Growth, nitrogen and energy utilization of juveniles from four salmonid species: diet, species and size effects

Aquaculture 234 (2004) 393 – 414 www.elsevier.com/locate/aqua-online

Growth, nitrogen and energy utilization of juveniles from four salmonid species: diet, species and size effects P.A. Azevedo a, S. Leeson a, C.Y. Cho a,b, D.P. Bureau a,* a

Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 b Ontario Ministry of Natural Resources, Guelph, Ontario, Canada N1G 2W1 Received 14 September 2003; received in revised form 3 January 2004; accepted 5 January 2004

Abstract The effect of dietary digestible protein/digestible energy (DP/DE) ratio on growth, feed efficiency (FE), digestibility, nitrogen (N) and energy utilization, and body composition of juveniles from four salmonid species reared in freshwater was investigated in a series of trials. Another objective of this study was to investigate how the FE, and N and energy utilization changed as fish grew. Four diets were formulated to be isoenergetic (DE=20 MJ/kg) but contain different DP/DE ratios, 24, 22, 20, and 18 g/MJ, achieved through reduction of DP level (53% to 39%) and increase of lipid level (19% to 26%). Diets were hand-fed to near-satiety to triplicate groups of lake trout (initial body weight (IBW), 47 g) and Atlantic salmon (IBW, 25 g) for 280 days at 13 jC and to rainbow trout (IBW, 47 g) and chinook salmon (IBW, 24 g) for 84 and 140 days, respectively, at 12 jC. Within species, weight gain was not affected by DP/DE ( P<0.05). However, a significant decrease in FE was observed with decreasing DP/DE for all species, except chinook salmon. Digestible N retention efficiency (NRE) increased linearly ( P<0.05) with decreasing DP/DE ratio while there was no diet effect on digestible energy retention efficiency (ERE) for all species. The effect of diet on FE, N and energy utilization as fish grew was investigated for lake trout and Atlantic salmon. As lake trout and Atlantic salmon grew, significant linear decreases in FE, NRE and ERE, irrespective of diet, were observed. The decrease in NRE and ERE as fish grew correlated with decreasing dressed carcass yields, and significant increases in body dry matter and lipid contents. More research is needed in order to gain further insight into species and size effects on feed and nutrient utilization. D 2004 Elsevier B.V. All rights reserved. Keywords: Trout; Salmon; Diet; Feed; Protein; Energy; Growth

* Corresponding author. Tel.: +1-519-924-4120x53668; fax: +1-519-767-0573. E-mail address: [email protected] (D.P. Bureau). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.01.004

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1. Introduction Considerable changes in the formulation and composition of diets for farmed salmonids have occurred over the past 15 to 20 years. The nutrient composition of these diets has changed towards increased lipid (from 8 –12% up to 30 or even 40%) and energy contents (>20 MJ/kg) and significant reduction in carbohydrate contents (from 40% down to 10– 15%) (Hardy, 2002; Storebakken, 2002). The development of these high energy diets, at least for Atlantic salmon, has been motivated by efforts to improve feed efficiency, reduce feed requirement (due to feed quota), feed cost, and reduce N waste outputs (Johnsen and Wandsvik, 1991; Johnsen et al., 1993; Hillestad et al., 2001; Storebakken, 2002). Besides Atlantic salmon, many other salmonid species are being cultured around the world, e.g., rainbow trout, chinook salmon and coho salmon. The same feeds are generally fed to various salmonid species (Cho, 1990; Cho, 1992) yet evidence suggests that different salmonid species utilize feeds of similar composition with different efficiencies (Berg and Bremset, 1998; Rasmussen and Ostenfeld, 2000; Refstie et al., 2000). Moreover, commercial feeds for salmonids vary widely in terms of proximate composition (protein, lipid, carbohydrate). To assist in feed composition choice for optimal feed utilization for each specific fish species, there is consequently a need to examine how efficiencies of feed, nutrient, and energy utilization are affected by species and feed composition. Several abiotic and biotic factors are known to affect nutrient and energy utilization. For example, many studies have reported that digestible protein to digestible energy ratio (DP/DE) of the diet significantly affect N utilization (Silver et al., 1993; Alsted, 1991; Johnsen and Wandsvik, 1991; Johnsen et al., 1993; Hillestad and Johnsen, 1994, Lanari et al., 1995; Einen and Roem, 1997; Grisdale-Helland and Helland, 1997; Ruohonen et al., 1998; Steffens et al., 1999). Comparison of the results from various studies suggests that N and energy utilization efficiency appears to differ between species (Berg and Bremset, 1998; Rasmussen and Ostenfeld, 2000; Refstie et al., 2000). Krogdahl et al. (2004) reported however, similar N retention efficiency (NRE, N gain/digestible N intake) and energy retention efficiency (ERE, recovered energy/DE intake) between Atlantic salmon and rainbow trout of the same age fed the same diets and under the same reared conditions. The effect of species on the nutrient and energy utilization by salmonids remains unclear and further investigation is needed. Values reported in the literature suggest that smaller fish and/or juvenile stages of fish generally have higher FE, NRE and ERE than larger and/or post-juvenile fish and their optimum dietary DP/DE appears to be different (Ronsholdt, 1995; Einen and Roem, 1997). No study have however, investigated how FE, NRE and ERE changes as fish grows, nor how these changes are affected by diet and fish species. Investigation of these changes is required for better definition of feed formulation for different fish species and fish sizes. The objective of this study was to determine the effects of DP/DE ratio, fish species and fish size on the efficiency of utilization of feed, N and energy for body weight gain, carcass yield and proximate composition, for juveniles from four salmonid species, lake trout, Atlantic salmon, rainbow trout and chinook salmon reared in freshwater. The second objective of this study was to investigate the effect of species and diet on the efficiency of

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utilization of feed, N and energy utilization for body gain, N and energy retention, respectively, as fish grew.

