Effects of pretreating a plant-based diet with phytase on diet selection and nutrient utilization in European sea bass

Aquaculture 319 (2011) 417–422

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Effects of pretreating a plant-based diet with phytase on diet selection and nutrient utilization in European sea bass R. Fortes-Silva ⁎, F.J. Sánchez-Vázquez, F.J. Martínez Department of Physiology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain

a r t i c l e

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Article history: Received 11 March 2011 Received in revised form 15 July 2011 Accepted 20 July 2011 Available online 27 July 2011 Keywords: Anti-nutritional factors Feeding behavior Self-selection Phytase Phosphorus Dicentrarchus labrax

a b s t r a c t As most animals, fish possess an impressive array of behavioral mechanisms to deal with challenges posed by the composition of the food they consume. Such responses can be behavioral, through both food selection and regulation of the amounts eaten, which helps to alleviate the deleterious effects of such nutritional imbalances. The purpose of the two experiments presented was to examine the capacity of groups of European sea bass to differentiate between isoproteic diets containing soybean meal (SM0), soybean meal + 1500 FTU/kg of phytase (SM1) and fish meal (FM) to detect any preference and also the effect of each diet on nutrient utilization and growth in the carnivorous species studied. Ninety-nine fish (mean initial weight 265 ± 10.5 g, S.E.M) were divided into three self-selection treatments (three replicates) based on different diets (FM vs SM0, T1); (FM vs SM1, T2) and (SM0 vs SM1, T3) using self-feeders (Experiments 1). Preferences were expressed as relative changes in feed demands for a specific feeder and diet selection was expressed as the percentage of diet, considering the total of diets as 100%. Another seventy-two fish (mean initial weight 255.0 ± 11.0 g, S.E.M) were randomly distributed among nine tanks to analyze body composition (P and Ca retention) and growth parameters (Experiment 2). In this case, fish were fed a single diet (FM or SM0 or SM1) in the same laboratory condition as Experiment 1. Averaged over all groups of Experiment 1, the preference tests demonstrated the capacity of European sea bass to discriminate between a diet with FM (P b 0.05) and a diet containing exogenous phytase SM1 (P b 0.05), their final choices being 15.8% SM0vs 84.2% FM; 26.7% SM1vs 73.3% FM; 9.8% SM0vs 90.2% SM1. In Experiment 2, the partial replacement of fish meal by soybean meal in diets (SM0 and SM1) resulted in reduced weight gain and lower specific growth rate (SGR), while, P and Ca retention increased significantly in fish fed diets SM1 and FM. It is believed that the metabolic consequences of the plant meal intake (SM0) negatively affected the feed acceptance in the studied species, and that exogenous phytase increased the acceptance of the plant diet. Finally, we showed that seabass defended an “intake target” by allowing them to select among different diets to satisfy their nutritional needs. © 2011 Elsevier B.V. All rights reserved.

1. Introduction There is wide evidence that fish can regulate diet intake to satisfy their nutritional requirements when allowed to self feed (Aranda et al., 2000; Fortes-Silva et al., 2011; Sánchez-Vázquez et al., 1998, 1999; Vivas et al., 2006). In previous studies, fish showed an ability to selfselect diets containing feeding stimulants (Adron and Mackie, 1978), methionine (Hidalgo et al., 1988), zinc (Cuenca et al., 1993), vitamin C (Paspatis et al., 1997) and dietary oil (Fortes-Silva et al., 2010a). The results showed that the fish were able to discriminate levels of the test substance among diets using self-selection methodology. Alternative protein sources have been studied intensively in several fish species. However, studies have also pointed to adverse effect of plant-based diet on growth performance and feed utilization,

