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*
Institute of Marine Biology, National Sun Yat-sen University, Kaohsiung 804 Taiwan, Republic of China and
Tainan Branch, Taiwan Fisheries Research Institute, Tainan County 724 Taiwan, Republic of China
2To whom correspondence should be addressed. E-mail: hychen{at}mail.nsysu.edu.tw
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • fish • grouper • fatty acids • docosahexaenoic acid • eicosapentaenoic acid
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Marine fish, such as turbot (Scophthalmus maximus) (2
), red seabream (Chrysophrys major) (3
), Asian seabass (Lates calcarifer) (4
), and Japanese flounder (Paralichthys olivaceus) (5
), cannot effectively elongate and desaturate the 18-carbon fatty acids and, thus, require the presence of highly unsaturated fatty acids (HUFA),3
especially docosahexaenoic acid (DHA) [DHA, 22:6(n-3)] and eicosapentaenoic acid (EPA) [EPA, 20:5(n-3)] in diets to maintain normal metabolism and growth. Freshwater fish such as salmonids, in contrast, can convert 18-carbon fatty acids to longer chain, more unsaturated fatty acids (6
) and, thus, 18:3(n-3) and 18:2(n-6) or both are important dietarily. The milkfish (Chanos chanos), an euryhaline tropical omnivorous fish, has the ability to bioconvert 18-carbon fatty acid into HUFA (7
). Weight gain of the milkfish was effectively improved when the diet contained 2% 18:3(n-3) alone, although the highest weight gain was achieved when the diet contained a combination of 1.0% 18:3(n-3), 0.5% EPA, and 0.5% DHA (8
).
DHA are an absolute requirement for growth and survival in juvenile turbot (9
). Although the bioconversion of EPA to DHA is possible in marine fish, the conversion rate is usually too low to meet the high demand for DHA in rapidly growing and developing larvae and juveniles (10
). Most nutritional studies in HUFA used lipids or esters of both EPA and DHA to supply (n-3)HUFA (11
–14
). These studies focused on the quantitative (n-3)HUFA requirement for juvenile marine fish. Optimal levels of (n-3)HUFA in several fishes have been established, including 0.5% for red seabream (11
), 0.8% for turbot (12
), and 1.0–1.7% for Asian bass (13
) and gilthead seabream (14
). This information, however, could not predict whether EPA or DHA is more important in satisfying the dietary needs of fish for essential fatty acid (EFA). Although DHA seemed to play an important role in enhancing growth of juvenile marine fish (15
,16
), information about this is scarce.
The grouper is an economically important food fish in tropical and subtropical areas. It is still mostly fed with trash fish in Taiwan and other Asian countries. Its nutritional requirements are still largely unknown except the optimal dietary protein levels (17
–19
). Being a carnivorous tropical marine fish, the grouper (n-3)HUFA requirement and metabolism are probably different from those of cold-water or warm-water marine fish in which dietary requirements are better understood.
