- Email: [email protected]
Effect of dietary lipid on growth performance and body composition of brown trout (Salmo trutta) reared in seawater
Aquaculture
ELSEVIER
123 (1994) 361-375
Effect of dietary lipid on growth performance and body composition of brown trout (Salmo trutta) reared in seawater Jacqueline Arzel”y*,Francisco X. Martinez Lopezb, Robert MCtailleP, Germaine Stephan”, Mich6le Viaud, Gilles Gandemefl, Jean Guillaume” “IFREMER, Centre de Brest, BP 70,29280 Plouzank, France bDepartment of Physiology & Pharmacology, Universityof Murcia, Facultad de Biologia, Murcia, Spain ‘CNE VA,BP 70,29280 PlouzanP,France ‘INRA, LEIMA, BP 527.44026 Nantes, France “INRA-IFREMER, Centre de Brest, BP 70,29280 Plouzank, France Accepted 4 February 1994
Abstract An experiment was carried out with 12 groups of a fast-growing strain of brown trout reared in seawater. Each treatment was fed to triplicate groups of 158 fish of 1.6 kg average body weight reared in 60-m3 floating cages. Four experimental diets corresponding to two levels (2 1 versus 29W) and two sources of added lipid (corn, i.e. vegetable, versus cod liver, i.e. marine) were tested. Crude protein content was similar (about 52%) in all diets. All fish were fed the same amount of calculated digestible energy. The level of fat had a slight but significant effect on growth rate and feed conversion; the higher dietary lipid level led to a faster daily growth index ( + 4.8%) and better feed conversion ( - 12%). Protein utilization, estimated by both protein efficiency ratio and productive protein value, was also improved in the high-lipid compared to low-lipid treatments. The same factor also significantly influenced fat and muscle water content which were higher and lower, respectively, in the high-lipid compared to low-lipid treatments. The source of added lipid did not influence growth rate, feed conversion, or protein eMiciency. Lipid source had no effect on body composition except in the case of liver which contained more lipid and less water in the fish fed cod liver oil. The source of lipid had very pronounced effects on the fatty acid (FA) profile of muscle and liver lipids but the variations mainly concerned saturated (in liver only), monounsaturated and 18:2n-6 FAs (in both tissues). On the other hand, n-3 polyunsaturated FAs were significantly but only *Corresponding
author.
0044-8486/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0044-8486( 94)00038-P
362
J. Arzel et al. /Aquaculture 123 (1994) 361-375
slightly modified by the source of lipid, corresponding for both tissues to that found in other salmonids. The level of 20:4n-6 remained constant. The elongation product of 18:2n-6, namely 20:2n-6,was incorporated in muscle and liver lipids while the 46 desaturation product, 20:3n-6, was only observed in liver.
1. Introduction Since the early work of Phillips and Hammer ( 1965) and Phillips et al. ( 1965 ), very little has been published concerning the nutrition of European or brown trout. Recently, studies have been undertaken to evaluate potential differences in the nutritional requirements of brown trout as compared to those of rainbow trout (Gabaudan et al., 1989; Arzel et al., 1992; Arzel and Guillaume, 1994). Both the protein requirement and the influence of lipid and energy levels on growth have been found to differ from those reported for rainbow trout. The potential beneficial effect of high-lipid diets has not been demonstrated in juvenile brown trout while few data are available on the effect of diet on body composition. Therefore, an experiment was conducted on large fish reared in seawater in order to study the influences of both dietary lipid content (21 versus 29%) and dietary lipid source (vegetable versus marine) on growth performance, body composition, fatty acid profile and quality of flesh. All diets contained adequate essential fatty acids to meet the requirement of salmonids. The results concerning fillet composition and quality were recently presented elsewhere (Arzel et al., 1993). Results on growth performance, body composition and FA composition of liver and muscle lipids are detailed in this paper. 2. Material and methods Twenty-month-old monosex (all female) brown trout (Sulmo truttu) were hatched and reared in the IFREMER-INRA experimental hatchery (Sizun, France). They were transferred into seawater at the IFREMER-INRA experimental station (Camaret, France), at the age of 1 year. They belonged to a fastgrowing strain. Fish with an average body weight of 1.6 kg were randomly allotted to 12 floating cages of 60 m3 volume. Each of the experimental diets was randomly assigned to three cages (three replicates per treatment). The experiment was conducted for 110 days from November 199 1. Water temperature was recorded each day and varied from 13 ‘C to 8’ C, while salinity was rather constant and close to 35 ppt. The four diets (Table 1) differed by quantity and quality of added lipids: 8 or 16% of vegetable (“V”, corn) or marine (“M”, cod liver) oil was added to a premix containing about 13% lipid from the various dietary ingredients, mainly fish meal and fish protein concentrate. The four diets were about isoproteic ( 52%). Digestible energy levels were calculated using the following coefficients in kJ g- ‘: fish protein, 19.7, wheat protein (supposed to be less digestible), 17.6, lipids,
363
J. Arzel et al. /Aquaculture 123 (1994) 361-375 Table 1 Composition
and proximate analysis of the diets (I dry weight)
Ingredients
Norwegian fish meal ( 70) ’ Soluble fish protein concentrate (83)* Wheat middlings ( 15 ) Wheat gluten (80) Cooked potato starch Oil (corn (V) or cod liver (M)) Soybean lecithin Vitamin premix3 Mineral premix4 Analysis Dry matter Lipid5 Protein5 Ash’
Diets v21
V29
M21
M29
60 8 10.35
60 8
60 8 10.35
60 8
10 8 1 1.65 1
2.35 10 16 1 1.65 1
10 8 1 1.65 1
2.35 10 16 1 1.65 1
91.6 21.5 53.7 8.1
90.8 28.5 50.9 7.5
91.4 21.4 54.0 8.1
90.8 29.4 50.7 7.5
(0.5) (0.3) (0.2) (0.1)
(0.3) (0.9) (0.8) (0.2)
(0.4) (0.2) (0.3) (0.1)
(0.5) (1.2) (0.4) (0.2)
‘Norseamink@, Norsildmel, Minde, Norway. *CPSP@; CTPP, Boulogne s/Mer, France. 3The vitamin mixture contained the following (per kg premix); retinyl acetate, lo6 IU; cholecalciferol, 10’ H-l; all-rat-a-tocopheryl acetate, 4000 mg; menadione sodium bisullite, 100 mg; thiamin, 1000 mg; riboflavin, 2500 mg; D-calcium pantothenate, 5000 mg; niacin, 1O4 mg; pyridoxine, 1000 mg; vitamin Bi2, 6 mg; folic acid, 500 mg; biotin, 100 mg; myo-inositol, lo5 mg. 4The mineral mixture contained the following (per kg premix); KCl, 9.0 g; KI, 4 mg; CaHP04-2H20, 50 g; NaCl, 4 g; CUS0,,‘5H20, 0.3 g; ZnS04*H20, 0.4 g; CoSO4-7H20, 2 mg; FeS04-7HZ0, 2 g; MnS04-H20, 0.3 g; CaC03, 2 1.5 g; MgCO,, 12.4 g; NaF, 0.1 g. 5As % of dry matter (s.e.m.).
33.5, cooked starch, 12.6 and raw starch, 5.0. The different diets were named V2 1, V29, M2 1 and M29 according to the source of the added oil and to the lipid content (21 versus 29). V2 1 and V29 as well as M21 and M29 were also named “V” and “M” diets, respectively. All fish were fed the same theoretical energy ration using feeding tables taking into account fish mean biomass and water temperature. In treatments V29 and M29, the feed ration was 12% lower than in treatments V2 1 and M2 1. Daily rations were distributed using automatic belt conveyor feeders. For adjustment of ration, biomass was measured every month and extrapolated every week using a theoretical growth model. Growth was estimated using the daily growth index calculated according to Kim and Kaushik ( 1992), an index derived from the Iwama and Tautz ( 198 1) model: DGI = [ (final weight) ‘I3- (initial weight ) ‘I31loo/number
of days
Ten fish were taken at the beginning of the experiment for body composition
364
J. Arzel et al. / Aquaculture 123 (1994) 361-3 75
analyses. At the end of the experiment live fish per cage, i.e. 15 per treatment, were chosen around the mean of body weight for the same analyses. From each fish, the liver and other viscera as well as a large sample of epiaxial muscle were withdrawn. The muscle sample was a mixture of three sub-samples taken at the level of the adipose fin, the dorsal fin and the anterior part. Each of these samples, as well as the remainder of the fish, was submitted to chemical analysis. Muscle and liver lipids were also analysed for fatty acid composition. Whole body composition was calculated from that of each “compartment” which had been analyzed separately. For analysis, samples of tissues were ground using a meat grinder and a highspeed cutter for whole carcass and muscle or liver, respectively. Viscera analyses were performed on fresh-ground samples because of difficulties of grinding. Freeze-dried samples were reground with a high-speed cutter and the water content was determined after dessication at 105 ‘C for 24 h. The lipid contents were determined using a Soxtec@ (Tecator) . In the case of muscle and liver lipid, a second extraction, according to Folch et al. ( 1957) was performed for FA analysis. The protein content was determined using a Tecator digestion system 40@ and a Kjeltec Auto 1030 analyzer@. Ash content was measured by burning in a mume furnace at 550°C for 12 h. Fatty acids (FAs) were assayed after transesterification according to Morrisson and Smith ( 1964) using a gas chromatograph (Packard model 427) equipped with a capillary column (Carbowax 20 M@) using helium as the carrier gas at constant temperature ( 19OOC). A Ross injector was used. The temperature of injector and detector was 220°C. The FA composition was calculated using an integrator Enica 2 1; heneicosanoic acid (C 2 1:O) was used as an internal standard. Data were analyzed by a two-way analysis of variance to test the effects of both lipid content and lipid quality. In the case of the FAs, one-way was preferred to two-way analysis of variance because FA composition was similar in M21 and M29 diets and different in V21 and V29 diets. Furthermore, for liver only one factor (source) was tested. In case of significant differences, the Newman-Keuls test was used to compare the means. For survival and fatty acid, expressed in %, data were transformed into arc sin ,/.