2. Materials and methods 2.1. Diets Four diets were formulated to be isoenergetic (DE=20 MJ/kg) but contain different DP/DE ratios (Table 1). The formulated DP/DE ratios were 24, 22, 20 or 18 g/MJ brought about by decreases in DP and increases in dietary lipids and carbohydrates (Table 1). Dietary ingredients were obtained from a local feed manufacturer (Martin Feed Mills, Elmira, Ontario). Acid-washed diatomaceous silica (Celite AW521, Celite, Lompoc, CA) was included in the diet to serve as a digestibility indicator. The diets were mixed using a Hobart mixer (Hobart, Don Mills, Ontario, Canada) and pelleted to appropriate size using a laboratory steam pellet mill (California Pellet Mill, San

Table 1 Composition of experimental diets Ingredients (g/100 g as is basis)

Fish meal, herring, 68% CP, 10% ash Blood meal, spray dried, 84% CP Corn gluten meal, 60% CP Wheat middlings, 17%CP Whey, 10% CP Celite AW521a CaHPO4 L-lysine Vitamin premixb Mineral premixc Fish oil, herring Total Determined diet composition, dry matter basis Dry matter (%, as is basis) Crude protein (%) Crude fat (%) Ash (%) Gross energy (MJ/kg) a

Diet 1

2

3

4

34 10 34 – 4.8 1 – 0.2 1 1 14 100

29 10 29 6.1 5.5 1 – 0.4 1 1 17 100

25 10 25 9.2 7.5 1 0.3 0.5 1 1 19.5 100

22 10 22 11 8.9 1 0.5 0.6 1 1 22 100

94.1 57.0 20.0 8.6 24.0

93.6 51.1 22.2 7.9 24.4

94.9 46.3 23.8 7.7 24.7

94.8 43.1 25.6 7.5 24.9

Celite AW521 (acid-washed diatomaceous silica) is a source of acid-insoluble ash. Provides per kg of diet: retinyl acetate, 3750 IU; cholecalciferol, 3000 IU; all-rac-a-tocopheryl acetate, 75 IU; menadione sodium bisulfite, 1.5 mg; 1-ascorbic acid (Stay C), 75 mg; cyanocobalamine, 0.03 mg; d-biotin, 0.21 mg; choline chloride, 1500 mg; folic acid, 1.5 mg; niacin, 15 mg; d-calcium pantothenate, 30 mg; pyridoxine HCl, 7.5 mg; riboflavin, 9 mg; thiamin HCl, 1.5 mg; Astaxanthin (Carophyll – Pink, Hoffman-La Roche), 75 mg. c Provides per kg of diet: sodium chloride (NaCl, 39% Na, 61% Cl), 1200 mg; ferrous sulfate (FeSO4
7H2O, 20% Fe), 13 mg; manganese sulfate (MnSO4, 36% Mn), 32 mg; zinc sulfate (ZnSO4.7H2O, 40% Zn), 60 mg; copper sulfate (CuSO4
5H2O, 25% Cu), 7 mg; potassium iodide (KI, 24% K, 76% I), 8 mg. b

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Francisco, CA, USA). The pelleted feed was subsequently dried in a forced air drier at room temperature for 24 h and then sieved. Part of the fish oil was incorporated in the mash prior to pelleting and part was sprayed on the top of the dry pellets. The diets were kept at 4 jC until used and only the amount required for each week was kept at room temperature. 2.2. Fish and experimental conditions Lake trout (Salvelinus fontinalis, offspring from captive brood stock) and Atlantic salmon (Salmo salar, anadromous strain, offspring from captive brood stock originating from LaHave River, New Brunswick, Canada) were obtained from two stations of the Ontario Ministry of Natural Resources, Harwood Fish Culture Station (Harwood, Ontario) and Ringwood Fish Culture Station (Ringwood, Ontario), respectively. Rainbow trout (Oncorhynchus mykiss, Ontario domestic strain, fall spawning) and chinook salmon (Oncorhynchus tshawytscha, offspring from wild brood stock captured in Lake Ontario tributaries) were obtained from a private hatchery (Rainbow Springs Trout Hatchery, Thamesford, Ontario, Canada) and from the Ontario Ministry of Natural Resources Ringwood Fish Culture Station, respectively. In trial A, lake trout (0+ age, IBW, 47.4F0.5 g) and Atlantic salmon (0+ age, IBW, 24.5F0.3 g) were randomly distributed to 12 rectangular fiberglass tanks (50 l) each, with three tanks per diet. Each tank was considered an experimental unit. In trial B, rainbow trout (0+ age, IBW, 47.4F0.5 g) and chinook salmon (0+ age, IBW, 24.1F0.3 g) were also distributed according to the same experimental design and maintained in the same rearing conditions and feeding protocol as lake trout and Atlantic salmon. The fish in both trials were hand-fed to near-satiety three times daily between 0900 and 1600 h. The tanks were supplied with filtered well water at 1.5 l/min. Water temperature was maintained between 12 and 13 jC by injecting hot water into the incoming waterline. Each tank was individually aerated. Mortality and temperature were checked daily. Fish were weighed every 28 days. Fish were acclimatised to the experimental conditions for a period of 2 weeks, during which time they were fed a commercial trout diet (Martin Mills Feeds, Elmira, Ontario, Canada). The animals were kept in accordance with the guidelines of the Canadian Council on Animal Care (CCAC, 1984) and the University of Guelph Animal Care Committee. 2.3. Fish sampling and chemical analysis On the first day of the experiment, fish (5 to 12 fish/sample) from each species were randomly selected and anaesthetised with tricaine methane sulfonate (2 g/10 l of water), sacrificed with a sharp blow to the head and frozen at 20 jC, until analysis. This procedure was repeated at 112-, 196- and 280-days of feeding trial for lake trout and Atlantic salmon, at 84-days for rainbow trout and at 140-days for chinook salmon. The whole fish were weighed and gutted. The viscera (including liver and gonads) and carcass (including kidney) were weighed and pooled for each tank and then frozen for analysis of chemical composition.