The present study was designed to determine the dietary requirements of grouper for DHA and EPA with a goal to differentiate the relative importance of DHA and EPA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The basal diet (Table 1
) contained 52.3 g/100 g diet of crude protein solely from fishmeal and 10.9 g/100 g diet of crude lipid from various lipid sources. Digestible energy was calculated to be 13.6 MJ (3247 kcal)/kg based on the physiological values of 18.83 kJ (4.5 kcal)/g for protein (20
), 14.60 kJ (3.49 kcal)/g for carbohydrate (21
), and 35.61 kJ (8.51 kcal)/g for lipid (22
). Protein (P)/energy (E) ratio was 36.81 mg/kJ (154 mg/kcal), which is equal to the optimal P/E ratio reported by Shiau and Lan (19
). Nine diets were evaluated with one diet being the control and one the reference. All diets were of the same composition except the lipids that accounted for 10 g/100 g of the diet. Compositions of the lipids mixture used in the test diets are listed in Table 2
. DHA [22:6(n-3)] and EPA [20:5(n-3)] were supplemented singly or in combination at 10 g/100 g of the lipid mixture. Tristearin (99% purity; Sigma, St. Louis, MO) was added to adjust the differences due to the purity of the EPA and DHA oils used. The control diet with no DHA or EPA supplement contained 1 g/100 g diet of a mixture of trilinolenin and trilinolein and 9 g/100 g diet of tristearin (Sigma). The reference diet contained 10 g/100 g diet of a mixture of natural oils, cod liver oil (ICN Biochemicals, Inc., Aurora, OH), linseed oil and safflower oil (Sigma) at a ratio of 2:1:1 (wt/wt/wt), thus containing linolenoate, linolenate, EPA, and DHA. The DHA/EPA ratio and fatty acid composition of the reference diet were typical of compositions of commercial grouper feeds, while the control diet was free of DHA and EPA supplements and contained trace amounts of DHA and EPA but a fair quantity of linoleic acid and linolenic acid. The fatty acid compositions of the test diets are shown in Table 3
. DHA and EPA were from two different sources: purified DHA and EPA (>99%) were purchased from Nu-Chek Prep (Elysain, MN). The DHA oil used in the study was a microalgae-derived product, DHASCO, from Martek Biosciences (Columbia, MN) and contained 40.87% DHA, 13.86% 16:0, 11.18% 18:1(n-9), 11.03% 18:0 and other fatty acids. The EPA oil (Shin-Hu, Taipei, Taiwan) contained 27.8% EPA and 18.6% DHA, 16.7% 16:0, 13.9% 18:1 and other fatty acids. Fishmeal, cornstarch and 
-starch were extracted four times with hot ethanol (95%; 1:1, wt/v). Vitamin E (all-rac--tocopherol) at a level of 400 mg/kg was mixed with the lipids before thoroughly mixed with other powder ingredients. The dough was extruded to a pellet size of 6 mm x 5 mm. The test diets were freeze-dried and packed in vacuum in small quantities and stored at -20°C until use.
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Grouper, Epinephelus malabaricus, were artificially propagated and raised at the hatchery of the Tainan Branch of the Taiwan Fisheries Research Institute. Before the feeding trials, all fish were fed a lipid-free diet for 4 wk, which acclimated the fish to the experimental conditions and depleted their lipid reserves. After acclimation, 270 apparently healthy fish were individually weighed (average weight 11.5 ± 0.7 g) and randomly assigned so that each of the nine dietary treatments had three replicates. Each initial fish group of 10 fish was housed in an outdoor 290-L flow-through rectangular tank. Seawater in each tank was well aerated and water was replaced at a rate of 600 mL/min. All tanks were covered by black polythene plate to reduce light levels. During the 12-wk feeding trial, the water temperature ranged between 18 and 27°C and the salinity ranged between 32 and 34 parts per thousand.
The fish were hand fed once daily at 0800–0900. Uneaten food and fecal matter, if any, were removed before each feeding. Initial feeding rate was 
5 g/100 g of body and fish were fed in excess thereafter.
The fish were weighed at the beginning and the end of the feeding trial. Weight gain (W, %) and survival rate (S, %) were calculated as W = [(Wt - Wo)/Wo] x 100 and S = (Nt/No) x 100, respectively. In the equations, Wt is the final fish weight, Wo is the initial fish weight, Nt is the survival fish number at the end, and No is the initial fish number.
Sampling procedure.
Fish were anesthetized with 0.4 g/L benzocaine (Sigma) before sample collections. At the termination of the feeding trial, three fish from each tank (nine fish per dietary treatment) were randomly selected, killed, and the liver and muscle were dissected and weighed. Samples from the same tank were then pooled, resulting in three replicates per treatment, minced and extracted for lipid analysis. Muscle was collected from the ventral strip posterior to the dorsal fin. The experiment was conducted in accordance with the guidelines of the University regarding research on experimental animals.
Lipid extraction and analysis.
