Haematokrit 210

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.

For the determination of the hematocrit heparinized blood was collected in a micro-hematocrit tube and centrifuged at 12,000 × g by a micro-hematocrit centrifuge (Haematokrit 210; Hettich Lab Technology, Tuttlingen, Germany).
According to Pendl (18 (link)) blood smears of EDTA blood were prepared (two per bird), stained with Pappenheim solution, and counted. Leucocytes and thrombocytes were counted in 20 fields of vision (at 1,000 × magnification) and at least 200 leucocytes were differentiated according to their morphological characteristics.
Blood gas traits (pCO₂; pO₂; TCO₂; BE_ecf; ${\text{HCO}}_3^{-}$ ), blood pH, blood electrolytes (sodium, potassium, chloride, ionized calcium), and the metabolites glucose and lactate were determined by the analyzer GEM® Premier 4,000 (Instrumentation Laboratory, Munich, Germany) in heparinized blood. Further metabolites and liver enzymes were analyzed by an automatic clinical chemistry analyzer in blood serum (Eurolyser CCA180, Eurolab, Austria).

The following variables were analyzed with both analyzers: impedance and optical RBC counts (RBC‐I, RBC‐O), hematocrit (HCT), hemoglobin concentration (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width standard deviation and coefficient of variation (RDW‐SD and RDW‐CV, respectively), impedance, optical and fluorescence platelet counts (PLT‐I, PLT‐O, PLT‐F, respectively), mean platelet volume (MPV), plateletcrit (PCT), platelet distribution width (PDW), platelet large cell ratio (P‐LCR), immature platelet fraction (IPF), reticulocyte count and percentage (RET), and low‐, medium‐, and high‐reticulocyte fluorescence ratio (LFR, MFR, HFR), immature reticulocyte fraction (IRF), reticulocyte hemoglobin equivalent (RET‐He), nucleated red blood cell count (NRBC), mature RBC hemoglobin equivalent (RBC‐He), WBC, counted on the white cell nucleated (WNR) or WBC differential (WDF) channel, and WBC differential count (DIFF). Manual 200‐cell WBC DIFF and NRBC counts (expressed as the number of NRBCs per 100 leukocytes) were performed by the first author (E200 microscope, Nikon, Kobe, Japan) and packed cell volumes (PCVs) were measured by the centrifugation of blood in microhematocrit tubes (Haematokrit 210, Hettich).