Clot activator tube

Samples were collected upon enrolment in the study for both groups. A standard venipuncture method was used. Blood was drawn into serum separator gel and clot activator tubes (Becton Dickinson, Vaud, Switzerland). Collected samples were then centrifuged at 4000× g for 10 min, in 10 to 30 min after collection. The obtained sera were stored into sterile centrifuge Eppendorf tubes and stored at −20 °C until the tests were performed.
Chemiluminescence on Immulite 2000 analyzer (Siemens Healthcare Diagnostics, Malvern, PA, USA) was used to determine T. gondii IgG antibodies. All tests were performed in accordance with the manufacturer’s protocol (including quality controls). Results were interpreted based on the manufacturer’s criteria. A value <6.5 IU/mL was considered negative; ≥6.5 IU/mL to 7.99 IU/mL was considered equivocal; ≥8 IU/mL, positive. For the purpose of this study, equivocal results were considered negative.

CVCs were flushed with saline from a 10 mL PosiFlush saline syringe (BD Biosciences, San Jose, CA, USA) prior to blood draw. The daily collection of blood samples from each CVC occurred between DPE-3 and DPE 10 with a 2.5 mL volume collected with a 5 mL syringe distributed into 1 mL sodium citrate MiniCollect tubes (Greiner Bio-One, Monroe, NC, USA), 1 mL clot activator tubes (BD Biosciences), and 0.5 mL K2EDTA MAP tubes. Saline was used to again flush the CVC line, which was then maintained by flushing with PosiFlush pre-filled heparin lock syringes (BD Biosciences). These syringes were left attached to the CVC cage-side port until the next sample collection. Animals were anesthetized with ketamine HCl at 10 mg/kg (VedCo, Saint Joseph, MO, USA) or a combination of tiletamine HCl and zolazepam HCl at 3 mg/kg (Fort Dodge Animal Health, New York, NY, USA) on days where sampling through the CVC was complicated due to kinked lines. When animals were anesthetized, blood was drawn via the saphenous vein.

Whole blood samples (8–10 mL) were obtained from anesthetized group mares. Following skin disinfection, samples were collected by jugular venipuncture into clot activator tubes (BD Vacutainer Systems, Plymouth, UK) and centrifuged for 15 min at 1500× g for serum separation. Serum samples were stored in 2 mL tubes (Eppendorf, São Paulo, Brazil) at −3 °C until use. Samples were collected at different time points. The first sample was collected before the anesthetic procedure and used to determine basal gastrin concentrations. The second sample was collected in the recovery room following 90 min of general anesthesia after the mares had been placed in lateral recumbency. The last sample was collected 4 months after anesthesia, 90 min after morning feeding, and without prior fasting. Serum gastrin concentrations were measured using a commercial double-antibody radioimmunoassay (RIA) designed for human use, at an external commercial laboratory (Diagnostics Clinical Analysis, DAC, Pirassununga, SP, Brazil), using a previously validated method of measuring gastrin in equine serum [15 (link)]. The test was performed to commercial standards and internal calibration.