4.1. Dietary Intervention and Sample Collection

The present study was approved by the Animal Care and Use Committee of Auvergne (CEMEA Auvergne) and the Ministère de l’Enseignement Supérieur et de la Recherche (01276.01). C57bl/6J mice were bred for 3 generations and housed in the animal facility the INRAE research center of Theix (Saint-Genes Champanelle, France) with a growing diet (A03 diet from Safe diets, Augy, France) supplemented with 1% (w:w) of fish oil (Omegavie® EPA, 70 TG, Polaris, Quimper, France) containing 75% of n-3 PUFA (omega 3 lineage), mainly in the form of EPA which represented 8% of the fatty acids in the final diet or 1% (w:w) of high-oleic sunflower oil (control lineage) containing 83.5% of oleic acid (Lesieur, Coudekerque Branche, France) as a control condition. The supplementation of the control group with 1% of sunflower oil aimed at matched the caloric supplementation related to the addition of fish oil in the omega 3 lineage. The details of the breeding strategy and lipid composition of the diets were described previously [23] and are summarized in Figure 6. Eight F3 male mice from oleic/control (HFoleic) and EPA/omega 3 (HFepa) lineages were matched for body weight (n = 8 per group) and fed a high-fat, high-sucrose diet (HFD, 24% of fat, 20% of sucrose) providing 45% of energy from fat (RD 12,451 from Research diet, Brogaarden Gentofte, Denmark) for 17 weeks. These animals constituted the HFoleic and HFepa groups, respectively. A reference group of F3 male mice (n = 8) from the control lineage was fed with a low-fat reference diet (Ref) providing 10% of energy from fat (RD 12450H from Research diet) during the challenge as a reference group. Animals were maintained under a temperature-controlled environment and 12 h–12 h light-dark cycle throughout the study. All the procedures were followed to reduce the number and manipulation of the animals in the study.

Design of the dietary intervention to obtain F3 animals from an n-3 PUFA and control lineage and explore the effect of a high-fat diet (HFD) in each lineage. MUFA, monounsaturated fatty acids; SFA, saturated fatty acids; PUFA, polyunsaturated fatty acids. HFD, high-fat diet; LFD, control low fat diet.

At the end of the feeding period, during the fasting period, the animals were sacrificed under anesthesia with 4% isoflurane. Visceral adipose tissue (VAT) from the epididymal area was harvested, snap-frozen in liquid nitrogen, and stored at −80 °C until use. Total RNA was extracted using TRIzol^® reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Each total RNA sample was assessed for quantification and integrity using the Agilent Bioanalyzer (Agilent, Santa Clara, CA USA). Only RNA samples exhibiting a RIN index > 6.5 were used in the current study (Ref, n = 6; HFoleic, n = 6; HFepa, n = 8) for microarray experiments.