Dietary carbohydrate, iron and zinc interactions in Atlantic salmon (Salmo salar)

Aquaculture 219 (2003) 597 – 611 www.elsevier.com/locate/aqua-online

Dietary carbohydrate, iron and zinc interactions in Atlantic salmon (Salmo salar) Bente Vangen, Gro-Ingunn Hemre* Institute of Nutrition, Directorate of Fisheries, P.O. Box 185, N-5804 Bergen, Norway Received 17 January 2002; received in revised form 2 May 2002; accepted 2 May 2002

Abstract Atlantic salmon (initial weight 312 g) were fed four experimental diets containing either low (5%) or high (10%) levels of wheat starch, combined with either a low (6 mg kg
1) or high (30 mg kg
1) iron and zinc level, in order to elucidate interactions between dietary starch and iron plus zinc. No difference in specific growth rate or protein productive value as a consequence of the variations in dietary starch was observed. Protein efficiency ratio was significantly lower in fish fed with 5% compared to 10% starch diets, indicating higher deposition of nonprotein components as a consequence of the higher starch level. Dietary iron plus zinc significantly influenced final weight, condition factor and hepatosomatic index, with lower values in fish fed with higher mineral levels. The present dietary manipulations did not affect blood haematology, with all values being within normal ranges for Atlantic salmon. Plasma levels of glucose, protein, cholesterol or triacylglycerols were not affected by the dietary treatments. Low activities of plasma aspartate amino transferase and alanine amino transferase indicated, along with no mortality, no negative effects on liver function or fish health due to dietary treatments. Glycogen levels in whole body, liver and spleen were increased in fish fed with 10% compared to 5% starch diets. Liver iron concentration was affected by both dietary starch and mineral levels. Whole body homogenates showed significantly higher zinc levels in fish fed with higher iron and zinc diets, whereas plasma zinc levels were affected by dietary starch, iron and zinc levels, which showed a significant interaction effect. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Atlantic salmon; Diet; Carbohydrate; Zinc; Iron; Growth; Organ nutrient concentrations; Haematology

1. Introduction No dietary requirement for carbohydrate has been demonstrated in fish; however, certain species exhibit reduced growth rates when fed with carbohydrate-free diets (Hilton *

Corresponding author. Tel.: +47-5523-8000; fax: +47-5523-8095. E-mail address: [email protected] (G.-I. Hemre).

0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 ( 0 2 ) 0 0 2 0 6 - 5

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and Hodson, 1983; Wilson, 1994; Hemre et al., in press). The optimal level of dietary carbohydrate varies with species and has been reported to range from 7% to 20% for Atlantic salmon and other carnivorous fish (Furuichi and Yone, 1982; Shimeno and Mommsen, 1991; Hemre et al., in press). For Atlantic salmon, there was no difference in growth when diets ranged from 5% to 20% starch, whereas feed utilisation and digestibility were reduced when levels exceeded 10% (Hemre et al., 1995a). In animal tissue, iron is predominantly found in compounds containing a porphyrin nucleus (Coultate, 1996). Oxygen is bound to porphyrin – iron-containing molecules either in haemoglobin (Hb) or in myoglobin (Mb). In severe anaemia, iron deficient erythropoiesis may develop and result in reduced haematocrit (Hct) and blood Hb concentrations. Haematological analysis has commonly been used to detect iron deficiency in fish (Kawatsu, 1972; Sakamoto and Yone, 1978; Gatlin and Wilson, 1986; Andersen et al., 1996). When feeding high starch diets to Atlantic salmon, Hct and Hb concentrations decrease, speculated to be an interaction effect between starch and iron (Hemre et al., 1995b). Aerobic metabolism depends on iron because of its role in the functional groups of most enzymes of the Krebs cycle as an electron carrier in cytochromes. Also, the cytochrome P450 family of enzymes depends on iron as an essential element (Nebert and Gonzalez, 1987). Zinc is an essential trace element for fish, present in all organs, tissues and fluids, acting as a stabiliser of membranes and cellular components (Chvapil, 1973); however, there is no specific zinc stored in the body (King and Keen, 1994). Zinc is a constituent of a great number of zinc-dependent enzymes participating in synthesis and degradation of carbohydrates and lipids (Lall, 1989; Sandstrøm, 1997). There is also a strong relationship between zinc and insulin, where zinc takes part in the synthesis, secretion and activity of insulin (Coulston and Nandona, 1980; May and Contoreggi, 1982; Faure et al., 1992). Zinc deficiency in fish leads to growth retardation, which is found to be especially pronounced if other minerals are also lacking (Hughes, 1985; Lall, 1989). In zinc-deficient rainbow trout fry, Ogino and Yang (1978) found severe reduction in the zinc content of the vertebrae, while iron content of the various tissues was increased, indicating an interaction between zinc and iron. Inadequate zinc supply may also result in impaired digestibility of protein and carbohydrates, increased moisture content and depressed protein and lipid concentrations (Ogino and Yang, 1978; Satoh et al., 1987). Aiming to elucidate if small variations in dietary starch influenced the utilisation and retention of iron and zinc, Atlantic salmon were fed with diets containing either 5% or 10% starch, combined with either low or high levels of iron plus zinc. As a primary response, parameters of growth, feed utilisation and protein retention were recorded. Thereafter, several organs were analysed for concentrations of macronutrients, iron and zinc. Haematological parameters, plasma nutrient concentrations and plasma enzyme activities were also recorded.

