2.5. Aorta Oil Red O staining and serum lipids

Mixed arterial and venous blood was collected at the time of necropsy in serum separator tubes (BD Vacutainer, BD). A minimum of 1 ml blood was obtained from each rat for lipid and liver enzyme analysis. Blood was allowed to clot at room temperature for 30 minutes before centrifugal serum separation. Aliquots of serum were frozen at ‐80°C immediately after centrifugal serum separation, kept frozen until analysis, and analyzed within 1 week of collection. Standard serum lipid panels, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were analyzed at a clinical laboratory (ARUP laboratories). Serum was analyzed from 6–14 offspring per sex per group.

Abdominal adipose tissue was dissected from the abdominal aorta. The abdominal aorta was stained en face for 7 min with Oil Red O (Sigma‐Aldrich) and rinsed twice for 7 min each time in isopropanol (Sigma‐Aldrich). A ruler was placed next to the aorta and photographs were taken. The pictures of the stained aortas were analyzed with Bioquant True Color Windows Image Analysis System (R&M Biometrics) for the number of areas stained with Oil Red O and for the percent of the abdominal aorta stained with Oil Red O. Aortas were analyzed from 7–10 offspring per sex per group. Analysis of stained aortas was performed by two people who were blinded to the rat sex and group.