Lipids were extracted from tissues or diets using a modified Folch method (23
). Tissue was homogenized and extracted repeatedly with mixtures of methanol and chloroform (1:2 and 2:1, v/v). Crude lipid extracts were pooled and evaporated completely under nitrogen, and 0.05% butylated hydroxytoluene was added to a final concentration of 50 g/L and stored at -20°C before analysis. Silica cartridge (Sep-Pak; Waters Associates, Milford, MA) was used to separate the crude lipid extracts into polar (phospholipids) and neutral fractions (mostly triglycerides) as described by Bitman et al. (24
). The amounts of polar and neutral fractions were determined gravimetrically. The neutral fraction was saponified with 50% KOH for 40 min. The saponified lipids and polar fractions were transmethylated with BF3 in methanol to yield methyl esters (25
).
Fatty acid methyl esters (FAME) were analyzed using a gas chromatograph (Hewlett-Packard 5890A; Hewlett-Packard, Palo Alto, CA) equipped with a flame ionization detector using a 30-m x 0.25-mm ID fused silica capillary column (SP-2380; Supelco, Bellefonte, PA). The flow rate of carrier gas helium was 20 cm/s. The thermal gradient program was initially 150°C for 2 min, followed by increment of 4°C/min to 180°C, 180°C for 18 min, 4°C/min increase to 250°C, and finally 250°C for 5 min. Injector and detector temperatures were 250°C and 260°C, respectively.
FAME peaks were integrated, identified and quantified with the HP ChemStation 1201A PC software package through comparison with known standards (Nu-Chek 461; Nu-Chek; Supelco 37; Supelco). Fatty acids were quantified relative to an internal standard, nonadecanoate (19:0; Sigma), and expressed as the percentage of total fatty acids.
Statistical analysis.
Weight gain, survival rate and relative liver weight were expressed as means ± SEM, and other results were presented as means ± SD. All data were analyzed by one-way ANOVA using the SAS statistical procedures (SAS Institute, Cary, NC), and significant differences between treatment means were tested by Tukey’s test. Significance of difference was accepted at P < 0.05 (26
). Data (fatty acid composition) that were identified as nonhomogeneous using the Bartlett’s test, were arc-sine transformed before ANOVA analysis. General linear models were carried out to assess the incorporation of dietary fatty acids into body tissues.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Significant differences were observed in fish growth among the dietary treatments (Table 4
), but not in fish survival or relative liver weight. Purified EPA and DHA (pD3 and pD2) were not better than the EPA and DHA oils (for example, D3 and D2). Juvenile grouper fed diet D3 that had a high DHA/EPA ratio achieved the best weight gain, which was significantly better than those of fish fed diets with low DHA/EPA ratios (D.7 and pD.3) and was the only treatment that promoted a significantly greater growth than control diet. Weight gains of D1, D2 and D3 groups were significantly greater (P < 0.05) from those fed diet with the lowest DHA/EPA ratio (pD.3). The lowest weight gain was observed in fish fed pD.3, the diet with the lowest DHA/EPA ratio, and was not different from the group fed the reference diet. Fish fed D.7 had a weight gain not different from the group fed the control diet with no supplemental DHA or EPA. The groups fed diets with DHA/EPA ratios < 1 (D.7 and pD.3; 0.94 from empirical analyses) generally suffered higher mortality than the other dietary groups. None of the groups exhibited any apparent deficiency symptoms that have been described in the literature. Mortality could not be attributed to the dietary treatments.
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The relative importance of NL and PL and ratios of NL/PL from the liver and muscle showed similar trends (Table 5
). The test fish had drastic changes in their lipid compositions during the feeding trial. NL became the predominant lipid fraction (>70%) both in liver and muscle. After being fed a lipid-free diet for 4 wk, the initial fish showed a muscle lipid dominated by PL (84%). Fish fed the reference diet containing only natural oils had a high level of NL in the liver, which was significantly higher than those of the fish fed diets with lower DHA/EPA ratios (D.7 and pD.3), but not other groups. All groups had similar tissue NL/PL except the reference diet group, which had a significantly higher ratio. The initial fish had a NL/PL much lower than the fish after dietary treatments.