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Fish carcass were allowed to thaw and then autoclaved. After cooling, a few drops of liquid antioxidant (Ethoxyquin, Novus Int., Canada, Mississauga, Ontario) were added to each pan. The fish carcass samples were then ground into homogeneous slurry in a Waring kitchen blender, transferred into shallow dishes, frozen, and subsequently lyophilised. These samples were then reground and stored at 20 jC prior to analysis. 2.4. Digestibility trial Lake trout (IBW, 32 g, 60 fish/tank) and Atlantic salmon (IBW, 24 g, 55 fish/tank) were obtained from the same source as fish used in feeding trial. The fish were stocked in a digestibility system equipped with feces settling columns (Guelph system) described by Cho et al. (1982). Lake trout and Atlantic salmon were maintained at 14 jC during the feces collection period (digestibility trial #1). Rainbow trout (IBW, 136 g, 25 fish/tank) and chinook salmon (IBW, 40 g, 30 fish/tank) from the feeding trial were transferred to a

Table 2 Apparent digestibility coefficients (ADC) of DM, CP, GE and of CL for lake trout and Atlantic salmon fed diets of varying DP/DE ratios for 280 days at 13 jC ADC (%) DM

CP

GE

CL

Lake trout Diet 1, DP/DE=24 Diet 2, DP/DE=22 Diet 3, DP/DE=20 Diet 4, DP/DE=18

84.3 81.9 78.8 79.3

94.3 94.3 93.8 94.6

88.7 86.0 82.3 82.4

89.3 86.8 81.9 83.8

Significancea Linear Quadratic

P<0.05 N.S.

N.S. N.S.

P<0.05 N.S.

N.S. N.S.

Atlantic salmon Diet 1, DP/DE=24 Diet 2, DP/DE=22 Diet 3, DP/DE=20 Diet 4, DP/DE=18

82.9 79.2 76.0 77.8

93.6 93.4 92.5 93.9

86.5 82.3 78.7 80.0

84.9 80.8 76.3 78.8

Significancea Linear Quadratic S.E.M.b

P<0.05 P<0.05 1.2

N.S. P<0.05 0.3

P<0.05 N.S. 1.6

N.S. N.S. 3.1

Effects of Species Diet SpeciesDiet

P<0.05 P<0.05 N.S.

P<0.05 P<0.05 N.S.

P<0.05 P<0.05 N.S.

P<0.05 N.S. N.S.

a

Significance = significance of the orthogonal linear and quadratic contrasts of dependent variables across diets. DM—dry matter, CP—crude protein, GE—gross energy, CL—crude lipid. b S.E.M. = standard error mean (n = 3); N.S. = not statistically significant ( Pz0.05).

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similar digestibility system as described above and fish were maintained at 11 jC (digestibility trial #2). Fecal samples were collected as described by Cho et al. (1982). A total of three samples per diet for each species were collected with the exception of two samples per diet for chinook salmon. 2.5. Chemical analysis Feed ingredients, diets, fish carcasses and fecal samples were analyzed for dry matter and ash according to AOAC (1995), crude protein (CP, %N6.25) using a Kjeltech autoanalyzer (Model #1030, Tecator, Hoganas, Sweden), lipid using the method of Bligh and Dyer (1959), and gross energy (GE) using a Parr 1271 automated bomb calorimeter (Parr Instruments, Moline, IL). The digestion indicator was determined using the acidinsoluble ash (AIA) indicator method of Atkinson et al. (1984). Table 3 Apparent digestibility coefficients (ADC) of DM, CP, GE and of CL for rainbow trout and chinook salmon fed diets with different DP/DE ratios for 84 and 140 days, respectively, at 12 jC ADC (%) DM

CP

GE

CL

Rainbow trout Diet 1, DP/DE=24 Diet 2, DP/DE=22 Diet 3, DP/DE=20 Diet 4, DP/DE=18 S.E.M.a

86.2 82.3 82.7 80.7 0.9

93.3 92.1 93.0 92.4 0.5

90.6 87.0 87.1 85.1 1.0

92.7 89.8 89.4 87.3 1.1

Significanceb Linear Quadratic

P<0.05 N.S.

N.S. N.S.

P<0.05 N.S.

P<0.05 N.S.

Chinook salmon Diet 1, DP/DE=24 Diet 2, DP/DE=22 Diet 3, DP/DE=20 Diet 4, DP/DE=18 S.E.M.a

87.8 86.4 85.0 84.2 1.1

94.7 94.7 94.0 94.3 0.6

92.1 91.0 90.0 89.4 1.2

94.4 94.6 93.7 93.6 1.2

Significanceb Linear Quadratic

P<0.05 N.S.

N.S. N.S.

N.S. N.S.

N.S. N.S.

Effects of Species Diet SpeciesDiet

P<0.05 P<0.05 N.S.

P<0.05 N.S. N.S.

P<0.05 P<0.05 N.S.

P<0.05 N.S. N.S.

a S.E.M. = standard error mean (n = 3 for trout and n = 2 for salmon); N.S. = not statistically significant ( Pz0.05). b Significance = significance of the orthogonal linear and quadratic contrasts of dependent variables across diets. DM—dry matter, CP—crude protein, GE—gross energy, CL—crude lipid.

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2.6. Calculations 2.6.1. Apparent digestibility coefficients (ADC) The apparent digestibility coefficients (ADC) for the nutrients and energy of the experimental diets were calculated as follows (Cho et al., 1982): ADC ¼ 1  ðF=D  Di=FiÞ where: D = % nutrient (or kJ/g gross energy) of diet; F = % nutrient (or kJ/g gross energy) of feces; Di = % digestion indicator (AIA) of diet; Fi = % digestion indicator (AIA) of feces.