3. Results The proximate analysis of the diets is given in Table 1 and the FA composition in Table 2. The digestible energy levels were estimated to be 16.7 and 18.8 kJ kg-’ for the low- and high-fat diets, respectively. The FA profile was rather similar in diets M2 1 and M29 which contained mostly lipids of marine origin. Larger differences were apparent between the composition of the M diets and the V21 and V29 diets, V21 being more similar to the M diets than V29. The greatest difference involved the polyunsaturated series: lower contents of n-3 FAs and higher contents of n-6 FAs were apparent in V2 1 and, which were greater, in the
J. Arzel et al. / Aquaculture 123 (1994) 361-375 Table 2 Fatty acid composition of the experimental carefully sampled aliquots) Fatty acid
365
diets, in % by weight of methyl esters (one analysis on
Diets v21
V29
M21
M29
,X saturated
18.5
16.1
20.7
19.4
C monoenes
36.5
35.8
47.0
49.9
18:2n-6 20:2n-6 20:4n-6 In-6
27.8 0.2 0.3 28.2
35.5 0.1 0.2 35.8
3.2 0.3 0.5 4.0
2.7 0.3 0.4 3.4
18:3n-3 18:4n-3 20:4n-3 20:5n-3 22:5n-3 22:6n-3 In-3
1.0 1.8 0.3 6.1 0.6 6.9 16.7
1.0 1.2 0.2 4.1 0.4 4.8 11.7
0.9 3.1 0.6 10.8 1.1 11.8 28.3
0.9 2.9 0.6 10.4 1.1 11.4 27.3
n-3/n-6
0.6
0.3
7.1
8.0
V29 diet than in the M diets. It should be noted that the n-3 FA level was relatively high even in the V diets which contained a high percentage of fish oil derived from fish meal. The sum of the monounsaturated FAs was similar in the V2 1 and V29 diets while it was clearly higher in the M diets than in the V diets. Growth performance, feed conversion, protein efficiency ratio (PER) and productive protein value (PPV= nitrogen retention as 96of intake) are given in Table 3. Survival rate was high in all treatments. A significant (PcO.05) but slight effect of lipid source was observed: survival rate was slightly higher for the V treatment. Growth was significantly improved by lipid level (P
2568 2610 2575 2579
(8) (24) (15) (35)
Final mean weight (g)
‘Mean with standard error of the mean in brackets. *P
Analysis of variance: Dietary lipid level Dietary lipid source Level X source
1621 1605 1614 1594
v21 V29 M21 M29
(7)’ (6) (17) (13)
Initial mean weight (8)
Diet
NS
NS *
97.5 98.8 94.9 96.4
(0.4) (0.6) (1.3) (0.9)
Survival rate (%)
tt NS NS
1.33 1.13 1.31 1.20
NS NS
(0.02) (0.03) (0.04) (0.03) (0.02) (0.03) (0.01) (0.05)
Apparent feed conversion
*
1.77 1.87 1.80 1.85
Daily growth index (%)
Table 3 Survival rate, daily growth index, feed conversion and protein efficiency ratio for fish fed the experimental
NS NS
**
1.40 1.74 1.33 1.65
(0.03) (0.05) (0.08) (0.07)
Protein efftciency ratio
diets
NS NS
n:
24.5 31.4 23.8 28.7
(0.5) (0.8) (1.2) (0.9)
Productive protein value (%)
NS NS
NS
NS
Nfj
D
NS
NS
-
-
-
_
1.34 (0.02)
1.32 (0.03) -2
(E)
-*
NS NS NS
(Z) 7.8 (0.3)
7.4 (0.3)
8.3 (0.3) (Z)
is less than 100%.
(E) 10.2 (0.4)
10.0 (0.4)
8.4 (0.6) (&
Ash
NS NS
*
7::;) 40.3 (1.4)
40.5 (0.8)
47.5 (1.7) :;:z,
Water
NS NS NS
(E)
(&
8.2 (0.3)
(&
(Z)
Protein
NS NS NS
42.9 (1.9) 43.1 (1.5) 44.9 (1.1) 43.5 (1.5) 45.1 (2.1)
Lipid
NS NS NS
0.93 (0.32) 0.81 (0.03) 0.79 (0.01) 0.79 (0.03) 0.76 (0.02)
Ash
NS NS NS
(CK) 1.4 (0.1)
NS
**
NS
$) 74.4 (0.3)
74.3 (0.2) 75.6 (0.2) 75.4 (0.2)
21.7 (0.1) 21.0 (0.1) 20.9 (0.1) 21.0 (0.1) 20.8 (0.1)
Lipid 1.2 (0.1) 1.3 (0.1) 1.4 (0.1)
69.3 (0.5)’ 69.6 (0.3) 68.3 (0.4) 69.2 (0.4) 68.3 (0.3)
Protein
(%)3
NS NS
*
t;::, 15.5 (0.1)
18.1 (0.3) 15.9 (0.2) 15.3 (0.2)
Protein
Composition Water
Water
(%)
% of body weight
Composition
% of body weight
Composition
(O/o)
Liver
Viscera (except liver)
NS
*
NS
(E)
(Z) 4.4 (0.2) 3.9 (0.2)
Lipid
analysis of I5 fish per dietary treatment)
Muscle
‘Standard error. *Missing data. ‘Glycogen not assayed; the sum of components
Analysis of variance Dietary lipid level Dietary lipid source Level X source
M29
M21
V29
v21
Initial
Treatment or diet
Table 4 Proximate analysis of whole muscle, viscera and liver and percentage of viscera and liver (individual
NS
*c
NS
$I,
(Z) 1.34 (0.01) 1.33 (0.01)
Ash
368
J. Arzelet al. /Aquaculture
123 (1994) 361-375
nor that of the interaction levelxsource was apparent. The same tendency was observed in the viscera, but the only significant effect was a lower water content (PC 0.05 ) induced by the high dietary fat level. In the liver, the influence of the tested factors on composition was quite different: dietary lipid quality influenced liver lipid, ash and water content, the vegetable oil inducing lower fat and higher water levels. A significant effect of lipid level on protein content was also observed in this organ (PC 0.05). The fatty acid composition of liver lipids is shown in Table 5 for fish fed diets V29 and M29, while the corresponding data for whole muscle are shown in Table 6 for fish fed the four experimental diets. In ail cases, there was a significant influence of dietary lipid on FA composition of tissues: compared to fish fed diet M29, the liver of fish fed diet V29 had a lower proportion of monounsaturated and n-3 FAs but a higher proportion of saturated and n-6 FAs. The content of total n-6 FA in the V29 group was four times as high as in the M29 group, due mainly to higher deposition of 18:2. But 20:2, which remained at low levels, was 4.5 times as high in the V29 as in the M29 group. 20:3n-6 was at an appreciable level in liver tissue and the relative difference between the V29 and M29 treatments was also significant (PcO.05); the content of 20:3n-6 was about ten times higher in fish fed the V29 diet than the M29 diet. Table 5 Fatty acid composition per dietary treatment) Fatty acid
of liver lipid, in % by weight of methyl ester (individual analyses of 9 samples
Diets
Analysis of variance
V29
M29
1 saturated
21.9 (1.5)
16.6 (1.0)
C monoenes
25.9 (1.3)
39.8 (1.9)
18:2n-6 20:2n-6 20:3n-6 20:4n-6 1 n-6’
11.6 2.1 1.9 3.0 19.4
(0.8) (0.3) (0.1) (0.4) (0.9)
1.8 0.6 0.2 2.2 4.9
(0.1) (0.0) (0.0) (0.2) (0.2)
18:3n-3 18:4n-3 20:4n-3 20:5n-3 22:5n-3 22:6n-3 T n-3
0.4 0.3 1.2 5.8 2.3 22.8 32.8
(0.1) (0.1) (0.3) (0.3) (0.2) (1.2) (1.4)
0.5 0.4 1.7 7.0 4.4 24.7 38.7
(0.0) (0.0) (0.1) (0.3) (0.3) (1.0) (1.1)
n-3/n-6 lP< 0.05. ‘Including 22:5n-6.
1.7 (0.1)
7.9 (0.2)
l
* * * * NS * NS
NS NS *
* NS *
J. Arzel et al. /Aquaculture
369
123 (1994) 361-375
Table 6 Fatty acid composition of whole muscle lipid, in % by weight of the methyl ester (individual of 15 samples per dietary treatment) Fatty acid
analysis
Diets v21
V29
M21
M29
1 saturated
20.9b (0.2)
19.P (0.3)
21.7’ (0.3)
20.6’ (0.1)
C monoenes
39.9b (0.2)
38.6” (0.2)
44.Y (0.3)
45.2a (0.2)
18:2n-6 20:2n-6 20:3n-6 2014~6 1 n-6
12.Ob (0.2) 0.8b (0.0) _*
18.0” (0.5) 1.P (0.1)
4.0’ (0.1) 0.4” (0.0)
3.9” (0.0) 0.4” (0.0)
0.4 (0.0) 13.2b (0.2)
0.4 (0.1) 19.4” (0.5)
0.4 (0.0) 4.8” (0.1)
0.4 (0.0) 4.7c (0.1)
18:3n-3 18:4n-3 20:4n-3 20:5n-3 22:5n-3 22:6n-3 1 n-3
1.1 (0.0) 1.9b (0.0) 1.4b (0.0) 4.6b (0.1) 2.3b (0.0) 14.7b (0.2) 26.0b (0.3)
1.1 (0.0) 1.7c (0.0) 1.2c (0.0) 4.0” (0.1) 1.8’(0.1) 12.8’ (0.2) 22.6” (0.3)
1.1 (0.0) 2.1” (0.0) 1.5” (0.0) 5.9” (0.1) 2.6” (0.0) 15.9” (0.3) 29.2” (0.4)
1.1 (0.0) 2.1” (0.0) 1.6” (0.0) 6.0” (0.1) 2.6” (0.0) 16.0” (0.2) 29.5” (0.2)
n-3/n-6
2.0
1.2
6.1
6.3
Values in the same row with the same superscript letters are not significantly different (P> 0.05). *Not quantified, values less than 0.1%.