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and 7). Concentrations of total monounsaturated fatty acids in the NL fraction of the liver and muscle of the initial fish were significantly higher than the test fish after the feeding trial, and so was the NL fraction in the muscle of the initial fish. Concentrations of total (n-3)HUFA in the initial, control and reference groups were significantly lower than the groups fed diets supplemented with DHA or EPA. Any combination of dietary EPA and DHA resulted in similar tissue (n-3)HUFA concentrations. The reference diet contained the lowest level of stearic acid (18:0; Table 3
), but the fish fed this diet did not show similarly low levels of the acid, except in the neutral fractions of the liver. As one of the indicators depicting EFA deficiency, oleate [18:1(n-9)] levels in the muscle of the initial fish were significantly higher than the fish fed any other diets supplemented with DHA and/or EPA. Levels of 20:1(n-9) in liver and muscle were also significantly higher in the initial fish than in the experimental fish, with the exception of the polar fractions in the liver.
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). The dietary DHA and EPA levels were complementary. The levels of eicosatrienoate [20:3(n-3)] and EPA were a direct reflection of the dietary EPA. Increased levels of dietary EPA resulted in a significant accumulation in liver and muscle of 20:3(n-3) and EPA. Tissue levels of DHA were also strongly influenced by dietary DHA incorporation. Dietary EPA and DHA levels affected the levels of docosapentaenoate [22:5(n-3)] in muscle, but not in liver (Table 8)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and juvenile striped jack (16
). Fish evaluated in both studies were either fish larvae or juveniles, which greatly need essential nutrients for rapid tissue synthesis and metabolism. DHA is indispensable in the development of neural tissues, such as the brain and retina (27
,28
). Similar to earlier findings in red sea bream (15
), the (n-3)HUFA requirement of the juvenile grouper was dependent on the DHA/EPA ratios in the diets.
The levels of EFA required by warmwater fish are, in general, limited to 0.5–2.0 g/100 g of the dry diet (29
). Excessive dietary (n-3)HUFA are detrimental to fish growth (30
). By incorporating 1 g/100 g HUFA in the diets of the grouper, the present study ensured the adequacy of EFA. No symptoms of EFA deficiency or related detrimental effects were observed in any of the dietary groups during the feeding trial. The growth of the fish fed the control diet that contained trace levels of DHA and EPA and substantial amounts of linoleic acid, and linolenic acid was only inferior to the fish fed diet D3 among the seven test diets. The trace amounts of DHA and EPA in the control diet seemed to satisfy the EFA requirement of E. malabaricus, a typical tropical marine fish. The EFA requirements of the milkfish (Chanos chanos), another tropical marine fish, also could be met by dietary supplement of either (n-3)HUFA or 18:3(n-3) (8
). The milkfish fed a diet with 2% 18:3(n-3) alone or combination of 0.5% EPA and 0.5% DHA showed enhanced growth that was lower than the growth when the fish were fed a diet with the combination of 1% 18:3(n-3), 0.5% EPA, and 0.5% DHA. In addition, juvenile milkfish did not exhibit EFA deficiency symptoms when fed diets with 1% 18:3(3n-3) alone or 0.5–1.0% (n-3)HUFA alone. Our control diet contained 1% of 18:3(n-3) and 18:2(n-6) in combination (3:1, wt/wt) and a residual amount of DHA and EPA derived from solvent-extracted fishmeal. When fed the control diet, the grouper grew better than the groups fed the diet with the lowest DHA level (pD.3) or the reference diet, but fish growth was only significantly poorer than the group fed the highest DHA diet (D3). It is not clear whether the trace amounts of residual DHA and EPA from fishmeal in the control diet served the needs of the fish for EFA or 18:3(n-3) and 18:2(n-6) alone satisfied the EFA requirements of the fish or whether combinations of both served to fulfill the needs. Limited bioconversion of 18:3(n-3) to EPA or DHA is possible in many marine fishes (2
–5
). Future studies with DHA and EPA-free diets will more clearly define their roles in satisfying the EFA requirements of the marine fish.
