Zebrafish Calorie Restriction Protocol

Adult zebrafish (AB line, ZIRC, Eugene, OR, USA) were kept at 28°C under a 14-h light:10-h dark cycle, and water conditions of environmental quality were maintained according to an established protocol [68 ]. Zebrafish at 3.5 mpf were assigned into two dietary groups (overfeeding and maintenance groups) with approximately five fish per 2-L tank. Zebrafish in the overfeeding group were fed three times per day with freshly hatched Artemia (corresponding to 60 mg cysts/fish/day). Zebrafish in the maintenance group were fed freshly hatched Artemia (corresponding to 5 mg cysts/fish/day) once per day. For calorie restriction, the zebrafish were fed freshly hatched Artemia (corresponding to 2.5 mg cysts/fish/day) for 2 weeks after being overfed for 8 weeks. Commercial flake food (Hikari Tropical Fancy Guppy, Kyorin, Hyogo, Japan) consists of 9% fat, 17% carbohydrate and 59% protein, whereas Artemia nauplii consist of 22% fat, 16% carbohydrate and 44% protein (dry weight basis).
The percentage of consumed Artemia was estimated by counting Artemia before and after feeding. Briefly, 1 mL of water was collected from 1700 mL of water containing freshly hatched Artemia corresponding to 100 or 8.5 mg cysts. The numbers of hatched Artemia were counted three times to determine a mean count. After counting, the samples were returned to the 2-L tank. Then, five fish were transferred to the tank for feeding. After 2 hours, the number of Artemia was counted as before feeding. Zebrafish fed 5 or 60 mg of Artemia per day consumed about 80 or 50% of the provided Artemia, respectively. Zebrafish fed 2.5 mg of Artemia per day consumed almost all of the provided Artemia.