Aquaculture nutrition, tập 16, số 6, 2010

Aquaculture Nutrition 2010 16; 559–568 doi: 10.1111/j.1365-2095.2009.00685.x Research Institute of Fish Culture and Hydrobiology, University of South Bohemia, Vodnany, Czech Republic Due to distinctive feeding habits and digestive physiology determination of nutrient digestibility is more demanding with crustacean than fish species A study was conducted to validate the use of linear prediction equations established with fish species to predict apparent digestible protein (ADP) and lipid (DL) contents [g kg)1 dry matter (DM)] of feed ingredients (ADP = )10.0731 + 0.8942 CP, DL = )1.5824 + 0.8654 CL) and compound diets (ADP = )51.4001 + 0.9872 CP, DL = )2.7303 + 0.9123 CL) from dietary crude protein (CP) and lipid (CL) contents for crustaceans (shrimp, lobster, crab) Observed values (n = 91) obtained in 17 studies, which evaluated CP digestibility of 26 feed ingredients for eight crustacean species, presented a linear relationship (R2 = 0.9213, RMSE = 54.1245) with predicted values Predicted values overestimated observed values with a mean prediction error (MPE) of 0.1315 However, observed DL values of 17 feed ingredients (n = 31; studies = 5; species = 5) were overestimated with a MPE of 0.4500 Similar trends than above were found with prediction of the ADP (n = 185; studies = 32; species = 11; R2 = 0.8047; MPE = 0.1002) and DL (n = 64; studies = 11; species = 3; R2 = 0.6907; MPE = 0.2372) contents of compound diets KEY WORDS: WORDS: crustaceans, digestible protein, digestible lipid, prediction Received 26 June 2008, accepted 27 February 2009 Correspondence: James Sales, Research Institute of Fish Culture and Hydrobiology, University of South Bohemia, Zatisi 728, 38925 Vodnany, Czech Republic E-mail: james_sales_1@hotmail.com Measurement of nutrient digestibility of diets and feed ingredients, which is of utmost importance to nutritionists Ó 2009 Blackwell Publishing Ltd and feed formulators to optimize nutritional value and cost of diets (Smith et al 2007) and in the application of waste management (Cousin et al 1996), is more problematic with crustaceans than fish Compared to fish, crustaceans are slow eaters (Ishikawa et al 1997), resulting in leaching losses of nutrients before feed is consumed Furthermore, collection of faeces by stripping or dissection, commonly used with fish, are no feasible methods with crustaceans (Smith & Tabrett 2004) The conventional ÔGuelphÕ system, based on settlement of faeces (Cho et al 1982) and widely used in digestibility studies with fish, creates difficulties with crustaceans due to their feeding habits and coprophagy (Martı´ nez-Palacios et al 2001) Several crustacean species, such as the common prawn (Palaemon serratus) and spot shrimp (Pandalus platyceros) (Forster & Gabbott 1971), hybrid lobster (Homarus sp.) (Bordner et al 1983), American lobster (Homarus americanus) (Leavitt 1985), red swamp crayfish (Procambarus clarkii) (Brown et al 1986) and common yabby (Cherax destructor) (Jones & De Silva 1997a), have the ability to selectively partition some components of its diet during digestion, complicating the use of markers in digestibility studies In addition, the regurgitation of part of the indigestible feed after a meal has been reported in some crustaceans (Forster & Gabbott 1971; Newman et al 1982) Recommendations for the study of feed digestibility in crustaceans include in vitro techniques applied as screening devices of the suitability of feed ingredients for inclusion in shrimp diets (Lee & Lawrence 1997; Lazo et al 1998) Digestive proteases from the animal under study, and enzymes extracted from animals fed the same diet that will be evaluated, can better access the digestibility of protein than commonly used or commercial enzymes that are not present in the shrimpÕs digestive system, or are acting on a different pH than in the shrimpÕs digestive gland (Lan & Pan 1993; Divakaran et al 2004; Lemos et al 2004) The pH-stat method, based on the degree of hydrolysis using digestive enzymes, has the potential for estimating the digestibility of alternative protein sources for inclusion in shrimp feeds (Ezquerra et al 1997), but was found to be inadequate with shrimp feeds that have partially been hydrolysed (Co´rdova-Murueta & Garcı´ aCarren˜o 2002) However, it was found with fish that apparent digestible protein (ADP; Sales 2008) and lipid (DL; Sales 2009) contents in feed ingredients and compound diets can be predicted with high accuracy from its dietary contents across a wide range of species, feed types, nutrient levels, life stages and rearing conditions with the use of linear regression techniques With crustaceans a similar analysis is hampered by a lack of studies presenting suitable values Despite the assumption that static empirical models should only be applied within ranges, thus also species, used for development (Sales 2008, 2009), the present study was aiming at validation of prediction equations established for the prediction of ADP and DL contents of fish feeds for crustacean species Databases were created from studies (Tables & 2) presenting dietary contents and digestibility coefficients of crude protein (CP) and crude lipid (CL) for feed ingredients (Table 3) and compound diets (Table 4) evaluated with several crustacean species Characteristics of datasets are presented in Table Tuan et al (2006) used three, four and five replicates in the same study to evaluate CP digestibility of different feed ingredients with mud crabs Irvin & Williams (2007) collected faeces in tropical spiny lobsters with a balloon glued to surround the anal pore, as described by Irvin & Tabrett (2005) Whereas Brown et al (1986) and Akiyama et al (1989) utilized single protein source diets to determine apparent protein digestibility of feed ingredients in red swamp crayfish and Pacific white shrimp, respectively, all other studies applied the reference Table Studies used for information on dietary contents and apparent digestibility of crude protein and crude lipid in feed ingredients evaluated with crustaceans Reference Shrimp Akiyama et al (1989) Bautista-Teruel et al (2003) Cruz-Sua´rez et al (2001) Cruz-Sua´rez et al (2007) Davis et al (2002) Ezquerra et al (1997) Herna´ndez et al (2008) Kumaraguru Vasagam et al (2007) Merican & Shim (1995) Smith et al (2007) Water type Temperature (°C) Feed