Purified EPA and DHA did not promote growth better than did the impure EPA and DHA oils. The fish fed the diets containing purified EPA and DHA (pD3, pD2, and pD.3) grew more poorly than the fish fed the diet supplemented with EPA and DHA oils or the control diet. The obvious differences between the purified DHA/EPA diets and the impure diets, especially D3 and D2, were the levels of 18:3(n-3) and 18:2(n-6). If the difference in fish growth was caused by the difference in the levels of the two fatty acids, it would suggest that the grouper had a dietary need for these two fatty acids, or perhaps for 18:3(n-3), which is not known to be essential in other species of marine fish. The inferior growth of the purified DHA/EPA groups may, in contrast, have been due to by the reduced digestibility or availability of DHA and EPA in the purified forms.
Relative liver weights did not differ among dietary groups, indicating that this variable was not affected by the dietary lipid composition. HSI of red drum was reported to increase with increasing dietary lipid level (31
), and changed little when dietary lipid levels were 7% and beyond (32
). There is no reported optimal lipid level for the grouper (33
), warm-water marine fish in general showed good growth performance when dietary lipid level ranged between 8% and 12%. In this study, the amount of lipids (11%) in the diets was within the upper levels used for marine fishes and might satisfy the dietary lipid requirements of grouper through the adequate supply of EFA and dietary energy.
NL, mostly triacylglycerols, is the predominant class of reserve lipid and always mobilized before PL during starvation (34
). In the current study, the initial fish that were fed for 4 wk a lipid-free diet were relatively low in NL and had low NL/PL ratios in both liver and muscle, indicating marked retention of the phospholipids when lipid supply was limited. Increasing dietary lipid level tends to enhance deposition of NL. Studies with red sea bream (35
) and Atlantic herring (36
) showed similar results when these fish were deprived of food.
Fatty acid compositions of fish, in general, are closely associated with diet (37
,38
). In the present study, in addition to the common linkage between tissue and dietary fatty acid composition, fatty acid compositions of muscle lipids were more sensitive than liver lipids in reflecting the dietary fatty acids. This relationship, however, was not necessarily true with saturated and monounsaturated fatty acids. The correlation coefficients between dietary and muscle concentrations of total saturated fatty acids (inclusive of stearic acid) were not significant (P = 0.62). Despite the variations in dietary composition, tissue compositions of saturated fatty acids were relatively uniform. The correlation coefficients between dietary and muscle concentrations of total monounsaturated fatty acids (inclusive of oleate) were also not significant (P = 0.80). Monounsaturated fatty acids originate mostly from de novo synthesis and only a small fraction is derived from the diet (39
). Being endogenous, they are markedly displaced by polyunsaturated fatty acids (PUFA) if sufficient PUFA are supplied in the diet (39
). Dietary PUFA have been reported to inhibit enzymes responsible for synthesis of monounsaturated fatty acids and decrease tissue levels of the acids (40
). Thus, the concentrations of total saturated fatty acids or monounsaturated fatty acids did not reflect their dietary origins. The grouper were self-regulatory in saturation or monounsaturation of the tissue lipids.
The grouper had unusually high tissue levels of 20:3(n-3) in this study. Both the control diet and the reference diet contained relatively high levels of 18:3(n-3). Fish fed with these two diets had, in addition to high tissue 18:3(n-3) levels, a relatively high level of 20:3(n-3) in both neutral and PL fractions. Accumulation of dietary 20:3(n-3) in the fish tissue could result in high tissue 20:3(n-3) levels. The possibility of bioconversion of 18:3(n-3) to 20:3(n-3), as being reported in turbot (41
) through elongation also could not be ruled out because of the levels of 18:3(n-3) in the diets. The tissue 20:3(n-3) levels were correlated with dietary EPA levels. If a high EPA level could inhibit the conversion of 20:3(n-3) to 20:4(n-3), then to EPA, tissue accumulation of 20:3(n-3) would occur as seen in the present results.
