Soybean and lupin seed meals as protein sources in diets for gilthead seabream (Sparus aurata): nutritional and histological implications

Aquaculture ELSEVIER

Aquaculture 130 (1995) 219-233

Soybean and lupin seed meals as protein sources in diets for gilthead seabream (Sparus aurata) : nutritional and histological implications L. Robaina”, M.S. Izquierdo”T*, F.J. Moyanob, J. SOCOJTO~, J.M. Vergara”, D. Monteroc, H. Ferrhndez-Palacios” “Dpto. Biologia, Univ. de Las Palmas de G. C., Campus Univ. Tafira 35017, Las Palmas de Gran Canaria, Canary Islands, Spain bDpto. Biologia Animal, Escuela Polithica Superior, Univ. de Almeria, 04071 Almeria, Spain ‘Institute Canario de Ciencias Marinas, Apdo. 56, Telde 35200, L.us Palmas de Gran Canaria, Canary Islands, Spain

Accepted 7 September 1994

Abstract The use of vegetable protein sources in diets for freshwater fish has been studied in more detail than for marine fish species. Two experiments were conducted to compare the effect of the partial substitution of fish meal by two different vegetable protein sources, soybean and lupin seed meals. Mean feed intake and growth were not significantly influenced by type or level of plant protein in the diet. Feed utilization indexes such as feed efficiency, protein efficiency ratio and protein productive values were not significantly affected by the type of plant protein in the diet, although a general reduction of these values was observed with increased inclusion of soybean meal. Histological studies showed an increased deposition of lipid and decreased glycogen deposits in the liver with increased levels of dietary soybean meal. Protein digestibility coefficients for lupin seed meal diets were similar to the control and 10% higher than those for the soybean meal diets. A significant reduction in trypsin activity was observed in fish fed the lupin seed meal diets, and for soybean meal diets when the substitution level reached 30%. Diets including plant protein showed a higher peak of ammonia excretion rate, which appeared 2 h later than that of the fish meal diet. Highest values of dissolved ammonia were registered in fish fed a soybean meal-based diet. These results suggest that properly treated lupin meals could be an important alternative dietary protein source for gilthead seabream. Keywords: Soybean meal; Lupin meal; Feeding and nutrition -

fish - protein sources; Sparus aurata; Lipoid

liver.

*Corresponding author. 0044-8486/95/$09.50

0 1995 Elsevier Science B.V. All rights reserved

SSDIOO44-8486(94)00225-8

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1. Introduction

Fish meal is the major protein source in feeds for intensive fish farming. In the last few years, a considerable amount of research on partial or complete substitution of dietary fish meal by other high protein sources has been conducted (Luquet and Kaushik, 1978; Pfeffer, 1982). Due to the lower price and higher market availability of vegetable protein sources with high protein content, the inclusion of these feedstuffs in freshwater fish feeds has increased substantially. The quality of a particular dietary protein source depends both on its digestibility and amino acid profile (Kaushik and Cowey, 199 1). Apart from amino acid composition, which is often unbalanced (Liener, 1980; NRC, 1981; Kaushik and Luquet, 1984; Tacon and Cowey, 1985)) endogenous antinutritional factors are the main factors limiting the use of high levels of vegetable feedstuffs in fish feeds (Chubb, 1982; Richardson et al., 1985; Gatlin and Phillips, 1989; Satoh et al., 1989). Many of these factors, such as protease inhibitors, can be inactivated by moist heat treatment, depending on feed particle size, duration of heat treatment and moisture conditions employed (Hanson, 1974; Rackis, 1974; Grant, 1989). Leguminous seeds, specially soybeans, have been used as an alternate protein source in fish feeds. Soybean products are commonly used as a protein source in the production of rainbow trout (Reinitz et al., 1978)) having also been included at low levels in diets for the production of large salmonids (Rumsey and Ketola, 1975; Spinelli et al., 1979; Krogdahl, 1989) and catfish (Liebowitz, 1981). However, other authors have found poor growth and protein utilization with inclusion of soybean meal (SBM) in diets fed to rainbow trout (Sandholm et al., 1976; Dabrowski et al., 1989), and channel catfish (Wilson and Poe, 1985). Less attention has been paid to the partial substitution of fish meal in diets for marine fish species. Growth performance of marine flatfish (Pleuronectesplatessa) improved when 40% of the dietary fish protein was substituted with SBM protein (Cowey et al., 197 1) . A concentrate of soybean protein with supplemental amino acids has been reported to be a useful protein source for young yellowtail when 30% of brown fish meal was substituted (Takii et al., 1989). But results of 40% substitution with SBM resulted in significantly poorer growth rates than lower replacement levels (Lee et al., 1991) in diets for this species. Extremely low feed intake levels of juvenile chinook salmon in seawater fed at 15% and 30% replacement levels of SBM and soy protein isolate have been reported by Hajen et al. (1993). Different authors have also found problems of palatability or the existence of different components affecting SBM utilization in salmon and seriola, other than the antitrypsin factor (Fowler, 1980; Lee et al., 1991) . Besides these variable results, SBM is not a readily available feedstuff worldwide. Thus, there is a need to investigate the possibility of using other vegetable protein sources with a nutritive value comparable to that of SBM. Lupin (Lupinus sp.) is regarded as one of the legumes with high potential, due to its high protein content (30-50%) and low market prices. Several recent studies suggest that lupin seed meal (LSM) may be a good alternative vegetable protein of high nutritive quality when used at levels up to 30% or 40% in rainbow trout diets (De la Higuera et al., 1988; Hughes, 1988; Gomes and Kaushik, 1989).