habit2 Pacific white shrimp Black tiger shrimp Blue shrimp Pacific white shrimp Pacific white shrimp Pacific white shrimp Pacific white shrimp Black tiger shrimp Litopenaeus vannamei Penaeus monodon Litopenaeus stylirostris Litopenaeus vannamei Litopenaeus vannamei Litopenaeus vannamei Litopenaeus vannamei Penaeus monodon Salt Salt Salt Salt Salt Salt Salt Salt 25–29 22–28 28 27–31 28 25 28 28 O O O O O O O O 22.3 14.5 2.7 1.6–2.0 8.0–10.0 3.5–4.0 5.2 4.0 Black tiger shrimp Black tiger shrimp Penaeus monodon Penaeus monodon Salt Salt 26–27 29 O O 20.7 23.5 Protein Protein Protein Protein Protein Protein Protein Protein, lipid Lipid Protein Red swamp crayfish Australian redclaw Procambarus clarkii Fresh Cherax quadricarinatus Fresh 24–27 27 O O 14.1 3.6 Protein Protein Australian redclaw Cherax quadricarinatus Fresh 27 O 3.6 Tropical spiny lobster Panulirus ornatus 28 C 700.0 Australian redclaw Red swamp crayfish Cherax quadricarinatus Fresh Procambarus clarkii Fresh 27 22 O O 94.0 40–50 mm Mud crab Scylla serrata Salt – C 148.0 Chinese hairy crab Mud crab Eriocheir sinensis Scylla serrata Fresh Salt 28 29 O C 5.1 96.0 12 Pavasovic et al (2007) Reigh et al (1990) Crab Catacutan et al (2003) Mu et al (2000) Tuan et al (2006) Scientific name 7 Crayfish Brown et al (1986) Campan˜a Torres et al (2005) Campan˜a Torres et al (2006a) Irvin & Williams (2007) Common name n1 Salt Size (g) Nutrient Lipid Protein, lipid Protein Protein Protein, lipid Protein Protein Number of means from study C, carnivorous; O, omnivorous Aquaculture Nutrition 16; 559–568 Ó 2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 559–568 Ó 2009 Blackwell Publishing Ltd 2 1 12 Crayfish Campan˜a Torres et al (2005) Irvin & Williams (2007) Jones & De Silva (1997a) Jones & De Silva (1997b) Pavasovic et al (2006) Reigh et al (1990) Ward et al (2003) Number of means from study C, carnivorous; O, omnivorous 10 10 2 22 4 6 18 5 Shrimp Ashmore et al (1985) Brunson et al (1997) Cabanillas-Beltra´n et al (2001) Catacutan (1991) Colvin (1976) Cruz-Sua´rez et al (2001) Cruz-Sua´rez et al (2007) Eusebio (1991) Fenucci et al (1982) Forster et al (2003) Goytortu´a-Bores et al (2006) Herna´ndez et al (2008) Kumaraguru Vasagam et al (2005) Kumaraguru Vasagam et al (2007) Lee & Lawrence (1985) Lin et al (2004) Lin et al (2006) Martı´nez-Palacios et al (2001) Ostrowski-Meissner et al (1995) Rivas-Vega et al (2006) Smith et al (1985) Sudaryono et al (1996) Sudaryono et al (1999a) Sudaryono et al (1999b) Taechanuruk & Stickney (1982) n1 Reference Australian redclaw Tropical spiny lobster Yabby Yabby Australian redclaw Red swamp crayfish Southern rock lobster Giant river prawn Atlantic white shrimp Pacific white shrimp Black tiger shrimp Indian white shrimp Blue shrimp Pacific white shrimp Black tiger shrimp Blue shrimp Pacific white shrimp Pacific white shrimp Pacific white shrimp Black tiger shrimp Black tiger shrimp Atlantic white shrimp Pacific white shrimp Pacific white shrimp Pacific white shrimp Pacific white shrimp Pacific white shrimp Pacific white shrimp Black tiger shrimp Black tiger shrimp Black tiger shrimp Giant river prawn Common name Cherax quadricarinatus Panulirus ornatus Cherax destructor Cherax destructor Cherax quadricarinatus Procambarus clarkii Jasus edwardsii Macrobrachium rosenbergii Penaeus setiferus Litopenaeus vannamei Penaeus monodon Penaeus indicus Litopenaeus stylirostris Litopenaeus vannamei Penaeus monodon Litopenaeus stylirostris Litopenaeus vannamei Litopenaeus vannamei Litopenaeus vannamei Penaeus monodon Penaeus monodon Penaeus setiferus Litopenaeus vannamei Litopenaeus vannamei Litopenaeus vannamei Litopenaeus vannamei Litopenaeus vannamei Litopenaeus vannamei Penaeus monodon Penaeus monodon Penaeus monodon Macrobrachium rosenbergii Scientific name Fresh Salt Fresh Fresh Fresh Fresh Salt Fresh Salt Salt Salt Salt Salt Salt Salt Salt Salt Salt Salt Salt Salt Salt Salt Salt Salt Salt Salt Salt Salt Salt Salt Fresh Water type 27 28 21–24 – 26–28 22 18 27 25–30 22 27–29 28–30 28 27–31 25–28 26–29 27 28 28 28 28 28 29–31 24–27 28 27 27 27 25–28 28–30 28 27–31 Temperature (°C) O C O O O O C O O O O O O O O O O O O O O O O O O O O O O O O O Feed habit2 3.6 700.0 8.3–16.0 7.0–15.0 29.6 40–50 mm 8.0–13.0 40–50 7.7 10.2 30–40 0.4–1.1 2.7 1.6–2.0 8.7 1.2–11.9 5.1 3.3 5.2 3.3 4.0 3.7, 9.8 11.5 8.6 10.2 1.5 15.4 8.4–11.0 4.7 4.3 4.1 >30 Size (g) Table Studies used for information on dietary contents and apparent digestibility of crude protein and crude lipid in compound diets evaluated with crustaceans lipid lipid lipid lipid lipid lipid lipid lipid lipid lipid Protein Protein, lipid Protein Protein Protein Protein Protein Protein Protein Protein, Protein, Protein Protein Protein Protein Protein Protein Protein, Protein, Protein, Protein, Protein Protein, Protein, Protein, Protein Protein Protein, Protein Protein Protein Protein Nutrient Table Dietary crude protein (CP) and lipid (CL) contents (g kg)1 dry matter; range) and apparent protein (APDC) and lipid (ALDC) digestibility coefficients (%; range) of individual feed ingredients Ingredient CP APDC Animal protein Blood meal Casein Crab meal Crustacean meal Fish meal Krill meal Meat meal Mussel meal Poultry meal Shrimp meal Squid meal 875.0 880.0–913.0 394.0 378.0 602.0–766.6 642.0 468.0–596.0 478.0 692.0–693.6 436.0–704.6 720.0–785.0 93.5 90.9–99.1 66.4 85.3 47.4–95.0 88.7 66.2–95.0 88.8 87.2–90.4 74.6–94.9 59.3–97.6 Plant protein Canola meal Copra meal Cow peas Lupin Mung beans Peas Soybean meal Soy concentrate 394.0–441.0 219.0 242.0–293.0 308.0–546.0 249.0–284.0 211.0–252.5 404.9–551.0 495.0–816.0 80.0–91.0 94.3 60.0–83.0 85.9–96.8 65.0–83.0 79.8–92.7 80.6–98.7 90.9–96.4 486.0 78.0 120.0–192.0 92.6 96.4 76.4–94.2 114.8–139.0 212.0 759.0 166.0–184.1 57.7–95.2 95.7 95.0–98.0 92.5–96.0 Grains BrewerÕs yeast Maize Rice bran Sorghum Wheat Wheat bran Wheat gluten Wheat midlings substitution method (Cho et al 1982), with from 150 to 500 g kg)1 of the reference diet replaced with the feed ingredient In the former two studies diets contained 60 and 40 g kg)1 dry matter (DM), respectively, of frozen squid blended in water as an attractant, which might have contributed to protein content Eleven of the 19 studies indicated in Table have incorporated the relative contribution of the nutrient to the combined diet in calculation of feed ingredient digestibility, as