2.4. Microstructural and Ultrastructural Studies

Histological analyses were carried out on the NT and tumbled pieces of raw meat to microscopically identify exactly which meat structures were damaged by the tumbling process. For this purpose, 1-cm³ meat samples were extracted from 2-cm-thick slice S4 at two different depths along the diameter of the slice. As detailed in Figure 2, the first level was close to the surface of the piece of meat (Surface, S) and the second (Centre, C) was located 2 cm down from the surface level, corresponding to the centre of the slice. These cubic samples were frozen in isopentane, cooled in liquid nitrogen, and stored at −80 °C until use. To carry out the histological analyses, several 10-µm-thick serial cross sections of fibres were prepared at −20 °C using a cryostat (Microm HM 560, Brignais, France). These histological sections were fixed on glass slides then air-dried at 20 °C. Sections were stained using haematoxylin-eosin-safron (HES) to evaluate the general structure of the muscle tissue, and with picro-sirius red to specifically analyze the endomysium and perimysium structures. To preserve the sections against negative post-stain change, the slides were mounted on a synthetic resin (Eukitt, Kindler GmbH & Co, Freiburg, Germany). Observations were made at 100× magnification on a transmission optical microscope coupled with a digital acquisition kit (Olympus BX61 microscope, Olympus DP 71 digital camera and Cell Sens software, Olympus France SAS, Rungis, France).

Further, to investigate ultrastructural modifications induced by tumbling in each tumbling condition, a 10 × 3 × 3 mm³ strip of muscle was immersed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 5.6 for several weeks. Small pieces (1 to 3 mm³) were cut from the strips and post-fixed in 1% osmium tetroxide in cacodylate buffer for 1 h at room temperature. The blocks were dehydrated though an increasing gradient of ethanol concentrations (70%, 95% and 100%) and embedded in epoxy resin (TAAB, Eurobio France). Semi-thin muscle sections of 1 µm thickness cut longitudinally to fibre direction (Reichert-Jung ultramicrotome, Unterschleissheim/Munich, Germany) were placed on a glass slide, stained with Toluidine Blue, and observed by light microscopy with the same Olympus BX 61 microscope as used for cryofixed samples. From the epoxy resin-embedded samples, ultra-thin sections of 90 nm thickness were cut longitudinally to fibre direction (Reichert-Jung ultramicrotome, Unterschleissheim/Munich, Germany), stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (Hitachi HM 7650, Tokyo, Japan) at 80 kV acceleration voltage. Micrographs were generated using a Hamamatsu AMT digital camera system (Hamamatsu, Japan) coupled to the microscope. Samples were prepared at the INRAE and observed at the Cellular Imaging Center for Health (CICS) laboratory (Université Clermont Auvergne, Clermont-Ferrand, France).