Micro osmometer 3300

Blood samples were analyzed for hematocrit (%) and hemoglobin concentration ([hemoglobin]; mg ml⁻¹). Hematocrit was determined as the fractional red cell volume after centrifugation of a sub-sample of blood in 80 μl heparinized microcapillary tubes at 10,000 rcf for 5 min in a hematocrit centrifuge (Haematokrit 210, Hettich, Tuttlingen, Germany). A handheld hemoglobin 201⁺ meter (Hemocue® AB, Ängelholm, Sweden) was used to determine hemoglobin concentration and the values were corrected for fish blood (Clark et al. 2008 ). Mean corpuscular hemoglobin concentration (MCHC, g dl⁻¹) was calculated according to Eq. 4. $$\text{MCHC =}\frac{\left[\text{hemoglobin}\right]}{\text{hematocrit}}\ast 10.$$
The whole blood was then centrifuged at 10,000 rcf for 5 min in a microcentrifuge (Eppendorf® 5415D, Sigma-Aldrich Sweden AB, Stockholm, Sweden), and the plasma was collected and frozen at − 18 °C for later analyses of plasma ion composition. The concentrations (i.e., [X]) of Na⁺, Cl⁻, K⁺ and Ca²⁺ were determined using an ISE comfort Electrolyte Analyzer (Convergent technologies, Cölbe, Germany) and plasma osmolality was determined with a freezing point osmometer (Micro osmometer 3300, Advanced instruments, Norwood, USA). All blood and plasma analyses were performed in duplicates and averaged.

Urine samples will be collected at the outset of each lab session to ensure participants are similarly hydrated for repeated body mass measurements. The samples will be analysed on the day for osmolality, via the freeze-point depression method (Micro Osmometer 3300, Advanced Instruments, MA, USA) and specific gravity (SUR-NE Clinical, Atago, Tokyo). Furthermore, during the 3-hour postprandial period which follows the breakfast meals, total urine output will be collected into containers prepared with 5 mL of a 10% thymol–isopropanol solution. At the end of the 3-hour period, the total volume of urine will be measured with a 1 mL sample extracted and frozen at −80 °C for subsequent analysis of urine urea concentration, as an indicator of protein excretion, to calculate the contribution of protein oxidation to whole body energy metabolism.