2. Materials and methods 2.1. Experimental diets Four experimental diets were formulated using either 10% or 5% suprex wheat starch (Condrico, the Netherlands), combined with either low (6 mg kg
1 each) or high (30 mg

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599

kg
1 each) levels of iron plus zinc. Groups HchoLM and HchoHM describe the high starch plus low or high mineral additions, respectively and groups LchoLM and LchoHM describe the low starch plus low or high mineral additions, respectively. The two carbohydrate levels were counterbalanced by dietary protein by weight. All diets were isoenergetic. Greater silver smelt (Argentina silus) constituted the protein source together with frozen squid mantle (Loligo pealeii). A small portion of coalfish (Pollachius virens) was added to the low carbohydrate diet to balance the protein content and a small portion of marine fish oil was added to the high carbohydrate diet to balance the lipid content. The minerals, iron and zinc, were added in crystalline form, iron(II) sulphate heptahydrate and zinc sulphate heptahydrate. Dry ingredients were thoroughly mixed before adding them to the homogenised wet ingredients (coalfish, squid and oil), and further mixed and homogenised using a Laska High-Speed Cutter (Falcon Food Equipment, Wellsroad, Halltrow, Bristol, Great Britain). The diets were then coagulated through heating at 80 jC Table 1 Ingredients, proximate composition and Zn and Fe concentrations in experimental diets Experimental diets LchoHM

LchoLM

HchoHM

HchoLM

36 699 84 175 – 3 3

36 699 84 175 – 3 3

83 811 96 – 2.4 3 3

83 811 96 – 2.4 3 3


1

Ingredients (g kg ) Wheat suprex Greater silver smelt Squid mantle Coalfish Marine fish oil Vitamin mixa Mineral mixa Minerals supplemented Iron(II) (mg kg
1)b Zinc(II) (mg kg
1)b

30 34

Proximate analyses (g kg
1) Starch Crude protein Dry matter Crude lipid Residue (%)c

55 530 585 287 7.5

Minerals analysed Iron (mg kg
1) Zinc (mg kg
1)

164 146

Ca2+ (g kg
1) PO42
(g kg
1)

16.7 13.7

6.0 6.8

51 518 583 293 7.7

73 55 17.5 12.2

34 39

104 439 591 286 6.0

140 136 14.8 11.4

6.0 6.8

106 443 624 279 6.1

61 48 13.4 10.3

Lcho and Hcho denote lower and higher dietary starch levels, while LM and HM denote lower and higher iron and zinc levels. a Vitamins and minerals (exclusive iron and zinc) were added according to NRC’s (1993) recommendations. b Iron and zinc were added as FeSO47H2O and ZnSO47H2O, respectively. c Ash plus fibre were calculated as residue (%).

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(1850 MHz in a microwave oven) before pelleting. Detailed composition of diets is given in Table 1. 2.2. Fish trial The fish experiment was carried out at Matre Aquaculture Reasearch Station, Matredal, Norway, and lasted for 8 weeks. Six hundred fish, with mean weight 312 g, were randomly and equally distributed into 12 tanks (1.51.5 m). Each diet was fed to triplicate tanks using ambient temperature and natural light regime from mid August to mid October. Water temperature declined from 15.5 in August to 13.5 jC in October when the experiment ended. Salinity increased from 21 to 28 gl
l during the experimental period. The feeding was by automatic feeders supplied with manual control. Feeding hours were during the light period. The experimental approach was using restricted feeding, adjusted after the least-eating fish tank. Feed wastes were collected, freeze-dried and weighed for each tank throughout the experiment. 2.3. Sampling procedure Initially, all fish in the experimental tanks were anaesthetised with metomidate (10 ml10 l
1), individually weighed and fork length measured. Twenty fish were randomly collected for analysis. Ten fish were pooled and used for whole body analysis. Blood was withdrawn from the caudal vessels of another 10 fish. Haematology was immediately performed, plasma separated at 3000g and stored at
80 jC until further analysis. The same fish were then killed with a sharp blow to the head, and dissected for pooled samples of liver, muscle and spleen. The collected organs were stored frozen at
20 jC until analysed. After 8 weeks of feeding, the fish were starved for 24 h prior to sampling. The procedure from the initial sampling was repeated, except during the final sampling; 30 fish from each tank were randomly collected for analysis using a pooled sample of 10 fish for whole body analyses. The remaining 20 fish were used for samples of blood, liver, muscle, spleen and vertebra. 2.4. Analytical procedures Diet, whole body and tissue samples were freeze-dried and homogenised prior to analyses. Diets and vertebrae were homogenised and pulverised using an ultracentrifugal mill (Retch Ultracentrifugalkva¨rn ZM100, Pihl, Bergen, Norway). Before dissection, vertebrae were heated for 2– 3 min in a microwave oven; thereafter, remaining muscles and blood vessels were thoroughly removed. In this procedure, deionised distilled water was used to rinse the vertebrae. Haematocrit (Hct), haemoglobin (Hb) and red blood cell count (Rbc) were determined in heparinised blood as described by Sandnes et al. (1988). Digestible carbohydrates in the diet and glycogen in the liver, muscle, spleen and whole body homogenates were analysed using an enzymatic method modified from Murat and Serfaty (1974) and Holm et al. (1986), as described by Hemre et al. (1989). Plasma samples were analysed by means of a Technicon RA1000 analyser. Technicon method No. SM4-0143K85 was used for plasma glucose determination, plasma protein

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was analysed according to the Biuret method, standardised for the RA1000 (Technicon method No. SM4-0147K82). Plasma total lipid, cholesterol levels and liver dehydrogenase complex (LDH) activity were quantified according to Technicon method No. SM40173H88, No. SM4-0139K82 and No. SM4-0145K82, respectively. The methods described by Sandnes et al. (1988) were used to determine plasma aspartate aminotransferase (ASAT) and plasma alanine aminotransferase (ALAT) activity. All the components were measured spectrophotometrically at 340 nm. The standard material SETpoint Chemical Calibrator (Technicon Instruments, Tarrytown, NY, USA) was used to ensure the quality of the analyses, and was treated with the samples throughout the analyses. Dried samples of diets, whole body and homogenised tissues determined for iron and zinc concentrations, and calcium and phosphorous analyses in diet only were digested in a mixture of 2 ml 65% nitric acid (Suprapure, Merck) and 0.5 ml 30% H2O2 (Perhydrol, Merck) using a Milstone MLS 1200 microwave oven. Concentrations of iron, zinc and calcium in diets, and iron and zinc in tissue homogenates were determined by flame atomic absorption spectroscopy (AAS) (Perkin Elmer 3300). Phosphorous concentration in diet was determined by graphite furnace (GF AAS) (PE4110ZL equipped with Zeeman background correction). Standard reference materials (TORT-2; Lobster – Hepatopancreas – Homard, Institute of Environmental Chemistry, Ottawa, Canada) were used to ensure the quality of the analyses, and was treated together with the samples in all series throughout the analyses. Total zinc concentrations in plasma were determined by flame atomic absorption spectrometry, as described for element analyses of tissues. The standard reference material Seronorm (Trace Elements Serum, Nycomed Pharma, Oslo, Norway) was used to ensure the quality of the analyses, and was treated together with the samples throughout the analyses. 2.5. Calculations Specific growth rate (SGR), ln(W1
W2)/(T2
T1)100; SGRð%Þ ¼

  lnW2
lnW1 e
1  100 T2
T1

Protein efficiency ratio (PER), (W2
W1)/g protein intake; protein productive value (PPV), g protein growth/g protein intake; W1 ¼ initial weight,

W2 ¼ final weight,

ðT2
T1 Þ ¼ duration of the experiment in days feed conversion ratio (FCR), g feed intake/(W2
W1); Condition factor, (weight, g/(length, cm)3)100; Hepatosomatic index (HSI), (liver weight, g/live weight, g)100; Mean cell volume (MCV), Mean cell haemoglobin (MCH) and Mean cell haemoglobin concentration (MCHC) were calculated according to Sandnes et al. (1988).