The arachidonic acid content was slightly but not significantly influenced by diet. Concerning the n-3 series, the group fed V29 had a significantly lower level than the group fed M29 (PC 0.05), the relative difference amounting to 15%, but the relative difference between groups was not the same for each fatty acid. It was greatest ( - 17%) and significant (PcO.05) for eicosapentaenoic acid (EPA 20:5n-3) while it was lowest ( - 8%) and not significant for docosahexaenoic (DHA 22:6n-3). In the whole muscle, the FA composition was similar in the groups fed M21 and M29, but different from the V2 1 and V29 groups; the results for the V21 group were intermediate between those from the V29 and M2 1 groups (or M29). The influence of a dietary supply of n-6 and n-3 FAs was very pronounced in this tissue. The content of total n-6 FA was about four times higher in the groups fed corn oil than in those fed cod liver oil; the concentration of 20:3n-6 was negligible while that of 18:2 was very high, being the highest of all FAs in the V29 group. The n-3 FA content was lower than that observed in liver tissue. The n-3 FA content was significantly (PcO.05) influenced by dietary treatment. The whole body composition of fish fed the four dietary treatments is shown in Table 7. Significant differences among treatments were found only for water and lipid contents which were significantly influenced by the level of dietary lipid
370
J. Arzel et al. /Aquaculture
123 (1994) 361-375
Table 7 Composition of whole body (calculated from the chemical analysis of the various compartments), samples per dietary treatment Dietary treatment
Water
Protein
Lipid
Initial v21 V29 M21 M29
65.2 66.0 64.1 65.2 64.7
18.4 17.5 18.1 17.9 17.4
15.5 14.5 16.2 15.0 15.9
Analysis of variance: Quantity Quality Quantity x Quality
(0.5) (0.3) (0.2) (0.4) (0.4)
89
NS
NS
NS
*
PI
(0.1) (0.1) (0.1) (0.1) (0.1)
15
Ash (0.4) (0.2) (0.2) (0.4) (0.4)
-1 1.77 (0.03) 1.81 (0.03) 1.72 (0.02)
PI
NS NS
-
‘Missing data.
(PC 0.0 1) , body lipid increasing by 9% while water decreased by 4% in the groups fed high fat compared to the groups fed low fat.
4. Discussion The analyses show that all experimental diets were very rich in lipid, including essential FAs, the levels of which clearly exceeded the requirement’ reported for salmonids (Kanazawa, 1985 ). The V29 diet which contained a high level of corn oil, also contained sufficient EFAs due to lipid supplied by fish meal. Protein level was also above the requirement of this species (Arzel et al., 1992). The similarity of the FA profiles of diets M2 1 and M29 is obviously due to the marine fish oil contained in both diets, while the differences between the FA profiles of diets V2 1 and V29 can be explained by the differences in the relative proportions of vegetable oil and fish meal in these diets. The lack of difference between growth rate and survival of fish fed the “V” or “M” diets seems to indicate that the EFA supply did not induce any adverse effect such as those observed by Takeuchi and Watanabe ( 1979) on rainbow trout fed 3% n-3 PUFA (60% of dietary lipid). In fact the n-3 PUFA of the diets tested varied from 2.9 to 6.8% of diets or 9.5 to 24.3% of lipid. According to Watanabe and Takeuchi ( 1989) and Lochmann and Gatlin ( 1993), the requirements for EFA are dependent on dietary lipid content and should be expressed as a percentage of total dietary lipid. The requirement amounts to about 10% of lipid in rainbow trout, and an excess of EFA exerts adverse effects when fed at four times the requirement level. If the requirement of brown trout is of the same order of magnitude as that of rainbow trout, no detrimental effect was indeed to be expected. The n-6/n-3 imbalance described by Yu and Sinnhuber ( 1976) is unlikely to have exerted an effect in our trial. Strictly speaking, a larger number of experimental diets (with various FA profiles) would have been needed to con-
J. Arzelet al. /Aquaculture 123 (1994) 361-37.5
371
firm this assessment. The significant but rather limited effect of dietary lipid level on growth rate is probably due to the feeding method, a theoretically equal amount of energy being allowed in all cases. Satiation feeding would probably have led to higher energy intake and higher weight gain in the groups fed V29 and M29 compared to the groups fed V2 1 and M2 1. In fact, fish fed to satiation usually regulate their food intake according to dietary energy level in such a way that their energy intake is nearly constant, but very high energy levels, i.e. very high lipid contents, lead to an overconsumption of dietary energy (Cho, 1987). The observed difference in daily growth index (4.8%) could be due to the inaccuracy of estimating the DE rations. The better feed conversion in high-lipid fed groups is presumably a consequence of the higher energy level of these feeds. The better efficiency of protein utilization in high-lipid (29%) compared to low-lipid diets (2 I%), evidenced by both PER and PPV, reflects a sparing effect of protein by energy or by lipid which has been well demonstrated in several species of fish (Takeuchi et al., 1978; Bromley, 1980; Cho and Kaushik, 1985). In carnivorous fish, this effect is particularly marked when lipid is used as an energy source (Lee and Putnam, 1973; Shimeno and Kajiyama, 1980; Kaushik and OlivaTeles, 1986; Watanabe and Takeuchi, 1989). The effect of dietary lipid on the chemical composition of tissues was significant only for muscle and whole body where opposite changes of water and fat were observed. It was not apparent in liver, the lipid content of which was always low (a result similar to that found by Gunstone et al., 1978, in a sample of wild Salmo trutta) and it was not significant in viscera. Neither was an increase observed in viscera proportion in fish fed the high-lipid diets. Thus, high-lipid diets did not induce an increase in visceral fat deposition, which has been established in rainbow trout (Ogino et al., 1976; Watanabe, 1987). This lack of influence can be explained by a tendency of brown trout to remain lean compared to rainbow trout ( Arzel et al., 199 1) and to the isoenergetic feeding plan. More surprising is the significant effect of lipid quality on liver lipid content: lipids of marine origin seem to be more easily stored in the liver than those which are partly of vegetable origin. A rather similar effect was observed in rainbow trout (Corraze, 1993, personal communication). Takeuchi and Watanabe ( 1979 ) found a decrease of liver weight in fish fed excess n-3 EFAs. However, the phenomenon observed in the present study is different and remains unexplained. In liver, a significant effect of dietary lipid level on protein content was also found, this phenomenon was difftault to explain, all the more since glycogen was not assayed and analysis of liver was not complete. The FA composition of body tissues reflects to a large extent that of dietary FAs as shown by the similarity of the FA pattern in the groups fed M2 1 and M29 and the dissimilarity of this pattern in the groups fed V21 and V29, both for dietary lipids and body lipids. However, the composition of tissue lipid does not simply reflect that of dietary lipid. Rather some FAs appear to be maintained within defined ranges which depend on animal species, and on the nature of the tissue being studied. Even if n-6 FAs were largely deposited in muscle of fish fed the V21 and V29 diets, the 20:4n-6 content appeared to remain very constant;
312
J. Arzel et al. / Aquaculture 123 (I 994) 361-3 75
the only n-6 FA deposited in large amounts was 18:2n-6. The lack of conversion of 18:2n-6 to 20:4n-6 is attributable to the inhibition of the 66 desaturase by the excess of PUFAs which could present a feedback effect on desaturation as shown by Brenner ( 197 1) and Urger et al. ( 198 1) in rainbow trout. The n-3 long-chain FAs (20:5n-3 and 22:6n-3) were much less variable in tissues than in diets. A more or less constant FA pattern was observed in muscle and, still more clearly, in liver total lipid. This latter phenomenon is similar to that described in coho salmon by Yu and Sinnhuber ( 198 1), and in rainbow trout by Greene and Selivonchick ( 1990) who wrote that: “This consistent pattern suggests that a physiologically optimum level of long-chain fatty acids is maintained in rainbow trout”. Similar results were also found in Atlantic salmon by Thomassen and Rnrsjnr ( 1989) with addition of various vegetable oils. Such a pattern characteristic of fish metabolism is illustrated by the EPA/DHA ratio which amounts to 0.25 and 0.28 in liver lipid, and 0.3 1 and 0.38 in muscle lipids for the groups fed V29 and M29, respectively, instead of 0.85 and 0.9 1 in the corresponding diets. But differences between tissues were obvious for this pattern; i.e. liver lipid compared to muscle lipid had higher n-3 FA content, lower EPA/DHA ratio and higher 20:4n-6 content. This phenomenon is probably due to the high percentage of phospholipid in liver; phospholipids generally have a higher content in PUFA and a lower short-chain FA/long-chain PUFA ratio than the triacylglycerols (Henderson and Tocher, 1987). The analysis of n-3 FAs such as 18:3, 18:4 and 20:4 did not reveal any effect of treatment on elongation of 18:3n-3 which was certainly low in all cases. In rainbow trout, this elongation was found during n-3 PUFA deficiency (Owen et al., 1975; Yu and Sinnhuber, 1976) but, using diets high in n-3 PUFA, Greene and Selivonchick ( 1990) did not observe any bioconversion of 18:3 into long chain n-3 PUFA. Furthermore, Greene and Selivonchik ( 1987) insisted on the fact that salmonids such as chum, coho and chinook salmon did not desaturate and elongate 18:3n-3 as efficiently as rainbow trout. Regarding the n-6 FAs, several authors have found large deposition of 18:2n-6 in salmonids fed diets rich in vegetable oil, comparable to the observations made in the present study, but bioconversion of 18:2n-6 into 20:4n-6 was effective only in the case of n-3 PUFA deliciency (Henderson and Tocher, 1987 ) . In the present study, a significantly higher level of 20:2n-6 was observed in fish fed the V2 1 and V29 diets, compared to the M21 and M29 diets in muscle, while in liver the same significant difference was observed for the fish fed V29 and M29 (P< 0.05). 20:3n-6 was also more concentrated in liver lipid of groups fed the V29 diet compared to those fed the M29 diet. Therefore, some elongation of 18:2n-6 occurred in both tissues, while the products of desaturation by 46 desaturase could be seen only in liver, a phenomenon described in the rat by Brenner ( 197 1). These results can be interpreted as an inhibition of bioconversion of 18-carbon precursors to long-chain PUFA by a large dietary supply of n-3 PUFA (LRger et al., 1981). Further analyses performed on phospholipids and other lipid fractions will help explain the influence of diet on lipid metabolism in brown trout. In conclusion, the present study showed that a large excess of n-3 PUFA appar-
J. Arzel et al. /Aquaculture 123 (1994) 361-375
313
ently does not induce any detrimental effect on growth, feed conversion, body composition or health of seawater-reared brown trout, at least under the conditions of this trial. The incorporation of a large quantity of vegetable oil was shown to significantly affect the organoleptic quality of flesh: smoked fillets of V-fed fish displayed a poorer flesh cohesion and a higher in-mouth fatty texture (Arzel et al., 1993). But it did not influence growth performance or feed utilization. It also clearly modified body lipid FA pattern in a manner rather similar to that described in other salmonids. Due to the shortness of the experiment, no conclusion can be drawn on the effects of the addition of vegetable oil on the health of brown trout; phenomena such as those described in Atlantic salmon by Bell et al. ( 199 1)) i.e. cardiac lesions under stressing conditions, possibly due to increased synthesis of eicosanoids, remain to be studied in brown trout in conditions different from those of this trial.