⁎ Corresponding author. Tel.: + 34 968 364931; fax: + 34 968 363963. E-mail address: [email protected] (R. Fortes-Silva). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.07.023

especially in carnivorous species (Cain and Garling, 1995; Hughes and Soares, 1998; Kauskik et al., 1995; Li and Robinson, 1997; Oliva-Teles et al., 1994; Vielma et al., 2000). Such results may be explained by the fact that plant-based feed may contain certain anti-nutritional factors: non available P, (Liebert and Portz, 2005); insoluble protein and vitamin, (Papatryphon et al., 1999); low diet digestibility, (Liu et al., 1998) and poor palatability (Lim and Persyn, 1989). Virtually all plant crops contain secondary compounds, including protease inhibitors, tannins, lectins, phytate, dietary fiber and starch (Drew et al., 2007), which limit their use in some fish species. Phytase is an enzyme that can be added to plant-based diets to hydrolyze non-digestible phytate, improve nutritional efficiency and protein digestibility (Sugiura et al., 2001) and reduce the cost of aquafeed production and the pollution potential of excreted phosphorus (Beegle, 2000; Sardar et al., 2007). Increasing mineral bioavailability would affect body composition and bone strength (Liebert and Portz, 2007; Sardar et al., 2007) and, in some cases, growth parameters (Jackson et al., 1996; Li and Robinson, 1997). Moreover, the

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improvement in micro minerals and essential amino acids by phytase can be beneficial for the health status of monogastric organisms, such as broiler chickens (Liu and Ru, 2010). However the effects of phytase on food acceptance and on the feeding behavior in carnivorous fish are unknown. According to Bedford (2000) the chemical effects of exogenous enzymes, such as phytase are well understood, but the manner in which their benefits to the animal are brought about is still under debate. Understanding the capabilities of commercially reared fish species to regulate intake may have beneficial economic effects, but also improve fish welfare and benefit the environmental (Rubio et al., 2005; Simpson and Raubenheimer, 2001; Yacoob et al., 2001). Our first purpose, in Experiment 1, was to evaluate the ability of European sea bass (Dicentrarchus labrax) to self-select between a diet composed of fish meal as protein source, another of soy meal and a third diet composed of soy meal plus exogenous phytase enzyme. A second aim, in Experiment 2, was to analyze the effect of each experimental diet offered separately on bone mineral composition (phosphorus, P and calcium, Ca) and growth parameters. P and Ca belong to the so-called major elements or macro minerals and are of special interest for many functions in animal metabolism (Flachowsky, 2010).

Table 1 Ingredients and chemical composition of the experimental diets. Diets

SM0

SM1

FM

Fish meala Soybean mealb Starch Fish oil Cellulose Mineral premixc Vitamin premixd Dicalcium phosphate DL-methionine L-Lysine Phytase (FTU/kg)

43.0 31.0 10.0 7.9 0.6 1.0 1.0 3.1 2.1 1.0 –

43.0 31.0 10.0 7.9 0.6 1.0 1.0 3.1 2.1 1.0 1500

63.0 – 21.2 7.6 2.5 1.0 1.0 3.0 1.3 – –

Proximate analysis Dry matter (%) Crude protein DE (kJ/g DM) Crude lipids Ash (% DM) Calcium (g/kg DM) Total phosphorus (g/kg DM) Phytate phosphorus (g/kg DM) Available phosphorus (g/kg DM)