Table 4 Growth, feed intake and feed efficiency of lake trout (IBW=47 g) and Atlantic salmon (IBW=24 g) fed diets of varying DP/DE ratios for 280 days at 13 jC Gain (g/fish)

TGCa

Feed intake (g/fish)

FEb (gain/feed)

Lake trout Diet 1, DP/DE=24 Diet 2, DP/DE=22 Diet 3, DP/DE=20 Diet 4, DP/DE=18

346 335 315 369

0.105 0.103 0.098 0.108

282 292 289 328

1.23 1.14 1.09 1.12

Significancec Linear Quadratic

N.S. N.S.

N.S. N.S.

N.S. N.S.

P<0.05 P<0.05

Atlantic salmon Diet 1, DP/DE=24 Diet 2, DP/DE=22 Diet 3, DP/DE=20 Diet 4, DP/DE=18

328 344 338 316

0.117 0.120 0.119 0.115

273 294 295 284

1.20 1.17 1.14 1.11

Significancec Linear Quadratic S.E.M.d

N.S. N.S. 22

N.S. N.S. 0.00382

N.S. N.S. 15

P<0.05 N.S. 0.024

Effects of Species Diet SpeciesDiet

N.S. N.S. N.S.

P<0.0001 N.S. N.S.

N.S. N.S. N.S.

N.S. P<0.05 N.S.

a

TGC=thermal-unit growth coefficient. FE=feed efficiency. c Significance=significance of the orthogonal linear and quadratic contrasts of dependent variables across diets. d S.E.M.=standard error mean (n=3); N.S.=not statistically significant ( Pz0.05). b

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2.6.2. Growth rate, thermal-unit growth coefficient (TGC) TGC ¼ 100  ½ðFBW1=3  IBW1=3 Þ  ðsum T  DÞ1  where: FBW=final body weight (g); IBW=initial body weight (g); sum TD = sum degrees Celsiusdays (Iwama and Tautz, 1981; Cho, 1992). 2.6.3. Feed efficiency (FE) FE ¼ live body weight gain=dry feed intake where: feed intake=total dry feed/sum (fishday); live body weight gain=(FBW/final number of fish)(IBW/initial number of fish); FBW=final body weight (g); IBW=initial body weight (g).

Table 5 Growth, feed intake and feed efficiency of rainbow trout (IBW=47 g) and chinook salmon (IBW=24 g) fed diets with different DP/DE ratios for 84 and 140 days, respectively, at 12 jC Gain (g/fish)

TGCa

Feed intake (g/fish)

FEb (gain/feed)

Rainbow trout Diet 1, DP/DE=24 Diet 2, DP/DE=22 Diet 3, DP/DE=20 Diet 4, DP/DE=18 S.E.M.c

146 152 146 152 4

0.221 0.227 0.220 0.226 0.00374

108 116 115 124 2

1.35 1.32 1.27 1.23 0.021

Significanced Linear Quadratic

N.S. N.S.

N.S. N.S.

P<0.05 N.S.

P<0.05 N.S.

Chinook salmon Diet 1, DP/DE=24 Diet 2, DP/DE=22 Diet 3, DP/DE=20 Diet 4, DP/DE=18 S.E.M.c

35 38 39 39 3

0.062 0.065 0.067 0.067 0.00361

36 36 39 42 1

0.97 1.03 0.98 0.93 0.058

Significanced Linear Quadratic

N.S. N.S.

N.S. N.S.

P<0.05 N.S.

N.S. N.S.

a

TGC = thermal-unit growth coefficient. FE=feed efficiency. c S.E.M. = standard error mean (n = 3). d Significance = significance of the orthogonal linear and quadratic contrasts of dependent; N.S. = not statistically significant ( Pz0.05). b

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Table 6 Retention efficiency of digestible N and energy for lake trout and Atlantic salmon fed diets of varying DP/DE ratios for 280 days at 13 jC NRE (%DNI)

ERE (%DEI)

Lake trout Diet 1, DP/DE=24 Diet 2, DP/DE=22 Diet 3, DP/DE=20 Diet 4, DP/DE=18

41.7 43.8 46.0 49.2

48.8 51.1 48.6 53.1

Significancea Linear Quadratic

P<0.05 N.S.

N.S. N.S.

Atlantic salmon Diet 1, DP/DE=24 Diet 2, DP/DE=22 Diet 3, DP/DE=20 Diet 4, DP/DE=18

43.3 47.0 49.6 52.7

49.6 50.4 48.4 51.7

Significancea Linear Quadratic S.E.M.b

P<0.0001 N.S. 1.2

N.S. N.S. 2.2

Effects of Species Diet SpeciesDiet

P<0.05 P<0.0001 N.S.

N.S. N.S. N.S.

N.S. = not statistically significant ( Pz0.05); NRE=N retention efficiency; DNI=digestible N intake; ERE=energy retention efficiency; DEI=digestible energy intake. a Significance=significance of the orthogonal linear and quadratic contrasts of dependent variables across diets. b S.E.M.=standard error mean (n=3).

2.6.4. Digestible N and energy retention efficiency (NRE and ERE) NRE; % ¼ ½½ðFBW  N contentfinal Þ  ðIBW  N contentinitial Þ=DNI  100 ERE; % ¼ ½½ðFBW  energy contentfinal Þ  ðIBW  energy contentinitial Þ=DEI  100 where: NRE=digestible N retention efficiency; ERE=digestible energy retention efficiency; FBW=final body weight; IBW=initial body weight; DNI=digestible N intake; DEI=digestible energy intake. 2.6.5. Dressed carcass yield (DCY) DCY; % ¼ ðdressed carcass weight=live body weightÞ  100

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2.7. Statistical analysis All data were analyzed using the GLM procedure from SAS (1990) and the Brown and Forsythe’s test (SAS, 1990) was used to test for homogeneity of variances for all the dependent variables prior to any other statistical analysis. Variable transformation was adopted if heterogeneity of variances was observed. Trial A was analyzed as a 24 factorial, in a randomized complete design with the species (2) and dietary DP/DE ratios (4) being the treatment factors and each tank being the experimental unit (three replicates per treatment combinations of species and diet). The trial with rainbow trout and chinook salmon had different durations for each species and, therefore, the statistical analysis was done by species and reduced to one treatment effect, i.e., diet. Weight gain, TGC, feed intake, FE, NRE and ERE, DCY and whole body composition were calculated for the entire experiments and the responses were averaged on a tank (experimental unit) basis. In trial A, these dependent variables were analyzed initially by analysis of covariance (ANCOVA) with initial fish body weight or final body weight, in the case of whole body composition, as the covariate factor and the main effects of species, diet and speciesdiet interaction, as well as interactions of covariate with each of the previous effects.