Acknowledgments
The authors would like to express their gratitude to the technicians of the Salmoniculture Experimentale IFREMER-INRA at Sizun and Camaret, mainly Mr. Luc Lebrun and Mr. HervC Le Delliou of the IFREMER Laboratoire de Nutrition at Brest for their technical assistance, as well as Dr. S.J. Kaushik of the INRA Laboratoire de Nutrition des Poissons at Saint-Pee-sur-Nivelle for careful reading of the manuscript.
References Arzel, J. and Guillaume, J., 1994. Donnees sur la nutrition et l’alimentation de la truite fario. Piscicult. Fr. (in press). Arzel, J., Metailler, R. and Guillaume, J., 1991. The relative efficiency of a combination of 2 protein and 2 lipid levels during the growth of brown trout (Salmo trutta) in different conditions. Communication CIEM, Comite Mariculture, F:5. La Rochelle, France, October 199 1, 17 pp. Arzel, J., Mttailler, R., Huelvan, C., Faurt, A. and Guillaume, J., 1992. The specific nutritional requirements of brown trout (Salmo tnrtta) . Blivisindi (Iceland Agr. Sci. ), 6: 77-92. Arzel, J., Cardinal, M., Comet, J., Mttailler, R., Sttphan, G. and Guillaume, J., 1993. Nutrition of brown trout (Salmo trutta) reared in seawater. Effect of dietary lipid on growth performances, body composition and fillet quality. World Aquaculture Conference, Torremolinos, Spain, 26-28 May 1993 (poster). Bell, J.G., McVicar, A.H., Park, M.T. and Sargent, J.R., 1991. High dietary linoleic acid affects the fatty acid composition of individual phospholipids from tissues of Atlantic salmon (Salmo salar): association with stress susceptibility and cardiac lesion. J. Nutr., 12 1: 1163- 1172. Brenner, R.R., 197 1. The desaturation step in the animal biosynthesis of polyunsaturated fatty acid. Lipids, 6: 561-575. Bromley, P.J., 1980. Effect of dietary protein, lipid and energy content on the growth of turbot (Scophthalmus maximus L.). Aquaculture, 19: 359-369. Cho, C.Y., 1987. La energia en la nutrition de 10s peces. In: M. de la Higuera (Editor), Nutrition en Acuicultura, II. J. Espinosa de 10s Monteros y U. Labarta (Publishers), Madrid, pp. 197-243.
374
J. Arzel et al. /Aquaculture 123 (1994) 361-375
Cho, C.Y. and Kaushik, S.J., 1985. Effects of protein intake on metabolizable and net energy values of fish diets. In: C.B. Cowey, A.M. Mackie and J.G. Bell (Editors), Nutrition and Feeding in Fish. Academic Press, London, pp. 95- 117. Folch, J., Lees, N. and Sloane Stanley, G.H., 1957. A simple method for the isolation and purification of total lipid from animal tissues. J. Biol. Chem., 226: 497-509. Gabaudan, J., Metailler, R. and Guillaume, J., 1989. Nutrition comparee de la truite arc-en-ciel (Oncorhynchus mykiss), de la truite fario (Salmo trutta) et du saumon coho (Oncorhynchus kisutch). Effet des taux de proteines totales et de lipides. Communication CIEM, Comite Mariculture, F:5. The Hague, Netherlands, October 1989,19 pp. Greene, D.H.S. and Selivonchick, D.P., 1987. Lipid metabolism in fish. Prog. Lipid Res., 26: 58-85. Greene, D.H.S. and Selivonchick, D.P., 1990. Effects of dietary vegetable, animal and marine lipids on muscle lipid and hematology of rainbow trout (Oncorhynchus mykiss). Aquaculture, 89: 165182. Gunstone, F.D., Wijesundera, R.C. and Scrimgeour, C.M., 1978. The component acids of lipids from marine and freshwater species with special reference to furan-containing acids. J. Sci. Food Agric., 29: 539-550. Henderson, R.J. and Tocher, D.R., 1987. The lipid composition and biochemistry of freshwater fish. Prog. Lipid. Res., 26: 281-347. Iwama, G.K. and Tautz, A.F., 1981. A simple growth model for salmonids in hatcheries. Can. J. Fish. Aquat. Sci., 38: 649-656. Kanazawa, A., 1985. Essential fatty acid and lipid requirement of fish. In: C.B. Cowey, A.M. Mackie and J.G. Bell (Editors), Nutrition and Feeding in Fish. Academic Press, London, pp. 281-298. Kaushik, S.J. and Oliva-Teles, A., 1986. Effect of digestible energy on nitrogen and energy balance in rainbow trout. Aquaculture, 50: 89-l 0 1. Kim, J.D. and Kaushik, S.J., 1992. Contribution of digestible energy from carbohydrates and estimation of protein/energy requirements for growth of rainbow trout (Oncorhynchus mykiss). Aquaculture, 106: 161-169. Lee, D.J. and Putnam, G.B., 1973. The response of rainbow trout to varying protein/energy ratio in a test diet. J. Nutr., 103: 9 16-922. LRger, C., Fremont, L. and Boudon, M., 198 1. Fatty acid composition of lipids in the trout. I. Influence of dietary fatty acids on the triglyceride fatty acid desaturation in serum, adipose tissue, liver, white and red muscle. Comp. Biochem. Physiol., 69B: 99-105. Lochmann, R.T. and Gatlin, D.M., III, 1993. Evaluation of different types and levels of triglycerides, singly and in combination with different levels of n-3 highly unsaturated fatty acid ethyl esters in diets of juvenile red drum Sciaenops ocellatus. Aquaculture, 114: 113-l 30. Morrisson, W.R. and Smith, L.M., 1964. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol. J. Lipid Res., 5: 600-608. Ogino, C., Chiou, J.Y. and Takeuchi, T., 1976. Protein nutrition in fish. VI. Effects of dietary energy sources on the utilization of protein by rainbow trout and carp. Bull. Jpn. Sot. Sci. Fish., 46: 385388. Owen, J.M., Adron, J.W., Middleton, C. and Cowey, C.B., 1975. Elongation and desaturation of dietary fatty acids in turbot Scophthalmus maximus L. and rainbow trout Salmo gairdneri Richardson. Lipids, 10: 528-531. Phillips, A.M., Jr. and Hammer, G.L., 1965. Modified pelleted dry mixtures as complete foods for brown trout. Fish. Res. Bull., N.Y., 28: 23-28. Phillips, A.M., Jr., Livingston, D.L. and Poston, H.A., 1965. The effect of three supplemental fats on the growth rate and body chemistry of brown trout. Fish. Res. Bull., N.Y., 28: 28-34. Shimeno, S. and Kajiyama, H., 1980. Effects of calorie to protein ratios in formulated diets on the growth, feed conversion and body composition of young yellowtail. Bull. Jpn. Sot. Sci. Fish., 46: 1083-1087. Takeuchi, T. and Watanabe, T., 1979. Effect of excess amounts of essential fatty acids on growth of rainbow trout. Bull. Jpn. Sot. Sci. Fish., 45: 15 17-l 5 19. Takeuchi, T., Yokoyama, M., Watanabe, T. and Ogino C., 1978. Optimum ratio of dietary energy to protein for rainbow trout. Bull. Jpn. Sot. Sci. Fish., 44: 729-732.
J. Atzelet al. IAqwcultute
123 (1994) 361-375
375
Thomassen, M.Y. and Rosjo, C., 1989. Different fats in feed for salmon: influence on sensory parameters, growth rate and fatty acids in muscle and heart. Aquaculture, 79: 129-135. Watanabe, T., 1987. Requerimientos de acidos grasos y nutrition lipidica en 10s peces. In: M. de la Higuera (Editor), Nutrition en Acuicultura, II. J. Espinosa de 10s Monteros y U. Labarta (Publishers), Madrid, pp. 99-165. Watanabe, T. and Takeuchi, T., 1989. Implications of marine oils and lipids in aquaculture. In: R.G. A&man (Editor), Marine Biogenic Lipids, Fats and Oils, Vol. II. CRC Press, Boca Raton, FL, pp. 457-479. Yu, T.L. and Sinnhuber, R.O., 1976. Growth response of rainbow trout (Salmo gait&w-i) to dietary w3 and 06 fatty acids. Aquaculture, 8: 309-3 17. Yu, T.C. and Sinnhuber, R.O., 198 1. Use of beef tallow as an energy source in coho salmon (Oncothynchus kisutch) rations. J. Fish. Res. Board Can., 38: 367-370.
