Feed intake, growth, and protein utilisation by post-smolt Atlantic salmon (Salmo salar) in response to graded levels of fish protein hydrolysate in the diet

Aquaculture 239 (2004) 331 – 349 www.elsevier.com/locate/aqua-online

Feed intake, growth, and protein utilisation by post-smolt Atlantic salmon (Salmo salar) in response to graded levels of fish protein hydrolysate in the diet Sta˚le Refstie a,b,*, Jan J. Olli c, Ha˚kon Standal d a

AKVAFORSK (Institute of Aquaculture Research AS), N-6600 Sunndalsøra, Norway b Aquaculture Protein Centre (APC), N-6600 Sunndalsøra, Norway c Jaruma AS, N-9730 Karasjok, Norway d Denofa AS, N-1630 Gamle Fredrikstad, Norway

Received 29 March 2004; received in revised form 8 June 2004; accepted 8 June 2004 Available online

Abstract This study investigated how partial dietary replacement of fish meal (FM) by a novel fish protein hydrolysate (FPH) affected feed intake, growth, feed efficiency, nutrient retention, and nutrient digestibility by Atlantic salmon in the early seawater stage. FM was replaced by FPH in increments, producing four extruded diets containing 0%, 5%, 10%, and 15% FPH. Each diet was fed to quadruplicate groups of 163-g salmon maintained in 8.3 jC seawater. The experiment lasted 68 days, divided into three periods. The feed consumption was higher in groups fed 10% and 15% FPH than in those fed 0% FPH, with intermediate intake in groups fed 5% FPH. This was mirrored by the growth, and the groups fed 0%, 5%, 10%, and 15% FPH reached respective individual weights of 323, 350, 362, and 377 g. The retention of protein, which ranged from 48% to 53%, was higher in groups fed 5% and 15% FPH than in those fed 0% FPH. The protein retention was lowest in the groups fed 10% FPH. The retentions of individual amino acids largely mirrored the overall protein retention. The differences in apparent digestibility of protein and individual amino acids were slight, but generally highest when feeding 5% and 15% FPH, lowest when feeding 0% FPH, and intermediate when feeding 10% FPH. In conclusion, the tested FPH proved an efficient feeding stimulant in Atlantic salmon and was highly digestible and well utilised for growth. D 2004 Elsevier B.V. All rights reserved. Keywords: Feedstuffs; Fish meal – fish protein hydrolysate; Feed intake – growth – digestibility – retention; Nitrogen – protein – amino acids; Atlantic salmon Salmo salar

* Corresponding author. AKVAFORSK (Institute of Aquaculture Research AS), N-6600 Sunndalsøra, Norway. Tel.: +47-71-69-53-22; fax: +47-71-69-53-01. E-mail address: [email protected] (S. Refstie). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.06.015

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1. Introduction Enzymatic hydrolysis of scrap fish and fish body parts (by-products) produces fish protein hydrolysates (FPH) rich in soluble low molecular weight peptides (Hertrampf and Piedad-Pascual, 2000; Liaset et al., 2000). Berge and Storebakken (1996) showed that moderate inclusion (3.3 g kg 1) of FPH in a low-temperature dried (LT) fish meal (FM)-based diet resulted in faster growth by Atlantic salmon fry. This demonstrated beneficial effects of FPH when used as a dietary supplement for salmon in this life stage. Whether FPH stimulated feed intake, improved the overall utilisation of protein for growth, or both was, however, unclear. Both FPH and fish meal are made from wild-caught fish. In terms of amino acid composition of the protein, there is little variation among fish protein products produced from different fish species (Hertrampf and Piedad-Pascual, 2000). The protein quality and thus the nutritional value of fish protein products are, however, highly dependent both on raw material freshness and drying conditions. The properties of any FPH furthermore depend on the selected raw materials as well as the hydrolysation process. LT fish meal made from fresh raw materials is highly digestible by fish (Anderson et al., 1992, 1995, 1997) and is well utilised for growth (Anderson et al., 1993, 1997; Aksnes and Mundheim, 1997; Aksnes et al., 1997). However, the amino acids in pre-digested fish protein are more readily absorbed by Atlantic salmon than those in intact fish protein, resulting in a faster and higher postprandial peak of essential amino acids in the plasma (Espe et al., 1993, 1999; Espe and Lied, 1994). Likewise, fish meal protein is more readily absorbed by rainbow trout than protein from malt flour and soybean meal, apparently due to more efficient gastric digestion of the fish meal (Yamamoto et al., 1998). Thus, one might expect a more gradual absorption of amino acids from combinations of feed ingredients that are digested at different rates (e.g., FPH, fish meal, and soy) than from single ingredients or combinations of similar ingredients. This, in turn, appears to result in a more economic utilisation of the amino acids for growth (Espe and Lied, 1994; Espe et al., 1999). Partial hydrolysis of fish protein to produce FPH may also produce components that stimulate the feed intake of Atlantic salmon. Substances like amino acids (Mearns, 1986; Jones, 1989, 1990) and various fractions of aqueous shrimp extracts (Mearns et al., 1987) function as chemo-attractants in salmonid fish. Such compounds are used with success as dietary feeding stimulants in striped bass (Papatryphon and Soares, 2000, 2001). Also, soluble peptides may exhibit flavouring characteristics, which depend on their amino acid composition (reviewed by Ney, 1979). Stale fish contain unpalatable components (e.g., trimethylamine), and the biological value of the protein is reduced as amino acids are deaminated to release volatile ammonia or decarboxylated to produce water-soluble biogenic amines (Pike et al., 1990; Pike and Hardy, 1997). Drying processes furthermore induce thermal damage on protein by causing cross-binding of peptide-bound amino acids, binding of peptide-bound amino acids to other nutrients and oxidation products, and/or amino acid oxidation (Nielsen et al., 1985; Finley and Phillips, 1988; Davidek et al., 1990). These reactions destroy the involved amino acids, and the bindings reduce the overall protein digestibility (Ljøkjel et al., 2000).