Table 7 Retention efficiency of digestible N and energy for rainbow trout and chinook salmon fed diets of varying DP/DE ratios (n=3) for 84 and 120 days, respectively, at 12 jC NRE (%DNI)

ERE (%DEI)

Rainbow trout Diet 1, DP/DE=24 Diet 2, DP/DE=22 Diet 3, DP/DE=20 Diet 4, DP/DE=18 S.E.M.a

49.3 51.8 51.5 54.1 1.7

50.4 55.6 52.1 57.3 2.1

Significanceb Linear Quadratic

N.S. N.S.

N.S. N.S.

Chinook salmon Diet 1, DP/DE=24 Diet 2, DP/DE=22 Diet 3, DP/DE=20 Diet 4, DP/DE=18 S.E.M.a

34.9 40.1 41.9 42.9 2.7

31.2 35.7 38.0 36.9 2.2

Significanceb Linear Quadratic

N.S. N.S.

N.S. N.S.

N.S. = not statistically significant ( Pz0.05); NRE=N retention efficiency; DNI=digestible N intake; ERE=energy retention efficiency; DEI=digestible energy intake. a Significance=significance of the orthogonal linear and quadratic contrasts of dependent variables across diets. b S.E.M.=standard error mean (n=3).

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If the covariate effect or the interaction of covariate factor with treatment effects was not significant, the previous model was simplified by removing the covariate effect and any interaction effect of covariate with treatment factors. When significant effects were found ( P<0.05) for treatment factors, the orthogonal polynomial (linear and quadratic) contrasts were done for each dependent variable (Steel et al., 1997). In addition to the above, FE, NRE and ERE were analyzed by a repeated measures ANOVA model to evaluate their responses over time. The model included diet, species and dietspecies interaction. The repeating variable was measured in each period between two consecutive samplings of fish. ADC were analyzed as two 24 factorials, in a randomized complete design with species (2) and diets (4) being the treatment factors and each collection unit being the experimental unit (n=3). A critical level of P<0.05 was adopted for all the tests.

3. Results Brown and Forsythe’s test (SAS, 1990) results were not significant ( P>0.05) for all the dependent variables tested meaning that variances were homogenous and that data transformation was not required for ANOVA. Covariate effect (body weight) was removed from the model as either its effect was not significant or it significantly interacted ( P<0.05) with treatment effects (diet and/or species) for all the dependent variables tested. 3.1. Digestibility The ADCs for dry matter, crude protein, gross energy and lipid are indicated in Tables 2 and 3 ADC of nutrient and energy were significantly higher for lake trout compared to Atlantic salmon (Table 2) and significantly higher for chinook salmon compared to rainbow trout (Table 3). ADC of DM and GE of all species linearly decreased ( P<0.05) with decreasing dietary DP/DE ratio while no effect of diet was observed on ADC of CP. ADC of lipids linearly decreased with decreasing dietary DP/ DE ratio in rainbow trout only ( P<0.05, Table 3). 3.2. Growth, feed intake and feed efficiency Overall body weight gain and growth rate (TGC) of all species did not differ between diets ( P>0.05; Tables 4 and 5. Feed efficiency decreased ( P<0.05, Tables 4 and 5) with decreasing DP/DE for all species except chinook salmon. The decreasing FE was paralleled by an increase in feed intake as DP/DE ratio decreased but this increase was only significantly linear ( P<0.05) for rainbow trout and chinook salmon. 3.3. Digestible nitrogen and energy retention efficiencies NRE was significantly lower (Table 6) for lake trout compared to Atlantic salmon (45% vs. 48%, respectively) and increased linearly ( P<0.05) as DP/DE decreased for these two

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Fig. 1. Feed, N and energy utilization responses (averageFstandard error mean across diets, n=12) of lake trout and Atlantic salmon fed four diets with different DP/DE ratios as fish grew during a period of 280 days. (A) Feed efficiency (FE, meanFstandard error mean (S.E.M.), n=12); (B) N retention efficiency (NRE, meanFS.E.M., n=12); (C) energy retention efficiency (ERE, meanFS.E.M., n=12); DNI=digestible N intake; DEI=digestible energy intake; live body weight calculated as the average live body weight between two consecutive sampling periods; mean values of FE, NRE and ERE were calculated based on ratios of feed, N and energy gains, respectively and feed, digestible N (DNI) and digestible energy intake (DEI) between two consecutive sampling periods.

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species. Diet had no effect on NRE of rainbow trout and chinook salmon. Rainbow trout had numerically higher NRE of 52% (average across diets) compared to 40% for chinook salmon (Table 7). Mean ERE was 50% for lake trout and Atlantic salmon and there were no species or diet effects ( P>0.05, Table 6). Mean ERE of rainbow trout and chinook salmon were 54% and 36%, respectively. Diet had no effect ( P>0.05) on ERE in either species. 3.4. Response of nitrogen and energy utilization as fish grew The first order polynomial for FE, NRE and ERE responses over time were significant indicating linear decreases in these variables (Fig. 1; Table 8) as lake trout and Atlantic salmon grew. These changes were independent of dietary DP/DE ratio ( P>0.05, Table 8). The linear decreases in FE and NRE as fish grew were significantly different between these two species. There was a significant species effect on the linear and quadratic decrease of FE with body weight by lake trout and small Atlantic salmon. FE decreased from 1.23 to 1.02 as lake trout grew from 100 g to more than 300 g while in salmon the FE decreased from 1.32 to 1.21. Lake trout NRE decreased from 49% to 43% as fish grew from around 100 g to more than 300 g, while Atlantic salmon showed a decrease from 58% to 38% while they grew from around 60 to 300 g. Furthermore, the rate of NRE decrease with body weight for salmon was significantly different as fish grew (second order polynomial was significant, P<0.05, Table 8). There was no species or diet effect on ERE change as fish grew ( P>0.05, Fig. 1 and Table 8). The decrease in NRE as fish grew agrees with the significant linear and quadratic decrease of FE observed for these two species as they grew ( P<0.0001, Table 8).