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2.6. Statistics Statistical analyses were carried out using Statistica 4.5 (Statsoft, USA 1993). Each experimental group was run in triplicate, resulting in n=3 for each dietary treatment. The data were examined using a two-way ANOVA to find significant differences among dietary groups. A Tukey honest significant difference (HSD) test was used to rank means when dietary groups differed significantly. Differences among groups were considered to be statistically different when P was <0.05. Normal distribution was checked using normal plot of within-cell residuals. Homogeneity of variances was tested using Levene’s test (ANOVA on absolute variances), and was considered homogenous when P was > 0.05. In some few cases, the normal distribution or homogeneity of variances was not completely fulfilled. For these variables, it was assumed that the populations sampled were normally distributed and had homogenous variances (the central limit theorem). Because none of these data showed extreme deviations from the limits set for significance, they were treated in the same manner as the other results.

3. Results No mortality or incidents of cataract were observed during the experiment. Final weights were significantly ( P<0.05) higher, and CF and HSI significantly lower ( P<0.05) in fish fed with diets low in iron and zinc compared to fish fed with diets high in iron and zinc, while a variation in starch level from 5% to 10% did not influence growth (Table 2). No difference could be detected in specific growth rate. Feed consumption was equal for all tanks, reminding on the restricted feeding regime with all leftover feed sampled and measured gravimetrically. Protein utilisation, measured as total growth towards protein intake (PER), was highly improved ( P<0.05) by the higher carbohydrate/lower protein Table 2 Weight (w/w), specific growth rate (SGR), condition factor, feed conversion, PER (protein efficiency ratio), PPV (protein productive value) and HSI (hepatosomatic index) of Atlantic salmon fed four experimental diets for 8 weeks; low carbohydrate high Zn+Fe (LchoHM), low carbohydrate plus low Zn+Fe (LchoLM), high carbohydrate plus high Zn+Fe (HchoHM) and high carbohydrate plus low Zn+Fe (HchoLM) Diet

Initial weight (g) Final weight (g) SGR (%) Condition factor (CF) Feed conversion ratio (FCR) PER PPV HSI (%)

ANOVA

LchoHM

LchoLM

HchoHM

HchoLM

CHO

Me2+ CHOMe2+

312(13) 444(10)a 0.61(0.09) 1.17(0.01)a 0.75(0.09) 2.49(0.01)a 0.49(0.06) 0.59(0.02)a

316(0.4) 450(9.4)b 0.61(0.03) 1.20(0.02)b 0.75(0.08) 2.60(0.01)a 0.45(0.03) 0.61(0.02)b

310(3.5) 431(4.8)a 0.57(0.02) 1.18(0.02)a 0.83(0.07) 2.76(0.00)b 0.47(0.04) 0.59(0.01)a

312(6.2) 450(5.2)b 0.63(0.04) 1.20(0.01)b 0.73(0.04) 3.11(0.00)b 0.55(0.05) 0.62(0.02)b

n.s. n.s. n.s. n.s. n.s. <0.05 n.s. n.s.

n.s. <0.05 n.s. <0.05 n.s. n.s. n.s. <0.05

n.s. n.s. n.s. n.s. n.s. n.s. 0.05 n.s.

Initial values: CF=1.13(0.02), HSI=0.95(0.11). Me2+=divalent ions (iron and zinc), CHO=carbohydrate, CHOMe2+=interaction between carbohydrate and divalent ions. Results are means from three tanks with their standard deviation given in parenthesis (n.s.=not significant). Means in the same row not sharing a common superscript are significantly different.

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level, with no effect from the variable zinc plus iron concentrations, while protein retention (PPV) was not affected by the present dietary treatments. Glycogen stored in whole body, liver and spleen homogenates differed significantly ( P<0.05 and less) between the groups fed with 5% and 10% dietary starch (Table 3). Liver glycogen was also significantly affected ( P<0.05) by a coupled effect of starch and iron plus zinc levels in the diet (Table 3). Varying levels of the divalent metal ions (Fe2+, Zn2+) in the diet gave significant responses in whole body, liver and plasma mineral concentrations. Zinc concentration in whole body homogenates was significantly ( P<0.001) increased in fish fed with diets high in zinc plus iron. Liver iron concentration was affected by both starch ( P<0.005) and iron plus zinc ( P<0.05) levels in the diet. Iron concentration in liver increased with increasing

Table 3 Glycogen (g kg
1 dw), iron (mg kg
1 dw) and zinc (mg kg
1 dw) concentrations in whole body, muscle, liver, spleen and vertebrae and plasma zinc concentration (mg l
1) of Atlantic salmon fed four experimental diets for 8 weeks; low carbohydrate high Zn+Fe (LchoHM), low carbohydrate plus low Zn+Fe (LchoLM), high carbohydrate plus high Zn+Fe (HchoHM) and high carbohydrate plus low Zn+Fe (HchoLM) Initial

Dietary groups

ANOVA

LchoHM

LchoLM

HchoHM

HchoLM

CHO

Me2+

CHOMe2+

Whole body Glycogen 1.2(0.1) Iron 38(3) Zinc 130(7)

4.6(0.6)a 58(9) 122(11)a

5.3(0.0)a 49(10) 103(1)b

7.2(0.2)b 48(3) 129(2)a

7.6(1.2)b 44(4) 107(4)b

<10
3 n.s. n.s.

n.s. n.s. <10
3

n.s. n.s. n.s.