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The most significant problem associated with thermal damage on fish meal is the formation of disulfide bindings between entities of cysteine (Opstvedt et al., 1984). Lysine may also bind to reducing sugars (Maillard reaction; Davidek et al., 1990) if sufficient carbohydrates are present. The objectives of the present work were to evaluate effects on (1) feed intake, growth, and feed efficiency, (2) retention of protein, amino acid, and energy, and (3) digestibility of macronutrients, energy, and amino acids by Atlantic salmon in the early seawater stage when LT fish meal was partly replaced by a novel FPH in a high-energy diet. The FPH was produced from fresh raw materials, and gentle heating and drying prevent thermal protein damage. The protein content in the diets was kept low to maximise the general utilisation of protein for growth. All diets contained 10% extracted soybean meal to maximise and stress the utilisation of sulfur-containing amino acids (methionine + cysteine). We chose to include from 0% to 15% FPH in the diets in increments, thereby providing up to 36% of the dietary protein as FPH.

2. Materials and methods 2.1. Dietary protein ingredients The tested fish protein hydrolysate (FPH; Denofa Fish Peptides, Denofa, Fredrikstad, Norway) was produced from fresh raw body parts of pollock. The pollock raw material was enzymatically hydrolysed with Protamex (Novozymes, Bagsværd, Denmark) according to the manufacturing instructions. The water-soluble protein fraction was spray-dried to prevent thermal damage to the protein. The low-temperature dried (LT) fish meal (FM; Norse LT-94, Vedde Herring Oil Factory, Langeva˚g, Norway) had the following specifications: 1.6 g kg 1 TVN, 0.89 g kg 1 cadaverine, 0.29 g kg 1 histamine, 25.9% of crude protein (CP) water-soluble protein, 10.8% oil, 3.5% salt, and 10.6% salt-free ash. The meal was made from herring (92%), blue whiting (6%), and Norway pout (2%), and the CP of the meal was 91.6% digestible by mink. The soybean meal (DenoSoy, Denofa) was de-fatted by hexane extraction and mildly toasted. The wheat was of the Norwegian Bastian variety and had a falling number of 330. Composition of the LT-FM and FPH is given in Table 1. 2.2. Diets Four diets were produced by high-pressure moist extrusion at the Centre for Feed ˚ s, Norway). The diets were formulated to contain 38% CP, 38% lipid, and Technology (A from 9% to 11% starch [dry matter (DM) basis]. All diets contained 10% extracted soybean meal and from 17% to 19% wheat. LT-FM was replaced by FPH in increments, producing diets containing 0% (FM), 5% (FPH-05), 10% (FPH-10), or 15% (FPH-15) FPH (Table 2). The FPH-containing diets were balanced by adding extra wheat. The pellet size of the feeds was 4 mm. All diets contained 100 mg yttrium oxide (Y2O3, Sigma, St. Louis, MO, USA) per kilogram dry mix as an inert marker to permit apparent digestibility measurements. The amino acid composition of the diets is given in Table 3.

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Table 1 Composition of the dietary protein ingredients

Dry matter (DM) (g kg

1

)

In DM (kg 1) Crude protein (CP; N  6.25) (g) Protein (sum amino acids)c (g) Lipid (g) Starch (g) Ash (g) In CP (%; g 16 1 g N) d Essential amino acids Arginine Histidine Isoleucine Leucine Lysine Methionine Methionine + cysteine Phenylalanine Phenylalanine + tyrosine Threonine Valine Nonessential amino acids Alanine Aspartate + asparagine Cysteine Glutamate + glutamine Glycine Proline Serine Tyrosine

LT fish meala

Fish protein hydrolysate (FPH)b

934.9

977.4

725.2 573.5 141.2 3.1 151.0

902.9 698.1 55.9 0.7 64.3

6.56 2.65 4.26 7.33 7.67 2.70 3.62 3.77 6.94 4.42 5.23

6.24 2.10 3.87 6.58 7.24 2.73 3.58 3.45 6.30 4.16 4.54

6.34 8.88 0.91 12.48 6.92 4.48 4.66 3.17

6.24 8.79 0.85 12.65 8.53 4.88 4.93 2.85

a Norse LT-94 (Norsildmel, Fyllingsdalen, Norway; 91.6 N-digestibility by mink, 1.6 mg ammonia kg 1.6 mg cadaverine kg 1, 1.6 mg histamine kg 1). b Denofa, Fredrikstad, Norway. c Sum of dehydrated amino acids (as when peptide-bound), excluding tryptophan. d Free amino acids (after hydrolysing the protein for amino acid analysis).