described by Forster (1999) Although crustacean meal, mussel meal and mung beans were absent in the development of the equation to predict ADP contents of feed ingredients for fish (Sales 2008), and crustacean meal, krill meal, mussel meal, copra meal, cow peas, lupin and mung beans were not included in the equation for DL prediction (Sales 2009), values for these ingredients were included in the present evaluation Studies using purified and semi-purified diets (Bordner et al 1983; Shiau & Chou 1991; Shiau et al 1991a,b; Shiau & Peng 1992; Koshio et al 1993; Cousin et al 1996; Gonza´lezPen˜a et al 2002; Guo et al 2006) were excluded from the dataset on compound diets With ADP contents of purified CL ALDC 46.0 48.0 59.0–132.0 161.0 96.0 53.0 92.1 53.2 42.0–83.5 66.0 87.2 63.7 39.0 44.5–170.0 87.2 41.4–87.8 64.0 11.0–15.0 95.0 8.3–11.0 95.0 77.0–106.0 33.2 70.0–130.0 13.0–192.5 40.5–92.1 47.0 130.0 53.0 15.0–35.0 94.5 93.3 85.6 90.6–95.0 proteins usually higher than that of practical protein sources (Shiau et al 1992), the former have no application in practical diet formulation on an industrial scale In studies evaluating feed ingredient digestibility through substitution only the reference diet was included in the compound diet dataset if containing practical feed ingredients Several studies evaluating CP digestibility of compound diets used three, four and five replicates (Fenucci et al 1982; Smith et al 1985; Jones & De Silva 1997b), whereas the study of Pavasovic et al (2006) has 10 replicates per treatment Martı´ nezPalacios et al (2001) used both siphoning and settling (modified Guelph system) to collect faeces for determination of CP and CL digestibility All values were converted to a DM basis Although DM loss of diets has been determined in several studies (Fenucci et al 1982; Taechanuruk & Stickney 1982; Brown et al 1986; Reigh et al 1990; Brunson et al 1997; Sudaryono et al 1999a,b; Cruz-Sua´rez et al 2001, 2007; Herna´ndez et al 2008), uncorrected digestibility estimates were used in datasets Aquaculture Nutrition 16; 559–568 Ó 2009 Blackwell Publishing Ltd Table Frequency and level (range) of inclusion of major protein supplying ingredients (protein database) and lipid sources (lipid database) in compound diets Ingredient Protein database Animal protein ingredients Crabmeal Crayfish meal Crustacean meal Fish meal Fish solubles Krill hydrolysate Krill meal Lobster meal Meat meal Mussel meal Scallop meal Shrimp meal Snail meal Squid meal Plant protein ingredients Cowpeas Kelp meal Lupin Lupin concentrate Maize gluten Peanut meal Soybean concentrate Soybean lechitin Soybean meal Wheat gluten Lipid database Lipid sources Coconut Cod liver Fish Palm Peanut None Table Description of databases Number of studies Ingredients Inclusion frequency (% of diets) Inclusion level (g kg)1) 2.2 1.1 6.5 93.5 21.6 1.1 1.1 0.5 4.9 6.5 0.5 60.5 0.5 51.4 20.0–147.0 100.0–359.1 50.0–51.0 32.0–778.0 20.0 60.0 330.0 83.0 100.0–378.0 10.0–50.0 130.0 29.0–729.0 304.9 5.0–127.0 3.8 4.3 4.3 0.5 3.2 5.9 22.2 14.6 39.5 17.3 150–295.0 33.9–59.4 100.0–705.0 240.0 32.0–37.7 257.6–290.0 2.5–124.0 4.2–60.8 43.8–895.9 22.0–160.0 3.1 34.4 45.3 3.1 3.1 10.9 50.0–70.0 3.0–30.0 8.2–70.0 50.0–70.0 50.0–70.0 ADP and DL contents (g kg)1 DM) were predicted from dietary CP and CL contents (g kg)1 DM) according to Sales (2008, 2009), respectively: Feed ingredients ADP = )10.0731 + 0.8942 CP DL = )1.5824 + 0.8654 CL Compound diets ADP = )51.4001 + 0.9872 CP DL = )2.7303 + 0.9123 CL Simple linear regression analysis, conducted as described by Sales (2008, 2009) with the software STATISTICA (data analysis software system, Version 7.1; StatSoft, Tulsa, OK, Aquaculture Nutrition 16; 559–568 Ó 2009 Blackwell Publishing Ltd Compound diets Characteristic Protein Lipid Protein Lipid Studies Number of means Number of species Replicates Two Three Four Five Marker Chromic oxide Total collection Yttrium oxide Faeces collection Balloon Filtering net or scooping Settling column Siphoning Tweezer Not indicated Protein determination Dumas (Ebeling 1968) Kjeldahl (AOAC 1990) Lowry method (Lowry et al 1951) Not indicated Lipid determination Chloroform/metanol (Folch et al 1957; Bligh & Dryer 1959) Ether (AOAC 1990) Not indicated 17 91 31 32 185 11 11 64 3 1 17 13 2 11 1 28 4 19 1 11 20 10 3 USA), was used to evaluate the relation between predicted (y) and observed (x) values A further measurement of the error of predicted relative to observed values (Theil 1966) was done by calculation of the mean square prediction error (MSPE): MSPE ẳ n X Oi Pi ị2 =n i¼1 where n is the number of experimental observations, and Oi and Pi the observed and predicted values, respectively The accuracy of prediction for different equations was evaluated by the mean prediction error (MPE): pffiffiffiffiffiffiffiffiffiffiffiffiffiffi MSPE MPE ¼ O where O is the mean of the observed values ECT ẳ X P X O ị ER ẳ SP r SO ị2 ED ẳ r2 Þ Â SO with X P and X O the mean predicted and observed values, respectively, SP and SO the standard deviations of the predicted and observed values, respectively, and r the correlation coefficient between predicted and observed values Although differences, most often contradictory, in CP digestibility between plant and animal protein sources have been reported with crustaceans (Brown et al 1986; Reigh et al 1990; Ahamad Ali 1992; Catacutan et al 2003; Campan˜a Torres et al 2006b), no improvement of the accuracy of prediction equations for ADP or DL contents was found by Sales (2008, 2009) when distinguishing between dietary plant and animal feed ingredients Due to this, and the limited number of values available, no separation according to type of feed ingredient was done Notwithstanding an intercept and slope different from and 1, respectively, 92% of the variation in ADP content could be explained by a linear regression model (Fig 1) Limited dispersion of points will cause small standard errors and high values for test statistics calculated for the intercept and slope This will result in values that are likely to be significant from zero and one, respectively (Mitchell 1997) However, the latter author also stated that the variation explained by the regression (R2) is of no relevance to validation, seen that there is no aim to predict from the fitted line In contrast, deviations, calculated as prediction minus observation, will