Kmc006

Overnight fasting blood samples were collected from 6 randomly selected hens in each treatment group, consisting of hens at 58 and 65 weeks of age from a wing vein. On those same days, a second blood sample was collected 30 min after re-feeding 20 g of feed. Commercial kits were used to determine plasma triacylglycerol (TG, Sigma-Aldrich, St. Louis, MO, USA), glucose and NEFA (non-esterified fatty acid, Wako Chemicals, Osaka, Japan) levels. Plasma insulin and interleukin-1β (IL-1β) concentrations were determined by a validated commercial ELISA kit (Cat.# 10-1249-01, Uppsala, Sweden, KMC006; Biosource International, Camarillo, CA, USA, respectively) [28 (link),29 (link)].

Plasma IL-6 and IL-1β levels were determined by validated commercial ELISA kits (Cat.# KMC0011, and KMC006; Biosource International, Camarillo, CA, USA, respectively) [44 (link)]. Paraffin embedded left ventricle sections were stained with (hematoxylin and eosin, H&E) to examine leukocyte infiltration or by trichrome Masson to visualize tissue fibrosis as described previously [1 (link),2 (link)]. Macrophage infiltration was further visualized by antigen retrieval using an avian-specific mouse monoclonal antibody conjugated with FITC (Clone KUL01, Abcam, Cambridge, UK) [45 (link)]. Three sections per hen and 5 images from each section were used for intensity quantification using Image-J software (NIH, Bethesda, MD, USA).