ELSEVIER
123 (1994) 361-375
Effect of dietary lipid on growth performance and body composition of brown trout (Salmo trutta) reared in seawater Jacqueline Arzel”y*,Francisco X. Martinez Lopezb, Robert MCtailleP, Germaine Stephan”, Mich6le Viaud, Gilles Gandemefl, Jean Guillaume” “IFREMER, Centre de Brest, BP 70,29280 Plouzank, France bDepartment of Physiology & Pharmacology, Universityof Murcia, Facultad de Biologia, Murcia, Spain ‘CNE VA,BP 70,29280 PlouzanP,France ‘INRA, LEIMA, BP 527.44026 Nantes, France “INRA-IFREMER, Centre de Brest, BP 70,29280 Plouzank, France Accepted 4 February 1994
Abstract An experiment was carried out with 12 groups of a fast-growing strain of brown trout reared in seawater. Each treatment was fed to triplicate groups of 158 fish of 1.6 kg average body weight reared in 60-m3 floating cages. Four experimental diets corresponding to two levels (2 1 versus 29W) and two sources of added lipid (corn, i.e. vegetable, versus cod liver, i.e. marine) were tested. Crude protein content was similar (about 52%) in all diets. All fish were fed the same amount of calculated digestible energy. The level of fat had a slight but significant effect on growth rate and feed conversion; the higher dietary lipid level led to a faster daily growth index ( + 4.8%) and better feed conversion ( - 12%). Protein utilization, estimated by both protein efficiency ratio and productive protein value, was also improved in the high-lipid compared to low-lipid treatments. The same factor also significantly influenced fat and muscle water content which were higher and lower, respectively, in the high-lipid compared to low-lipid treatments. The source of added lipid did not influence growth rate, feed conversion, or protein eMiciency. Lipid source had no effect on body composition except in the case of liver which contained more lipid and less water in the fish fed cod liver oil. The source of lipid had very pronounced effects on the fatty acid (FA) profile of muscle and liver lipids but the variations mainly concerned saturated (in liver only), monounsaturated and 18:2n-6 FAs (in both tissues). On the other hand, n-3 polyunsaturated FAs were significantly but only *Corresponding
author.
0044-8486/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0044-8486( 94)00038-P
362
J. Arzel et al. /Aquaculture 123 (1994) 361-375
slightly modified by the source of lipid, corresponding for both tissues to that found in other salmonids. The level of 20:4n-6 remained constant. The elongation product of 18:2n-6, namely 20:2n-6,was incorporated in muscle and liver lipids while the 46 desaturation product, 20:3n-6, was only observed in liver.
1. Introduction Since the early work of Phillips and Hammer ( 1965) and Phillips et al. ( 1965 ), very little has been published concerning the nutrition of European or brown trout. Recently, studies have been undertaken to evaluate potential differences in the nutritional requirements of brown trout as compared to those of rainbow trout (Gabaudan et al., 1989; Arzel et al., 1992; Arzel and Guillaume, 1994). Both the protein requirement and the influence of lipid and energy levels on growth have been found to differ from those reported for rainbow trout. The potential beneficial effect of high-lipid diets has not been demonstrated in juvenile brown trout while few data are available on the effect of diet on body composition. Therefore, an experiment was conducted on large fish reared in seawater in order to study the influences of both dietary lipid content (21 versus 29%) and dietary lipid source (vegetable versus marine) on growth performance, body composition, fatty acid profile and quality of flesh. All diets contained adequate essential fatty acids to meet the requirement of salmonids. The results concerning fillet composition and quality were recently presented elsewhere (Arzel et al., 1993). Results on growth performance, body composition and FA composition of liver and muscle lipids are detailed in this paper. 2. Material and methods Twenty-month-old monosex (all female) brown trout (Sulmo truttu) were hatched and reared in the IFREMER-INRA experimental hatchery (Sizun, France). They were transferred into seawater at the IFREMER-INRA experimental station (Camaret, France), at the age of 1 year. They belonged to a fastgrowing strain. Fish with an average body weight of 1.6 kg were randomly allotted to 12 floating cages of 60 m3 volume. Each of the experimental diets was randomly assigned to three cages (three replicates per treatment). The experiment was conducted for 110 days from November 199 1. Water temperature was recorded each day and varied from 13 ‘C to 8’ C, while salinity was rather constant and close to 35 ppt. The four diets (Table 1) differed by quantity and quality of added lipids: 8 or 16% of vegetable (“V”, corn) or marine (“M”, cod liver) oil was added to a premix containing about 13% lipid from the various dietary ingredients, mainly fish meal and fish protein concentrate. The four diets were about isoproteic ( 52%). Digestible energy levels were calculated using the following coefficients in kJ g- ‘: fish protein, 19.7, wheat protein (supposed to be less digestible), 17.6, lipids,
363
J. Arzel et al. /Aquaculture 123 (1994) 361-375 Table 1 Composition
and proximate analysis of the diets (I dry weight)
Ingredients
Norwegian fish meal ( 70) ’ Soluble fish protein concentrate (83)* Wheat middlings ( 15 ) Wheat gluten (80) Cooked potato starch Oil (corn (V) or cod liver (M)) Soybean lecithin Vitamin premix3 Mineral premix4 Analysis Dry matter Lipid5 Protein5 Ash’
Diets v21
V29
M21
M29
60 8 10.35
60 8
60 8 10.35
60 8
10 8 1 1.65 1
2.35 10 16 1 1.65 1
10 8 1 1.65 1
2.35 10 16 1 1.65 1
91.6 21.5 53.7 8.1
90.8 28.5 50.9 7.5
91.4 21.4 54.0 8.1
90.8 29.4 50.7 7.5
(0.5) (0.3) (0.2) (0.1)
(0.3) (0.9) (0.8) (0.2)
(0.4) (0.2) (0.3) (0.1)
(0.5) (1.2) (0.4) (0.2)
‘Norseamink@, Norsildmel, Minde, Norway. *CPSP@; CTPP, Boulogne s/Mer, France. 3The vitamin mixture contained the following (per kg premix); retinyl acetate, lo6 IU; cholecalciferol, 10’ H-l; all-rat-a-tocopheryl acetate, 4000 mg; menadione sodium bisullite, 100 mg; thiamin, 1000 mg; riboflavin, 2500 mg; D-calcium pantothenate, 5000 mg; niacin, 1O4 mg; pyridoxine, 1000 mg; vitamin Bi2, 6 mg; folic acid, 500 mg; biotin, 100 mg; myo-inositol, lo5 mg. 4The mineral mixture contained the following (per kg premix); KCl, 9.0 g; KI, 4 mg; CaHP04-2H20, 50 g; NaCl, 4 g; CUS0,,‘5H20, 0.3 g; ZnS04*H20, 0.4 g; CoSO4-7H20, 2 mg; FeS04-7HZ0, 2 g; MnS04-H20, 0.3 g; CaC03, 2 1.5 g; MgCO,, 12.4 g; NaF, 0.1 g. 5As % of dry matter (s.e.m.).
33.5, cooked starch, 12.6 and raw starch, 5.0. The different diets were named V2 1, V29, M2 1 and M29 according to the source of the added oil and to the lipid content (21 versus 29). V2 1 and V29 as well as M21 and M29 were also named “V” and “M” diets, respectively. All fish were fed the same theoretical energy ration using feeding tables taking into account fish mean biomass and water temperature. In treatments V29 and M29, the feed ration was 12% lower than in treatments V2 1 and M2 1. Daily rations were distributed using automatic belt conveyor feeders. For adjustment of ration, biomass was measured every month and extrapolated every week using a theoretical growth model. Growth was estimated using the daily growth index calculated according to Kim and Kaushik ( 1992), an index derived from the Iwama and Tautz ( 198 1) model: DGI = [ (final weight) ‘I3- (initial weight ) ‘I31loo/number
of days
Ten fish were taken at the beginning of the experiment for body composition
364
J. Arzel et al. / Aquaculture 123 (1994) 361-3 75
analyses. At the end of the experiment live fish per cage, i.e. 15 per treatment, were chosen around the mean of body weight for the same analyses. From each fish, the liver and other viscera as well as a large sample of epiaxial muscle were withdrawn. The muscle sample was a mixture of three sub-samples taken at the level of the adipose fin, the dorsal fin and the anterior part. Each of these samples, as well as the remainder of the fish, was submitted to chemical analysis. Muscle and liver lipids were also analysed for fatty acid composition. Whole body composition was calculated from that of each “compartment” which had been analyzed separately. For analysis, samples of tissues were ground using a meat grinder and a highspeed cutter for whole carcass and muscle or liver, respectively. Viscera analyses were performed on fresh-ground samples because of difficulties of grinding. Freeze-dried samples were reground with a high-speed cutter and the water content was determined after dessication at 105 ‘C for 24 h. The lipid contents were determined using a Soxtec@ (Tecator) . In the case of muscle and liver lipid, a second extraction, according to Folch et al. ( 1957) was performed for FA analysis. The protein content was determined using a Tecator digestion system 40@ and a Kjeltec Auto 1030 analyzer@. Ash content was measured by burning in a mume furnace at 550°C for 12 h. Fatty acids (FAs) were assayed after transesterification according to Morrisson and Smith ( 1964) using a gas chromatograph (Packard model 427) equipped with a capillary column (Carbowax 20 M@) using helium as the carrier gas at constant temperature ( 19OOC). A Ross injector was used. The temperature of injector and detector was 220°C. The FA composition was calculated using an integrator Enica 2 1; heneicosanoic acid (C 2 1:O) was used as an internal standard. Data were analyzed by a two-way analysis of variance to test the effects of both lipid content and lipid quality. In the case of the FAs, one-way was preferred to two-way analysis of variance because FA composition was similar in M21 and M29 diets and different in V21 and V29 diets. Furthermore, for liver only one factor (source) was tested. In case of significant differences, the Newman-Keuls test was used to compare the means. For survival and fatty acid, expressed in %, data were transformed into arc sin ,/.