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The aim of this work was to study the partial substitution of sardine meal by two vegetable protein sources (SBM and LSM) in diets for juvenile gilthead seabream by establishing the maximum levels of dietary inclusion and comparing the nutritional quality of these two types of legume seed meals. The first experiment was conducted to study fish growth performance, feed utilization, liver histology and digestive enzyme activities. The second experiment assessed the metabolic and digestive utilization of the diets by measuring NH: -N excretion of fish fed on these diets and apparent digestibility coefficients for dietary protein and lipid.

2. Material and methods Diets Soybean meal and lupin seeds (Lupinus angustijolius) were obtained from a local importer. Trypsin inhibitor activity (Liu and Markakis, 1989) and protein solubility in 0.2% KOH (Araba and Dale, 1988) were tested for SBM, and results indicated that meal obtained had received an appropriate heat treatment. Lupin seeds were kept in fresh water for 24 h, to eliminate excess alkaloids, before being dried and ground. Two groups of experimental diets were formulated by replacing fish meal protein with either SBM or LSM protein at levels of lo,20 and 30%. A diet containing fish meal as the Table 1 Formulation

and composition

of the experimental

diets

Ingredients (% as fed)

C

SlO

s20

s30

LlO

L20

L30

Fish meal Soybean meal Lupin meal Fish oil EPA 42’ Starch Dextrin Vitamin premix* Mineral premix* CMC” Cellulose

76.61

68.95 10.08 _ 6.59 _

61.29 20.16

53.63 30.24

61.29

53.63

7.79 4.08 1.36 2.00 0.40 0.50 -

68.95 11.54 4.13 0.85 4.08 1.36 2.00 0.40 0.50 6.19

23.08 2.30 I .68 4.08 2.00 0.40 0.50 3.31

34.62 0.43 2.54 4.08 1.36 2.00 0.40 0.50 0.44

4.08 1.36 2.00 0.40 0.50 9.06

4.08 1.36 2.00 0.40 0.50 6.04

7.19 _ 4.08 1.36 2.00 0.40 0.50 3.02

Moisture Protein Ether extract Ash

6.06 55.78 13.23 14.90

5.95 57.13 12.71 13.76

8.67 55.35 13.25 11.96

9.36 55.45 13.80 12.16

6.34 56.32 12.58 11.98

7.83 55.88 11.83 11.99

10.20 52.92 11.43 11.37

Gross energy (kj /g)

18.12

18.93

18.47

16.95

16.83

16.77

18.07

Proximate composition

5.99

1.36

(%)

(a) Triacylglyceride mixture containing 42% n-3 HUFA. (b) Carboxymethyl cellulose. *Commercial mixtures supplied by A.T.P., A/S (Vergara,

1992).