Muscle Glycogen Iron Zinc

1.4(0.2) 11(4) 22(2)

5.9(1.0) 12.1(1.1) 22.0(3.4)

5.8(0.0) 11.4(0.6) 20.3(1.6)

6.7(0.6) 11.9(0.1) 23.5(3.6)

5.9(0.6) 11.1(0.7) 20.4(2.6)

n.s. n.s. n.s.

n.s. n.s. n.s.

n.s. n.s. n.s.

Liver Glycogen Iron Zinc

8.6(0.1) 330(20) 112(9)

74(12)a 282(39)a 165(48)

101(10)a 212(30)ab 156(31)

189(15)b 194(24)b 164(43)

184(8)b 156(26)b 168(52)

<10
6 <0.005 n.s.

n.s. <0.05 n.s.

<0.05 n.s. n.s.

Spleen Glycogen Iron Zinc

0.4(0.1) 910(60) 120(20)

1.3(0.51)a 1021(150) 131(61)

1.1(0.38)a 891(109) 142(66)

2.4(1.3)b 890(75) 158(73)

2.9(1.1)b 810(85) 183(84)

<0.05 n.s. n.s.

n.s. n.s. n.s.

n.s. n.s. n.s.

Vertebra Iron Zinc

nm nm

12.2(0.06) 73(10)

12.0(0.5) 57(9)

12.3(0.7) 73(12)

12.4(1.2) 71(5)

n.s. n.s.

n.s. n.s.

n.s. n.s.

Plasma Zinc

18(4)

24.8(0.1)a

19.1(0.05)b

26.8(0.1)c

19.7(0.06)d

<0.001

<0.001

<0.001

CHO=carbohydrate, Me =divalent ions (iron and zinc), CHOMe2+=interaction between carbohydrate and divalent ions, nm=not measured. Results are means from three tanks with their standard deviation given in parenthesis (n.s.=not significant). Means in the same row not sharing a common superscript are significantly different. 2+

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Table 4 Haematocrit (Hct), haemoglobin (Hb), red blood cell count (Rbc), mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC) in Atlantic salmon fed four experimental diets differing in carbohydrate, iron and zinc levels for 8 weeks: low carbohydrate high Zn+Fe (LchoHM), low carbohydrate plus low Zn+Fe (LchoLM), high carbohydrate plus high Zn+Fe (HchoHM) and high carbohydrate plus low Zn+Fe (HchoLM) Initial

Hct (%) Hb (g 100 ml
1) Rbc (1012 l
1) MCV (10
15 l
1) MCH (10
6 l
1) MCHC (g 100 ml
1)

45(4) 8(1) 1.2(0.1) 392(6) 69(5) 18(1)

Dietary groups

ANOVA

LchoHM

LchoLM

HchoHM

HchoLM

CHO

Me2+

CHOMe2+

42(1) 7.2(0.2) 1.1(0.1) 362(2) 63(0) 17(1)

40(4) 7.1(0.2) 1.2(0.0) 350(40) 62(2) 18(2)

42(2) 7.1(0.3) 1.1(0.0) 372(9) 62(1) 17(0)

41(2) 6.9(0.2) 1.2(0.1) 360(20) 60(2) 17(0)

n.s. n.s. n.s. n.s. n.s. n.s.

n.s. n.s. n.s. n.s. n.s. n.s.

n.s. n.s. n.s. n.s. n.s. n.s.

CHO=carbohydrate, Me2+=divalent ions (iron and zinc), CHOMe2+=interaction between carbohydrate and divalent ions. Results are means from three tanks with their standard deviation given in parenthesis (n.s.=not significant).

iron level in the diet. In addition, groups fed with high starch diet showed reduced liver iron concentrations ( P<0.05) compared to groups fed with low starch diet. Plasma zinc concentration responded to dietary zinc treatment and differed significantly among all groups ( P<0.001). Also, an interaction among carbohydrate and iron and zinc was detected (Table 3). Plasma zinc concentration increased with increasing dietary zinc levels ( P<0.05) and with increasing dietary carbohydrate ( P<0.05). Neither glycogen iron nor zinc levels in muscle nor vertebra were significantly affected by the dietary treatments. No statistical differences were found between dietary treatments with respect to Hct, Hb, Rbc concentrations or the indices MCV, MCH and MCHC (Table 4). Plasma nutrient (glucose, total protein, triacylglycerols, cholesterol) concentrations were all in the lower range of normal values for Atlantic salmon at final sampling, and Table 5 Plasma concentrations of glucose, total protein, triacylglycerides, cholesterol, ASAT (aspartate amino transferase), ALAT (alanine amino transferase) and LDH (lactate dehydrogenase) in Atlantic salmon fed four experimental diets differing in carbohydrate, iron and zinc level for 8 weeks: low carbohydrate high Zn+Fe (LchoHM), low carbohydrate plus low Zn+Fe (LchoLM), high carbohydrate plus high Zn+Fe (HchoHM) and high carbohydrate plus low Zn+Fe (HchoLM) Initial

Glucose (mM) Total protein (g l
1) Triacylglycerol (mM) Cholesterol (Amol l
1) ASAT (U l
1) ALAT (U l
1) LDH (U l
1)103

1.3(0.4) 9.5(1.4) 0.3(0.2) 1.3(0.2) 37(9) 7(3) 398 (239)

Dietary groups

ANOVA

LchoHM

LchoLM HchoHM HchoLM

CHO Me2+ CHOMe2+

5.4(0.1) 55.4(4.3) 3.60(0.74) 10.8(1.1) 227(19) 46(8) 5.2 (1.7)

6.9(3.2) 55.4(1.7) 5.0(2.0) 14.7(5.7) 271(110) 48(15) 5.0(1.8)

n.s. n.s. n.s. n.s. n.s. n.s. n.s.

8.8(4.7) 57.3(1.1) 5.7(1.8) 14.2(6.1) 308(137) 58(25) 4.8(1.1)

7.3(2.6) 63(17) 4.67(0.30) 12.3(3.5) 234(62) 41(2) 3.4(0.9)

n.s. n.s. n.s. n.s. n.s. n.s. n.s.

n.s. n.s. n.s. n.s. n.s. n.s. n.s.

CHO=carbohydrate, Me2+=divalent ions (iron and zinc), CHOMe2+=interaction between carbohydrate and divalent ions. Results are means from three tanks with their standard deviation given in parenthesis (n.s.=not significant).