1

, 900

2.3. Fish, rearing conditions, and sampling The experiment was carried out at AKVAFORSK, Sunndalsøra, Norway. Seawater adapted Atlantic salmon (Salmo salar) were fed the experimental diets for a total of 68 days, divided into three periods of 27, 28, and 13 days, respectively. Prior to the experiment, the fish were fed commercial diets (Skretting, Stavanger, Norway). At the start of the experiment, 16 groups of salmon (163 g, 60 fish/group) were randomly distributed from two holding tanks to fibreglass tanks (1  1  0.6 m3, water depth 40– 50 cm) supplied with seawater. The water temperature during the experimental

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Table 2 Formulation and composition of the experimental diets Diet code

FM

FPH-05

FPH-10

FPH-15

Formulation (g kg ) Fish meala Fish protein hydrolysate (FPH)b Fish oil Soybean mealc Wheatd Constant ingredientse

411.1 0.0 313.0 102.2 170.4 3.3

344.9 51.2 320.1 102.5 178.1 3.3

279.0 102.8 327.5 102.8 184.6 3.3

211.6 154.3 334.4 102.9 193.6 3.3

Composition Dry matter (DM) (g kg

948.7

945.0

946.8

943.7

383.9 315.9 388.7 86.9 74.7 26.2

383.5 312.9 382.5 92.1 67.2 26.5

386.1 321.6 384.4 102.4 62.7 26.5

384.3 329.2 376.4 106.5 58.6 26.5

1

1

)

In DM (kg 1) Crude protein (N  6.25) (g) Protein (sum amino acids)f (g) Lipid (g) Starch (g) Ash (g) Gross energy (MJ) a

Norse LT-94 (Norsildmel, Fyllingsdalen, Norway). Denofa, Fredrikstad, Norway. c DenoSoy (Denofa, Fredikstad, Norway). d Bastian variety (falling number of 330, protein content of 15.2). e Constant ingredients (in g kg 1): 2.9 g vitamin and mineral premix (G.O. Johnsen, Oslo, Norway); 0.3 g phosphorylated vitamin C (G.O. Johnsen); 0.1 g Y2O3 (Sigma). f Sum of dehydrated amino acids (as when peptide-bound), excluding tryptophan. b

period was 8.3 F 0.5 jC (mean F S.D.; see Fig. 1), and the O2 saturation of the outlet water ranged from 81% to 97%. Each diet was fed to quadruplicate groups of fish. The fish were fed continuously (24 h day 1) by electrically driven disc feeders, and uneaten feed was collected daily from the outlet water in wire mesh strainers. The feeding rate was planned to be 15% in excess and was adjusted according to the recorded overfeeding every 3 days (Helland et al., 1996). The fish were weighed in bulk at the start of the experiment and at days 27, 55, and 68. The fish were deprived of feed for 1 day prior to each weighing except at day 68. At day 68, the fish were stripped as described by Austreng (1978) to collect faeces for digestibility determination before the final weighing. The faecal samples were pooled per tank and immediately frozen at 20 jC. Before distributing fish to the experimental tanks, 20 fish were sampled from the holding tanks. These fish were euthanised in water with a lethal concentration of tricaine methanesulfonate (MS 222, Argent Chemical Laboratories, Redmond, WA, USA), weighed individually, and immediately frozen at 20 jC as four pooled samples of five fish each. At day 69, this procedure was repeated, sampling five fish per tank.

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Table 3 Amino acid composition of the experimental diets Diet code

FM

FPH-05

FPH-10

FPH-15

6.72 2.62 4.15 7.20 7.16 2.58 3.67 4.04 7.25 4.38 5.10

6.70 2.54 4.18 7.25 7.08 2.58 3.68 4.06 7.24 4.31 5.02

6.74 2.32 4.16 7.20 7.18 2.50 3.63 4.12 7.35 4.38 4.92

6.78 2.31 4.19 7.22 7.11 2.63 3.72 4.14 7.40 4.41 5.05

5.92 9.05 1.09 14.75 6.51 4.93 5.03 3.21

5.83 9.06 1.11 14.99 6.60 5.20 5.04 3.18

5.84 9.19 1.12 15.06 6.84 5.18 5.19 3.23

6.08 9.29 1.09 15.43 7.21 5.35 5.31 3.26

1

In CP (%; g 16 g N) Essential amino acidsa Arginine Histidine Isoleucine Leucine Lysine Methionine Methionine + cysteine Phenylalanine Phenylalanine + tyrosine Threonine Valine Nonessential amino acidsa Alanine Aspartate + asparagine Cysteine Glutamate + glutamine Glycine Proline Serine Tyrosine a

Free amino acids (after hydrolysing the protein for amino acid analysis).

The experiment was conducted in accordance with laws and regulations that control experiments and procedures in live animals in Norway, as overseen by the Norwegian Animal Research Authority. 2.4. Calculations Crude protein (CP) was calculated as N  6.25. Protein was estimated after hydrolysing the protein for amino acid analysis as the sum of dehydrated amino acids (as when peptide-bound), not including tryptophan. Specific growth rate (SGR) was calculated as: SGR = 100  (ln W1 ln W0)  D 1, where W0 and W1 are the initial and final weights (tank means), respectively, and D represents the number of feeding days. Thermal-unit growth coefficient (TGC) was calculated according to Iwama and Tautz (1981), modified by Cho (1992) as: TGC=(W11/3 W01/3)  (ADj) 1, where ADj is the thermal sum (feeding days  average temperature, jC). Feed intake was estimated by subtracting uneaten feed from fed feed on a dry matter basis. Recovery of uneaten feed was estimated as described by Helland et al. (1996), and the recorded uneaten feed was corrected for dry matter losses during feeding and collection. Feed efficiency ratio (FER) was calculated as: FER = G  F 1, where G is the weight gain and F is the consumption of dry matter from the feed. Instantaneous (daily) feed intake (percentage of body weight) was estimated as: 100  Fd  [Wd 1+( Fd 1  FERp)] 1,