give an indication of how far the model fails Predicted apparent digestible protein (g kg–1 DM) The MSPE can be differentiated (Benchaar et al 1998) into: (1) error in central tendency (ECT) as measurement of the deviation of the mean of predicted values from the mean of observed values, (2) error due to regression (ER) presenting the difference of the least squares regression coefficient from one, and (3) error due to disturbance (ED) illustrating the variation in observed values that is not accommodated by a least squares regression of observed on predicted values 900 y = 40.2352* + 0.9123*x R = 0.9213, RMSE = 54.1245 800 700 600 500 400 300 200 100 0 100 200 300 400 500 600 700 800 900 1000 Determined apparent digestible protein (g kg–1 DM) Figure Linear relationship between predicted and observed apparent digestible protein values for feed ingredients (n = 91) * Different (P < 0.05) from for intercept and for slope to simulate observed values (Mitchell 1997) This is accommodated in MPE analysis, which account for deviations of predicted values from observed values caused by mean bias, linear bias and random variation (Oldick et al 1999) A MPE of 0.1315 derived with the ADP equation (Table 6) is comparable to a value of 0.1064 obtained for this measurement when evaluating the prediction equation with independent studies on fish species (Sales 2008) With most of the MSPE attributed to the ED (>99%), the error was caused by a failure to predict the pattern of fluctuations across observed pffiffiffiffiffiffiffiffiffiffiffiffiffiffi values With a mean overestimation ( MSPE) of 56.4118 g kg)1 DM (Table 6), overestimations at both ends of the data range have been found, with neutral or underestimated values in the middle (Fig 1) Predicted DL values showed a strong linear relationship (R2 value above 0.8000) with observed values, with an intercept and slope not significantly different from and 1, respectively (Fig 2) However, a MPE of 0.4500 (Table 6) presented evidence of a high overestimation of observed values by the prediction equation, with a relative high proportion of the MSPE assigned to deviation from the regression slope (ER) Due to a lack of values from independent studies the DL prediction equation has not been validated to observed values with fish species (Sales 2009) Similar to tendencies found with feed ingredients, predicted ADP values for compound diets were highly related to observed values (Fig 3), presented an overestimation of Aquaculture Nutrition 16; 559–568 Ó 2009 Blackwell Publishing Ltd Table Mean prediction errors (MPEs) and components of the mean square prediction error (MSPE) between predicted and observed values (g kg)1 dry matter) Proportion of MSPE Ingredients Protein Lipid Compound diets Protein Lipid MPE Bias1 Error in central tendency Error due to regression Error due to disturbance 56.4118 20.5422 0.1315 0.4500 2.6075 8.1650 0.0021 0.1580 0.0011 0.3496 0.9967 0.4924 30.8516 14.0427 0.1002 0.2372 3.3632 8.3252 0.0119 0.3515 0.1274 0.0019 0.8607 0.6466 Predicted, observed Predicted apparent digestible lipid (g kg–1 DM) 180 140 y = 1.6331 + 1.1431x Predicted apparent digestible lipid (g kg–1 DM) pffiffiffiffiffiffiffiffiffiffiffiffiffi MSPE R = 0.8117, RMSE = 18.8371 160 140 120 100 80 60 40 20 0 20 40 60 80 100 120 –1 Determined apparent digestible lipid (g kg 140 Predicted apparent digestible protein (g kg–1 DM) y = 16.5420 + 0.9572x R = 0.8047, RMSE = 30.7087 500 400 300 200 100 0 100 200 300 400 500 120 100 80 60 40 20 0 20 40 60 80 100 120 Determined apparent digestible lipid (g kg–1 DM) DM) Figure Linear relationship between predicted and observed apparent digestible lipid values for feed ingredients (n = 31) 600 y = 25.1376* + 0.7160*x R = 0.6907, RMSE = 9.8839 600 Determined apparent digestible protein (g kg–1 DM) Figure Linear relationship between predicted and observed apparent digestible protein values for compound diets (n = 185) Aquaculture Nutrition 16; 559–568 Ó 2009 Blackwell Publishing Ltd Figure Linear relationship between predicted and observed apparent digestible lipid values for compound diets (n = 64) * Different (P < 0.05) from for intercept and for slope observed values, and have a relatively low MPE value with the ED contributing to most to the MSPE (Table 6) However, the linear relationship between predicted and observed DL values was characterized by an R2 value of less than 0.7000 (Fig 4), and a MPE value of almost 0.2400 (Table 6) With 35% of the MSPE caused by deviation of the mean predicted value from the mean of observed values, elimination of a portion of the error by a mean correction factor would be possible As also valid with feed ingredients, the inadequacy of equations to accurately predict DL values in compound diets for crustacean species could partly be attributed to a limited number of independent values and studies available, causing bias in the evaluation A further contributor could have been the extrapolation of equations outside ranges used for development, although this had no effect on the prediction of ADP content However, Ishikawa et al (1997) stated that the digestion and absorption of dietary lipids might be different between crustaceans and fish Unlike vertebrates, crustaceans are unable to synthesize cholesterol (Kanazawa et al 1971) and bile acids from acetate and cholesterol (Holwerda & Vonk 1973), assuming that the mechanism for assimilation of dietary lipids is different from that found in other animals (Teshima & Kanazawa 1983) Furthermore, assimilation of free fatty acids is more effective than that of triacylglycerols (Glencross et al 1997) This study presents evidence that the ADP contents of a wide variety of feed ingredients and compound diets could be predicted in crustacean species, reared under a range of different dietary, environmental and physiological conditions, with a high degree of accuracy from dietary CP contents with the use of a linear prediction equations developed with fish species This permits easy and rapid obtainable results, which could be used in practical diet formulation for crustacean species, and will eliminated the use of lengthy, tedious and troublesome digestibility experiments that could be subjected to considerable error However, prediction equations for DL contents established with fish species were found to be unsuitable for crustaceans, probably