Rmh 3000

All CF-1 and FVB mice in this study were maintained at 20–26 °C, with dark/light cycles of 12 h, and fed with pellet (Prolab RMH-3000, LabDiet) and water ad libitum. Mice were sacrificed by an overdose of inhalatory isoflurane anesthesia. Experimental procedures were approved by the Bioethics Committee at Universidad de Concepcion, Chile and followed the norms imposed by the Bioethics Committee of the National Research and Development Agency, Chile (ANID), as well as the guidelines of the European Council Directive for the Care of Laboratory Animals.

Mechanically air-dried, whole plants of a proprietary strain of Chondrus crispus, cultivated intensively on-land, were obtained from Acadian Seaplants Limited (Dartmouth, Nova Scotia, Canada). The seaweed samples were finely ground and passed through a 60-mesh sieve (0.25 mm in diameter). The fructo-oligo-saccharide (FOS) powder, extracted from chicory roots, was provided by Cargill (Wayzata, MN), as Oliggo – Fiber™ DS2 inulin, with an average degree of polymerization less than 10. C. crispus and FOS were mixed with a standard basal feed (RMH 3000, LabDiet, St. Louis, MO, USA), respectively, at the ratio of 0.0 (plus 2.5 % corn starch), 0.5 (plus 2.0 % corn starch) or 2.5 % (dry w/w). The mixed feed was then pelleted (4.7 mm in diameter, 1.0-1.5 cm in length) using a feed mill facility located at the Faculty of Agriculture, Dalhousie University, Truro, Nova Scotia. Diets were prepared just before the trial and stored under dry and cool conditions.

All mouse experiments were conducted in compliance with procedures reviewed and approved by the Case Western Reserve University Institutional Animal Care and Use Committee. Studies utilized female mice that were on a C57BL/6J genetic background. Stra6^-/- and Isx^-/- mice were generated as previously described (16 (link), 22 (link)). Isx^-/-/Stra6^-/-DKO mice were generated in the vivaria at Case Western Reserve University. WT mice were obtained from the Jackson Laboratory. Mice were bred and raised on a standard chow diet containing ∼15,000 IU vitamin A/kg diet (Prolab RMH 3000, LabDiet, St. Louis, MO) in a room on a 12:12 h light/dark cycle with ad libitum access to food and water. After weaning, mice (n=4–5) were maintained on a diet supplemented with vitamin A (4,000 IU vitamin A, retinyl acetate) or BC (25 mg/kg) for a period of 8 weeks. Specific diets were prepared by Research Diets (New Brunswick, NJ). At the end of the dietary intervention, mice were anesthetized by using a cocktail of ketamine (20 mg/ml) and xylazine (1.7 mg/ml). Blood samples were collected via cardiac puncture. Mice were transcardially perfused with 20 ml of PBS and sacrificed by cervical dislocation. Tissues were immediately harvested for analysis or snap frozen in liquid nitrogen and stored at ˗80°C until further use.