3. Results The proximate analysis of the diets is given in Table 1 and the FA composition in Table 2. The digestible energy levels were estimated to be 16.7 and 18.8 kJ kg-’ for the low- and high-fat diets, respectively. The FA profile was rather similar in diets M2 1 and M29 which contained mostly lipids of marine origin. Larger differences were apparent between the composition of the M diets and the V21 and V29 diets, V21 being more similar to the M diets than V29. The greatest difference involved the polyunsaturated series: lower contents of n-3 FAs and higher contents of n-6 FAs were apparent in V2 1 and, which were greater, in the
J. Arzel et al. / Aquaculture 123 (1994) 361-375 Table 2 Fatty acid composition of the experimental carefully sampled aliquots) Fatty acid
365
diets, in % by weight of methyl esters (one analysis on
Diets v21
V29
M21
M29
,X saturated
18.5
16.1
20.7
19.4
C monoenes
36.5
35.8
47.0
49.9
18:2n-6 20:2n-6 20:4n-6 In-6
27.8 0.2 0.3 28.2
35.5 0.1 0.2 35.8
3.2 0.3 0.5 4.0
2.7 0.3 0.4 3.4
18:3n-3 18:4n-3 20:4n-3 20:5n-3 22:5n-3 22:6n-3 In-3
1.0 1.8 0.3 6.1 0.6 6.9 16.7
1.0 1.2 0.2 4.1 0.4 4.8 11.7
0.9 3.1 0.6 10.8 1.1 11.8 28.3
0.9 2.9 0.6 10.4 1.1 11.4 27.3
n-3/n-6
0.6
0.3
7.1
8.0
V29 diet than in the M diets. It should be noted that the n-3 FA level was relatively high even in the V diets which contained a high percentage of fish oil derived from fish meal. The sum of the monounsaturated FAs was similar in the V2 1 and V29 diets while it was clearly higher in the M diets than in the V diets. Growth performance, feed conversion, protein efficiency ratio (PER) and productive protein value (PPV= nitrogen retention as 96of intake) are given in Table 3. Survival rate was high in all treatments. A significant (PcO.05) but slight effect of lipid source was observed: survival rate was slightly higher for the V treatment. Growth was significantly improved by lipid level (P
2568 2610 2575 2579
(8) (24) (15) (35)
Final mean weight (g)
‘Mean with standard error of the mean in brackets. *P
Analysis of variance: Dietary lipid level Dietary lipid source Level X source
1621 1605 1614 1594
v21 V29 M21 M29
(7)’ (6) (17) (13)
Initial mean weight (8)
Diet
NS
NS *
97.5 98.8 94.9 96.4
(0.4) (0.6) (1.3) (0.9)
Survival rate (%)
tt NS NS
1.33 1.13 1.31 1.20
NS NS
(0.02) (0.03) (0.04) (0.03) (0.02) (0.03) (0.01) (0.05)
Apparent feed conversion
*
1.77 1.87 1.80 1.85
Daily growth index (%)
Table 3 Survival rate, daily growth index, feed conversion and protein efficiency ratio for fish fed the experimental
NS NS
**
1.40 1.74 1.33 1.65
(0.03) (0.05) (0.08) (0.07)
Protein efftciency ratio
diets
NS NS
n:
24.5 31.4 23.8 28.7
(0.5) (0.8) (1.2) (0.9)
Productive protein value (%)
NS NS
NS
NS
Nfj
D
NS
NS
-
-
-
_
1.34 (0.02)
1.32 (0.03) -2
(E)
-*
NS NS NS
(Z) 7.8 (0.3)
7.4 (0.3)
8.3 (0.3) (Z)
is less than 100%.
(E) 10.2 (0.4)
10.0 (0.4)
8.4 (0.6) (&
Ash
NS NS
*
7::;) 40.3 (1.4)
40.5 (0.8)
47.5 (1.7) :;:z,
Water
NS NS NS
(E)
(&
8.2 (0.3)
(&
(Z)
Protein
NS NS NS
42.9 (1.9) 43.1 (1.5) 44.9 (1.1) 43.5 (1.5) 45.1 (2.1)
Lipid
NS NS NS
0.93 (0.32) 0.81 (0.03) 0.79 (0.01) 0.79 (0.03) 0.76 (0.02)
Ash
NS NS NS
(CK) 1.4 (0.1)
NS
**
NS
$) 74.4 (0.3)
74.3 (0.2) 75.6 (0.2) 75.4 (0.2)
21.7 (0.1) 21.0 (0.1) 20.9 (0.1) 21.0 (0.1) 20.8 (0.1)
Lipid 1.2 (0.1) 1.3 (0.1) 1.4 (0.1)
69.3 (0.5)’ 69.6 (0.3) 68.3 (0.4) 69.2 (0.4) 68.3 (0.3)
Protein
(%)3
NS NS
*
t;::, 15.5 (0.1)
18.1 (0.3) 15.9 (0.2) 15.3 (0.2)
Protein
Composition Water
Water
(%)
% of body weight
Composition
% of body weight
Composition
(O/o)
Liver
Viscera (except liver)
NS
*
NS
(E)
(Z) 4.4 (0.2) 3.9 (0.2)
Lipid
analysis of I5 fish per dietary treatment)
Muscle
‘Standard error. *Missing data. ‘Glycogen not assayed; the sum of components
Analysis of variance Dietary lipid level Dietary lipid source Level X source
M29
M21
V29
v21
Initial
Treatment or diet
Table 4 Proximate analysis of whole muscle, viscera and liver and percentage of viscera and liver (individual
NS
*c
NS
$I,
(Z) 1.34 (0.01) 1.33 (0.01)
Ash
368
J. Arzelet al. /Aquaculture
123 (1994) 361-375
nor that of the interaction levelxsource was apparent. The same tendency was observed in the viscera, but the only significant effect was a lower water content (PC 0.05 ) induced by the high dietary fat level. In the liver, the influence of the tested factors on composition was quite different: dietary lipid quality influenced liver lipid, ash and water content, the vegetable oil inducing lower fat and higher water levels. A significant effect of lipid level on protein content was also observed in this organ (PC 0.05). The fatty acid composition of liver lipids is shown in Table 5 for fish fed diets V29 and M29, while the corresponding data for whole muscle are shown in Table 6 for fish fed the four experimental diets. In ail cases, there was a significant influence of dietary lipid on FA composition of tissues: compared to fish fed diet M29, the liver of fish fed diet V29 had a lower proportion of monounsaturated and n-3 FAs but a higher proportion of saturated and n-6 FAs. The content of total n-6 FA in the V29 group was four times as high as in the M29 group, due mainly to higher deposition of 18:2. But 20:2, which remained at low levels, was 4.5 times as high in the V29 as in the M29 group. 20:3n-6 was at an appreciable level in liver tissue and the relative difference between the V29 and M29 treatments was also significant (PcO.05); the content of 20:3n-6 was about ten times higher in fish fed the V29 diet than the M29 diet. Table 5 Fatty acid composition per dietary treatment) Fatty acid
of liver lipid, in % by weight of methyl ester (individual analyses of 9 samples
Diets
Analysis of variance
V29
M29
1 saturated
21.9 (1.5)
16.6 (1.0)
C monoenes
25.9 (1.3)
39.8 (1.9)
18:2n-6 20:2n-6 20:3n-6 20:4n-6 1 n-6’
11.6 2.1 1.9 3.0 19.4
(0.8) (0.3) (0.1) (0.4) (0.9)
1.8 0.6 0.2 2.2 4.9
(0.1) (0.0) (0.0) (0.2) (0.2)
18:3n-3 18:4n-3 20:4n-3 20:5n-3 22:5n-3 22:6n-3 T n-3
0.4 0.3 1.2 5.8 2.3 22.8 32.8
(0.1) (0.1) (0.3) (0.3) (0.2) (1.2) (1.4)
0.5 0.4 1.7 7.0 4.4 24.7 38.7
(0.0) (0.0) (0.1) (0.3) (0.3) (1.0) (1.1)
n-3/n-6 lP< 0.05. ‘Including 22:5n-6.
1.7 (0.1)
7.9 (0.2)
l
* * * * NS * NS
NS NS *
* NS *
J. Arzel et al. /Aquaculture
369
123 (1994) 361-375
Table 6 Fatty acid composition of whole muscle lipid, in % by weight of the methyl ester (individual of 15 samples per dietary treatment) Fatty acid
analysis
Diets v21
V29
M21
M29
1 saturated
20.9b (0.2)
19.P (0.3)
21.7’ (0.3)
20.6’ (0.1)
C monoenes
39.9b (0.2)
38.6” (0.2)
44.Y (0.3)
45.2a (0.2)
18:2n-6 20:2n-6 20:3n-6 2014~6 1 n-6
12.Ob (0.2) 0.8b (0.0) _*
18.0” (0.5) 1.P (0.1)
4.0’ (0.1) 0.4” (0.0)
3.9” (0.0) 0.4” (0.0)
0.4 (0.0) 13.2b (0.2)
0.4 (0.1) 19.4” (0.5)
0.4 (0.0) 4.8” (0.1)
0.4 (0.0) 4.7c (0.1)
18:3n-3 18:4n-3 20:4n-3 20:5n-3 22:5n-3 22:6n-3 1 n-3
1.1 (0.0) 1.9b (0.0) 1.4b (0.0) 4.6b (0.1) 2.3b (0.0) 14.7b (0.2) 26.0b (0.3)
1.1 (0.0) 1.7c (0.0) 1.2c (0.0) 4.0” (0.1) 1.8’(0.1) 12.8’ (0.2) 22.6” (0.3)
1.1 (0.0) 2.1” (0.0) 1.5” (0.0) 5.9” (0.1) 2.6” (0.0) 15.9” (0.3) 29.2” (0.4)
1.1 (0.0) 2.1” (0.0) 1.6” (0.0) 6.0” (0.1) 2.6” (0.0) 16.0” (0.2) 29.5” (0.2)
n-3/n-6
2.0
1.2
6.1
6.3
Values in the same row with the same superscript letters are not significantly different (P> 0.05). *Not quantified, values less than 0.1%.