related to differences in the mechanisms of lipid digestibility between fish and crustaceans This study was financial supported by Research Plan No MSM 6007665809 of the University of South Bohemia Cˇeske´ Budeˇjovice, 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Table Ingredient and nutrient composition of experimental diets g kg)1 Ingredient )MM ) I )MM + I Soy protein concentratea Corn gluten mealb Wheat glutenc Soybean meald Fish meale Wheat flourb Blood mealf Menhaden oilg Lysine-HCL Taurine Dicalcium phosphate Trace Premixh Choline CL Stay-C Vitamin Premixi Inositol Potassium chloride Magnesium oxide Sodium chloride Analyzed composition, dry Crude protein, g kg)1 Crude fat, g kg)1 Ash, g kg)1 Calcium, g kg)1 Phosphorus, g kg)1 Magnesium, g kg)1 Zinc, mg kg)1 Copper, mg kg)1 Sodium, g kg)1 Potassium, g kg)1 Sodium/potassium ratio 146.6 146.6 169.1 169.1 70.4 70.4 158.4 158.4 – – 276.6 271.3 – – 134.4 134.4 12.6 12.6 5.0 5.0 25.5 25.5 1.0 1.0 1.1 1.1 0.3 0.3 4.0 4.0 – 0.3 – – – – – – matter basis 453 451.2 162 159 47.6 48.4 6.6 6.6 8.9 8.8 1.2 1.3 75 73 27 26 3.4 3.3 5.8 0.58 0.55 +MM + I FM 146.6 169.1 70.4 158.4 – 262.3 – 134.4 12.6 5.0 25.5 1.0 1.1 0.3 4.0 0.3 5.6 0.6 2.8 – – 192.2 309.7 281.1 99.3 111.0 – – – 1.0 1.1 0.3 4.0 0.3 – – – 446.1 161 56 7.1 1.9 69 27 4.6 8.8 0.52 420.5 169 76.2 13.1 11.1 2.2 80 23 4.2 9.1 0.46 Solae Profine VP, St Louis, MO, 720 g kg)1 crude protein Archer Daniels Midland, Decatur, IL, 600 g kg)1 crude protein c MGP Ingredients, Inc., Atchison, KS, 820 g kg)1 crude protein d Archer Daniels Midland, Decatur, IL, 470 g kg)1 crude protein e Peruvian anchovy, Silver Cup Fish Feeds, Murray, UT, 700 g kg)1 crude protein f Blood meal, 820 g kg)1 protein, Gavilon LLC, Omaha, NE, 810 g kg)1 crude protein g Menhaden oil, Omega Protein Inc., Houston TX h Contributed in mg kg)1 of diet; zinc 37; manganese, 10; iodine, 5; copper, i Contributed, per kg diet; vitamin A, 2880 IU; vitamin D, 2800 IU; vitamin E, 75 IU; vitamin K3, 0.5 mg; thiamin mononitrate, mg; riboflavin 4.6 mg; pyridoxine hydrochloride, 4.5 mg; pantothenate, DL-calcium, 23.3 mg; cyancobalamin, 0.01 mg; nicotinic acid, 10.9 mg; biotin, 0.16 mg; folic acid, 2.1 a b All ingredients were ground using an air-swept pulverizer (Model 18H, Jacobsen, Minneapolis, MN, USA) to a particle size of F R2 C.V 98.3 87.4b 1.09a 3.40a 51.6a 1.05 100.0 95.2a 0.85b 2.90b 31.5b 1.19 0.19 0.01 0.01 0.01 0.01 0.14 0.26 0.69 0.71 0.66 0.89 0.30 1.63 4.40 5.43 4.99 7.42 7.14 32.0c 36.5a 34.9b 36.5a 38.8a 35.5a 0.01 0.01 0.82 0.73 6.20 7.04 142.6ab 106.1ab 16.1b 736.7a 27.8a 139.7ab 111.6a 16.9a 727.4a 27.9a 149.6a 98.3b 18.7a 735.0a 27.0b 0.02 0.01 0.02 0.02 0.01 0.46 0.51 0.46 0.46 0.55 5.37 5.94 9.71 1.80 1.38 Means in the same row with the same superscript are not significantly different (P > 0.05) FCR = Feed conversion ratio FI = Feed intake HSI = Hepatosomatic index Aquaculture Nutrition 16; 654–661 Ó 2009 Blackwell Publishing Ltd No claim to original US government works There was no difference in ERE between trout fed the fish meal diet and trout fed +MM+I, 35.5% and 36.5%, respectively (Table 2) ERE were reduced when MM or inositol was removed from the diet Trout fed -MM+I had a ERE of only 31.7% while trout fed )MM)I only retained 27.9% of dietary energy (Table 2) Significant effects of diet on body composition were observed (Table 2) Trout fed the fish meal-based diet had less body lipid (980 g kg)1) and less energy (27.0 J kg)1) than trout fed +MM+I (111.6 g kg)1 lipid, 27.9 J kg)1, energy) There were no significant differences in body energy content among the trout fed the three plant-based diets Changing levels of MM and inositol did affect body protein, lipid, moisture, and ash content Trout fed +MM + I, )MM + I, or )MM ) I had 139.7, 142.6 and 132.8 g kg)1 body protein, respectively Body lipid content was similarly affected Trout fed )MM ) I had lower body lipids (98.1 g kg)1) than trout fed +MM + I (111.6 g kg)1) Trout fed )MM+I only had 106.1 g kg)1 body lipid which was intermediate to, and not different from, trout fed the other plant-based diets Body ash levels of 16.9 g kg)1 were higher for the trout fed the plant-based diet supplemented with both MM and inositol than for trout fed the other plant-based diets (16.1 and 15.2 g kg)1, )MM + I and )MM ) I, respectively) Body moisture of 727.4 and 736.7 g kg)1 was lower for the trout fed the plant-based diet supplemented with both MM and inositol or inositol alone, respectively, than for trout fed the unsupplemented plant-based diet which had body moisture content of 756.9 g kg)1 Ceroid was present in the majority of spleens from fish fed the fish meal diet Only trace amounts of ceroid deposition was seen in liver cells of trout fed the fish meal-based diet and was mostly present in macrophages lining blood sinusoids Ceroid was absent from both the livers and the spleens of trout fed any of the plant-based diets Significant lesions were observed in livers of trout fed the plant-based diet -MM-I relative to trout fed +MM + I (Fig 1) Diffuse necrosis was present in livers of several fish and perivascular inflammation were also common Bile duct proliferation varied from mild to severe and focal areas of leucocytic inflammation were often present Hepatocytes were swollen, cytoplasm was finely granular and nuclei varied in size and were often pelomorphic (Fig 2) Nuclear inclusions were also common Swollen hepatocytes with finely granular fluid cytoplasm undergoing necrosis were diffusely scattered throughout Degeneration of vessel walls was also seen Regenerative foci of immature hepatocytes were sometimes diffusely scattered throughout degenerate tissue Again, as compared to trout fed +MM+I, trout fed )MM+I had significant pathological changes in their livers Mild diffuse necrosis of hepatocytes and focal and Figure Liver section of fish fed diet )MM ) I shows perivascular inflammation Note degeneration of endothelial lining of vessel wall (arrow heads) Bile duct proliferation is diffusely scattered throughout liver tissue (arrows) Bar = 25 lm Staining by hematoxylin and eosin Figure Liver section of fish fed diet )MM ) I A swollen, fluidfilled hepatocyte with degenerate nucleus is seen in center of photo Note inclusion in swollen nucleus of adjacent cell (arrow) Swollen, degenerate cells are scattered throughout (D) Bar = 25 lm Staining