The animals were pair-fed (15 to 20 g/day) AIN-93 M powdered diet, as recommended [30], and received distilled water ad libitum. The principles of laboratory animal care (NIH publication No. 86-23, revised 1985) were followed, as well as the specific selleck chemicals llc national laws (n° 9.605/1998). All procedures were approved by the Ethics Committee of the

Federal University of Viçosa, Brazil. Creatine and caffeine supplementation Every day, the animals from the groups SCr and ECr were supplemented with creatine, while those from the groups SCrCaf and ECrCaf received creatine plus caffeine. Creatine was given using a two-stage procedure: loading and maintenance. During the loading stage (7 days), in the first week, a dosage of 0.430 g of powdered creatine monohydrate (Sigma) per kg of body weight per day was added to 15 g of the diet (AIN-93 M powdered diet) and given to the groups SCr, ECr, SCrCaf and ECrCaf. The maintenance stage lasted 5 weeks, starting from the second week, and a dosage of 0.143 g of creatine/kg body weight/day was added to 15 g of the diet and given to the groups SCr, SCrCaf, ECr and ECrCaf. Everolimus price From the second to the sixth week, a dosage of 10 mg of powdered

caffeine (Sigma) per kg body weight per day was given to animals from the groups SCaf, SCrCaf, ECaf and ECrCaf. Animals from the groups SPl and EPl received diet only. From the fourth week on, all animals received 20 g of the diet every day. Exercise training protocol During the first week, the

animals from the groups EPl, ECr, ECaf and ECrCaf swam for 30 min/day Pregnenolone in a tank (60 cm wide, 75 cm long, 80 cm deep) filled with water at 32 ± 1°C to adapt to the environment. The exercise training regime comprised vertical jumps from the bottom of the tank to the surface water. To augment the exercise intensity, an external load (% of body weight) was added to the animal by using plumber spheres in a lycra vest. The deepness of water was determined by an average percentage of the animals’ length (i.e. distance between the end of the posterior members and the nostril) (Table 1). The training program was conducted from the second to the sixth week of the experiment and the animals performed 4 sets of 10 jumps with 1 minute recovery time between sets, 5 days/week (Table 1). This exercise training regime and the working apparatus are currently used in our laboratory and elsewhere [31]. During the last training session, concentrations of blood lactate of the exercised animals were monitored in three moments.