Packed cell volume (PCV; L/L) and total plasma protein (TPP; g/L) concentration were manually determined on the day of sampling, while automated hematological analysis (Sysmex XT-2000iV, Sysmex, Kobe, Japan) of preserved blood specimens (Streck Cell Preservative; Streck, Omaha, USA) was performed 2–8 days later (21 (link)). Automated analysis provided indicators of red blood cell number additional to PCV (red blood cell [RBC; ×10^12/L] count and hemoglobin [Hb; g/L] concentration), as well as platelet (PLT; ×10^9/L) and total white blood cell (WBC; ×10^9/L) counts. Duplicate Wrights-Giemsa-stained blood smears prepared the day of sampling were later reviewed for manual differential WBC and nucleated RBC (nRBC) counts. Reticulocyte percentage was determined from new methylene blue-stained blood smear counts (Reticulocyte stain, Sigma-Aldrich, Germany) using a Miller Square (9:1 ratio) eyepiece reticule to count the equivalent of >1,000 RBC. In addition to nRBC, the absolute reticulocyte count for each pup was subsequently calculated (reticulocyte percentage x RBC count) as a measure of the intensity of RBC regeneration.
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