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Fig. 1. Instantaneous (daily) and cumulative dry matter (DM) intake (mean, n = 4) and water temperature.

where Fd represents feed intake at day d, Wd 1 and Fd 1 are weight and feed intake the previous day, respectively, and FERp is FER during the current experimental period. Retentions of N, protein, individual amino acids, and energy were calculated as 100  [(W1  C1) (W0  C0)]/[ F  Cdiet], where Cdiet is the content in the diets, and C0 and C1 are initial and final contents in the fish (pooled samples of five fish per tank), respectively. For calculation of retention, proline and lysine in fish were calculated as proline + hydroxyproline and lysine + hydroxylysine, respectively. Apparent digestibility was estimated by the indirect method, as described by Maynard and Loosli (1969), using Y2O3 as an inert marker (Austreng et al., 2000). Nitrogen and energy expenditures were budgeted based on consumption, gain, apparent digestibility, and retention. Metabolic losses were estimated as the difference between consumption and the sum of deposition and faecal excretion.

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2.5. Chemical analyses Faeces and homogenised fish were frozen ( 20 jC) and freeze-dried (Hetosicc Freeze drier CD 13-2 HETO, Birkerød, Denmark) prior to analyses. Feed ingredients, diets, and freeze-dried faeces were analysed for dry matter (105 jC to constant weight), ash (combusted at 550 jC to constant weight), nitrogen (Kjeltec Auto Analyser, Tecator, Ho¨gana¨s, Sweden), lipid [pre-extraction with diethylether and hydrolysis with 4 M HCl prior to diethylether extraction (Stoldt, 1952) in a Soxtec (Tecator) hydrolysing (HT-6) and extraction (1047) apparatus], starch [determined as glucose after hydrolysis by a-amylase and amylo-glucosidase, followed by glucose determination by the ‘‘GODPOD method’’ (Megazyme, Bray, Ireland)], and amino acids (as described below). Diets and faeces were further analysed for gross energy (Parr 1271 Bomb calorimeter, Parr, Moline, IL, USA) ˚ s, Norway, by inductively coupled plasma mass spectroscopy and yttrium [at Jordforsk, A (ICP-MS), as previously described by Refstie et al., 1997]. Freeze-dried fish were analysed for dry matter, nitrogen, amino acids, and energy. Amino acids were analysed at Aminosyraanalyscentralen (Uppsala, Sweden). The samples were hydrolysed for 24 h at 110 jC in 6 M HCl, which had 1 mg ml 1 phenol and norleucine (internal standard) added. Following in vacuo evaporation, the hydrolysate was dissolved in buffer and analysed with an Alpha-Plus amino acid analyser (LKB Vertriebs, Vienna, Austria), using the standard protein hydrolysate program with sodium citrate buffers and ninhydrin detection. The detected values for threonine and serine were corrected for destruction using respective recovery values of 0.96 and 0.90. Methionine and cysteine were determined separately as methionine sulfone and cysteic acid following oxidation with performic acid. The results were normalised on the basis of the recovery of norleucine. Tryptophan was not determined. 2.6. Statistical analyses The results were analysed by the general linear model procedure in the SAS computer software (SAS, 1985). Mean results per tank were subjected to one-way analysis of variance (ANOVA) with diet as the independent variable. Significant differences were indicated by Duncan’s multiple range test. The level of significance was P V 0.05, and the results are presented as mean F standard error of the mean (S.E.M.).

3. Results As shown in Table 1, LT-FM contained less protein but more lipid and ash than the FPH. The estimated level of crude protein (CP; N  6.25) was higher than the sum of protein-bound amino acids (excluding tryptophan) in both products. In the diets, the starch content reflected the inclusion of wheat (Table 2). As the ash content in the FPH was low (6.4%), the dietary ash content was reduced by the inclusion of FPH. Other differences in diet composition were marginal (Tables 2 and 3). No fish died during the experiment. During the first period, the growth rates (SGR and TGC  1000) were highest in the groups fed the diets with 10% and 15% FPH (FPH-10

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and FPH-15), intermediate in the groups fed the diet with 5% FPH (FPH-5), and lowest in the groups fed the diet with no FPH (FM; see Table 4). During the second period, the groups fed FM and FPH-05 grew at similar rates, but apart from this, the differences in growth pattern did not change. In the third period, there were no significant differences in growth rate between any groups. Overall (days 0– 68), the growth rates were highest in the groups fed FPH-10 and FPH-15, intermediate in the groups fed FPH-05, and lowest in the groups fed FM. The different treatment groups reached final body weights ranging from 323 to 377 g. As for the overall growth rates, final body weight was highest in the groups fed FPH-10 and FPH-15, intermediate in the groups fed FPH-05, and lowest in the groups fed FM. The consumption of feed dry matter (DM) paralleled the growth performances (Fig. 1; Table 5). During the first period, the feed efficiency ratio (FER) was similar in all treatment groups except in those fed FM, where it was lower. In the second and third periods, there were no significant differences in FER, although it was numerically higher in the groups fed FM and FPH-10 than in the groups fed FPH-10 and FPH-15 during the last period. When seen over all periods, there were no treatment differences in FER. The retention of consumed and digested N was higher in groups fed FPH-05 and FPH-15 than in those fed FM and FPH-10 (Table 5). The numerical figure was lowest in the FPH-10 group. The retention of consumed and digested protein (sum protein-bound amino acids, excluding tryptophan) was generally lower than that of N, but the ranking between treatment groups was similar. The retention of consumed energy was lower in groups fed FM than in any other treatment group, while the retention of digested energy was similar in all treatment groups.

