Introduction Materials and methods

1. Introduction

In Japan, red sea bream is one of the most popular food fish and a large culture industry has grown to meet the demand. However, this species can build up remarkable Ž . body fat depots Oku and Ogata, 2000 , resulting in higher lipid content of cultured red Ž . sea bream flesh compared to wild fish Morishita et al., 1988; Osato et al., 1991 . Ž . Ž Indeed, muscle lipid contents in cultured red sea bream 1.2–1.5 kg and wild fish 1.3 . Ž . kg were 4.7–9.0 and 1.3, respectively Morishita et al., 1988 . This has been associated with lower consumer satisfaction. The preferred lipid content depends on Ž local customs, yet a high level of fat leads to poor appearance and taste Fauconneau et . al., 1995 . The extent of marbling, intramuscular fat deposition in beef cattle has been related to Ž . blood retinol level Torii et al., 1996 . Retinol action on muscular fattening appears to be mediated by retinoic acid, which has an antiadipogenic action on preadipocytes present Ž . in muscle tissue Keller et al., 1993; Kamei et al., 1994 . A recent trend in the beef cattle industry in Japan has been for farmers to feed vitamin A-deficient diets to produce Ž richly marbled beef, which is valued as a high-quality product in Japan Takeyama et al., . 1996a,b . There is little information on the effects of retinoids on body lipid deposition in fish. Therefore, the present study was conducted to investigate the effects of dietary retinoids, especially retinoic acid, on body fat deposition of juvenile red sea bream. A 6-week feeding trial was carried out with diets containing different retinoid sources to examine the effects of these compounds on tissue and plasma lipid contents. Subse- quently, to confirm that dietary retinoic acid was internally absorbed, plasma retinoic acid levels were determined at 6-h post-feeding.

2. Materials and methods

Ž . Juvenile red sea bream Pagrus major were obtained from a private hatchery Ž . Nisshin Marine Tech, Aichi, Japan and transported to the indoor facilities of the National Research Institute of Aquaculture at Nansei, Mie. For 2 months prior to the start of the experiment, the fish were held in indoor tanks and fed commercial dry feeds Ž . C-series, Kyowa Hakko, Tokyo, Japan under a natural photoperiod. Fish were selected to be of uniform size and randomly assigned to eight tanks, 20 fish per tank. The fish were acclimated to the tank conditions for 1 week prior to the commencement of the feeding trial. At the beginning of the experiment, mean body weight of the fish was 8.6 0.0 g. A sample of 16 fish was taken from the common pool at the start of the Ž . experiment and frozen y808C for later proximate analysis. Each treatment was assigned to duplicate groups in a completely random design, and the treatments consisted of four diets. The control diet was a commercial dry feed Ž . C-series, Kyowa Hakko, Tokyo, Japan and retinoid diets were prepared by supplement- Ž 6 . ing the control diet with either retinyl acetate 1.42–1.80 = 10 IUrg or crystalline Ž . Ž . Ž all-trans-retinoic acid retinoic acid Wako, Osaka, Japan . Retinyl acetate 100 mgrkg . Ž . diet or retinoic acid 10 or 100 mgrkg diet was dissolved in 60 ml acetonerkg diet and top dressed on the control diet to prepare the respective retinoid diets. An equivalent amount of the carrier acetone was added to the control diet. All four diets were dried at 508C under vacuum in a rotary evaporator to remove acetone and then stored at y208C. Crude protein and crude fat contents of the test diets were 61.7 and 13.9 on a dry Ž basis, respectively. The control diet contained 5.8 mg retinyl palmitaterkg diet 10,500 . IUrkg diet as a basal level of the dietary retinoid. The fish were hand-fed to apparent satiation twice a day, 6 daysrweek for 6 weeks. Ž 3 . The tanks used in this study were square polyvinylchloride tanks 60 = 25 = 35 cm with a 52.5 l capacity, filled to 40 l. Each tank was supplied with seawater drawn from Gokasho Bay, Mie at the rate of 2 lrtankrmin. The water temperature ranged from 19.38C to 23.58C during the feeding trial. The fish were weighed individually after being anesthetized with 0.01 ethyl 3-aminobenzonate methansulfonic acid salt solution Ž . Aldrich Chemical Company, Milwaukee, WI, USA , at the beginning, at 3 weeks and the end of the feeding test. At the end of the feeding test, fish were starved for 48 h and then were sampled from each tank. Ten fish from each tank were frozen and stored for whole body analysis. The remaining fish in each tank were bled from the caudal vein with a heparinaized syringe. Plasma was separated by centrifugation, and the plasma and the bled bodies were stored at y808C for subsequent analysis. Following the feeding trial, determination of plasma retinoic acid levels was con- Ž . ducted using fish mean body weight 60 g from the same source as the fish in the feeding trial. The fish for plasma retinoic acid determination was fed only one time the Ž . test diets, that is, blood samples were taken from eight fish starved for 48 h initial and Ž . then the remaining fish eight fish per tank and four tanks were fed either the control or the respective retinoid diets. Blood samples were taken 6 h post-feeding by the protocol as described earlier. Plasma was separated by centrifugation and stored at y808C for subsequent analysis. Crude protein and crude fat of the test diets and the whole body were determined by Ž the Kjeldahl method and diethyl ether extraction, respectively two pooled samples of . five fish from each tank . The bled fish was stored at y808C, and then prior to lipid analysis, these were dissected and liver, visceral adipose tissue, and dorsal white muscle samples were taken. Hepatosomatic and visceral adiposomatic indices were calculated Ž according to the formula: organ index s 100 = liver or visceral adipose tissue . weightrbody weight . Lipid contents of dorsal white muscle, liver, and visceral adipose Ž tissue were determined by chloroform–methanol extraction two pooled samples of five . Ž . Ž . fish from each tank Folch et al., 1957 . Plasma non-esterified fatty acid NEFA and Ž . triacylglycerol levels individual fish were determined by diagnostic kits, NEFA C-Test Ž . Wako and Triglyceride E-Test Wako Wako , respectively. Ž . Plasma from four fish 0.25 ml plasma from each fish was pooled to one and duplicate samples for each dietary treatment were used for plasma retinoid determina- tion. Plasma retinol and retinoic acid were extracted according to the method of Takeda Ž . and Yamamoto 1994 . Retinoids were analyzed using a high-performance liquid Ž . chromatograph Pharmacia LKB Biotechnology, Uppsala, Sweden with gradient pump Ž 2249 and VWM 2141 spectrophotometric detector, and Wakosil-II5C18 HG ø 4.6 = 250 . Ž . mm; Wako column. As the mobile phases, mixtures of acetonitrile A , methyl Ž . Ž . alcohol–0.3 ammonium acetate solution 16:28, by vol.: B , and 2-propanol C were Ž . used. Retinoic acid was separated using a mixture of A and B 75:25, by vol. at a flow rate of 0.6 mlrmin at 308C and detected at 345 nm. Retinol was separated using a Ž . mixture of B and C 15:85, by vol. at a flow rate of 0.4 mlrmin at 308C and detected at Ž 325 nm. A vitamin-A–alcohol solution purity 98, Fluka Chemie, Buchs, Switzer- . Ž land and all-trans-retinoic acid purity 95, Biomol Research Lab., Plymouth . Meeting, PA, USA were used as the authentic standards for plasma retinoid determina- tion. Data were statistically compared between the control and the fish fed retinoids using Ž . Student’s t-test P - 0.05 , without any transformations of percentage data prior to the analysis.

3. Results