*2.3. Isometric Tension Measurements*

The soleus and EDL muscles were placed in a Petri dish covered with a transparent resin bottom (Sylgard, World Precision Instruments, Sarasota, FL. USA) where they were fixed with the help of entomological pins immersed in Krebs-Ringer solution (118 mM NaCl, 4.75 mM KCl, 1.18 mM MgSO4, 24.8 mM NaHCO3, 1.18 mM KH2PO4, 10 mM glucose, and 2.54 mM CaCl2) and carbogen gas (95% O2 and 5% CO2) was supplied. Excess connective and fatty tissue were removed under a stereoscopic microscope.

The muscle was mounted into a chamber for isometric tension measurements, with its proximal end attached to the bottom of the chamber and the distal end to the hook of an optical transducer, which was connected to an amplifier and this in turn to an analog–digital interface (World Precision Instruments, Sarasota, FL. USA) that allowed acquiring the tension generated by the muscle in a computer, using the MDAC software (World precision instruments, Sarasota, FL. USA). Two platinum electrodes were placed inside the recording chamber, which was connected to a stimulus isolation unit and an electric stimulator (Grass) in order to apply the protocol to induce fatigue, which consisted of 100 V pulses, 300 ms of duration, and a frequency of 45 Hz for soleus muscle and 50 Hz for EDL muscle. The stimulation was stopped once fatigue was presented. The fatigued muscles were stored at −80 ◦C to be subsequently homogenized for biochemical tests' performance (measurement of total reactive oxygen species, lipid peroxidation, and glutathione levels).

The muscle was stretched 1.3 times its resting length and left to perfuse in the physiological solution for 10 min before recording the isometric tension; the experiment was performed at a temperature of 25 ◦C.
