Osr6121

After overnight fasting, animals were anesthetized with an intraperitoneal injection of Zoletil 100 (75 mg/kg) and sacrificed. Blood was rapidly collected from the trunk into polypropylene tubes. Serum was isolated after 30 min of coagulation at room temperature and centrifuged at 2,000 × g for 15 min. Collected sera were stored at –70°C for later processing. Glucose concentrations in serum were measured by using a commercially available reagent (OSR6121, Beckman Coulter, Brea, CA, United States) on Olympus AU400 (BLOCK Scientific, North Bellport, NY, United States). The concentrations of serum free fatty acids (FFAs) were assessed by a commercially available kit (FA115, Randox Laboratories Ltd., Crumlin, United Kingdom) and serum triglycerides (TG) were analyzed with triglycerides reagent (Code 12528, Biosystem, Barcelona, Spain). Both measurements were performed on the semi-automatic biochemistry analyzer Rayto 1904-C (Rayto, Shenzhen, China).
After blood collection and transcardial perfusion, livers and retroperitoneal and perirenal depots of visceral adipose tissue (VAT) were carefully excised, weighed, and stored in liquid nitrogen until further analysis.

Immediately upon receipt of the blood samples from the field veterinarian, the blood samples were centrifuged at 3000 × g for 15 min at 4°C to facilitate harvesting the serum and plasma for insulin and glucose assays, respectively. A local veterinary diagnostic laboratory (IDEXX New Zealand, Palmerston North, New Zealand) measured plasma glucose concentration (OSR6121, Beckman-Coulter, Sydney, NSW, Australia) on an AU680 Chemistry Analyzer (Beckmancoulter, Sydney, NSW, Australia); whereas serum samples for insulin quantification were stored in -80°C until the end of the study and then shipped on dry ice to the University of Queensland (Gatton, QLD, Australia) for analysis.

On the day of the test, a nurse placed a catheter on the subject's arm, started the kinetic test for 120 min, and then took the catheter off. The subjects were instructed to eat the dairy dessert or pound cake within 5 to 10 min, under fasting conditions, with 150 ml of water at the clinical site. The kinetic test consisted of sampling venous blood at T-5 and T-1 min before the subject ate the meal, and then at T15, T30, T45, T60, T90 and T120 min after meal intake.
For the kinetic test, the blood samples were collected in sodium fluoride and potassium oxalate for glucose determination, and serum-separating tubes for insulin. The level of blood glucose was assessed by an enzymic UV test (hexokinase method) (AU480; Beckman Coulter) and commercially available glucose reagents (OSR6121; Beckman Coulter). The blood insulin level was assessed by an immunoradiometric assay (Cisbio Bioassays).
Each subject's compliance was checked by the study coordinator during the sessions (meal intake according to protocol and respect of the sampling time).