91.6 47.0 19.2 15.8 12.5 15.3 14.0 1.21 12.79

89.2 46.9 19.3 15.7 12.5 15.0 14.2 1.20 13.00

89.1 47.8 19.2 15.1 17.3 24.5 20.1 0.36 19.74

a

2. Material and methods 2.1. Animal housing Two different experiments were performed at the same time, in the same laboratory conditions. The juveniles of European sea bass used in the experiment were obtained from Doramenor, Murcia, Spain and placed in the fish laboratory of the University of Murcia, Cartagena, Spain. They were acclimatized for 1 month in a circular tank (150 L) with pumped sea water (connected to a recirculation unit) and suitable aeration, during which time they were fed with a commercial diet. Before the experimental period, fish were fed twice a day until near satiation under a 12L:12D photoperiod to accustom them to the laboratory conditions. The water temperature was 20–22 °C. The oxygen level was above 6 mg/L, the water exchange rate 100% per day and water salinity ranged between 31 and 32‰. Fish were reared and manipulated following Spanish legislation on Animal Welfare and the legislation concerning the protection of animals used for experimental purposes (EC Directive 86/609/EEC). 2.2. Experimental diets Three experimental diets (SM0, SM1 and FM) were formulated to contain around 47.0% crude protein as a source of soybean meal or fish meal. The basic diet (SM0) was made into 3 mm pellets using an electric pelleting machine, which basically consists of a mill that forces the wet diets (100 parts ingredients with 30 parts distilled water) through small holes in a die. Exogenous phytase was added to SM0 to produce SM1 (Table 1). The phytase used in the trial was obtained by fermentation from an Aspergillus niger strain (Natuphos®5000, BASF-Germany, EC 3.1.3.8) and contained no additives that could affect its palatability for fish. To avoid denaturing of the enzyme during feed processing, the phytase (1500 FTU) was diluted in 50 ml of distilled water and added to 1 kg of SM1 by mean of a spray and the resulting feed was dried at room temperature (Hughes and Soares, 1998). Dissolving phytase as liquid suspension to spray on to the feed pellet after feed processing could be a solution to the problem of denaturing (Hughes and Soares, 1998). Most phytases have an optimal activity in a pH range of 4.5–6.0 and a temperature range of 45–60 °C (Cao et al., 2007). Outside the optimal range of pH and temperatures the action of phytase is reduced (Lei and Stahl, 2000). All diets were processed taking into account the above parameters.

Standard fish meal with 70% of crude protein supplied by Skretting, Spain. Toasted, dehulled with 47% of crude protein supplied by El Granero, Madrid, Spain. Minerals (g or mg/kg diet): calcium carbonate (40% Ca), 2.15 g; magnesium oxide (60% Mg), 1.24 g; ferric citrate, 0.2 g; potassium iodide (75% I), 0.4 mg; zinc sulphate (36% Zn), 0.4 g; copper sulphate (25% Cu), 0.3 g; manganese sulphate (33% Mn), 0.3 g; dibasic calcium phosphate (20% Ca, 18%P), 5 g; cobalt sulphate, 2 mg; sodium selenite (30% Se), 3 mg; KCl, 0.9 g; NaCl, 0.4 g. d Vitamins (UI/kg in diet): Vitamin A, 100,000; D3, 150,000; E, 3000; B1, 1600; B2, 3200; B6, 3200; B12, 2,4; C, 15,000; K, 1000; d-Pantoten, 9600; Folic acid, 600; Niacin, 12000; Biotin, 28,0; Betaín, 80,000. b c

One FTU of phytase represented the amount of phytase that liberates inorganic phosphorus from a 1.5 mM solution of sodium phytate at a rate of 1 μmol min −1 at pH 5.5 and 37 °C. The same spray treatment was applied to all other diets, using distilled water only. The diets were stored at 4 °C until use. Dietary moisture was determined by drying the samples for 24 h at 110 °C; crude protein was estimated by the micro-Kjeldahl method (Nx6.25), crude fat by diethyl ether extraction, ash by heating at 450 °C for 24 h and nitrogen free extract (NFE) as the remainder. The digestible energy was calculated using the following estimated DE coefficients: 20.9 kJ/g for protein; 35.1 kJ/g for fat and 11.0 kJ/g for carbohydrate, values found in the literature for carnivorous fish (Hillestad et al., 1999).

2.3. Experiment 1 Ninety-nine fish (265 ± 10.5 g, S.E.M) were randomly distributed among nine tanks to evaluate the feeding behavior. The fish were divided into three self-selection treatments based on different diets (FM vs SM0, T1); (FM vs SM1, T2) and (SM0 vs SM1, T3) with 3 replicates each. Each tank had two self-feeders that delivered an average 0.72 g, each time a fish activated a string sensor located 0.5 cm below the water surface (Sánchez-Vázquez et al., 1996). This sensor was activated by fish pulling on the rubber tip, a system that allows the distribution of feed on demand as well as enabling the demands and rewards to be monitored. Every day the feed remaining in the feeder was weighed and the feeder recipient refilled. After feed demands had stabilized, feeds were switched between feeders. Feed intake was monitored in this way every day throughout the experiment. The presence of uneaten food pellets was visually checked every day and real feed intake was calculated by difference.

