identify or differentiate the types I, IIA, and IIX fibers and consisted of adapting the metachromatic staining method of ATPase activity in myofibers described by D'Angelis et al [10] for the preincubation in acid medium [11,12] at pH 4.45 to 4.55 for 5 to 6 minutes at 18 C to 20 C, followed by incubation in alkaline medium [13] at pH 10.50 to 10.55 for 25 minutes at 37 C. To verify the mATPase histochemical data, the indirect immunohistochemical method (peroxidase-antiperoxidase) [10] was used after incubation with monoclonal anti-slow myosin primary antibody (Clone NOQ7.5.4D; Sigma-Aldrich, Química do Brasil Ltda, São Paulo, SP, Brazil). The slow-twitch fibers (type I) and fast-twitch fibers (type II) were identified by the golden color of the diaminobenzidine precipitate formed in the antigen-antibody complex in the former and lack of color in the latter.…”

“…The slow-twitch fibers (type I) and fast-twitch fibers (type II) were identified by the golden color of the diaminobenzidine precipitate formed in the antigen-antibody complex in the former and lack of color in the latter. The oxidative potential of the skeletal muscle fibers was assessed through nicotinamide adenine dinucleotide tetrazolium reductase [10,14]. Type I and type IIA fibers were stained purple, and type IIX were stained light purple or had no coloration.…”

Effect of feeding pregnant gilts fermented potato extract protein on the prenatal development and semitendinosus muscle characteristics of newborn piglets

“…The frozen sections of the ST muscle tissue were stained with hematoxylin and eosin (H&E) and stained for myofibrillar adenosine triphosphatase (mATPase) after pre-incubation in an acidic or alkaline solution (pH 4.6 and 10.4), following a previously described method (D'Angelis et al, 2014). For mATPase staining, tissue slides were pre-incubated with an acidic (5 min) or alkaline solution (15 min) at room temperature (RT) and rinsed twice in the wash solution (0.18 M calcium chloride (CaCl 2 ) in 0.1 M sodium barbital solution).…”