Histological muscle characterization in hypertrophied Marchigiana cattle breed

Myostatin (MSTN), also known as GDF8 (Growth Differentiation Factor 8), is one of the major regulators of skeletal muscle development. Mutations on MSTN gene are responsible for double muscling phenotype in several livestock animals. This phenotype occurs at a high frequency in some cattle breeds such as Belgian Blue and Piedmontese, and it was also identified in Marchigiana cattle, one of the most important Italian beef cattle breeds. A transversion mutation at nucleotide 874 in exon 3 (g.874G>T) of MSTN gene was found in Marchigiana breed. In this work a Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR-RFLP) test was used to determine the genotype at MSTN locus. Besides, histological analysis were carried out to investigate on differences in muscle morphology between the three genotypes. Furthermore, considering the important role played by myostatin during myogenesis, a satellite cell specific marker (PAX7) was considered to verify if a myostatin deficiency leads to an increase in these stem cells. Ten bullocks aged between 18 and 24 months (except one which was 13 months old) were sampled. Blood and muscles were collected at slaughtering. All animals were genotyped at the MSTN locus. Three different muscles were characterized: semitendinosus, psoas major and longissimus dorsi. Formalin-fixed paraffinembedded sections of muscle tissues were stained with haematoxylin/eosin. Myofiber cross sectional area (CSA) was used for muscle comparison. Part of each muscle samples were lysed and analysed for the expression of PAX7 by Western blotting. Genotyping at MSTN locus showed that two bullocks were homozygous for the mutation (TT), five were heterozygous (GT) and three were normal (GG). The CSA values in homozygous (TT) and normal (GG) were quite similar but microscopy analysis revealed muscle hyperplasia in homozygous (TT) bullocks, in accordance with previous studies. Western blotting analysis showed different expression levels of PAX7 in the three genotypes, with an increase in the mutant homozygous one. These findings confirm that lack of myostatin influences the proliferation of muscle precursor cells during myogenesis. Further studies about the mechanisms by which myostatin inhibits muscle growth are needed to better understand the particular muscle development in the hypertrophied Marchigiana cattle breed.