Supplementation in trained rats Motriz, Rio Claro, v.19 n.4, p.709-716, Oct./Dec. 2013 711 The rats in the trained groups performed exercise on a treadmill at an intensity equivalent to the maximal lactate steady state (MLSS). To determine the MLSS, an exercise series of 25 minutes racing with rectangular intensity was performed on a treadmill at different speeds (15, 20, 25 and 30 m/min velocities), with 48 hours between each series. Blood samples from a small cut at the end of the tail (25 µL) were collected every five minute to measure the lactate levels. A single incision made before beginning the exercise series was sufficient to collect all samples. The blood lactate concentration corresponding to the MLSS was taken at the highest speed where there was no variation of blood lactate greater than 1.0 mmol/L between 10 and 25 min of exercise (Manchado et al., 2005; Araújo et al., 2011). The blood lactate concentration was determined by an enzymatic method (Hill et al., 1924). After the MLSS was determined, the animals ran on a treadmill at their MLSS intensities for 40 minutes/day for five days/week. Oral Glucose Tolerance Test - OGTT At 150 days old, an OGTT was performed on the animals after 12 hours of fasting. An initial blood sample was taken through a small cut at the end of the tail of each animal. Then, a glucose solution (80%) was administered to each animal through a polyethylene gastric catheter at a dose of 2 g/kg body weight. Blood samples were collected after 30, 60 and 120 min with heparinized capillaries (25 µL) to determine the glucose concentration. A single incision was sufficient to collect all samples. The blood glucose concentrations were determined by the glucose oxidase method (Nogueira et al., 1990) and insulin was determined using a radioimmunoassay (Hebert et al., 1965). The results were analyzed by calculating the area under the glucose curve during the test using the trapezoidal method (Mathews et al., 1990) and the software ORIGIN 6.0 (2000). Indicators of muscular glycidic metabolism: oxidation and glucose uptake, glycogen synthesis and lactate production by the soleus muscle At the end of the experiment, when the rats were fed and rested, they were exsanguinated after being anesthetized with carbon dioxide. At the time of euthanasia, the right soleus muscle was excised to measure the indicators of muscular glycidic metabolism (oxidation and glucose uptake, synthesis and concentration of glycogen and production of lactate). The right soleus muscle was isolated with minimal injury, and longitudinal slices weighing between 25 and 35 mg were placed in 20 ml siliconized scintillation vials containing 1.5 ml of Krebs-Ringer bicarbonate buffer. The vials were closed with rubber covers, sealed with plastic rings and subjected to 30 minutes of preincubation while stirred in a Dubnoff bath at 60 rpm and continuously gassed with O2 /CO2 (95%/5%). After the preincubation period, the Components AIN – 93M* (g_kg–1) Addition of 2% creatine** (g_kg–1) Addition of13% creatine*** (g_kg–1) Creatine 0.0 20.0 130.0 Cornstarch 465.7 444.7 335.7 Casein (85% protein) 140.0 140.0 140.0 Dextrin 155.0 155.0 155.0 Sucrose 100.0 100.0 100.0 Soybean Oil 40.0 40.0 40.0 Fiber 50.0 50.0 50.0 Mineral mix 35.0 35.0 35.0 Vitamin mix 10.0 10.0 10.0 L-cystine 1.8 1.8 1.8 Choline bitartrate 2.5 2.5 2.5 Kcal/Kg 3.802,77 3.802,77 3.802,77 * American Institute of Nutrition (AIN-93M) (Reeves et al., 1993). ** Creatine maintenance diet according to Demenice et al. (2009) *** Creatine loading diet adapted from Demenice et al. (2009) and according to Hultman et al. (1996) and Vandenberghe et al. (1997)
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