The arachidonic acid content was slightly but not significantly influenced by diet. Concerning the n-3 series, the group fed V29 had a significantly lower level than the group fed M29 (PC 0.05), the relative difference amounting to 15%, but the relative difference between groups was not the same for each fatty acid. It was greatest ( - 17%) and significant (PcO.05) for eicosapentaenoic acid (EPA 20:5n-3) while it was lowest ( - 8%) and not significant for docosahexaenoic (DHA 22:6n-3). In the whole muscle, the FA composition was similar in the groups fed M21 and M29, but different from the V2 1 and V29 groups; the results for the V21 group were intermediate between those from the V29 and M2 1 groups (or M29). The influence of a dietary supply of n-6 and n-3 FAs was very pronounced in this tissue. The content of total n-6 FA was about four times higher in the groups fed corn oil than in those fed cod liver oil; the concentration of 20:3n-6 was negligible while that of 18:2 was very high, being the highest of all FAs in the V29 group. The n-3 FA content was lower than that observed in liver tissue. The n-3 FA content was significantly (PcO.05) influenced by dietary treatment. The whole body composition of fish fed the four dietary treatments is shown in Table 7. Significant differences among treatments were found only for water and lipid contents which were significantly influenced by the level of dietary lipid
370
J. Arzel et al. /Aquaculture
123 (1994) 361-375
Table 7 Composition of whole body (calculated from the chemical analysis of the various compartments), samples per dietary treatment Dietary treatment
Water
Protein
Lipid
Initial v21 V29 M21 M29
65.2 66.0 64.1 65.2 64.7
18.4 17.5 18.1 17.9 17.4
15.5 14.5 16.2 15.0 15.9
Analysis of variance: Quantity Quality Quantity x Quality
(0.5) (0.3) (0.2) (0.4) (0.4)
89
NS
NS
NS
*
PI
(0.1) (0.1) (0.1) (0.1) (0.1)
15
Ash (0.4) (0.2) (0.2) (0.4) (0.4)
-1 1.77 (0.03) 1.81 (0.03) 1.72 (0.02)
PI
NS NS
-
‘Missing data.
(PC 0.0 1) , body lipid increasing by 9% while water decreased by 4% in the groups fed high fat compared to the groups fed low fat.
4. Discussion The analyses show that all experimental diets were very rich in lipid, including essential FAs, the levels of which clearly exceeded the requirement’ reported for salmonids (Kanazawa, 1985 ). The V29 diet which contained a high level of corn oil, also contained sufficient EFAs due to lipid supplied by fish meal. Protein level was also above the requirement of this species (Arzel et al., 1992). The similarity of the FA profiles of diets M2 1 and M29 is obviously due to the marine fish oil contained in both diets, while the differences between the FA profiles of diets V2 1 and V29 can be explained by the differences in the relative proportions of vegetable oil and fish meal in these diets. The lack of difference between growth rate and survival of fish fed the “V” or “M” diets seems to indicate that the EFA supply did not induce any adverse effect such as those observed by Takeuchi and Watanabe ( 1979) on rainbow trout fed 3% n-3 PUFA (60% of dietary lipid). In fact the n-3 PUFA of the diets tested varied from 2.9 to 6.8% of diets or 9.5 to 24.3% of lipid. According to Watanabe and Takeuchi ( 1989) and Lochmann and Gatlin ( 1993), the requirements for EFA are dependent on dietary lipid content and should be expressed as a percentage of total dietary lipid. The requirement amounts to about 10% of lipid in rainbow trout, and an excess of EFA exerts adverse effects when fed at four times the requirement level. If the requirement of brown trout is of the same order of magnitude as that of rainbow trout, no detrimental effect was indeed to be expected. The n-6/n-3 imbalance described by Yu and Sinnhuber ( 1976) is unlikely to have exerted an effect in our trial. Strictly speaking, a larger number of experimental diets (with various FA profiles) would have been needed to con-
J. Arzelet al. /Aquaculture 123 (1994) 361-37.5
371
firm this assessment. The significant but rather limited effect of dietary lipid level on growth rate is probably due to the feeding method, a theoretically equal amount of energy being allowed in all cases. Satiation feeding would probably have led to higher energy intake and higher weight gain in the groups fed V29 and M29 compared to the groups fed V2 1 and M2 1. In fact, fish fed to satiation usually regulate their food intake according to dietary energy level in such a way that their energy intake is nearly constant, but very high energy levels, i.e. very high lipid contents, lead to an overconsumption of dietary energy (Cho, 1987). The observed difference in daily growth index (4.8%) could be due to the inaccuracy of estimating the DE rations. The better feed conversion in high-lipid fed groups is presumably a consequence of the higher energy level of these feeds. The better efficiency of protein utilization in high-lipid (29%) compared to low-lipid diets (2 I%), evidenced by both PER and PPV, reflects a sparing effect of protein by energy or by lipid which has been well demonstrated in several species of fish (Takeuchi et al., 1978; Bromley, 1980; Cho and Kaushik, 1985). In carnivorous fish, this effect is particularly marked when lipid is used as an energy source (Lee and Putnam, 1973; Shimeno and Kajiyama, 1980; Kaushik and OlivaTeles, 1986; Watanabe and Takeuchi, 1989). The effect of dietary lipid on the chemical composition of tissues was significant only for muscle and whole body where opposite changes of water and fat were observed. It was not apparent in liver, the lipid content of which was always low (a result similar to that found by Gunstone et al., 1978, in a sample of wild Salmo trutta) and it was not significant in viscera. Neither was an increase observed in viscera proportion in fish fed the high-lipid diets. Thus, high-lipid diets did not induce an increase in visceral fat deposition, which has been established in rainbow trout (Ogino et al., 1976; Watanabe, 1987). This lack of influence can be explained by a tendency of brown trout to remain lean compared to rainbow trout ( Arzel et al., 199 1) and to the isoenergetic feeding plan. More surprising is the significant effect of lipid quality on liver lipid content: lipids of marine origin seem to be more easily stored in the liver than those which are partly of vegetable origin. A rather similar effect was observed in rainbow trout (Corraze, 1993, personal communication). Takeuchi and Watanabe ( 1979 ) found a decrease of liver weight in fish fed excess n-3 EFAs. However, the phenomenon observed in the present study is different and remains unexplained. In liver, a significant effect of dietary lipid level on protein content was also found, this phenomenon was difftault to explain, all the more since glycogen was not assayed and analysis of liver was not complete. The FA composition of body tissues reflects to a large extent that of dietary FAs as shown by the similarity of the FA pattern in the groups fed M2 1 and M29 and the dissimilarity of this pattern in the groups fed V21 and V29, both for dietary lipids and body lipids. However, the composition of tissue lipid does not simply reflect that of dietary lipid. Rather some FAs appear to be maintained within defined ranges which depend on animal species, and on the nature of the tissue being studied. Even if n-6 FAs were largely deposited in muscle of fish fed the V21 and V29 diets, the 20:4n-6 content appeared to remain very constant;
312
J. Arzel et al. / Aquaculture 123 (I 994) 361-3 75
the only n-6 FA deposited in large amounts was 18:2n-6. The lack of conversion of 18:2n-6 to 20:4n-6 is attributable to the inhibition of the 66 desaturase by the excess of PUFAs which could present a feedback effect on desaturation as shown by Brenner ( 197 1) and Urger et al. ( 198 1) in rainbow trout. The n-3 long-chain FAs (20:5n-3 and 22:6n-3) were much less variable in tissues than in diets. A more or less constant FA pattern was observed in muscle and, still more clearly, in liver total lipid. This latter phenomenon is similar to that described in coho salmon by Yu and Sinnhuber ( 198 1), and in rainbow trout by Greene and Selivonchick ( 1990) who wrote that: “This consistent pattern suggests that a physiologically optimum level of long-chain fatty acids is maintained in rainbow trout”. Similar results were also found in Atlantic salmon by Thomassen and Rnrsjnr ( 1989) with addition of various vegetable oils. Such a pattern characteristic of fish metabolism is illustrated by the EPA/DHA ratio which amounts to 0.25 and 0.28 in liver lipid, and 0.3 1 and 0.38 in muscle lipids for the groups fed V29 and M29, respectively, instead of 0.85 and 0.9 1 in the corresponding diets. But differences between tissues were obvious for this pattern; i.e. liver lipid compared to muscle lipid had higher n-3 FA content, lower EPA/DHA ratio and higher 20:4n-6 content. This phenomenon is probably due to the high percentage of phospholipid in liver; phospholipids generally have a higher content in PUFA and a lower short-chain FA/long-chain PUFA ratio than the triacylglycerols (Henderson and Tocher, 1987). The analysis of n-3 FAs such as 18:3, 18:4 and 20:4 did not reveal any effect of treatment on elongation of 18:3n-3 which was certainly low in all cases. In rainbow trout, this elongation was found during n-3 PUFA deficiency (Owen et al., 1975; Yu and Sinnhuber, 1976) but, using diets high in n-3 PUFA, Greene and Selivonchick ( 1990) did not observe any bioconversion of 18:3 into long chain n-3 PUFA. Furthermore, Greene and Selivonchik ( 1987) insisted on the fact that salmonids such as chum, coho and chinook salmon did not desaturate and elongate 18:3n-3 as efficiently as rainbow trout. Regarding the n-6 FAs, several authors have found large deposition of 18:2n-6 in salmonids fed diets rich in vegetable oil, comparable to the observations made in the present study, but bioconversion of 18:2n-6 into 20:4n-6 was effective only in the case of n-3 PUFA deliciency (Henderson and Tocher, 1987 ) . In the present study, a significantly higher level of 20:2n-6 was observed in fish fed the V2 1 and V29 diets, compared to the M21 and M29 diets in muscle, while in liver the same significant difference was observed for the fish fed V29 and M29 (P< 0.05). 20:3n-6 was also more concentrated in liver lipid of groups fed the V29 diet compared to those fed the M29 diet. Therefore, some elongation of 18:2n-6 occurred in both tissues, while the products of desaturation by 46 desaturase could be seen only in liver, a phenomenon described in the rat by Brenner ( 197 1). These results can be interpreted as an inhibition of bioconversion of 18-carbon precursors to long-chain PUFA by a large dietary supply of n-3 PUFA (LRger et al., 1981). Further analyses performed on phospholipids and other lipid fractions will help explain the influence of diet on lipid metabolism in brown trout. In conclusion, the present study showed that a large excess of n-3 PUFA appar-
J. Arzel et al. /Aquaculture 123 (1994) 361-375
313
ently does not induce any detrimental effect on growth, feed conversion, body composition or health of seawater-reared brown trout, at least under the conditions of this trial. The incorporation of a large quantity of vegetable oil was shown to significantly affect the organoleptic quality of flesh: smoked fillets of V-fed fish displayed a poorer flesh cohesion and a higher in-mouth fatty texture (Arzel et al., 1993). But it did not influence growth performance or feed utilization. It also clearly modified body lipid FA pattern in a manner rather similar to that described in other salmonids. Due to the shortness of the experiment, no conclusion can be drawn on the effects of the addition of vegetable oil on the health of brown trout; phenomena such as those described in Atlantic salmon by Bell et al. ( 199 1)) i.e. cardiac lesions under stressing conditions, possibly due to increased synthesis of eicosanoids, remain to be studied in brown trout in conditions different from those of this trial.