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sole protein source was used as the control. All diets were formulated to be approximately isocaloric in gross energy and similar in crude protein and lipid content (Table 1). Dietary essential amino acid profiles were calculated and compared with the essential amino acid profile of S. aurutu fingerlings as a reference of dietary requirements for amino acids (NRC, 1981; New, 1986; Hughes, 1988; Moyano, 1990; Vergara, 1992). No supplemental amino acids were added to the diets. A mixture of triacylglycerols containing 42% n-3 HUFA was added to the LSM diets to supply an adequate level of essential fatty acids (Ibeas et al., 1994). In the second experiment, 1% cellulose was substituted by chromic oxide as an inert indicator for apparent digestibility determinations (Austreng, 1978). After mixing all the ingredients, pelleting was performed in a 2 HP (Mobba, Milano, Italy) pellet mill with a 3 mm die. The diets were dried and stored frozen at - 20°C until used. Biochemical analysis Protein content (%N X 6.25) was determined by the Kjeldahl method and total lipids by petroleum ether extraction (Soxhlet technique). Gross energy content of the diets was determined using an IKA oxygen bomb calorimeter (Heitersheim, Germany). Gross energy content of the fish was calculated using theoretical indexes (Brafield, 1987). Statistical analysis In both experiments all the data were subjected to one-way analysis of variance (ANOVA), and differences between means compared by the Tukey test at a 95% interval of confidence (P < 0.05).

2.1. Experiment

1

Juvenile seabream (Sparus aurutu) of 40 g average initial weight were acclimated for 3 weeks to a control fish meal-based diet in 1000-l tanks. Then, fish were anaesthetized (MS222, Lab. Sandoz, Switzerland), weighed and stocked in 100-l fiberglass tanks in triplicate groups of 12 fish. Tanks were supplied with acontinuous flow ( 1 l/min) of filtered seawater ( 19 + 15°C). Each diet was fed to satiation, four times a day (09.00, 12.00, 15.00 and 18.00 h) . A constant photoperiod of 12 h L/ 12 h D was maintained. At the begining of the experiment, 10 fish were weighed, killed and frozen at - 80°C for subsequent proximate analysis. Growth performance and feed utilization Feed consumption was recorded daily and the fish were weighed every 2 weeks. At the end of the experimental period (60 days), the fish were anaesthetized and weighed after a 24-h fast. Three fish from each tank were removed and used for whole body composition analysis. The nutritional formulae employed were: feed efficiency (FE = body wt gain(g) / feed intake(g) as dry matter), protein efficiency ratio (PER= body wt gain(g) /protein intake(g)), and protein productive value (PPV= ( (% final body protein X final body wt) - (% initial body protein X initial body wt) / (total protein intake(g) X wt increase(g))) X 100).

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130 (I 995) 2 19-233

50cm

i

Fig. 1. Faeces collection

I

system: A, water outlet; B, valve; C, settling column; D, centrifugal

tube.

Enzyme activity Studies on digestive enzyme activity were conducted on three fish from each tank. A midgut portion of 1 cm, containing pyloric caeca and proximal intestine, was removed, weighed to the nearest mg, homogenized ( 1: 10 wt/vol) in cold Tris-HCI buffer (300 mM, pH 8.1) and centrifuged at 14 000 rpm (4°C 15 min). Trypsin activity was measured at 25°C with BAPNA (N-benzoyl-L-arginine-p-nitroanilide) as specific substrate. The reaction mixture consisted of 50 ~1 BAPNA (0.25 mM in Tris-HCI buffer, 0.1 M, pH 8.1, containing 20 mM CaCl,), 430 ~1 Tris-HCl buffer and 20 ~1 enzyme extract. The assay was carried out for 10 min and activity calculated from the increase in absorption at 25°C and 405 nm (Hofer and Uddin, 1985). Activity was expressed as mUnits/mg soluble protein. Soluble protein of extracts was determined according to Bradford ( 1976). Histological studies At the end of the experiment, livers from five fish from each tank were weighed for hepatosomatic index and fixed in 10% neutral-buffered formalin. Samples were stained with hematoxylin and eosin and periodic acid-Schiff (PAS) for histological examination (Martoja and Martoja-Pierson, 1970).