Table 8 Retention efficiency of feed, digestible N and energy for lake trout and Atlantic salmon at different body weights when fed diets of varying DP/DE ratios for 280 days at 13 jC

First order polynomial Effects of Species Linear trout Linear salmon Diet SpeciesDiet Second order polynomial Effects of Species Quadratic trout Quadratic salmon Diet SpeciesDiet

FE (gain/feed)

NRE (%DNI)

ERE (%DEI)

P<0.0001

P<0.0001

P<0.05

P<0.0001 P<0.05 P<0.0001 N.S. N.S. P<0.0001

P<0.0001 P<0.05 P<0.0001 N.S. N.S. N.S.

N.S. N.S. P<0.05 N.S. N.S. N.S.

P<0.0001 P<0.0001 P<0.05 N.S. N.S.

N.S. N.S. P<0.05 N.S. N.S.

N.S. N.S. N.S. N.S. N.S.

1

FE=feed efficiency; NRE=N retention efficiency; DNI=digestible N intake; ERE=energy retention efficiency; DEI=digestible energy intake; 1N.S.=not statistically significant ( Pz0.05); (P = 0.0537).

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3.5. Carcass traits and whole body composition Dressed carcass yield (DCY) averaged 89% for lake trout and Atlantic salmon (from 90% to 88%). Decreasing DP/DE ratio significantly reduced the DCY for Atlantic salmon ( P<0.05). Similar response was observed for lake trout, however, the decrease was not significant. The statistical analysis of body composition does not include final weight as covariate as its effect was not significant ( P>0.05). Body composition was not different between lake trout and Atlantic salmon with the exception of a higher CP content of Atlantic salmon compared to trout (18% vs. 17%, P<0.05, Table 9). Furthermore, diet had no effect on body composition of lake trout whereas a linear ( P<0.05) increase of whole body lipid content was observed by Atlantic salmon as DP/DE ratio decreased. DCY for rainbow trout was on average 87% and linearly decreased with decreasing DP/ DE ratio ( P<0.05). Water and CP contents of rainbow trout whole body linearly decreased ( P<0.05) whereas lipid content linearly increased ( P<0.05) as DP/DE ratio decreased.

Table 9 Whole body composition of lake trout (FBW=388 g) and Atlantic salmon (FBW=356 g) fed diets with different DP/DE ratios for 280 days at 13 jC Water %

CP %

Lipids %

Ash %

Lake trout Diet 1, DP/DE=24 Diet 2, DP/DE=22 Diet 3, DP/DE=20 Diet 4, DP/DE=18

71.8 70.0 71.6 69.7

17.0 17.0 17.2 16.6

8.5 9.8 9.4 9.7

2.0 2.0 2.0 2.0

Significancea Linear Quadratic

N.S. N.S.

N.S. N.S.

N.S. N.S.

N.S. N.S.

Atlantic salmon Diet 1, DP/DE=24 Diet 2, DP/DE=22 Diet 3, DP/DE=20 Diet 4, DP/DE=18

70.5 70.5 72.8 68.7

17.9 17.8 17.8 18.0

8.4 8.9 8.6 10.5

2.1 2.0 2.0 2.1

Significanceb Linear Quadratic S.E.M.b

N.S. P<0.05 0.8

N.S. N.S. 0.4

P<0.05 N.S. 0.5

N.S. N.S. 0.08

Effects of Species Diet SpeciesDiet

N.S. P<0.05 N.S.

P<0.05 N.S. N.S.

N.S. P<0.05 N.S.

N.S. N.S. N.S.

N.S.=not statistically significant ( Pz0.05); FBW=final body weight; CP=crude protein. a Significance=significance of the orthogonal linear and quadratic contrasts of dependent variables across diets. b S.E.M.=standard error mean (n=3).

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Table 10 Whole body composition of rainbow trout (FBW=196 g) and chinook salmon (FBW=62 g) fed diets with different DP/DE ratios for 84 and 140 days, respectively, at 12 jC Water %

CP %

Lipids %

Ash %

Rainbow trout Diet 1, DP/DE=24 Diet 2, DP/DE=22 Diet 3, DP/DE=20 Diet 4, DP/DE=18 S.E.M.a

72.5 71.4 71.5 70.1 0.7

17.5 16.9 16.4 16.2 0.3

7.8 9.6 9.9 11.6 0.3

2.1 2.0 2.0 2.0 0.06

Significanceb Linear Quadratic

P<0.05 N.S.

P<0.05 N.S.

P<0.0001 N.S.

N.S. N.S.

Chinook salmon Diet 1, DP/DE=24 Diet 2, DP/DE=22 Diet 3, DP/DE=20 Diet 4, DP/DE=18 S.E.M.a

76.3 74.6 74.1 74.1 0.7

17.0 16.4 16.9 16.7 0.2

5.2 6.0 6.9 7.1 0.2

2.4 2.3 2.3 2.4 0.05

Significanceb Linear Quadratic

N.S. N.S.

N.S. N.S.

P<0.0001 N.S.

N.S. N.S.

FBW=final body weight; CP=crude protein. a S.E.M.=standard error mean (n=3); N.S.=not statistically significant ( Pz0.05). b Significance=significance of the orthogonal linear and quadratic contrasts of dependent variables across diets.