Acknowledgments
The authors would like to express their gratitude to the technicians of the Salmoniculture Experimentale IFREMER-INRA at Sizun and Camaret, mainly Mr. Luc Lebrun and Mr. HervC Le Delliou of the IFREMER Laboratoire de Nutrition at Brest for their technical assistance, as well as Dr. S.J. Kaushik of the INRA Laboratoire de Nutrition des Poissons at Saint-Pee-sur-Nivelle for careful reading of the manuscript.
References Arzel, J. and Guillaume, J., 1994. Donnees sur la nutrition et l’alimentation de la truite fario. Piscicult. Fr. (in press). Arzel, J., Metailler, R. and Guillaume, J., 1991. The relative efficiency of a combination of 2 protein and 2 lipid levels during the growth of brown trout (Salmo trutta) in different conditions. Communication CIEM, Comite Mariculture, F:5. La Rochelle, France, October 199 1, 17 pp. Arzel, J., Mttailler, R., Huelvan, C., Faurt, A. and Guillaume, J., 1992. The specific nutritional requirements of brown trout (Salmo tnrtta) . Blivisindi (Iceland Agr. Sci. ), 6: 77-92. Arzel, J., Cardinal, M., Comet, J., Mttailler, R., Sttphan, G. and Guillaume, J., 1993. Nutrition of brown trout (Salmo trutta) reared in seawater. Effect of dietary lipid on growth performances, body composition and fillet quality. World Aquaculture Conference, Torremolinos, Spain, 26-28 May 1993 (poster). Bell, J.G., McVicar, A.H., Park, M.T. and Sargent, J.R., 1991. High dietary linoleic acid affects the fatty acid composition of individual phospholipids from tissues of Atlantic salmon (Salmo salar): association with stress susceptibility and cardiac lesion. J. Nutr., 12 1: 1163- 1172. Brenner, R.R., 197 1. The desaturation step in the animal biosynthesis of polyunsaturated fatty acid. Lipids, 6: 561-575. Bromley, P.J., 1980. Effect of dietary protein, lipid and energy content on the growth of turbot (Scophthalmus maximus L.). Aquaculture, 19: 359-369. Cho, C.Y., 1987. La energia en la nutrition de 10s peces. In: M. de la Higuera (Editor), Nutrition en Acuicultura, II. J. Espinosa de 10s Monteros y U. Labarta (Publishers), Madrid, pp. 197-243.
374
J. Arzel et al. /Aquaculture 123 (1994) 361-375
Cho, C.Y. and Kaushik, S.J., 1985. Effects of protein intake on metabolizable and net energy values of fish diets. In: C.B. Cowey, A.M. Mackie and J.G. Bell (Editors), Nutrition and Feeding in Fish. Academic Press, London, pp. 95- 117. Folch, J., Lees, N. and Sloane Stanley, G.H., 1957. A simple method for the isolation and purification of total lipid from animal tissues. J. Biol. Chem., 226: 497-509. Gabaudan, J., Metailler, R. and Guillaume, J., 1989. Nutrition comparee de la truite arc-en-ciel (Oncorhynchus mykiss), de la truite fario (Salmo trutta) et du saumon coho (Oncorhynchus kisutch). Effet des taux de proteines totales et de lipides. Communication CIEM, Comite Mariculture, F:5. The Hague, Netherlands, October 1989,19 pp. Greene, D.H.S. and Selivonchick, D.P., 1987. Lipid metabolism in fish. Prog. Lipid Res., 26: 58-85. Greene, D.H.S. and Selivonchick, D.P., 1990. Effects of dietary vegetable, animal and marine lipids on muscle lipid and hematology of rainbow trout (Oncorhynchus mykiss). Aquaculture, 89: 165182. Gunstone, F.D., Wijesundera, R.C. and Scrimgeour, C.M., 1978. The component acids of lipids from marine and freshwater species with special reference to furan-containing acids. J. Sci. Food Agric., 29: 539-550. Henderson, R.J. and Tocher, D.R., 1987. The lipid composition and biochemistry of freshwater fish. Prog. Lipid. Res., 26: 281-347. Iwama, G.K. and Tautz, A.F., 1981. A simple growth model for salmonids in hatcheries. Can. J. Fish. Aquat. Sci., 38: 649-656. Kanazawa, A., 1985. Essential fatty acid and lipid requirement of fish. In: C.B. Cowey, A.M. Mackie and J.G. Bell (Editors), Nutrition and Feeding in Fish. Academic Press, London, pp. 281-298. Kaushik, S.J. and Oliva-Teles, A., 1986. Effect of digestible energy on nitrogen and energy balance in rainbow trout. Aquaculture, 50: 89-l 0 1. Kim, J.D. and Kaushik, S.J., 1992. Contribution of digestible energy from carbohydrates and estimation of protein/energy requirements for growth of rainbow trout (Oncorhynchus mykiss). Aquaculture, 106: 161-169. Lee, D.J. and Putnam, G.B., 1973. The response of rainbow trout to varying protein/energy ratio in a test diet. J. Nutr., 103: 9 16-922. LRger, C., Fremont, L. and Boudon, M., 198 1. Fatty acid composition of lipids in the trout. I. Influence of dietary fatty acids on the triglyceride fatty acid desaturation in serum, adipose tissue, liver, white and red muscle. Comp. Biochem. Physiol., 69B: 99-105. Lochmann, R.T. and Gatlin, D.M., III, 1993. Evaluation of different types and levels of triglycerides, singly and in combination with different levels of n-3 highly unsaturated fatty acid ethyl esters in diets of juvenile red drum Sciaenops ocellatus. Aquaculture, 114: 113-l 30. Morrisson, W.R. and Smith, L.M., 1964. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol. J. Lipid Res., 5: 600-608. Ogino, C., Chiou, J.Y. and Takeuchi, T., 1976. Protein nutrition in fish. VI. Effects of dietary energy sources on the utilization of protein by rainbow trout and carp. Bull. Jpn. Sot. Sci. Fish., 46: 385388. Owen, J.M., Adron, J.W., Middleton, C. and Cowey, C.B., 1975. Elongation and desaturation of dietary fatty acids in turbot Scophthalmus maximus L. and rainbow trout Salmo gairdneri Richardson. Lipids, 10: 528-531. Phillips, A.M., Jr. and Hammer, G.L., 1965. Modified pelleted dry mixtures as complete foods for brown trout. Fish. Res. Bull., N.Y., 28: 23-28. Phillips, A.M., Jr., Livingston, D.L. and Poston, H.A., 1965. The effect of three supplemental fats on the growth rate and body chemistry of brown trout. Fish. Res. Bull., N.Y., 28: 28-34. Shimeno, S. and Kajiyama, H., 1980. Effects of calorie to protein ratios in formulated diets on the growth, feed conversion and body composition of young yellowtail. Bull. Jpn. Sot. Sci. Fish., 46: 1083-1087. Takeuchi, T. and Watanabe, T., 1979. Effect of excess amounts of essential fatty acids on growth of rainbow trout. Bull. Jpn. Sot. Sci. Fish., 45: 15 17-l 5 19. Takeuchi, T., Yokoyama, M., Watanabe, T. and Ogino C., 1978. Optimum ratio of dietary energy to protein for rainbow trout. Bull. Jpn. Sot. Sci. Fish., 44: 729-732.
J. Atzelet al. IAqwcultute
123 (1994) 361-375
375
Thomassen, M.Y. and Rosjo, C., 1989. Different fats in feed for salmon: influence on sensory parameters, growth rate and fatty acids in muscle and heart. Aquaculture, 79: 129-135. Watanabe, T., 1987. Requerimientos de acidos grasos y nutrition lipidica en 10s peces. In: M. de la Higuera (Editor), Nutrition en Acuicultura, II. J. Espinosa de 10s Monteros y U. Labarta (Publishers), Madrid, pp. 99-165. Watanabe, T. and Takeuchi, T., 1989. Implications of marine oils and lipids in aquaculture. In: R.G. A&man (Editor), Marine Biogenic Lipids, Fats and Oils, Vol. II. CRC Press, Boca Raton, FL, pp. 457-479. Yu, T.L. and Sinnhuber, R.O., 1976. Growth response of rainbow trout (Salmo gait&w-i) to dietary w3 and 06 fatty acids. Aquaculture, 8: 309-3 17. Yu, T.C. and Sinnhuber, R.O., 198 1. Use of beef tallow as an energy source in coho salmon (Oncothynchus kisutch) rations. J. Fish. Res. Board Can., 38: 367-370.