by hematoxylin and eosin Aquaculture Nutrition 16; 654–661 Ó 2009 Blackwell Publishing Ltd No claim to original US government works perivascular inflammation was noted in livers of several fish Swollen hepatocytes with granular cytoplasm were common and numerous in some fish These cells were fluid filled and often undergoing necrosis Nuclear inclusions were seen in hepatocytes of most trout Nucleoli were often swollen and highly eosinophilic Most spleens were normal, but did show moderate amounts of lipofuscin Inositol is frequently supplemented to purified, semi-purified, and experimental diets (NRC (National Research Council) 1993) Waagbo et al (1998) reported endogenous inositol levels in a practical diet with 40% fish meal to be 300 mg kg)1 inositol, and this level was sufficient for maximum growth of Atlantic salmon without supplementation In the current study, supplementing the plant-based diet with 300 mg inositol kg)1 diet did not significantly increase weight gain, but protein and energy retention efficiencies were significantly improved Reduced feed intake and slow growth have been reported as symptoms of an inositol deficiency (McLaren et al 1947), but no improvements in growth or differences in feed intake due to supplementation of 300 mg inositol kg)1 diet to a plant-based diet was detected in the present study In contrast, there was an increase in feed intake of trout fed the plant-based diet as compared to trout fed the fish meal based diet The more domesticated strains of rainbow trout available today may feed more aggressively than the relatively unselected strains of the past, and could explain the differences observed between the present trial and that of McLaren et al (1947) Kitamura et al (1967) reported decreases in amino-transferase activity in rainbow trout fed diets deficient in inositol Supplementing the plant-based diet with 300 mg inositol kg)1 diet significantly improved protein and energy retention efficiencies Even though feed intake remained high in the present study, a change in amino acid metabolism due to inositol deficiency may have resulted in less protein deposited Poor utilization of protein by trout fed the unsupplemented diet is supported by the observed reduction in protein retention Deng et al (2002) and Burtle & Lovell (1989) found no requirement for myo-inositol by juvenile sunshine bass or channel catfish, respectively In the study by Deng et al (2002), graded levels of myo-inositol were supplemented to purified diets and fed to sunshine bass An additional diet, with added antibiotic, to suppress intestinal synthesis of inostitol was also fed and no gross symptoms of deficiency or growth reductions were observed Burtle & Lovell (1989) reported similar results and both studies were supported by Aquaculture Nutrition 16; 654–661 Ó 2009 Blackwell Publishing Ltd No claim to original US government works analysis of liver and brain vitamin concentrations that also showed no differences among treatments Fish species evaluated in those two studies are considered warm water (catfish) and cool water (sunshine bass), in contrast to the coldwater trout used in the present study Additionally, weight gain in present study was approximately 1800% of initial weight for trout, compared to 600% for channel catfish (Burtle & Lovell 1989) and approximately 175% for sunshine bass (Deng et al 2002) Practical diets were fed in the present study along with a longer growth period, compared to the purified and semi-purified diets fed to the bass and catfish which may explain some of the observed differences Trout diets with moderate levels of fish meal (30%) will contain approximately 0.13% magnesium, and a diet with soy-protein concentrate as the primary protein source will contain approximately 0.06% magnesium Shearer (1989) estimated the magnesium requirement to be 0.14% using growth as the response criteria, but using whole body magnesium levels a requirement of 0.06% of the diet was estimated The later value is in agreement with estimates by Ogino & Chiou (1976) and Ogino et al (1978) Diets used in those studies, however, were semi-purified as compared to the practical type diets fed in the present study The plantbased MM supplemented diets in the current study contained 0.10% magnesium and the unsupplemented diet contained 0.07% magnesium Assuming less than complete absorption of magnesium from the diets, the plant-based, unsupplemented, diet could be marginally deficient in magnesium Sodium, potassium and chlorine are the most prevalent electrolytes in fish and other animals and are the ions utilized for calculating the cation–anion difference in animal feeds These minerals are abundant in fish meals and fish can readily absorb these minerals from the water, but waterborne levels are not always sufficient to meet requirements (Shearer 1988) Addition of sodium chloride up to 4% of a commercial type trout diet (fish meal based) had no effect on growth of rainbow trout in freshwater, and feeding dietary levels above 4% to trout decreased fish performance (Salman & Eddy 1988) Chiu et al (1987) found the levels of dietary potassium, sodium and chloride in semi-purified diets to be critical for optimal growth, feed efficiency and amino acid metabolism for rainbow trout Significant interactions of these dietary electrolytes were also observed, demonstrating the importance of proper balance in the diet (Chiu et al 1987) Dersjant-Li et al (2000) observed that dietary Na/K ratio also would be an important factor in balancing dietary minerals In the current study, the +MM diet had a Na/K ratio (0.52) which is intermediate to the FM diet (0.46) and unsupplemneted diet, -MM-I (0.58) Supplementation level with potassium was slightly below, and sodium was slightly above, the ratio of the FM diet Decreasing the sodium/ potassium ratio of the plant-base diets, by supplementing with sodium and potassium, did result in an increase in growth rate Cation–anion balance was also demonstrated as an important factor in the diet of African catfish (Dersjant-Li et al 1999, 2000) Even with the supplementation of both inositol and macrominerals, growth rate of trout fed the plant-based diet was approximately 8.2% less than trout fed the fish meal diet Kaushik et al (1995) replaced 100% of the fish meal in a 65% fish meal diet with soy protein concentrate and observed no