Dietary EPA and DHA levels showed good correlations with muscle 22:5(n-3) levels but not with liver 22:5(n-3) levels. In in vitro studies with isolated mammalian cells, extrahepatic cells were found to have limited abilities to desaturate or chain-elongate fatty acids (42
). The differential abilities, if occurred in the grouper, would result in different 22:5(n-3) levels in liver and muscle even when the dietary fatty acid compositions are the same. Fatty acid composition in tissues can reflect both the selectivity of the enzymes and the relative abundances of substrate fatty acids available to the enzymes (43
). Muscle may possess unique chain-elongating activities or elongase that are absent in liver. Another possible reason for the lack of correlation between dietary and liver 22:5(n-3) levels is that the grouper may be able to retroconvert DHA to 22:5(n-3). Thus, 22:5(n-3) in the liver of the grouper could be in part supplied through EPA elongation and in part via DHA retroconversion. It would then be transported from the liver to muscle.
The grouper were capable of directly incorporating dietary DHA and EPA into body tissues. Diets low in DHA tended to promote DHA deposition. DHA is favorably deposited in fish tissues at the expense of EPA (44
). When fed the control diet that was DHA- and EPA-depleted, the level of DHA in grouper tissue was generally higher than that of EPA.
Grouper fed the control diet, which was rich in both 18:3(n-3) and 18:2(n-6), but low in both DHA and EPA, had the lowest tissue DHA and EPA levels of all groups; their growth, however, was not lower than the fish fed diets containing medium levels of DHA/EPA (D2 and D1). This indicates that the grouper had limited capabilities to elongate and desaturate 18:3(n-3) to HUFA. Marine fish, such as plaice (Pleuronectes platessa) (45
), turbot (46
), red sea bream, and black sea bream (Mylio macrocephalus) (47
) can elongate or desaturate 18:3(n-3), but the conversion capacity is not sufficient to meet metabolic needs. The limited ability of marine finfish to convert 18:3(n-3) to PUFA or HUFA seems ubiquitous. E. malabaricus, a marine warm-water fish, is no exception.
Tissue levels of 20:1(n-9) were a good indicator of EFA deficiency in E. malabaricus. Occurrence of eicosatrienoic acid [20:3(n-9)] in fish tissue has been suggested to be an indicator of EFA deficiency (48
,49
), and so has oleate [18:1(n-9)] (11
,50
). No fish in the present study contained 20:3(n-9) before or after the feeding trial. The initial fish that were exposed to prolonged feeding of the lipid-free diet had significantly higher tissue levels of 20:1(n-9) than the fed groups. Muscle levels of 20:1(n-9) in the PL fractions in the grouper fed diets without DHA were also greater than those of the other dietary grouper. The activities of 
-5 or -6 desaturase that desaturate 20:1(n-9) to 20:3(n-9) were likely to be limited or lacking. Tissue oleate levels were relatively constant and were not a good indicator of EFA deficiency in grouper. The initial fish had a large accumulation of 20:1(n-9) in the NL fractions both in liver and muscle, suggesting that the 20:1(n-9) level in the NL fractions might be more suitable for evaluating the EFA status of juvenile grouper.
In conclusion, dietary DHA promoted growth of the juvenile grouper. DHA was superior to EPA as the EFA in the diets of the grouper. Tissue, especially muscle, DHA and EPA levels in the grouper were highly reflective of dietary levels of DHA and EPA, indicating that direct incorporation was likely. In addition, 20:1(n-9) concentration in NL fractions of muscle or liver seems to be an appropriate indicator of dietary EFA deficiency for the grouper.
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FOOTNOTES |
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3 Abbreviations used: DHA, docosahexaenoic acid; EFA, essential fatty acid; EPA, eicosapentaenoic acid; FAME, fatty acid methyl esters; HUFA, highly unsaturated fatty acid; NL, neutral lipid; PL, polar lipid; PUFA, polyunsaturated fatty acid.
Manuscript received 18 June 2001. Initial review completed 2 August 2001. Revision accepted 5 October 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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