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none changed as a consequence of the present dietary treatments (Table 5). All initial values were, however, extremely low. No variations were found in proximate compositions in any of the measured tissues: whole body, muscle or spleen (Table 6). Liver protein concentrations were lower in the high starch groups compared to the low starch groups ( P<0.05), with no effects from the variable zinc plus iron in the diets (Table 6). The higher dietary starch also affected the vertebra lipid level, with reduced lipid vertebra ( P<0.01) concentrations when given the higher dietary starch levels (Table 6). Table 6 Proximate composition (dry matter, protein and lipid given as g kg
1 dw, and percentage of fibres+ash) of whole body homogenates, muscle, liver, spleen and vertebra in Atlantic salmon fed four experimental diets differing in carbohydrate, iron and zinc levels for 8 weeks: low carbohydrate high Zn+Fe (LchoHM), low carbohydrate plus low Zn+Fe (LchoLM), high carbohydrate plus high Zn+Fe (HchoHM) and high carbohydrate plus low Zn+Fe (HchoLM) Initial

Whole body

ANOVA

LchoHM

LchoLM

HchoHM

HchoHM

CHO

Me2+

CHOMe2+

Whole body Dry matter Protein Lipid Fibres+ash

30.1(0.5) 59.0(0.8) 29.5(0.4) 6.9

30.6(0.9) 59.3(1.2) 33.5(1.6) 6.7(0.1)

30.5(0.7) 57.4(1.5) 35.9(4.4) 6.5(0.1)

30.6(0.5) 56.7(0.8) 34.3(1.2) 7.0(0.02)

30.9(0.3) 57.0(1.0) 37.3(3.6) 6.5(0.1)

n.s. n.s. n.s. n.s.

n.s. n.s. n.s. n.s.

n.s. n.s. n.s. n.s.

Muscle Dry matter Protein Lipid Fibres+ash

28.3(0.3) 71.4(0.3) 15.2(2.8) 5.0

26.9(0.3) 78.8(0.9) 17.0(0.9) 5.5(0.2)

26.9(0.4) 77.8(2.1) 17.3(1.2) 5.4(0.1)

26.7(0.5) 78.2(1.0) 17.8(0.9) 5.4(0.0)

27.4(0.3) 77.4(0.2) 18.3(1.3) 5.2(0.2)

n.s. n.s. n.s. n.s.

n.s. n.s. n.s. n.s.

n.s. n.s. n.s. n.s.

Liver Dry matter Protein Lipid Fibres+ash

67.2(5.5) 72.3(0.2) 2.9(0.02) 6.4

24.9(1.0) 68.2(1.6)a 8.2(0.9) 6.2(0.4)

25.2(0.9) 65.5(1.2)a 8.2(0.2) 6.3(0.4)

25.4(0.8) 58.3(0.5)b 9.0(1.8) 6.2(0.6)

25.5(0.6) 57.6(1.1)b 9.0(1.1) 6.1(0.4)

n.s. <0.001 n.s. n.s.

n.s. 0.05 n.s. n.s.

n.s. n.s. n.s. n.s.

Spleen Dry matter Protein Lipid Fibres+ash

27.3(2.5) nm nm nm

27.4(0.3) 66.9(0.8) 58.8(7.4) 5.3(0.2)

27.2(1.2) 67.0(7.3) 64.0(15.7) 5.3(0.2)

26.8(0.8) 67.6(2.3) 58.8(2.4) 5.8(0.2)

26.3(0.7) 64.4(2.7) 57.0(18.0) 39.9(0.1)

n.s. n.s. n.s. n.s.

n.s. n.s. n.s. n.s.

n.s. n.s. n.s. n.s.

Vertebra Dry matter Protein Lipid Fibres+ash

nm nm nm nm

44.0(1.5) 34.5(1.6) 20.4(0.3)a 40.3(0.8)

43.7(1.9) 34.5(1.4) 20.3(0.9)a 39.7(1.2)

41.3(2.3) 36.2(1.4) 18.9(0.5)b 40.7(1.2)

42.9(0.06) 36.0(1.6) 18.8(0.2)b 41.2(0.6)

n.s. n.s. <0.01 n.s.

n.s. n.s. n.s. n.s.

n.s. n.s. n.s. n.s.

Means in the same row not shearing a common superscript are significantly different ( P<0.05). CHO=carbohydrate, Me2+=divalent ions (iron and zinc), CHOMe2+=interaction between carbohydrate and divalent ions, nm=not measured. Results are means from three tanks with their standard deviation given in parenthesis (n.s.=not significant).

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4. Discussion Growth achieved in the present experiment was rather low compared to normal SGR values at this fish size and water temperature (Austreng et al., 1987). This was expected due to the restricted feeding regime and also explains the relatively low HSI values. In other studies using satiation feeding, salmon adjusts its feed intake when carbohydrate exerts larger parts of the diet, presumably to get an optimal protein to energy intake for maximal growth (Hemre et al., 1995a; Sveier et al., 1999). Despite less protein consumption by groups HchoHM and HchoLM compared to groups LchoLM and LchoLM, no reduction in feed conversion ratio due to higher starch and lower protein intake was found, indicating a protein-sparing effect from moderate starch levels in the diet, in accordance with other reports from carnivorous fish (Bergot, 1979; Degani et al., 1986; Wilson, 1994; Hemre et al., in press) and confirmed by higher PER values found in the Hcho groups. In the present experiment, PER and PPV values were high and in the same ranges as reported in several studies (Hemre et al., 1995a; Grisdale-Helland and Helland, 1997). The higher iron plus zinc levels resulting in the lowest growth was most obvious in group HchoHM, fed with higher carbohydrate level, indicating an interaction effect between dietary starch and mineral levels on weight gain. This might be explained by more intestinal residue starch in the Hcho groups, which might have influenced the absorption of several nutrients (Krogdahl, 2001, personal communication). The major difference in liver iron between dietary treatments was caused by the difference in dietary starch, confirming earlier results (Hemre et al., 1995a,b). Equal calcium and phosphorous concentrations in the experimental diets that exclude calcium or phosphorous, contributed to this difference between groups. Liver protein concentration responded to dietary protein level and may be explained by the liver’s role in protein metabolism. Through the portal vein, the liver receives most amino acids for redistribution and transformation. As both protein levels met the requirements for salmonids (NRC, 1993), no differences would be expected in protein deposition in other tissues, as was also the present results. Glycogen levels in whole body, liver and spleen reflected dietary starch levels confirming earlier findings (Hemre et al., 1995b). High levels of glycogen in a cell may be an indicator of diminished usage and pathological accumulation rather than a sign of increased metabolic activity (Ghadially, 1988), and are found to enhance the toxic effects of overloads of trace elements (Hilton and Hodson, 1983). The present glycogen levels were, however, lower than those reported for inactivated liver cells in salmonids (Hilton and Dixon, 1982; Bæverfjord, 1992). Iron levels found in the different organs were in accordance with results reported by Andersen et al. (1996), whereas Bjørnevik and Maage (1993) and Maage et al. (1991b) reported higher iron concentrations in liver and whole body of Atlantic salmon fed with diets containing similar starch and iron concentrations. Also, the spleen iron values were low with a concentration varying from 213 to 280 mg kg
1 (w/w), whereas Bjørnevik and Maage (1993) found iron levels up to 920 mg kg
1 (w/w) in fish fed diets with 60 mg kg
1 supplemented iron and 150 g kg
1 dietary dextrin. However, the dietary history of the fish in the experiments done by Bjørnevik and Maage (1993) was most likely different