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Feeder switched

2.4. Experiment 2

2.5. Data analysis Data were analyzed using SPSS software. Feed consumption rates were expressed as g/100 g BW day −1 and the relative diet selection was expressed as the percentage of diet, considering the total of diets as 100%. The results are expressed as the mean with the corresponding standard errors. Significant differences between average diet selections and the main effect between treatments were assessed by Student's t-test. The significant differences in the P or Ca content of bone and growth parameters were analyzed by ANOVA test, followed by Tukey test of multiple comparison at 95% significance level.

100 90 80

Selection (%)

Seventy-two fish with an initial body weight of 255.0± 11.0 g, S.E.M were randomly distributed among nine tanks to analyze body composition and growth parameters. Fish were fed 2% of BW twice a day at 9:30 and 13:00 h, in accordance with the feeding schedule for the growth trial, for 90 days. At the beginning, a pooled sample of 18 fish was stored in a freezer for bone analyses. Three groups (G1, G2 and G3) were fed one experimental diet, (FM, SM0 or SM1, respectively), in three replicates. All possible care was taken during feeding that no uneaten food should settle on the tank bottom. Food remains in the water were dried and weighed. For waste feed collection the Guelph system was used (Cho et al., 1982). At the end of the experiment, body weight was measured to ascertain whether growth performance showed the same pattern. Before measuring the body weight, the fish were starved overnight. Specific growth rate (SGR), carcass index (CI), feed efficiency (FE) and survival rate percentage were calculated as per standard procedures. The fish from each treatment were stored in a freezer and were used for body analyses 12 weeks after the experiment. Operculum samples were collected from 6 individuals randomly selected from each tank, and dipped in boiling distilled water for 30 s before removing the skin and connective tissues (Vielma et al., 2000). Bones and diets were dried for 24 h at 100 °C before being analyzed. After sample digestion in a microwave (Perkin Elmer), the phosphorus and calcium concentration were determined using ICP techniques (Dalsgaard et al., 2009); all analyses were performed in duplicate. Phytate in the feed ingredients was analyzed by the method described by Rama Rao et al. (2006). Available phosphorus was calculated by deducting phytate from total phosphorus (Sajjadi and Carter, 2004).

70 60 50

** *

*

*

*

* *

*** * *

* ***

***

**

40 30 20 10 0

1

3

5

7

9

11

13

15

17

19

21

23

25

Time (days) Fig. 1. Selection of two experimental diets, FM (▲) and SM0 (□), considering the total of diets as 100% and the corresponding standard errors (S.E.M) of three groups (11 sea bass per group), in the 25 days and two phases of the experimental period. After feed demand had stabilized (day 11), feeds were switched between feeders. The preference for the complete fish meal diet, represented by the asterisk, was significant (P b 0.05).

3.2. Experiment 2 Feed intakes were corrected for the amount of feed wasted. While there was no difference between SM0 and SM1 in feed efficiency (FE), the same parameter was improved with increasing levels of fish meal in the FM diet (P b 0.05, Table 2). No improvement in the SGR was observed for fish submitted to treatments SM0 and SM1 (P b 0.05, Table 2). Final weights fell when fish meal was replaced by soybean meal (SM0) and soybean meal + phytase (SM1); however, these parameters improved in fish fed the FM diet (Table 2). No significant difference was observed in the carcass index (CI) (P N 0.05) or mortality between groups (G1, G2 and G3). The proximate bone compositions of fish fed the different diets are presented in Fig. 4. Fish fed the FM diet in G1 group showed a higher bone P and Ca content than those fed diets SM0 and SM1 (P b 0.05). Moreover, the bones of fish fed diet SM1 in G3 had a higher P and Ca contents than the bones of fish fed SM0 in G2(P b 0.05). Likewise, fish fed diet FM showed better phosphorus retention efficiency than those fed the other diets (P b 0.05, Table 2) and higher Ca retention than the