decrease in growth Magnesium, sodium and potassium were supplemented to the fish meal free diet, but much larger fish were used in that study The initial size of the rainbow trout used by Kaushik et al (1995) was 83 g/f and only 270% gain was observed during the 12-week trial In the current 15-week trial, trout with an initial weight of 4.8 g/f were used and over 1900% gain was observed If larger fish were used in the current trial and less gain observed, differences in weight gain between the trout fed +MM+I and the trout fed FM may not have been detected Other studies have demonstrated beneficial effects of supplementing substances to plant-based diets that are present in fish meal but not normally considered dietary essential (Gaylord et al 2006; Aksnes et al 2007) In the present study, supplementation of sodium chloride, potassium chloride and magnesium oxide and inositol significantly improved growth of rainbow trout fed fish meal free diets, but not to a level equivalent to those fish fed the diet containing fish meal The addition of MM and I to a plant-based diet, also had beneficial effects on histopathological condition, and retention of both dietary protein and energy Even though growth was only marginally affected by individual addition of inositol or macro-minerals to the plant-based diet, the data indicate an additive effect of supplementation The results of this study suggest a marginal deficiency of macro-minerals and inositol, and that longer term feeding of fish meal free, plant-based diets without macro-minerals or inositol supplements would result in a reduced weight gain of rainbow trout Additional research is necessary to identify factors not examined in the current study that could explain the decreased performance of rainbow trout fed the plant-based diets We wish to thank ARS technicians April M Teague and Lorrie Van Tassel, ARS SCEP student, G Scott Snyder, and Univer- sity of Idaho personnel Mike Casten for their assistance with this study This study was funded by the USDA/Agricultural Research Service, Trout-Grains Project, # 5366-21310-00300D Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the United States Department of Agriculture (USDA) AOAC (Association of Official Analytical Chemists) (1995) Official Methods of Analysis of Official Analytical Chemists International, 16th edn Association of Official Analytical Chemists, Arlington, VA Aksnes, A., Mundheim, H., Toppe, J & Albrektsen, S (2008) The effect of dietary hyrdroxyproline supplementation on salmon (Salmo salar L.) fed high plant protein diets Aquaculture, 275, 242–249 Barrows, F.T., Gaylord, T.G., Stone, D.A.J & Smith, C.E (2007) Effect of protein source and nutrient density on growth efficiency, histology, and plasma amino acid concentration of rainbow trout (Oncorhynchus mykiss Walbaum) Aquacult Res., 38, 1747–1758 Burtle, G.J & Lovell, R.T (1989) Lack of response of channel catfish (Ictalurus punctatus) to dietary myo-inositol Can J Fish Aquatic Sci., 46, 218–222 Chiu, Y.N., Austic, R.E & Rumsey, G.L (1987) Interactions among dietary minerals, arginine and lysine in rainbow trout (Salmo gairdneri) Fish Physiol Biochem., 41, 45–55 Deng, D., Hemre, G & Wilson, R.P (2002) Juvenile sunshine bass (Morone chrysops x Morone saxatilis) not require dietary myoinositol Aquaculture, 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DC, USA Ogino, C & Chiou, J.Y (1976) Mineral requirements in fish Magnesium requirements of carp Bull Jpn Soc Sci Fish., 42, 71–75 Ogino, C., Takashima, F & Chiou, J.Y (1978) Requirements of rainbow trout for dietary magnesium Bull Jpn Soc Sci Fish., 44, 1105–1108 Refstie, S., Korsoen, O.J., Storebakken, T., Baeverfjord, G., Lein, I & Roem, A.J (2000) Differing nutritional responses to dietary soybean meal in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) Aquaculture, 190, 49–63 Refstie, S., Storebakken, T., Baeverfjord, G & Roem, A.J (2001) Long-term protein and lipid growth of Atlantic salmon (Salmo Aquaculture Nutrition 16; 654–661 Ó 2009 Blackwell Publishing Ltd No claim to original US government works salar) fed diets with partial replacement of fish meal by soy protein products at medium or high lipid level Aquaculture, 193, 91–106 Salman, N.A & Eddy, F.B (1988) Kidney function in response to salt feeding in rainbow trout (Salmo gairdneri Richardson) Comp 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feeds for rainbow trout Oncorhynchus mykiss (Walbaum) Aquacult Res., 31, 585–593 Waagbo, R., Sandnes, K & Lie, O (1998) Effects of inositol supplementation on growth, chemical composition and blood chemistry in Atlantic salmon, Salmo salar L., fry Aquacult Nutr., 4, 53–59 Yamamoto, T., Shima, T., Furuita, H & Suzuki, N (2002) Influence of feeding diets with and without fish meal by hand and by self-feeders on feed intake, growth and nutrient utilization of juvenile rainbow trout (Oncorhynchus mykiss) Aquaculture, 214, 289–305 Aquaculture Nutrition 2010 16; 662–670 doi: 10.1111/j.1365-2095.2010.00758.x 1,2 1 Faculty of Food Science and Nutrition, School of Health Sciences, University of Iceland, Reykjavı´k, Iceland; Icelandic Food Research and Innovation, Division of Biotechnology and Biomolecules, Sauda´rkro´kur, Iceland Proteome analysis was used to study the effects of feeding early Atlantic cod (Gadus morhua) larvae with a saithe (Pollachius virens) protein hydrolysate (SPH) Protein hydrolysates have previously been shown to beneficially affect fish larval development Feeding was initiated on day post hatch (ph) or as soon as the larvae opened their mouth and the protein expression was monitored days later or in 6-dph cod larvae The results demonstrated changes in the abundance of 13 protein spots in the cod larvae fed SPH Of these, seven protein spots were up-regulated and six protein spots showed down-regulation Five of the up-regulated proteins in cod larvae are known to be involved in energy metabolism A few early larval specific proteins were down-regulated in the SPHfed cod larvae possibly because of an enhanced development in this group relative to the control group Two trypsin isoforms were detected within the cod larval proteome The detection of the trypsin spots was made possible by co-electrophoresis of known cod trypsins with the cod larval protein extract Surprisingly, no difference in trypsin content was observed between the