Table 4 Specific growth rates (SGR), thermal-unit growth coefficients (TGC  1000), and body weights (mean F S.E.M., n = 4) Diet (days)

FM

FPH-05

FPH-10

FPH-15

SGR 0 – 27 28 – 55 56 – 68 0 – 68

1.02 F 0.06c 1.22 F 0.06y 0.97 F 0.05 1.09 F 0.06b

1.29 F 0.03b 1.25 F 0.08x,y 0.92 F 0.05 1.20 F 0.04a,b

1.39 F 0.02a,b 1.36 F 0.07x,y 0.83 F 0.04 1.26 F 0.04a

1.43 F 0.03a 1.45 F 0.03x 0.90 F 0.06 1.33 F 0.02a

TGC  1000 0 – 27 28 – 55 56 – 68 0 – 68

2.22 F 0.14c 3.05 F 0.18b 2.90 F 0.17 2.67 F 0.16b

2.85 F 0.06b 3.22 F 0.23b 2.83 F 0.15 2.99 F 0.11a,b

3.08 F 0.06a,b 3.55 F 0.19a,b 2.58 F 0.16 3.17 F 0.12a

3.18 F 0.07a 3.81 F 0.09a 2.83 F 0.20 3.36 F 0.07a

Weight (g) 0 27 55 68

163 F 1 210 F 3b 285 F 8c 323 F 11b

164 F 1 227 F 1a 310 F 7b 350 F 8a,b

163 F 1 231 F 2a 325 F 8a,b 362 F 11a

163 F 1 233 F 2a 335 F 5a 377 F 6a

a,b,c x,y

Different superscript letters denote significant differences within each row ( P < 0.05). Different superscript letters denote trends within each row ( P < 0.1).

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Table 5 Feed intake, feed efficiency ratio (FER), and retention of nitrogen (N), protein, and energy (mean F S.E.M., n = 4) Diet (days)

FM

FPH-05

FPH-10

FPH-15

29.0 + 0.4b 29.1 + 1.8b,c 10.0 + 0.4 88.0 F 3.9b,c

31.6 + 0.7a 32.9 + 1.1a,b 10.3 + 0.6 98.8 F 3.7a,b

32.6 + 0.6a 33.8 + 0.8a 11.2 + 0.3 104.0 F 2.9a

FER, g body weight increase/g feed intake (DM) 1.31 F 0.01a 0 – 27 1.15 F 0.04b 28 – 55 1.30 F 0.04 1.27 F 0.05 56 – 68 1.27 F 0.03 1.28 F 0.05 0 – 68 1.25 F 0.02 1.28 F 0.02

1.32 F 0.01a 1.23 F 0.04 1.10 F 0.05 1.23 F 0.02

1.32 F 0.01a 1.30 F 0.00 1.11 F 0.10 1.26 F 0.02

Retention of N (%) Consumed Digested

58.9 F 0.4a 67.9 F 0.4a

56.3 F 0.9b 65.4 F 1.0b

58.7 F 0.2a 67.5 F 0.4a

Retention of protein (sum amino acids)* Consumed 50.6 F 1.2a,b Digested 56.7 F 1.2x,y

52.0 F 1.0a 57.7 F 1.0x

48.3 F 0.8b 54.0 F 1.3y

52.8 F 1.1a 58.5 F 1.2x

Retention of energy (%) Consumed 43.4 F 0.8b Digested 63.4 F 0.4

48.3 F 1.3a 64.8 F 1.1

49.0 F 1.8a 66.7 F 1.9

50.2 F 1.1a 67.5 F 2.3

Feed intake (DM), % of initial weight 0 – 27 25.1 + 1.0c 28 – 55 27.4 + 1.6c 56 – 68 10.6 + 0.3 0 – 68 79.1 F 4.7c

57.0 F 0.3b 67.2 F 0.5a,b

a,b,c

Different superscript letters denote significant differences within each row ( P < 0.05). Different superscript letters denote trends within each row ( P < 0.1). * Sum of dehydrated amino acids (as when peptide-bound), tryptophan excepted. x,y

The final whole-body energy content was highest in the fish fed FPH-10 and FPH-15, intermediate in the fish fed FPH-05, and lowest in the fish fed FM (Table 6). The wholebody contents of CP and individual amino acids as related to CP were similar in all treatment groups. When measured as the sum of protein-bound amino acids, the wholebody protein content was, however, lowest in the FPH-10 groups, intermediate in the FM and FPH-05 groups, and highest in the FPH-15 groups. As shown in Fig. 2, the deposition of N per kilogram gain was similar in all groups. Likewise, the metabolic losses of energy per kilogram gain did not differ between treatment groups. Metabolic excretion of N per kilogram gain and deposition of energy per kilogram gain were ranked in parallel with the retention figures for N and energy, Table 6 Final energy and protein content of whole fish (mean F S.E.M., n = 4) Diet

FM 1

Energy (MJ kg ) Crude protein (CP) (g kg Protein* (g kg 1) a,b,c

FPH-05 c

1

)

8.2 F 0.1 173.8 F 1.5 126.9 F 1.2a,b

FPH-10 b

8.7 F 0.1 174.2 F 0.8 126.9 F 0.8a,b

FPH-15 a

9.1 F 0.2 174.6 F 2.4 125.5 F 1.5b

Different superscript letters denote significant differences within each row ( P < 0.05). * Sum of dehydrated amino acids (as when peptide-bound), excluding tryptophan.