3. Results 3.1. Experiment 1 100 90

Selection (%)

No mortality was registered during the experiment and no pellets were observed to accumulate at the bottom of the tanks. Fish quickly learned to operate self-feeders and to select among the diets, with low daily variations. Feed demands were around 2% body weight, while food delivery closely reflected the demand-feeding activity of the fish, and hence the amount of food actually ingested. At one extreme (T1), fish showed a specific preference (84.2%) for the fish meal diet, compared with 15.8% for the soy meal diet throughout the 25-day feeding period (arcsin of selection P b 0.05, Fig. 1). As expected, this selection pattern was also observed in T2 for the fish meal diet (73.3%) and soybean meal + phytase (26.7%), (P b 0.05, Fig. 2) at the end of trial. At the other extreme (T3) fish preferred the diet of soybean meal + phytase (90.2%) to the soybean meal only diet (9.8%), (P b 0.05, Fig. 3). Moreover, each treatment was followed by a validation phase (food switched between feeders) in order to verify whether changes in feeder preferences were driven by the diet composition. After switching the diets between feeders for all the treatments, fish quickly responded to the preferred diet, and the pattern of intake was reestablished.

Feeder switched

80 70 60

* *

50 40

* * *

*

*

* * * * *

*

* * * *

15

17

*

30 20 10 0

1

3

5

7

9

11

13

19

21

Time (days) Fig. 2. Selection of two experimental diets, FM (▲) and SM1 (1500 IU kg−1, ◊), considering the total of diets as 100% and the corresponding standard errors (S.E.M) of three groups (11 sea bass per group), in the 22 days and two phases of the experimental period. After feed demand had stabilized (day 10), feeds were switched between feeders. The preference for the complete fish meal diet, represented by the asterisk, was significant (P b 0.05).

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Feeder switched

120

Selection (%)

90

* * * * * *

80 70 60

**

*

50

**

Bone composition (g kg-1 DM)

100

a

80 60

P

40

Ca

20

40 0

30

K SM0

20 10 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39

Time (days) Fig. 3. Selection of two experimental diets, SM0 (□) and SM1 (1500 IU kg−1, ◊), considering the total of diets as 100% and the corresponding standard errors (S.E.M) of three groups (11 sea bass per group), in the 40 days and two phases of the experimental period. After feed demand had stabilized (day 24), feeds were switched between feeders. The preference for the complete phytase diet, represented by the asterisk, was significant (P b 0.05).

fish fed the SM0. Also, fish fed SM1 showed better phosphorus and Ca retention than those fed diet SM0 (P b 0.05, Table 2). 4. Discussion Despite the relatively large number of reports devoted to phytase, no studies exist on food selection in a carnivorous species given the freedom to choose a plant diet supplemented with phytase, or the effect of phytase supplementation on food intake. The differences in food selection observed in our study may reflect that nutritional needs provide the differences in nutritional value of the diets. In self-feeding reward conditions, fish quickly learn the relationship between ‘feed seeking’ and the nutritional or sensory characteristics of the reward (Geurden et al., 2007). Such behavior was observed in our results, and, after switching the food between feeders, fish only needed a few days to re-establish their previous preferences. We estimated that the available phosphorus content in FM might exceed the dietary phosphorus requirement of European sea bass.