DCY and body composition for chinook salmon were not affected by diet ( P<0.05). However, body lipid content of this species linearly increased as DP/DE ratio decreased ( P<0.05, Table 10).

4. Discussion Despite the large number of published studies on nutrition of salmonids, direct comparisons of N and energy utilization among species are scarce. Comparison of results from different studies is difficult because of confounding factors, such as different experimental design, different diet composition, feeding protocol, experimental conditions, etc. Most of these factors were controlled in this study thereby allowing direct comparison of different species within trial. The results from the present study clearly indicate that juvenile of different species and sizes have different feed efficiencies, different ADC of N and energy, as well as different N and energy utilization/retention. This is in agreement with the results from other comparative studies with salmonid fish (Berg and Bremset, 1998; Rasmussen and Ostenfeld, 2000; Refstie et al., 2000). In these studies, differences were observed

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in terms of nutrient digestibility, digestible nutrient and energy retention efficiencies, and carcass composition. However, findings from Krogdahl et al. (2004) partially disagrees with the previous results in the sense that despite differences in digestibility, Atlantic salmon and rainbow trout had similar digestible N and energy retention efficiency when fed the same diet and under the same rearing conditions. The differences between Krogdahl et al. (2004) findings and the results from the present and other studies may be due to differences between families or strains within a species (Medale, 1993; Corraze et al., 1993; Grisdale-Helland and Helland, 1998; Thodesen et al., 1999; Bonnet et al., 1999; Thodesen et al., 2001; Overturf et al., 2003). Differences in nutrient utilization between experiments may also be due to differences in the stage of development of the fish or due to seasonal variations in growth and feed utilization (Kadri et al., 1996; Morkore and Rorvik, 2001; Nordgarden et al., 2002). It could also be due to differences in environmental factors, such as water temperature and salinity, and/or interaction of these factors with species and fish size (Krogdahl et al., 2004). 4.1. Growth and body composition All diets supported similar growth within each species suggesting that DP/DE ratios from 18 to 24 g/MJ are sufficient to support good growth performance and results were similar to Iwama (1996), Cho (1990) and Cho (1992). Both rainbow trout and chinook salmon increased feed intake of isoenergetic diets as dietary protein was replaced by lipids and carbohydrates. This suggests that fish regulate their feed intake to meet their ‘‘growth target’’ and the associated ‘‘nutrient demands’’, rather than regulate their feed intake in relation to ‘‘energy requirement’’ as frequently assumed in the literature. The increase of feed intake with decreasing DP content of the diet may indicate that diets with lower DP content would be deficient in either one or more digestible amino acids for maximal growth for rainbow trout and chinook salmon while they were optimal for the other species. This also could be due to the possibility that amino acids play a greater role in meeting energy requirements of rainbow trout and chinook salmon compared to Atlantic salmon and lake trout. This warrants further investigation. Increasing the nonprotein energy content of the diets significantly reduced dressed carcass yield for all species except for chinook salmon. This agrees with many other studies where high lipid diets and low DP/DE ratios were reported to decrease carcass yield of juvenile fish (Alsted, 1991; Hillestad and Johnsen, 1994; Helland and GrisdaleHelland, 1998; Jobling et al., 1998; Rasmussen et al., 2000). Diet had a small but significant effect on whole body fish composition. The body lipid content significantly increased while the water content decreased as the dietary nonprotein content increased. This result agrees with many others where protein-sparing effect was associated with increasing fish body fat content (e.g., Hillestad and Johnsen, 1994; Ruohonen et al., 1998; Rasmussen, 2001; Steffens et al., 1999). Similarly to lake trout and Atlantic salmon, rainbow trout also had a significant increase of body lipid content and significant decrease of dressed carcass yield with increasing dietary lipid content and therefore decreasing DP/ DE ratio.

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4.2. Nutrient and energy digestibility ADC of N (protein) was high, 92 – 95% for all the species irrespective of diet reflecting the high quality protein sources used in our study. ADC of crude lipids of all diets for rainbow trout were similar (average 90%) to reported values for rainbow trout (Azevedo et al., 1998). The ADC of lipids for lake trout and Atlantic salmon averaged 86% and 80%, respectively. Our results are similar to those of Grisdale-Helland and Helland (1998) who reported ADC of lipids of about 80% for Atlantic salmon weighing approximately100 g reared in saltwater at 9.4 jC lower than 80%. The ADC of lipids for Atlantic salmon observed in the present study are lower than most values reported for this species (Aksnes, 1995; Hemre et al., 1995; Grisdale-Helland and Helland, 1997; Helland and GrisdaleHelland, 1998; Sveier et al., 1999; Hillestad et al., 2001). ADC of lipids in our study was determined with Atlantic salmon of 24 g compared to fish of greater weights (>80 g) used in the studies mentioned above. It is possible that ADC of lipids increases with increasing body weight. Results from Grisdale-Helland and Helland (1998) and Refstie et al. (2001) suggest that ADC of lipids can be significantly influenced by size of the fish. ADC of lipid for rainbow trout decreased from 93% to 87% as dietary lipid and carbohydrate inclusion rates increased. This minor effect could possibly be due to either the effects of the inclusion rates of dietary lipid or carbohydrate or both. Brauge et al. (1994) found that the ADC of lipid for rainbow trout decreased from 82% to 76% as dietary lipid was increased from 10% to 14%. Krogdahl et al. (2004) found that the ADC of lipids was significantly affected by the dietary starch inclusion rates. In the present study, the major carbohydrate sources were corn gluten mean and wheat middlings. The decrease of ADC of lipids observed in rainbow trout as well as lake trout and Atlantic salmon could be due to the increasing content of the soluble non-starch polysaccharides (NSP) in the diets as the added amounts of corn gluten meal and wheat middlings increased. Soluble NSP increase the viscosity of the digesta and the water content of the feces and therefore reduce the digestibility of lipids (Storebakken, 2002). On the other hand, the increase of either dietary lipids and/or carbohydrates did not cause any significant effect on ADC of lipid for chinook salmon. 4.3. Nutrient utilization FE was similar between lake trout and Atlantic salmon. Moreover, FE decreased with increasing the nonprotein energy in the diet for both species. This decrease in FE could be due to an intake of dietary protein (amino acids) below requirement for maximal protein deposition for the specific rearing conditions present. Fish increased their feed intake possibly to get the amino acids they needed for a certain target growth for the specific rearing conditions present. Despite of this, it is possible that the amino acid intake was still below requirements and weight gain below the target for the specific rearing conditions. To a certain extent fish was able to compensate for the lower dietary amino acid supply and achieve similar weight gain among diets by improving the protein (amino acid) utilization for growth with the lower dietary protein contents. This is supported by the higher NRE obtained with the lower protein diets compared to diets with higher protein content. Similar protein sparing effect by nonprotein energy sources have also been