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from the dietary history of the fish used in the present experiment. In recent years, iron level in commercial diets for Atlantic salmon has been a subject for debate. Salte et al. (1994) suggested that Atlantic salmon did not have a strict regulation of iron absorption, and that excess iron in tissues caused winter ulcers. This resulted in reduced or absence of iron supplements to commercial fish diets in Norway (Bjørnevik and Maage, 1993). However, differences in liver iron concentrations in the present trial imply that small variations in dietary starch can affect bioavailability of iron. The amount of bioavailable iron to the fish must therefore be considered before the degree of iron fortification is set. In the present study, dietary starch and mineral levels were without major effects on blood haematology, although there was a falling trend for MCHC with increasing dietary starch level. This is in contrast to the conclusions of carbohydrate influences on haematology by Hemre et al. (1995b), where MCHC and Rbc were the only haematological parameters not responding to dietary treatment. The present variation in carbohydrate was only 5% to 10%, and the parameters influenced in earlier studies (Hb and MCV) were first significantly decreased when starch exceeded 10% of the dietary dry matter. Hemre et al. (1996) found decreased Hb, explained as a long-term effect of increased dietary starch, maybe explained by reduced mineral absorption due to residue starch as found for rainbow trout (Hilton and Hodson, 1983). Andersen et al. (1997a,b) only found reduced Hb values when iron stored was severely depleted. Functional iron measured as blood haemoglobin concentration, in addition to iron stored in liver tissue, can help detect suboptimal iron prior to development of anaemia (Andersen, 1997). Early stages of iron deficiency may induce changes in various enzyme systems where iron is an essential element (Dallman, 1974; Galan et al., 1984), which cannot be concluded from the present results. Plasma ALAT and LDH values in the present study were higher than reported earlier, indicating mildly stressful conditions (Sandnes et al., 1988; Hemre et al., 1995b, 1996). Plasma ASAT level was quite low, showing no leakage of this enzyme. ASAT and ALAT belong to the non-plasma specific enzymes which are located within tissues (Tietz, 1976). Cell damage will, however, cause leakage to the enzymes, normally detected as elevated plasma levels (Maita et al., 1984). Plasma glucose concentrations in groups fed with low carbohydrate diets were similar to that found in other studies with carnivorous fish (Shimeno and Mommsen, 1991; Bæverfjord, 1992; Hemre, 1995b). In high starch groups, levels were somewhat elevated, however, within normal ranges and below expected kidney threshold (Lin et al., 2000; Deng et al., 2001). The kidney threshold for glucose is not yet established for Atlantic salmon. The high accumulation of liver glycogen in these groups indicates that liver is a major organ in regulation of blood glucose, as also indicated for other carnivorous fish (Hemre and Kahrs, 1997). Levels within the normal ranges of plasma total protein, triacylglycerols and cholesterol indicate a good nutritional status in the fish (Sandnes et al., 1988), while initial status indicates mild starvation before experimental set-up. This can be explained by a rather long adaptation period with low appetite before the study started. Muscle iron and zinc levels were within ranges found in previous studies (Maage et al., 1993) but showed no variations reflecting dietary levels. Maage et al. (1993) suggested that trace element concentrations in salmon fillets were not increased by dietary supplementation, while Lorentzen (1998) found that muscle could be enriched with selenium if given in a protein-bound form.

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Whole body, vertebrae and plasma zinc concentrations are widely used clinical indicators of zinc status in experimental animals (King, 1990; Maage and Julshamn, 1993). In the present experiment, an increase in plasma and whole body zinc concentrations with dietary zinc levels supports the use of plasma and whole body as zinc status indicators for Atlantic salmon. Whole body and plasma zinc concentrations in the present experiment are in accordance with the levels found by Maage and Julshamn (1993), indicating that the zinc status of the fish in the present experiment was satisfying. Also, the tendency of increased condition factor without any reductions in length growth in fish fed with low zinc diet indicates that the range around 6 mg kg
1 was sufficient to avoid shortbody dwarfism (Maage et al., 1991a). Plasma zinc concentration was strongly influenced by dietary starch, by dietary iron plus zinc levels and with a strong interaction effect, which may have resulted from increased blood glucose, which adds to increased insulin secretion. Stored insulin is found to contain a substantial amount of zinc (Reeves and O’Dell, 1983), and consequently, insulin release may be followed by an increased zinc concentration in plasma. Gatlin et al. (1991) and Maage and Julshamn (1993) found vertebrae to be a sensitive indicator of zinc status in fish. Maage and Julshamn (1993) also suggested that fish can mobilise at least some zinc from the vertebrae. The present findings, however, did not show any difference in vertebra zinc concentrations. The only organ showing a response in lipid concentration as dietary starch increased was vertebrae, indicating that vertebrae may use lipid for energy. Fish bones have cavities, which are often filled with adipose tissue (Kryvi and Totland, 1997), which can explain the relative high portion of lipid in this tissue. To our knowledge, data on the normal range of vertebrae proximate composition do not exist.