SPH-fed and the control larval groups KEY WORDS: 2-DE, Atlantic cod larvae, fish protein hydrolysate, LC/LC-MS, MALDI-TOF MS, Trypsins Received May 2009, accepted 16 December 2009 Correspondence: Ho´lmfrı´dur Sveinsdo´ttir, Matı´s ohf., Icelandic Food Research and Innovation, Division of Biotechnology and Biomolecules, Verid, Ha´eyri 1, 550 Sauda´rkro´kur, Iceland E-mail: holmfridur sveinsdottir@matis.is Early larvae of Atlantic cod (Gadus morhua) lack a functional stomach at first feeding (Hall et al 2004) Therefore, digestion of proteins at this developmental stage relies primarily Matı´s ohf., on the presence of pancreatic proteases such as trypsin and chymotrypsin in the cod larvae Previous analysis has demonstrated low amounts and activities of trypsin and chymotrypsin just prior to first feeding (1–3 days post hatch, dph) (Sveinsdo´ttir et al 2006), the critical time period at which the cod larvae must initiate feeding or face starvation Mouth opening of cod larvae occurs at approximately dph and after days the jaw becomes functional and food particles may be seen in the larval gut for the first time (Kjørsvik et al 1991) Thus, there is a possibility that small food particles in the seawater can enter the larval tract prior to active feeding These may therefore influence the expression of digestive enzymes at early larval stages Prehydrolysed proteins, i.e protein hydrolysates, have been shown to be beneficial for fish larval development Sea bass larvae (Dicentrarchus labrax) fed with a fish protein hydrolysate consisting of peptide chains of 10–20 amino acids (Cahu et al 1999) or a fish hydrolysate consisting of di- and tripeptides (Zambonino Infante et al 1997) demonstrated an improved larval growth and survival The specific ability of fish larvae to digest peptides could explain the beneficial effects of fish hydrolysates on larval development Young larvae appear to have high peptidase activities as shown in sea bass larvae where 15-day-old larvae had a 10-fold higher leucinealanine peptidase activity than 40-day-old larvae (Cahu & Infante 2001) Protein hydrolysates also appear to facilitate the maturation processes of the gastrointestinal tract Indeed, it has been shown that an increase in brush border membrane enzyme activity and a decrease in leucine-alanine peptidase occur earlier in larvae fed a diet containing protein hydrolysates than in larvae fed a diet containing native proteins (Cahu et al 1999) In addition, protein hydrolysates seem to modulate the activity of pancreatic proteases, especially that of trypsin (Cahu et al 2004) In this context, the concentration of the protein hydrolysate under study is of importance Proteomics is defined as Ôthe study of the entire proteome or a subset thereofÕ (Vilhelmsson et al 2006) where the Ó 2010 Blackwell Publishing Ltd proteome is the expressed protein complement of the genome Unlike the genome, the proteome varies among tissues, as well as with time, reflecting the organismÕs environment and its adaptation there to Therefore, proteome analysis may be used to identify a wide range of molecules and biochemical pathways involved in the responses of fish to environmental factors including food (Martin et al 2001, 2003; Vilhelmsson et al 2004) The aim of the study presented here was to use proteome analysis to study the response of Atlantic cod larvae to saithe (Pollachius virens) protein hydrolysate (SPH) feeding Modulation of trypsin in the cod larvae by the SPH was of special interest The experiment was carried out during the spawning season (May–June) 2005 at the Marine Research Institute at Stadur near Grindavı´ k, Iceland, a commercial production unit for cod hatchery Fertilized Atlantic cod eggs were obtained from wild caught brood stocks and transferred to the hatchery station at Stadur Two dph, the yolk-sack larvae were divided into two groups: C-group (control group) and SPH-group (treated group) and transferred to black conical rearing silos (150 L), three silos for each treatment Details of egg incubation and larvae rearing conditions at the hatchery station at Stadur have been published elsewhere (Steinarsson & Bjornsson 1999) Briefly, the rearing temperature was kept constant at °C during the first week ph The temperature was then gradually raised to 11 °C until day 24 ph The water in the silo was continuously aerated and light intensity was kept at 300 lux on the water surface throughout the experiment Feeding with microalgae (Isochrysis) and rotifers started on day ph The larvae were fed three times with rotifers and twice with microalgae per day At day ph, larval samples for proteome analysis were collected The larvae were rinsed thoroughly with distilled water and quickly frozen in liquid nitrogen At day 24 ph, samples for dry weight measurements were collected, 20 larvae from each group were rinsed in distilled water and dried in preweighed plastic trays for 48 h at 70 °C The SPH was obtained from the biotechnology company Pr mex Ltd., (Siglufjoărdur, Iceland) The SPH was prepared through enzymatic hydrolysis (ProtamexÒ, Novozymes, Aquaculture Nutrition 16; 662–670 Ó 2010 Blackwell Publishing Ltd Bagsvaerd, Denmark) of saithe muscle proteins (88 g protein 100 g saithe muscle)1) The product specifications as well as the chemical and amino acids analysis of the SPH is shown in Table Prior to larvae transfer on day ph, 5.0 g of a dried SPH (Prı´ mex) was dissolved in L seawater and added directly to the seawater in the silos Subsequently, the water flow was taken off for 30 After the beginning of first feeding (day ph), this was repeated prior to morning feeding until day ph For the control group, L seawater was added directly to the seawater in the silos and as in the SPH-group, the water flow was taken off for 30 An overview of the experimental design is showed in Fig Pooled larval samples were collected at day ph for proteome analysis, 12–15 larvae from each silo, in total 40 Table Chemical, mineral and amino acid analysis of the saithe protein hydrolysate (SPH) Saithe protein hydrolysate Chemical analysis Protein Fat Ash Moisture Mineral analysis Iron Arsenic Calcium Mercury Lead Amino acid analysis Alanine Cysteine Histidine Lysine Proline Tyrosine Arginine Glutamic acid Isoleucine Methionine Serine Valine Aspartic acid Glycine Leucine Phenylalanine Threonine g kg)1 88.0 (±2.0) 1.5 (±0.5) 2.0 (±0.5)