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reported by other studies (e.g., Johnsen et al., 1993; Hillestad and Johnsen, 1994, Einen and Roem, 1997; Grisdale-Helland and Helland, 1997). ADC of nutrients and energy were higher in lake trout compared to Atlantic salmon. However, salmon appeared to catabolize less amino acids than lake trout as indicated by a higher NRE for salmon compared to lake trout. The fact that the ERE was the same for both species and body lipid contents were similar suggests that these two species did utilize digestible energy with the same efficiency and this was independent of the nutrient utilized as energy source. The effect of diet on FE was different between rainbow trout and chinook salmon. FE of rainbow trout significantly decreased with decreasing DP/DE ratio similarly to the other juvenile salmonids in this study while FE of chinook salmon was not affected by diet. The poor feed intake by chinook salmon and consequently the low body weight gain achieved during the feeding trial could be the reason for the absence of statistical effects of diet on nutrient and energy utilization efficiency by this species. Rainbow trout, unlike lake trout and Atlantic salmon, did not show any significant protein sparing effect of lipid and carbohydrates when fed the same diets. This difference among salmonid species has extremely important practical consequences when formulating diets with optimum DP/DE ratio for different salmonid species. Any DP/DE ratio within the range of 18 to 24 g/MJ would be appropriate for juvenile rainbow trout while for juvenile lake trout and Atlantic salmon a lower DP/DE ratio of 20 or 18 g/MJ would be desired for optimal body weight gain and N retention efficiency. There was a trend toward increase in NRE with decreasing DP/DE ratio; however, this change was not significant for rainbow trout reinforces and confirms the previous suggestion that DP/DE ratio of less than 22 g/MJ may not be desirable for juvenile rainbow trout (Cho and Woodward, 1989; Cho and Kaushik, 1990). Rasmussen et al. (2000) also failed to observe any significant correlation between NRE and lipid intake by rainbow trout, indicating that protein sparing was not improved by adding lipid to already lipid rich diets (z20%). These authors suggested that protein retention increases upon intake of lipid-rich feeds due to a concomitant reduction in protein intake. In our study, however, rainbow trout increased feed intake as the dietary protein decreased and lipid contents increased. The same protein intake by this species irrespective of increased dietary lipid levels agreed with the fact that diet had no effect on NRE. The absence of protein sparing effect of nonprotein energy nutrients by rainbow trout and chinook salmon suggest that rainbow trout and chinook salmon may preferentially use more amino acids as energy sources than Atlantic salmon and lake trout. Further metabolic studies are required to investigate the basis of this preferential use of different nutrients for energy purposes. 4.4. Feed, nitrogen and energy utilization as fish grew FE and NRE decreased, as fish grew. This suggests that the utilization of feed was size dependent and that the optimal DP/DE ratio for growth and feed efficiency is expected to decrease, as fish grow. The results by Einen and Roem (1997) also confirm this idea. Although Einen and Roem (1997) used larger Atlantic salmon than in the current study, these authors found a decrease in optimal DP/DE for growth, i.e., from 19 g/MJ for fish between 1 and 2.5 kg to 16– 17 g/MJ for fish between 2.5 and 5 kg.

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The decrease of FE as lake trout and Atlantic salmon grew was associated with decreasing NRE and ERE. Lower NRE and ERE suggests that a greater proportion of energy that the fish consumed was used for metabolism and less for growth. Lower NRE also suggests that the conversion of dietary amino acids into body protein decreased as fish of these two species increased in size. Moreover, a lower FE as fish grew was also associated with a higher body dry matter and lipid contents as fish grew (results not shown).

5. Conclusion In conclusion, differences in FE among different salmonid species and fish of different sizes were partially due to differences in the digestibility of nutrients and energy and partially due to differences in the efficiency of utilization of amino acids. Digestible energy utilization for growth was independent of the energy source (proteinenergy vs. nonprotein energy). Because the efficiency of energy utilization for growth was the same for lake trout and Atlantic salmon and the DE intake was also similar between these two species it is suggested that the partitioning of DE intake for metabolism was also similar between these two fish. This could be investigated by either respirometry or via partitioning of metabolizable energy intake for maintenance, metabolism, and growth using statistical or more mechanistic approaches. Lower energy retention efficiency as fish grew could be related to greater maintenance needs and less energy available for growth. Since the different fish species and fish of different sizes had different body composition (protein and lipid contents), difference or changes in FE, NRE and ERE could be related to difference in the cost of protein and lipid deposition. These factors should be examined to gain further insight into the basis of species and fish size differences.

Acknowledgements The present work was supported by the Fundacßa˜o para a Cieˆncia e Tecnologia (FCT, Portugal), the Ontario Ministry of Natural Resources (OMNR), the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA), and AquaNet, the Network of Centres of Excellence in Aquaculture. The excellent technical assistance by Ursula Wehkamp, Greg Arndt, Jennifer Gibson, and Stephen Gunther is highly appreciated. Special thanks to Drs. Ian McMillan and J.C. Plaizier for their help with the statistical analysis of the data.

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