5. Summary Dietary starch level was mirrored in whole body, liver and spleen. Both dietary starch and iron did influence liver and spleen iron concentrations. Adding 5% or 10% dietary starch did not result in any changes in haematological parameters, neither did the present variations in iron plus zinc. Dietary zinc influenced zinc concentrations in plasma and whole body. Additionally, plasma zinc concentration was influenced by both dietary starch and iron levels. Muscle composition did not change as a consequence of the present dietary manipulations. Acknowledgements The authors thank Mrs. Jenny Sleire for her skillful technical assistance during the fish trial. References Andersen, F., 1997. Studies on iron nutrition in Atlantic salmon (Salmo salar) with respect to requirement, bioavailability, interactions and immunity. Dr. Scient. thesis, University of Bergen, Norway.

B. Vangen, G.-I. Hemre / Aquaculture 219 (2003) 597–611

609

Andersen, F., Maage, A., Julshamn, K., 1996. An estimation of dietary iron requirement of Atlantic salmon, Salmo salar L., parr. Aquac. Nutr. 2, 41 – 47. Andersen, F., Lorentzen, M., Waagbø, R., Maage, A., 1997a. Bioavailability and interactions with other micronutrients of three dietary iron sources in Atlantic salmon, Salmo salar, smolts. Aquac. Nutr. 3, 65 – 71. Andersen, F., Lygren, B., Maage, A., Waagbø, R., 1997b. Interaction between two dietary levels of iron and two forms of ascorbic acid and the effect on growth, antioxidant status and some non-specific immune parameters in Atlantic salmon (Salmo salar) smolt. Aquaculture 161, 437 – 451. ˚ sga˚rd, T., 1987. Growth rate estimates for cultured Atlantic salmon and rainbow Austreng, E., Storebakken, T., A trout. Aquaculture 60, 157 – 160. Bergot, F., 1979. Carbohydrate in rainbow trout diets: effects of the level and source of carbohydrate and the number of meals on growth and body composition. Aquaculture 34, 543 – 547. Bæverfjord, G., 1992. Digestible and indigestible carbohydrates in rainbow trout diets: III. Effects on growth, digestibility, pancreatic hormones, liver glycogen deposition and liver morphology. PhD Thesis, Norwegian College of Veterinary Medicine, Oslo, Norway. Bjørnevik, M., Maage, A., 1993. Effects of dietary iron supplementation on tissue iron concentrations and haematology in Atlantic salmon (Salmo salar). Fisk. Dir. Skr., Ser. Ernær. 6, 35 – 45. Chvapil, M., 1973. New aspects in the biological role of zinc: a stabilizer of macromolecules and biological membranes. Life Sci. 13, 1041 – 1049. Coulston, L., Nandona, P., 1980. Insulin-like effect of zinc in adipocytes. Diabetes 29, 665 – 667. Coultate, T.P. (Ed.), 1996. FOOD The Chemistry of its Components. The Royal Society of Chemistry, Cambridge, UK. Dallman, P.R., 1974. Tissue effects of iron deficiency. In: Jacobs, A., Worwood, M. (Eds.), Iron in Biochemistry and Medicine, vol. 1. Academic Press, New York, pp. 437 – 475. Degani, G., Viola, S., Levnon, D., 1986. Effects of dietary carbohydrate source on growth and body composition of the European eel (Anguilla anguilla L.). Aquaculture 52, 97 – 104. Deng, D.F., Refstie, S., Hung, S.S.O., 2001. Glycemic and glycosuric responses in white sturgeon (Acipenser transmontanus) after oral administration of simple and complex carbohydrates. Aquaculture 199, 107 – 117. Faure, P., Roussel, A., Coudray, C., Richard, M.J., Halimi, S., Favier, A., 1992. Zinc and insulin sensitivity. Biol. Trace Elem. Res. 32, 305 – 310. Furuichi, M., Yone, Y., 1982. Availability of carbohydrate in nutrition of carp and red sea bream. Bull. Jpn. Soc. Sci. Fish. 48, 945 – 948. Galan, P., Hercberg, A., Touitou, Y., 1984. The activity of tissue enzymes in iron-deficient rat and man: an overview. Comp. Biochem. Physiol. 77B, 647 – 653. Gatlin, D.M., Wilson, R.P., 1986. Characterisation of iron deficiency and the dietary iron requirement of fingerling channel catfish. Aquaculture 52, 191 – 198. Gatlin, D.M., O’Connel, J.P., Scarpa, J., 1991. Dietary zinc requirement of the red drum, Scianeops ocellatus. Aquaculture 92, 359 – 365. Ghadially, F.N. (Ed.), 1988. Ultrastructural Pathology of the Cell and Matrix, vol. 2, 3rd edn. Butterworths, London, UK, p. 1340. Grisdale-Helland, B., Helland, S., 1997. Replacement of protein by lipid and carbohydrate in diets for Atlantic salmon (Salmo salar) at the end of the freshwater stage. Aquaculture 152, 167 – 180. Hemre, G.-I., Kahrs, F., 1997. 14C-injection in Atlantic cod (Gadus morhua), metabolic responses and excretion via the gill membrane. Aquac. Nutr. 2, 3 – 8. Hemre, G.-I., Lie, Ø., Lied, E., Lambertsen, G., 1989. Starch as an energy source in feed for cod (Gadus morhua): digestibility and retention. Aquaculture 80, 261 – 270. Hemre, G.-I., Sandnes, K., Lie, Ø., Torrisen, O., Waagbø, R., 1995a. Carbohydrate nutrition in Atlantic salmon: I. Growth and feed utilization. Aquac. Res. 26, 149 – 154. Hemre, G.-I., Sandnes, K., Lie, Ø., Waagbø, R., 1995b. Blood chemistry and organ nutrient composition in Atlantic salmon (Salmo salar) fed graded amounts of wheat starch. Aquac. Nutr. 1, 37 – 42. Hemre, G.-I., Waagbø, R., Hjeltnes, B., Aksnes, A., 1996. Effect of gelatinized wheat and maize in diets for large Atlantic salmon (Salmo salar) on glycogen retention, plasma glucose and fish health. Aquac. Nutr. 2, 33 – 39. ˚ ., 2002. Carbohydrates in fish nutrition: effects on growth, glucose Hemre, G.-I., Mommsen, T.P., Krogdahl, A metabolism and hepatic enzymes. Aquac. Nutr., in press.

