Anatomical site and divergent functionalities of muscles can be related to differences in IMF content, metabolism and adipogenic gene expression. Then, potential differences in different muscles in beef cattle were studied. As a second objective, the main sources of experimental variability associated to RT-qPCR results were analyzed following a nested design in order to implement appropriate experimental designs minimizing gene expression variability. To perform the study Longissimus thoracis (LT), Semitendinosus (SM), Masseter (MS), Sternomandibularis (ST) and subcutaneous adipose tissue (SAT) samples of Pirenaica young bulls (n = 4) were collected for IMF, collagen and protein quantification, analysis of adipocyte size distribution and gene expression (PPARG, CEBPA, FAPB4 and WNT10B). A greater IMF content was observed in MS and SM muscles, which had a bimodal adipocyte size distribution while it was unimodal in the muscles LT and ST. This suggest that the different IMF accretion in the muscles studied might be related to different rates of hyperplasia and hypertrophy and that IMF might develop later in LT and ST muscles. The former differences were not mirrored by the expression of the genes analyzed, which might be related to the different contribution of mature and non-mature adipocytes to the total gene expression. When comparing IMF and SAT gene expression, late and early developing tissues respectively, expression of PPARG, CEBPA and FABP4 was higher in the SAT, in agreement with bigger cell size and numbers. The variability study indicates that the analytical factors that add higher variability to the gene expression are the sampling and RT and therefore, it would be appropriate to include those replicates in the design of future experiments. Based on the results, the use of MS and SM muscles could allow less expensive experimental designs and bigger sample size that could permit the detection of lower relevant differences in gene expression.

Adipogenesis, myogenesis and fibrogenesis are related processes that can contribute to meat quality. Therefore, extending the knowledge of these processes would facilitate the identification of molecular markers that predict intramuscular fat accretion. The main purpose of this work, based on previous results, was to further study the expression of key genes related to adipogenic, myogenic, fibrogenic processes and some cytokines in Longissimus thoracis (LT) and Masseter (MS) muscles of Pirenaica and Holstein young bulls. Longissimus thoracis and MS muscles from Pirenaica (n = 4) and Spanish Holstein (n = 4) were sampled for proximate analysis, determination of adipocyte size distribution and expression of key candidate genes. Fat percentage was lower in LT than in MS muscle in Pirenaica young bulls (P = 0.023) and was higher in LT muscle in Holstein than in Pirenaica young bulls (P = 0.007). Gene expression analysis revealed that the mRNA level of myogenic differentiation 1 (MYOD) was higher in LT than in MS muscles in both groups of animals (P < 0.001) and that myostatin (MSTN) expression was also higher in LT than in MS muscle in Holstein bulls (P = 0.001). On the other hand, MSTN and PPARG showed higher expression in LT and MS in Pirenaica young bulls (P = 0.026), while the expression of fatty acid-binding protein 4 (FABP4) was higher in Holstein young bulls, also in both muscles (P < 0.001). The results suggested that the development of intramuscular adipose depot was directly related to the expression of adipogenic genes, such as FABP4, but inversely related to the expression of the cytokine MSTN and the myogenic gene MYOD, genes which showed a muscle-specific expression.