224

2.2. Experiment

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2

Seabream juveniles of 72 g average initial weight were acclimated for 1 week to a control fish meal-based diet, in 100-l fiberglass tanks (modified from Cho et al., 1985) (Fig. 1) designed to allow a quick and easy recovering of faeces in triplicate groups of 10 fish. Tanks were provided with a continous flow ( 1 l/min) of filtered seawater at 19 + 0.5”C. Digestibility The experimental diets were fed to satiation, twice a day (09.00 and 15.00 h). Faecal matter was collected at 12-h intervals, centrifugated for 20 min at 10 000 rpm and freezedried prior to analysis. Proximal composition of faeces was used to calculate apparent digestibility coefficients (ADC) for protein and lipid. Ammonia excretion Measures of dissolved ammonia (DA) were carried out at 0,2,4,6 and 8 h after feeding (09.00 h) for an 8-h period on two consecutive days. Water flow was interrupted during the sampling period. Water samples were processed using a flow injector analyser (Tecator, FIA star 5010). Values previously determined by Porter et al. ( 1987) for this species were used as a basis for calculation of total excretion in a 24-h period by extrapolating the values obtained in the present experiment.

3. Results

3.1. Experiment

I

Growth per$ormance and feed utilization Acceptance of all the experimental diets was good and no mortality was observed during the experimental period. Daily mean feed intake ranged from 1.19 to 1.34 g/ 100 g fish and it was not influenced by the type or level of plant protein in the diet. Similarly, weight gain of fish expressed in terms of weight increase was not significantly (P < 0.05) affected by the type or substitution level of plant protein used in the diets (Table 2). The highest weight increase was observed in fish fed diet SlO, being significantly higher than that of fish fed diet L30. Hepatosomatic indexes (HSI) did not differ among fish fed the experimental diets (Table 2). Values for FE of the experimental fish were similar in all cases and no effect of the type of plant protein used was observed. FE for fish fed the SlO and LlO diets was significantly higher than in those fed the L20 diet. PER values were not significantly influenced by type or level of plant protein in diet, showing quite similar values in all the experimental groups. PER was significantly (P < 0.05) higher in fish fed the S20 diet than in those fed the L20 diet. Protein retention, as measured by PPV (Table 2), was not significantly influenced by the type of plant protein in the diet. Values obtained for fish fed diets LlO or S20 were lower than that of the control group.

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Table 2 Weight increase, nutritive utilization of feed and protein, protein retention, hepatosomatic of whole fish fed the experimental diets’

225

index and composition

Parameter

C

SlO

s20

s30

LlO

L20

L30

Mean initial body weight (g) Mean final body weight (g ) Weight increase (% initial weight) FE PER PPV HSI

38.27”

40.29a

39.38”

37.008

38.55”

38.68”

39.63a

60.09”b

64.46”

62.02k

56.49a

60.2gab

59.06ab

61 .46”c‘

56.70ab

59.96b

57.50ab

52.67”b

56.428b

52.77ab

.51.76a

0.61ab l.15a” 24.91” l.31a

0.63b 1.16”” 22.39”= 1.29”

0.61ab l.21b 19.70ab 1.28”

0.55”b l.12ab 21.95‘-” 1.38”

0.63b l.lgab 19.01” 1.45”

0.53” 1.03” 24.87* 1.60”

0.56”b 1.19a” 27.666 1.38”

56.96“’ 29.39”b 67.65” 26.82

52.87”” 32.55b 66.88” 26.22

53.6@ 30.16ab 68.25a 23.75

51.98” 30.46”b 66.98’ 24.63

58.22’ 27.60” 68.57” 22.40

57.67d 28.56” 68.43a 24.65

Fish composition (g/ 100 g live weight) 56.08bc“ Protein 31.8gb Fat Moisture 67.06” Energy 25.92 retention’ (%)

‘Means in the same row with same superscript are not significantly ‘Calculations based on values provided by Brafield ( 1987).

different (P < 0.05).