Table 2 Effect of diets on growth parameters and P and Ca retention. Parameter (unit)

Initial weight (g) Final weight (g) Specific growth rate (%)1 CI(%)2 FE3 Survival (%) P bone retention (%)4 Ca bone retention (%)4

Experimental diets SM0

SM1

FM

257.3 ± 9 301.1 ± 18a 0.18 ± 0.09a 94.6 ± 1.1 0.55 ± 0.04a 100.0 36.3 ± 0.8a 33.8 ± 1.0a

253.9 ± 10 306.6 ± 15a 0.22 ± 0.08a 93.8 ± 1.2 0.56 ± 0.04a 100.0 38.0 ± 0.6b 37.0 ± 0.9b

254.4 ± 12 378.2 ± 11b 0.47 ± 0.07b 93.1 ± 1.5 0.67 ± 0.05b 100.0 47.0 ± 1.4c 39.1 ± 1.9b

FBW = final body weight. IBW = initial body weight. N = concentration in diet. Nf = final concentration of nutrient in fish. Ni = initial concentration of nutrient in fish. Within the same row, means with different superscript letters differ significantly (P b 0.05). Absence of superscript indicates no significant difference between treatments. 1 SGR (Specific growth rate, %)= (Ln final weight − Ln initial weight)× 100 / no days. 2 CI (Carcass index, %) = (gutted weight / body weight) × 100. 3 FE (Feed efficiency) = Weight gain / dry feed intake. 4 Nutrient retention (%) = 100 × (FBW × Nf − IBW × Ni) / (feed intake × N) (Storebakken et al., 1998).

c

b

100

M

L SM1

FM

Fig. 4. P and Ca concentration in bone of European sea bass fed three experimental diets containing two protein sources (fish meal, FM; soybean meal, SM0 or soybean meal supplemented with phytase, SM1). Means with different superscript letters differ significantly (P b 0.05).

Therefore, it was to be expected that fish select the FM diet. Moreover, we believe that the reduced selection of SM0 in (SM0 vs FM, T1) and (SM0 vs SM1, T3) was most probably related to the anti-nutritional factors of soybean and the nutrients provided by the two other diets (see results of Exp. 2). Our results agree with those of Fortes-Silva et al. (2010b), who found that tilapia, an omnivorous species, prefer a diet supplemented with exogenous phytase. These authors concluded that tilapia was capable of discriminating the effect of phytase levels in a plant-based diet and that fish decrease their feed intake when exogenous phytate is supplied in the diet. Food selection and nutrient utilization may be species specific and also depend on the feed ingredients used (Cao et al., 2007). However, according to Oliva-Teles et al. (1998), supplementation with phytase may prove valuable in sea bass diets that include high levels of plant feedstuffs. Voluntary feed intake increases when dietary phosphorus increases from severely suboptimal to optimal levels (Pimentel-Rodrigues and Oliva-Teles, 2001; Rodehutscord, 1996). However, when the phosphorus available is very low, an inhibitory response in food intake is observed (de la Higuera, 2001). Decreased feed intake in fish fed deficient diets may prevent or delay the onset of metabolic disorders (de la Higuera, 2001). This association between self-feeding and nutritional reward has been used successfully to assess the feeding behavior of several fish species in relation with dietary nutrient or energy intake (Geurden et al., 2007; Rubio et al., 2009; Sánchez-Vázquez et al., 1999; Vivas et al., 2003; Yamamoto et al., 2001). Those authors agree with Boujard et al. (2000) and Almaida-Pagán et al. (2006) that a nutrient regulation mechanism acts on a short-term basis, while a satiation mechanism, involving metabolic and neurohormonal pathways, is probably responsible for the regulation of feed intake. Alternatively (or additionally) a negative feedback control of food intake may occur (Forbes, 2001) as a result of ‘discomfort’ generated in the digestive tract and in the rest of the body as a consequence of feeding. The phytate phosphorus content of SM0 (with 31% of Soybean meal) was around 1.21 g kg −1, of SM1 1.20 g kg −1 and of FM 0.36 g kg −1, respectively. This phytate concentration in soybean meal can readily form chelates with divalent minerals, such as calcium, iron, zinc and magnesium (Apines et al., 2003). The formation of mineral–phytate complexes is associated with a reduction in mineral bioavailability. According to Biswas et al. (2007), phytase supplementation at 2000 FTU in a diet with 30% soybean meal + 40% fish meal (14.5 g/kg total P in diet) beneficially reduces not only the dependence on fish meal as a primary source of protein, but also lowers phosphorus discharge from red sea bream (Pagrus major) diets without compromising growth. Moreover, according to these authors soybean meal levels higher than 30% in the diet depress growth performance in red sea bream irrespective of phytase supplementation. Also, another study revealed that diet with 1 g kg −1 of phytate + 1000FTU kg −1 improved phosphorus retention and decreased the phosphorus load (Cao et al., 2008).

