2.3. Cryotherapy

The cryochamber (type Wroclaw, Zagrobelny-Raczkowski-Strank, Wrocław, Poland) was divided into two compartments held at different temperatures (− 60°C and − 90°C) measured before cryostimulation and at the level of animals. The animals were weighed daily before WBC. The air in both chambers contained 22% oxygen and 78% nitrogen. The walls of the chamber were lined with multilayer thermal insulation for use at low temperatures. The cryochamber was equipped with three entrances. The doors had a window, which allowed for the visual inspection of the procedure. The humidity and temperature were monitored by an operating system. The rats were exposed to temperatures − 60 and − 90°C using the two-stage cryogenic chamber every day at the same time between 9 am and 10 am for 5 and 10 consecutive days (Figure 1). During exposition to very low temperature in cryochamber rats were kept in wooden cages placed on the floor. There were the following study groups: − 60/10, − 60/5, − 90/10, and − 90/5, corresponding to the temperature (− 60°C and − 90°C) and number of 1 min sessions applied (5 or 10). The eating behavior and physical activity were controlled daily. There were no signs of complications after cryotherapy sessions in all subjected animals.