Fish composition Final body protein was significantly influenced by the type and level of plant protein used in diets (Table 2). Thus, substitution of more than 10% dietary fish meal protein by LSM significantly increased final body protein when compared to the same levels of substitution by SBM. Moreover, increasing the level of vegetable protein in such diets was associated with a higher protein content of the fish. Calculated values of energy retention, which relates total energy deposition in fish and total gross energy intake, ranged between 22 and 26% for all the experimental groups. Enzyme activity Values of trypsin-like specific activity showed wide variation among the experimental groups, ranging from 8 to 180 mUnits/mg soluble protein (Table 3). Values of protease activity in fish fed the LSM diets decreased as the substitution level increased, although no Table 3 Protein and lipid ADC and trypsin activity (mUnits/mg

soluble protein) for the different diets’

Parameter

C

SlO

s20

s30

LlO

L20

L30

ADC protein ADC lipid

92.95bc 92.59”

93.63’ 93.16a

86.22a 95.89’

87&tpb 97.5lC

95.49’ 97.16*

94.55’ 93.95ab

92.96bc 95.35&

0.15”

o.02ab

0.04k

0.01a

0.01”

Trypsin activity

0.126

o.09cd

‘Means in the same row with same superscript

are not significantly

different (P < 0.05).

226

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Plate I. Hepatic and pancreatic tissues of fish fed the experimental S30; (d) diet L20; (e) diet L30.

diets: (a) control diet; (b) diet S.20; (c) diet

significant differences were apparent. A significant (P < 0.05) reduction in trypsin activity was observed in fish fed the SBM diets when the substitution level reached 30%. Histological studies

Liver samples of fish from the different treatments were compared to those of the control group, which did not show any alterations from normal histology (Plate la). A greater number of lipid droplets around the pancreatic tissue was observed in the liver of fish fed the SBM diets (Plates 1b and 1c) , and eccentric located nuclei were present when the level of SBM in the diet was equal to or higher than 20%. Deposition of PAS-positive substances in liver sections decreased and was more diffuse as the proportion of SBM increased in the

228

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130 (1995) 219-233

600

500

4 I= 400 ? 2300 ? z

200

k?

0

4

2

24

8

6

Time (h) Fig. 2. Accumulated

production

of dissolved ammonia

(mg N-NH:

/kg fish).

diet. Moreover, areas with high levels of hepatocyte vacuolization and disorganization were present in some samples from fish fed the S30 diets. Although the liver histology of fish fed the LSM diets was similar to the control, a small number of lipid droplets together with some reduction of glycogen deposits in the cytoplasm were present in the liver of fish fed diets L20 and L30 (Plates Id and le) .

50

f”10 0

u

Y 0

2

4

i 8

6

Time (h) Fig. 3. Time patterns of dissolved ammonia

(mg N-NW

/kg fish)

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229

2

Digestibility ADC values for protein and lipid (Table 3) showed significant differences among the experimental diets, the highest values being obtained in fish fed the ADC values for LSM protein were about 10% higher than those for SBM protein. differences in lipid ADC values were not related to the level or type of legume present in the diet (Table 3).

(P < 0.05) LSM diets. Significant seed meals

Ammonia excretion Figs. 2 and 3 show the accumulated production of dissolved ammonia (DA) and time patterns of DA excretion for each group of fish, respectively. Clear differences in time and amount of peak production of DA were noticeable when comparing results obtained for fish fed fish meal protein with those for fish fed the diets including vegetable proteins. Peak DA excretion rate was reached 4 h after feeding the fish meal diet, whereas the same peak was reached 2 h later in the rest of the fish. A significant (P < 0.05) negative correlation was established ( - 0.829) between NH,+ -N excretion and digestibility of protein for all the fish fed diets including either SBM or LSM.

4. Discussion The good acceptance of all the experimental diets was of interest, since a low intake of feeds containing increasing levels of some vegetable feedstuffs has been reported by several authors (Jackson et al., 1982; Roselund, 1986). This observation seems to be associated with the high sensitivity of fish to the organoleptic properties of their diets (Bone and Marshall, 1982). In juvenile chinook salmon, extremely low feed intake levels of diets containing 15 and 30% of SBM and soy protein isolate were related to the poor palatability of these products (Hajen et al., 1993). The poor palatability of SBM has also been reported in other fish species (Fowler, 1980). The good feed intake of LSM in diets of the present experiment agrees with the results previously reported in salmonids (De la Higuera et al., 1988; Gomes and Kaushik, 1989; Hughes, 1991). Furthermore, in contrast to SBM (Liener, 1966), the antinutritional factors in lupin seed meal which affect palatability (mainly alkaloids) are easily removed by washing in fresh water. Thus, nutritional evaluation of both vegetable proteins was not conditioned by low feed intake. Similar nutritional values were observed for both plant proteins, based on the FE, PER and PPV values. Nevertheless, an overall decrease in these values was observed as the level of SBM in diet increased, although no significant differences were detected within diets of each series. The high ADC values obtained for protein of LSM diets were in agreement with those reported for other fish species such as rainbow trout or eel (De la Higuera et al., 1988; Hidalgo, 1988). A significant (P < 0.05) reduction in trypsin activity and protein ADC was observed for the SBM diets when the substitution levels increased. The presence of trypsin inhibitors in the SBM used in this experiment must be discounted because trypsin inhibitor activity

