Haemacolor

An aliquot of gently homogenized blood was used to perform total white blood cells (WBC) counts. The remaining blood was centrifuged at 10,000 × g for 10 min at 4°C and plasma stored at −80°C until assayed. Blood smears were air dried and stained with Wright’s stain (Haemacolor; Merck) after fixation with formol-ethanol (10% of 37% formaldehyde in absolute ethanol). The slides were examined (1,000×). At least 200 leukocytes were counted per smear and classified as thrombocytes, lymphocytes, monocytes, and neutrophils. Detection of peroxidase activity was carried out as described by Afonso et al. (24 (link)) in order to facilitate identification of neutrophils. The relative percentage and absolute value (×10⁴ ml⁻¹) of each cell type was subsequently calculated.

The haematological profile was assessed according to Machado et al. [28 (link)] by analysing the haematocrit, haemoglobin content (SPINREACT, Girona, Spain), and total peripheral white blood cell (WBC) and red blood cell (RBC) counts. Mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were calculated based on the haematocrit and haemoglobin values. Blood smears were produced from fresh blood samples. Smears were firstly fixed with formol–ethanol (10% of 37% formaldehyde in absolute ethanol) and afterward stained with Wright’s stain (Haemacolor; Merck, Rahway, NJ, USA). Neutrophils were identified according to their peroxidase activity, which was detected using the method described by Afonso et al. [29 (link)]. The slides were examined under oil immersion (×1000), and at least 200 leucocytes were counted and classified as thrombocytes, lymphocytes, monocytes and neutrophils. Each cell type’s absolute concentration and relative proportion were subsequently calculated.

Blood parameters were accessed according to Peres et al.[32] . Briefly, total white (WBC) and red (RBC) blood cell count was made from blood dilutions using a hemocytometer. Fresh heparinized blood was used for haematocrit (HT) and its value was determined by the micro centrifugation (10,000 x g for 10 min, at room temperature) of haematocrit tubes. Drabkin´s solution was used for haemoglobin determination (HB; SPINREACT kit, ref. 1,001,230, Spain). The mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), and mean corpuscular haemoglobin (MCHC) were calculated as follows:•

MCH (pg cell⁻¹) = (HB/RBC) x 10

•

MCV (µm³) = (HT/RBC) x 10

•

MCHC (g 100 mL⁻¹) = (HB/HT) x 100

Immediately after blood collection, blood smears were made from fresh blood, air-dried, fixed with formol-ethanol (3.7% formaldehyde in absolute ethanol), and stained with Wright's solution (Haemacolor; Merck). Neutrophils were detected by their peroxidase activity as described by Afonso et al.[33] (link). The slides were carefully analyzed under oil immersion (1,000x), and at least 200 leucocytes were counted and classified as thrombocytes, lymphocytes, monocytes, and neutrophils. The absolute value (x 10⁴ µL⁻¹) of each cell type was calculated.

Blood smears were made immediately after blood collection, air-dried, fixated with formol–ethanol (3.7% formaldehyde in absolute ethanol), and stained with Wright’s stain (Haemacolor; Merck). Then, the glass slides were examined (1000×) under oil immersion, and at least 200 leucocytes were counted and classified as lymphocytes, thrombocytes, monocytes, and neutrophils. Neutrophil identification was performed by the detection of peroxidase activity according to Afonso et al. [40 (link)]. The absolute value (×10⁴ μL⁻¹) of each cell type was subsequently calculated.
Blood parameters were assessed according to Peres et al. [41 (link)]. Total red (RBC) and white (WBC) blood cell counts were performed using a hemocytometer. Fresh heparinized blood was spun for haematocrit (Ht) using microhematocrit tubes (10,000× g for 10 min at room temperature). Drabkin’s solution was used for haemoglobin determination (HB; SPINREACT kit, ref. 1001230, Girona, Spain). The mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC) were calculated as follows:

The hematological profile was verified, and the hematocrit percentage (HT), total red blood cell (RBC) counts, and total and differential white blood cell (WBC) counts were performed according to Machado et al. [52 (link)]. The hemoglobin (HG) content (Hemoglobin—Drabkin—colorimetric, Spinreact) was quantified. The mean corpuscular volume (MCV), the mean corpuscular hemoglobin (MCH), and the mean corpuscular hemoglobin concentration (MCHC) were calculated as described by Machado et al. [52 (link)]. From the homogenized blood, blood smears were prepared following Machado et al.’s [52 (link)] protocol, and the identification of neutrophiles, through the presence of peroxidase activity, was carried out according to Afonso et al. [53 (link)]. Additionally, the blood smears were stained with Wright’s stain (Haemacolor, Merck) to perform differential WBC counts, as described by Machado et al. [52 (link)].

Blood was collected from the caudal vein using heparinized syringes. The hematological profile consisted of total white (WBC) and red (RBC) blood cells counts and blood smears for differential WBC counting as described by Machado, et al. [36 (link)].
Blood smears were initially fixed with formol-ethanol (formaldehyde at 4%) and afterward stained with Wright’s stain (Haemacolor; Merck, Darmstadt, Germany). Neutrophils were identified according to their peroxidase activity, which was detected using the method described by Afonso, et al. [37 (link)]. The slides were examined under oil immersion (1000×) and at least 200 leucocytes per slide were counted and classified as thrombocytes, lymphocytes, monocytes, and neutrophils. The relative proportion of each cell type was subsequently calculated.

Vaginal smear samples were stained with Haemacolor^® (Merck KGaA, Darmstadt, Germany) stain, and based on the percentage of the cornified superficial cells, the estrus cycle stages were evaluated [44 (link)].