9.1 F 0.1a 175.8 F 1.4 130.9 F 1.3a

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Fig. 2. Budgeted nitrogen and energy utilisation per kilogram gain (mean, n = 4), where different letters denote significant differences between bars ( P < 0.05). Metabolic losses are estimated as the difference between consumption and the sum of deposition and faecal excretion.

respectively (see Table 5). For both N and energy, the faecal losses per kilogram gain were ranked in parallel with the apparent digestibility estimates (see Table 8). Retentions of both consumed and digested essential amino acids were generally highest in the groups fed the FPH-15 diet (Table 7), while the retention figures were generally lowest in the groups fed FPH-10. However, these differences were only significant ( P < 0.05) for histidine and lysine. For both consumed and digested histidine, there was a slight but nonsignificant gradual increase in retention when including up to 10% FPH in the diet, but a markedly and significant increase when increasing the FPH inclusion from 10% to 15%. The retention of digested cysteine was very high, ranging from 78% to 82%.

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Table 7 Retention of essential and semi-essential amino acids (mean F S.E.M., n = 4) Diet

FM

FPH-05

FPH-10

FPH-15

Retention of consumed amino acids (%) Arginine 44.3 F 1.3 Cysteine 51.6 F 1.4 Histidine 49.6 F 1.9b Isoleucine 54.8 F 1.8x,y Leucine 52.1 F 1.8x,y Lysine 61.1 F 1.8b Methionine 59.8 F 3.1 Phenylalanine 48.8 F 1.8x,y Threonine 53.8 F 1.3x,y Tyrosine 56.6 F 2.7 Valine 51.9 F 1.8

41.8 F 4.7 56.5 F 4.4 51.9 F 1.4b 55.2 F 1.1x,y 51.8 F 1.1x,y 63.3 F 1.3a,b 64.4 F 1.2 50.7 F 1.4x,y 56.2 F 1.9x,y 56.3 F 1.7 53.3 F 1.1

42.0 F 0.4 50.8 F 1.7 52.9 F 1.1b 52.6 F 1.2y 49.4 F 1.1y 59.0 F 1.5b 62.0 F 0.9 47.3 F 0.7y 52.1 F 1.1y 52.8 F 1.3 52.4 F 1.0

46.3 F 1.2 55.4 F 1.1 60.0 F 1.5a 59.1 F 2.1x 55.5 F 1.8x 66.6 F 1.9a 65.3 F 2.2 52.8 F 1.5x 57.5 F 1.4x 58.1 F 2.1 56.5 F 1.7

Retention of digested amino acids (%) Arginine 47.6 F 1.2 Cysteine 82.1 F 2.7 Histidine 55.3 F 1.9b Isoleucine 61.3 F 1.9 Leucine 57.7 F 1.9x,y Lysine 65.9 F 1.8a,b Methionine 66.2 F 3.2 Phenylalanine 55.1 F 1.9 Threonine 61.6 F 1.3 Tyrosine 63.7 F 3.0 Valine 58.3 F 2.0

44.6 F 5.0 80.9 F 5.2 57.9 F 1.7b 61.1 F 1.0 56.6 F 1.1x,y 67.7 F 1.3a,b 71.0 F 1.4 56.2 F 1.4 64.0 F 2.0 63.1 F 1.6 59.5 F 1.0

45.0 F 0.5 78.1 F 1.8 59.8 F 1.2b 58.7 F 1.3 54.5 F 1.2y 63.6 F 1.6b 69.0 F 1.2 52.6 F 0.8 59.9 F 1.3 59.7 F 1.4 58.9 F 1.1

49.4 F 1.2 81.7 F 2.3 67.3 F 1.8a 65.4 F 2.5 60.7 F 2.1x 71.5 F 2.2a 71.6 F 2.3 58.2 F 1.7 65.6 F 1.8 64.9 F 2.6 63.0 F 2.1

a,b,c x,y

Different superscript letters denote significant differences within each row ( P < 0.05). Different superscript letters denote trends within each row ( P < 0.1).

The faecal DM content was highest in the groups fed FM, intermediate in the groups fed FPH-05 and FPH-10, and lowest in the groups fed FPH-15 (Table 8). The apparent digestibility estimated for N, lipid, and energy was lower in the groups fed FM than in any other treatment group. The apparent digestibility of protein did not differ among treatment groups, although the numerical trend was similar as for N. The apparent digestibility of starch and ash was higher in groups fed FM and FPH-05 than in those fed FPH-10 and FPH-15. The digestibility of starch was inversely related to the dietary starch level. Except for phenylalanine, there were no significant treatment effects on the estimates for apparent digestibility of individual essential amino acids (Table 8). The digestibility of phenylalanine was lower in the groups fed FM than in any other treatment group. For leucine and arginine, the digestibility estimates tended ( P < 0.1) to be highest in the groups fed FPH-05 and FPH-15, intermediate in the groups fed FPH-10, and lowest in the groups fed FM. Among the nonessential amino acids, there were significant treatment effects on apparent digestibility estimates for alanine, cysteine, glycine, and proline. The alanine digestibility was highest in the groups fed FPH-15, intermediate in the groups fed FPH-05, and lowest in the groups fed FM and FPH-10. The glycine digestibility was highest in the groups fed FPH-15, intermediate in the groups fed FPH-10 and FPH-05, and lowest in the

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Table 8 Digestibility of macronutrients and individual amino acids (mean F S.E.M., n = 3) Diet Faecal DM (%) Apparent digestibility (%) Nitrogen Protein (sum amino acids)* Lipid Starch Ash Energy Essential amino acids Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine Nonessential amino acids Alanine Aspartate + asparagine Cysteine Glutamate + glutamine Glycine Proline Serine Tyrosine a,b x,y

