Ethylene diamine tetraacetic acid (edta)

Blood samples for haematological analyses were collected in 3.0 ml tubes containing EDTA (Terumo, Leuven, Belgium) and analysed within 90 minutes of sampling. Haematology parameters were measured employing a Sysmex XE-2100 haematology analyser (Sysmex, Kobe, Japan).

Plasma and serum were prepared using EDTA and T-MG tubes, respectively [Terumo Medical Corporation, Elkton, MD]. EDTA tubes used to prepare plasma for hormone analysis contained 50 μM DPP IV inhibitor [Millipore, St. Charles, MO] and a protease inhibitor cocktail [Sigma-Aldrich, St. Louis, MO]. Clinical chemistry analysis was performed by standard techniques using an Olympus AU640 Clinical Chemistry analyzer (Olympus America Inc., Melville, NY). Insulin, amylin, leptin, ghrelin, GIP and PYY were analyzed using Milliplex MAP Mouse Gut Hormone Panel and MSD assays were used for measurement of aGLP-1 and tGLP-1 (Meso Scale Discovery, Gaithersburg, MD). Glycated hemoglobin (HbA1c) in blood hemolysates was analyzed with a Primus ultra2 liquid affinity chromatograph (Trinity Biotech, Jamestown, NY).

Samples were obtained from 189 camels from three herds in Wd-Alhlio, Alshagrab and Khor Wd-Omer, which are located around El-Showak in Kassala state (n = 148), and from Tumbool market (at a camel slaughterhouse) in El-Gazira state (n = 41) where camels are brought from western Sudan, across the natural barrier of the River Nile (see map in Fig. 1). After obtaining the consent of the camels’ owners, 3 ml of blood was drawn from the jugular vein into vacutainer tubes with EDTA (Terumo, Tokyo, Japan). The samples were labelled with a unique code and were placed in a cool box at 4 °C until they were transported to the laboratory.Fig. 1

A map of Sudan: The locations of the sampling areas from different herds are shown with black stars. The black dot indicates Tumbool slaughterhouse. Source: http://www.d-maps.com/carte.php?num_car=1310&lang=en. With some modifications

All of the blood samples were collected before surgery, or before newly scheduled chemotherapy. Three mL of peripheral venous blood was collected and transferred to a 5 mL blood collection tube containing EDTA (TERUMO, Tokyo, Japan). In a Tosoh enrichment tube (Figure 1a), 3.5 mL Ficoll (GE Healthcare, Chicago, IL, USA), 3 mL blood sample, 3 mL fixative solution, and 180 μL Tirofiban were added and mixed well. After standing for 15 min at room temperature (RT), 75 μL of RosetteSep reagent Human CD45 Depletion Cocktail (STEMCELL technologies, Vancouver, BC, Canada) was added to deplete the white blood cells and the red blood cells. After centrifugation (2000× g, 10 min, 24 °C), the concentrated mononuclear layer was collected, mixed with a lysing solution, then centrifuged at 300× g for 10 min at 24 °C. After removing the supernatant, the pellet was resuspended in 30 mL of polyethylene glycol bovine serum albumin (PEG-BSA) and centrifuged at 300× g for 5 min at 24 °C. This procedure was repeated three times. After removing the supernatant, the final pellet was suspended in PEG-BSA.

Venous blood samples were drawn from the cubital vein of a seated subject after an 8-h overnight fast. Five mL of blood samples were collected into tubes that contained ethylenediaminetetraacetic acid (EDTA; Terumo, Tokyo, Japan). The tubes were put on ice immediately and kept there for a period of about 15 min. Next, the tubes were incubated at 4 °C by centrifugation at 3000 rpm for 15 min. This was followed by separation of the plasma into tubes, which were stored at −80 °C for a period of 2 weeks to 2 months until the desired analysis for amino acids was performed.

Sixty Holstein–Frisian dairy cows were enrolled in early post-partum (26.5 ± 1.5 days in milk). The total mixed ration (TMR) and the chemical composition of lactation diet was previously reported in the study of Fiore et al. [22 (link)]. Blood samples, analyzed for this research, were the same collected in the study of Fiore et al., taken from the high producing dairy farm located in Padua, Italy (45°36′ N. 11°40′ E. 23 m above sea-level) [18 (link),22 (link),23 (link)].
Blood samples were collected from the coccygeal vein using a vacutainer system. The BHB was measured using the Nova Biomedical Express digital reader (Nova Biomedical, Runcorn, UK) with specific BHB test strips (Stat Strip Ket, Nova Biomedical, Runcorn, United Kingdom), before taking the blood sample.
The purpose of the measurement of ketone bodies in the farm was to select dairy cows with a concentration of BHB greater than 1.0 mmol/L. The quantity of ketone bodies above this numerical value is often present in asymptomatic animals, but it is an excellent indicator of the risk of developing metabolic disorder in the postpartum [13 (link)].
The blood samples were collected for each bovine enrolled. Two samples were collected in vacuum tubes containing EDTA (5 mL; Terumo Venoject, Leuven, Belgium) and one in Venosafe tubes containing Clot Activator (9 mL; Terumo Venosafe, Leuvel, Belgium).

Blood samples for haematological analyses, including haemoglobin, leukocyte count and platelet count, were collected in 3.0 mL tubes containing EDTA (Terumo, Leuven, Belgium) and analysed within 90 minutes of sampling. Haematological parameters were measured with the Sysmex XE-2100 haematology analyser (Sysmex, Kobe, Japan).