610

B. Vangen, G.-I. Hemre / Aquaculture 219 (2003) 597–611

Hilton, J.W., Dixon, D.G., 1982. Effect of increased liver glycogen and liver weight on liver function in rainbow trout, Salmo gairdneri Richardson: recovery from anaesthesia and plasma 35S-sulphobromophatlein clearance. J. Fish Dis. 5, 185 – 195. Hilton, J.W., Hodson, P.V., 1983. Effects of increased dietary carbohydrate on selenium metabolism and toxicity in rainbow trout (Salmo gairdneri). J. Nutr. 113, 1241 – 1248. Holm, J., Bjo¨rck, I., Drews, A., Asp, N.-G., 1986. A rapid method for the analysis of starch. Sta¨rke 38, 224 – 283. Hughes, S.G., 1985. Nutritional eye diseases in salmonides: a review. Prog. Fish-Cult. 47, 81 – 85. Kawatsu, H., 1972. Studies on the anemia of fish: V. Dietary iron deficient anemia in brook trout, Salvelinus fontinalis. Bull. Freshwater Fish. Res. Lab. 22, 59 – 66. King, J.E., 1990. Assessment of zinc status. J. Nutr. 120, 1474 – 1479. King, J.C., Keen, C.L., 1994. Zinc. In: Shils, M.E., Olson, J.A., Shike, M. (Eds.), Modern Nutrition in Health and Disease, vol. 1(2), 8th edn. Williams and Wilkins, Baltimore, pp. 214 – 230. Kryvi, H., Totland, G.K., 1997. Fiskeanatomi. Høyskoleforlaget AS. Norwegian Academic Press, Kristiansand S., Norway (in Norwegian). Lall, S.P., 1989. The minerals. In: Halver, J.E. (Ed.), Fish Nutrition, vol. 1, 2nd edn. Academic Press, San Diego, pp. 219 – 257. Lin, S.-C., Liou, C.-H., Shiau, S.-Y., 2000. Renal threshold for urinary glucose excretion by tilapia in response to orally administered carbohydrates and injected glucose. Fish Physiol. Biochem. 23, 127 – 132. Lorentzen, M., 1998. Trace elements in practical diets to Atlantic salmon (Salmo salar L.). Dr. Scient. thesis, University of Bergen, pp. 65+5 papers. Maage, A., Julshamn, K., 1993. Assessment of zinc status in juvenile Atlantic salmon (Salmo salar) by measurement of whole body and tissue levels of zinc. Aquaculture 117, 170 – 191. Maage, A., Lorentzen, M., Bjørnevik, M., Julshamn, K., 1991a. Zinc requirement of Atlantic salmon (Salmo salar) fry during start feeding. In: Kaushik, S.J., Luquet, P. (Eds.), Fish Nutrition in Practice, Biarritz, France, June 24 – 27, INRA, Paris 1993, p. 873. Maage, A., Sveier, H., Julshamn, K., 1991b. A comparison of tissue levels of four essential trace-elements in wild and farmed Atlantic salmon (Salmo salar L.). Fisk. Dir. Skr., Ser. Ernær. IV, 111 – 116. Maage, A., Lorentzen, M., Bjørnevik, M., Julshamn, K., 1993. Is it possible to increase the trace element concentration in salmon fillets by supplementation of the feeds? XV International Congress of Nutrition, Sept. 26 – Oct. 1, Adelaide, Australia. Maita, M., Shiomitsu, K., Ikeda, Y., 1984. Health assessment by the climogram of hemachemical constituents in cultured yellowtail. Bull. Jpn. Soc. Sci. Fish. 51, 206 – 211. May, J.M., Contoreggi, C.S., 1982. The mechanism of the insulin-like effects of ionic zinc. J. Biol. Chem. 257, 4362 – 4368. Murat, J.C., Serfaty, A., 1974. Simple enzymatic determination of polysaccharide (glycogen) content of animal tissue. Clin. Chem. 20, 1576 – 1577. National Research Council (NRC), 1993. Nutrient Requirements in Fish. National Academy Press, Washington, DC, USA, p. 103. Nebert, D.W., Gonzalez, F.J., 1987. P450 genes: structure, evolution and regulation. Annu. Rev. Biochem. 56, 945 – 993. Ogino, C., Yang, G.-Y., 1978. Requirement of rainbow trout for dietary zinc. Bull. Jpn. Soc. Sci. Fish. 44, 1015 – 1018. Reeves, P.G., O’Dell, B.L., 1983. The effect of zinc deficiency on glucose metabolism in meal-fed rats. Br. J. Nutr. 49, 441 – 452. Sakamoto, S., Yone, Y., 1978. Requirement of Red Sea bream for dietary iron-II. Bull. Jpn. Soc. Sci. Fish. 44, 223 – 225. Salte, R., Rørvik, K.A., Reed, E., Norberg, K., 1994. Winter ulcers of the skin in Atlantic salmon, Salmo salar L., pathogenesis and possible aetiology. J. Fish Dis. 17, 661 – 665. Sandnes, K., Lie, Ø., Waagbø, R., 1988. Normal ranges of some blood chemistry parameters in adult farmed Atlantic salmon, Salmo salar. J. Fish Biol. 32, 129 – 136. Sandstrøm, B., 1997. Bioavailability of zinc. Eur. J. Clin. Nutr. 51, 17 – 19. Satoh, S., Takeuchi, T., Watanabe, T., 1987. Availability to rainbow trout of zinc in white fish meal and of various zinc compounds. Nippon Suisan Gakkaishi 53, 595 – 599.

B. Vangen, G.-I. Hemre / Aquaculture 219 (2003) 597–611

611

Shimeno, M.A., Mommsen, T.P., 1991. Effects of nutritional state on in vivo lipid and carbohydrate metabolism of coho salmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol. 81, 473 – 483. Sveier, H., Wathne, E., Lied, E., 1999. Growth, feed and nutrient utilisation and gastrointestinal evacuation time in Atlantic salmon (Salmo salar L.): the effect of dietary fish meal particle size and protein concentration. Aquaculture 180, 265 – 282. Tietz, N. (Ed.), 1976. Fundamentals of Clinical Chemistry Saunders, Philadelphia, USA. Wilson, R.P., 1994. Utilization of dietary carbohydrate by fish. Aquaculture 124, 67 – 80.