230

L. Robaina et al. /Aquaculture 130 (1995) 219-233

analysis indicated that the SBM had received appropriate heat treatment. The reduction of these parameters may have been associated with the presence of phytates. Phytate is unavailable to most finfish and terrestrial monogastric animals because of the lack of phytase, which catalyzes the conversion of phytate to its moieties. Phytic acid has been reported to reduce protein digestibility in rainbow trout (Spinelli et al., 1983) as well as Zn bioavailability in salmonids (Richardson et al., 1985) and channel catfish (Satoh et al., 1989). Since soybean meal has approximately 75% of phosphorus (P) in the form of phytic acid ( 1.0-l 5% of the meal), it is possible that P availability decreased as the level of SBM in the experimental diets increased. The increase in lipid deposition in muscle and liver, as well as the decrease in liver glycogen, have been reported as signs of P deficiency in fish (Sakamoto and Yone, 1978). This is in agreement with the histological results obtained in the current study, where liver of fish fed high levels of soybean meal showed higher lipid deposition with a decrease in glycogen deposits. A further consideration is the possible binding capacity of soybean agglutinin (SBA) to the brush border membrane of the enterocytes. Apart from protease inhibitors, SBA may also contribute to the. toxic effect of fullfat soybean and soybean products in diets for salmonids (Hendriks et al., 1990). When fish were fed the LSM diets, intestinal trypsin activity decreased as the dietary substitution level increased. These results, compared with those for the control diet, suggest the presence of some kind of antitrypsic factor in the lupin meal. However, no antitrypsin factors have been reported in lupin, and the results obtained for protein digestibility indicate that the lupin meal was well digested. Although there were no significant differences in weight increase, the overall reduction in fish growth when plant protein levels increased may be due to different factors. Antinutritional factors may limit the use of high levels of vegetable feedstuffs in fish feeds (Chubb, 1982; Richardson et al., 1985; Gatlin and Phillips, 1989). Plant protein sources may include antinutritional factors other than trypsin inhibitors. Some oligosaccharides and starches may be mistaken as available carbohydrates, as has been reported for soybean and lupin meals (Saini, 1989). In fact, a decrease in nutrient utilization, mediated by soybean carbohydrates has been reported in salmonids (Arnesen et al., 1989). The results obtained for the production of DA agreed with previous reports for gilthead seabream (Porter et al., 1987) and sockeye salmon (Brett and Zala, 1975). In both cases, a marked increase in ammonia excretion occurred, peaking 4-4.5 h after feeding. The delay in ammonia excretion observed in fish fed diets containing vegetable proteins may have been related to a longer time required for the digestion, absorption and metabolism of the plant proteins. Total daily excretion of DA, calculated from values obtained during an 8-h period, was 40% higher in fish fed the S30 diet than in those fed the control diet, suggesting a higher deamination of dietary protein for the highest level of SBM replacement. This fact is related to the non-significant but lower protein deposition and PPV in the fish. In conclusion, the type or level of vegetable protein fed to fish in this experiment did not significantly affect nutritive utilization of the diets. Nevertheless, lower values of digestibility and histological alterations observed in fish fed on soybean diets suggested that properly treated lupin meals could be an important alternative to the use of SBM in diets for gilthead seabream juveniles. Level of LSM protein, in such a case, must not exceed 20% of total protein to prevent lipid deposition in the liver.

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Acknowledgements We express our gratitude to Professor Takeshi Watanabe, Tokyo University of Fisheries, for his valuable suggestions during this study. The experiments were partially supported by a grant from the Las Palmas de Gran Canaria University (Beta de Tercer Ciclo) .

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