FM

FPH-05 a

FPH-10 b

FPH-15 b

9.9 F 0.2

9.1 F 0.1c

11.2 F 0.1

10.3 F 0.3

84.9 F 0.5b 89.3 F 0.4 67.7 F 2.7b 67.8 F 0.7a 14.0 F 2.8a 68.5 F 1.6b

86.8 F 0.4a 90.1 F 0.4 76.6 F 2.8a 66.5 F 0.5a 13.4 F 1.6a 74.6 F 1.6a

86.1 F 0.3a 89.5 F 0.1 76.3 F 1.4a 63.7 F 0.9b 19.6 F 0.8b 73.4 F 0.7a

86.9 F 0.3a 90.3 F 0.3 77.9 F 2.6a 62.1 F 0.4b 25.3 F 1.5b 74.4 F 1.3a

93.0 F 0.3y 89.7 F 0.6 89.4 F 0.3 90.3 F 0.3y 92.8 F 0.4 90.2 F 0.6 88.5 F 0.3b 87.4 F 0.5 89.0 F 0.4

93.8 F 0.3x 89.7 F 0.6 90.4 F 0.5 91.4 F 0.3x 93.4 F 0.3 90.7 F 0.5 90.1 F 0.4a 87.8 F 0.6 89.6 F 0.6

93.4 F 0.0x,y 88.5 F 0.1 89.6 F 0.1 90.8 F 0.1x,y 92.8 F 0.1 89.8 F 0.3 90.0 F 0.1a 87.0 F 0.2 89.0 F 0.2

93.8 F 0.2x 89.2 F 0.3 90.3 F 0.4 91.4 F 0.3x 93.2 F 0.4 91.2 F 0.3 90.7 F 0.2a 87.6 F 0.4 89.8 F 0.4

90.6 F 0.3b 83.4 F 0.6 63.0 F 1.6c 92.8 F 0.3 85.5 F 0.4c 89.1 F 0.3c 88.2 F 0.5 89.0 F 0.5

91.3 F 0.3a,b 84.1 F 0.5 69.7 F 1.0a 93.5 F 0.3 87.1 F 0.4b 90.4 F 0.3a 88.8 F 0.5 89.2 F 0.6

90.9 F 0.1b 83.5 F 0.1 65.0 F 0.9b,c 92.8 F 0.1 86.7 F 0.3b 89.4 F 0.1b,c 88.3 F 0.1 88.5 F 0.1

91.9 F 0.2a 84.8 F 0.4 67.8 F 1.0a,b 93.2 F 0.3 88.4 F 0.2a 90.0 F 0.2a,b 89.0 F 0.2 89.6 F 0.4

Different superscript letters denote significant differences within each row ( P < 0.05). Different superscript letters denote trends within each row ( P < 0.1). * Sum of dehydrated amino acids (as when peptide-bound), excluding tryptophan.

groups fed FM. The ranking among treatments was similar for digestibility of proline and cysteine, being highest in the groups fed FPH-05 and FPH-15, intermediate in groups fed FPH-10, and lowest in groups fed FM.

4. Discussion The major finding of this experiment is that inclusion of up to 15% of enzymatically hydrolysed and spray-dried fish protein hydrolysate (FPH) in diets based on lowtemperature (LT) dried fish meal (FM) and soybean meal stimulates growth in Atlantic salmon. The main response to FPH appears to be increased feed intake. However, even at 5% inclusion, the utilisation of dietary protein for growth is increased. FPH is as digestible as LT-FM by Atlantic salmon.

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Moderate dietary inclusion of enzymatically hydrolysed FPH has also previously been shown to have a definitive positive effect on growth by Atlantic salmon (Berge and Storebakken, 1996). As a similar effect was not observed in turbot, Oliva-Teles et al. (1999) suggested that a major cause for this response is high palatability of the FPH by salmon. This is in line with the present results. Bitter flavouring agents reduce the palatability of feed by salmonid fish (Warren, 1963; Schreck and Moffitt, 1987; Hustvedt et al., 1991; Toften et al., 1995). Unpleasant bitterness may also be a problem with many protein hydrolysates. This is caused by soluble peptides with a high content of amino acids with hydrophobic functional groups (Ney, 1979). The concentration of such peptides in a hydrolysate may be controlled by the specificity of the enzymes used for hydrolysis and the separation steps in downstream processing (Adler-Nissen, 1986). The present FPH was specifically produced to avoid bitter flavour. Hydrolysed fish protein has long been used in commercial salmon diets in the form of fish silage produced by acidic hydrolysis and autolysis (Espe and Lied, 1999). This is a process that might potentially destroy essential amino acids, particularly tryptophan (Jackson et al., 1984; Shahidi et al., 1995). High dietary inclusion of fish silage has repeatedly been shown to affect growth negatively (Espe et al., 1992, 1999; Heras et al., 1994; Sveier et al., 2001). Espe et al. (1999) indicated a positive effect of moderate substitution of fish meal by fish silage, with an optimum at about 10% replacement. However, this did not improve the digestibility or retention of the dietary nitrogen, as opposed to the improved nitrogen utilisation when substituting fish meal by the present FPH. When compared to previous results obtained with Atlantic salmon of comparable size at similar temperatures (Austreng et al., 1987), the different treatment groups grew from 9% (fed no FPH) to 33% (fed 15% FPH) faster than expected. The slower growth (lower TGC) during the first than the second period was probably caused by an initial lag period when the fish adapted to the experimental conditions. The generally slow growth during the last period indicates that the experimental conditions became suboptimal. This was probably due to a combined effect of large fish size, which exceeded 300 g, and high fish density, which exceeded 30 kg (m 3) 1. The retentions of nitrogen and energy were high when compared to the normal range reported for Atlantic salmon of comparable size (Grisdale-Helland and Helland, 1995, 1997; Thodesen et al., 1999; Nordrum et al., 2000; Refstie et al., 2000; Refstie and Tiekstra, 2003). The final whole-body energy content was also high in all treatment groups. The average content of energy in animal (crude) protein and lipid is 23.6 and 39.5 kJ g 1, respectively (Brody, 1945). In whole body, other components contribute insignificant energy. Based on these assumptions, the calculated final lipid content in fish fed the diets with 0%, 5%, 10%, and 15% FPH averaged 198, 210, 220, and 221 g kg 1, respectively. Such a lipid level is high in salmon of the given size (Shearer et al., 1994) and may indicate deposition of lipid in adipocyte-rich tissues and escalating fattening. This probably resulted from low protein and high lipid content in the diets, as previously demonstrated by Refstie et al. (2001). Fattening is a highly energy-inefficient growth form, so this may also in part explain the tendency towards lower FER in the groups fed 10% and 15% FPH during the last period.

