*2.2. Sample Preparation*

As part of the health examination while under general anesthesia, blood samples were collected from the jugular, cephalic or saphenous vein of each animal. Blood was collected in plain tubes (Sistema BD Vacutainer ®, Becton Dickinson Italia SPA, Italy) and serum was obtained by centrifugation at 10 min at 2500× *g*. Written owner consent for use of surplus blood samples, and use of data for scientific purposes, was obtained during consultations. The study design was approved by University of Milan Animal Welfare Bioethical Committee (Approval number OPBA 31/2019).

#### *2.3. Total Serum Protein*

Total serum protein concentration was measured by spectrophotometry using the colorimetric biuret method (Hagen Diagnostica S.R.L., Via Pratese 13 Firenze) on a Cobas Mira Classics Roche automated chemistry analyzer (Roche S.p.A., Mannheim, Germany).

#### *2.4. Agarose Gel Electrophoresis*

Sera samples were refrigerated at 4 ◦C and were analyzed within 8 hours of sampling. Protein fractions were analyzed using a semiautomated agarose gel electrophoresis (AGE) system (with HYDRAGEL Kit β1-β2 (SEBIA, Issy-les-Moulineaux, France). Serum was electrophoresed for 7 minutes at 33 volts hours and stained with diluted Amidoschwarz dye at pH 2 (4 g/<sup>L</sup> Amidoschwarz dye and 6.7% ethylene glycol). The AGE procedure was conducted according to the manufacturer's instructions, and commercial human serum was used as the control (normal control serum, Sebia, Evry, France). Using the computer software Phoresis for Windows 2000 or XP Pro (Sebia), the electrophoretic curve for each sample was displayed. Protein fractions were determined as the percentage optical absorbance, and the absolute concentration in g/dL was automatically calculated from the total serum protein concentration. Albumin to globulin (A/G) ratios were also calculated. The same operator analyzed all samples. To establish the inter-assay-accuracy of agarose gel electrophoresis on tiger serum, sera from two healthy tigers, one Bengal Tiger, 9A, F and one Siberian Tiger 1,1A, F, were tested 3 times on the same day, in the same laboratory and interpreted in duplicate by two operators. Coe fficient of variability (CV) computed as SD/mean × 100 was calculated for each protein fraction.