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Our results pointed to a significant increase (P b 0.05, Table 2) of P and Ca in the bone of sea bass fed FM and SM1, suggesting that the availability of P and Ca is closely related with the results for diet selection in Experiment 1. Nwanna et al. (2007), observed higher P and Ca deposits in carp fed diets containing phytase. Likewise, Vielma et al. (2002), Storebakken et al. (1998) and Sajjadi and Carter (2004) reported that pre-treatment of soy proteins with phytase hydrolyzed the phytate, making the P in soybean meal more available and increasing feed efficiency in carnivorous species. Also in our results, phosphorus and calcium retention in fish fed the negative control diet without phytase was statistically lower than in the fish fed diets supplemented with phytase or the fish meal diet. Other results concerning the supplementation fish diets with phytase (Cheng and Hardy, 2002; Forster et al., 1999; Sugiura et al., 2001) including results for European sea bass (Oliva-Teles et al., 1998) showed that phytase can improve P retention and reduce the phosphorus discharge into water, considered one of the main pollution elements in aquatic environments. Improvements in growth parameters and diet utilization (FE) were observed only in fish fed the FM diet. The final weight and SGR of fish fed the negative control diet without phytase were statistically the same in the fish fed diets supplemented with phytase. Moreover, CI and survival did not differ between treatments. These results are similar to those obtained in European sea bass by Felip et al. (2006) and Oliva-Teles et al. (1998), who observed that the addition of 2000 FTU/kg did not enhance the performance of European sea bass. Similarly, supplementation of the diets with phytase did not significantly affect SGR or mortality in rainbow trout (Dalsgaard et al., 2009). Depressed growth was observed when soybean meal replaced 30% or more fish meal protein in flounder (Paralichthys olivaceus) and Korean rockfish (Sebastes schlegeli) (Choi et al., 2004; Lim et al., 2004). In an evaluation of the growth parameters of sea bass fed five different levels of soybean meal replacing protein obtained from fish meal and using self-feeders, Kaushik et al. (2004) found that all groups had very good growth with no significant differences between groups in this respect nor as regards, feed efficiency and daily nitrogen gains. Also according to several authors, the partial replacement of fish meal by plant-protein sources is feasible in European sea bass (Tibaldi et al., 1999; Tulli et al., 1999). It seems, then, that the results concerning plant-protein use in carnivorous species are contradictory; however, the beneficial effect of phytase supplementation on nutrient improvement in most fish species seems to be confirmed. Given the global needs for fish meal for aquaculture, there is an increasing demand for alternative protein sources in aquafeeds for carnivorous fish. In marine species, Kauskik et al. (1995) and Watanabe et al. (1999) observed a number of adverse effects in fish fed non-fish meal diets, besides a modified food intake. From a comparative point of view, more insight is needed into the specific effects of plant proteins plus phytase on food selection and diet intake, since these factors are species specific and might affect, among other factors, food acceptance and feed efficiency. 5. Conclusions The results of this experiment confirmed that European sea bass fed a plant diet or plant diet plus phytase (1500 FTU/kg) did not show improved growth parameters compared with fish fed a fish meal diet, but phytase significantly improved the nutritional proprieties of a plant diet, increasing P and Ca retention in bone. This study is the first to demonstrate that a carnivorous species is capable of distinguishing a diet containing exogenous phytase, and that the European sea bass has a general preference for the diet that provides better nutritional value. These findings should be considered when discussing the food intake regulation mechanisms in fish and the supplementation of diets with phytase.

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