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Although the apparent digestibility of N was lower in fish groups fed the FM diet than in all fish groups fed diets with FPH, the difference was slight. Furthermore, the treatment groups did not differ significantly in terms of apparent digestibility of protein (sum amino acids excluding tryptophan) that is useful for muscle growth, although the digestibility was numerically lowest when feeding the diet with no FPH. This discrepancy may be due to higher nonprotein nitrogen (NPN) content in the FPH, suggesting that certain NPN components are readily absorbed. The availability of several amino acids appeared lower in the LT-FM than in the FPH. This was seen from low apparent digestibility when feeding the diet with no FPH, particularly of cysteine. Formation of disulfide bindings and subsequently reduced cysteine digestibility is an important indicator of thermal damage on fish protein (Opstvedt et al., 1984). Thus, the lowered amino acid availability may follow from more severe drying conditions and thus more thermal damage on the LT-FM protein than the FPH protein. However, the low retention of N, protein (sum amino acids), and certain individual amino acids by the fish fed 10% FPH stands out as illogical. It does not appear to be related to the nutritional qualities of the FPH. It does, however, correspond with low apparent digestibility of cysteine and low FER in periods 2 and 3. Furthermore, the energy (and thus lipid) contents in fish fed the diets 10% and 15% FPH were identical, although the fish fed 10% FPH gained 15 g (but nonsignificantly) less individual weight. The sum of these observations may indicate slightly more processing damage on the protein in the diet with 10% FPH, resulting in increased amino acid catabolism and conversion to lipid. If so, this may have been caused by different extrusion conditions during diet manufacture (Sørensen et al., 2002). The apparent digestibility of lipid was extraordinary low in all treatment groups, ranging from 68% to 78%. This may in part be caused by the soybean meal in the diets, which at 10% inclusion supplied c 11% of the dietary protein. Dietary soybean meal has repeatedly been shown to restrict the overall digestibility of lipid by Atlantic salmon (Refstie et al., 1998, 1999, 2000, 2001; Storebakken et al., 1998). The cause(s) for this are still uncertain, but appear related to soybean meal intolerance by salmon, which is manifested by enteritis in the distal intestine (Ingh et al., 1991, 1996; Baeverfjord and Krogdahl, 1996). High dietary lipid level may furthermore have worsened negative effect(s) on the digestion and absorption of lipid. FPH markedly improved the lipid digestibility even at 5% dietary inclusion, but the reason(s) for this remain unclear. Tryptophan content in feed ingredients and feeds was not determined in the present experiment, as the analytic method used cannot measure tryptophan accurately in starch-rich substrates. Consequently, the content of protein (sum of peptide-bound amino acids) in the ingredients and diets was underestimated, but only to a small extent, as fish protein only contains 0.8 –1.2 g 16 1 g N of tryptophan (Hertrampf and Piedad-Pascual, 2000). A very high retention of digested cysteine (78 –82%) indicates that cysteine was actively synthesised from serine and methionine by the transsulfuration pathway (Yokoyama et al., 1994). Reduced availability of dietary cysteine due to thermal protein damage may increase the conversion of methionine to cysteine. As fish protein is rich in methionine (Hertrampf and Piedad-Pascual, 2000), this is unproblematic when feeding LT fish meal-based diets. Methionine is, however, the first limiting amino acid in vegetable

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protein meals (e.g., soybean meal) that is used for fish (reviewed by Refstie and Storebakken, 2001). Fish-based ingredients with high cysteine availability due to gentle drying may prove valuable to spare methionine when feeding diets with high levels of vegetable protein. To conclude, the tested FPH proved to increase the feed intake in Atlantic salmon. Enhanced feed intake was more pronounced at 10% and 15% than at 5% dietary inclusion. The availabilities of several amino acids were higher in the FPH containing diets than in the control diet, resulting in more efficient utilisation of the protein for growth. Thus, the salmon responded by faster growth when including up to 15% FPH in the diet, thereby supplying up to 36% of the dietary protein.

Acknowledgements The authors want to acknowledge the skilful technical assistance of F. Johnsen at FishFeed and of F. Nerland and A. Valset at AKVAFORSK. Denofa provided financial support for the experiment.

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