Effects of salinity on food intake and macronutrient selection in European sea bass

Physiology & Behavior 85 (2005) 333 – 339

Effects of salinity on food intake and macronutrient selection in European sea bass V.C. Rubio*, F.J. Sa´nchez-Va´zquez, J.A. Madrid Department of Physiology, Faculty of Biology, University of Murcia, Campus de Espinardo, Murcia 30100, Spain Received 17 June 2004; received in revised form 19 April 2005; accepted 26 April 2005

Abstract Salinity is one of the most relevant environmental parameters in regards to fish physiology, modifying food intake and growth performance in many fish species; however, its possible effects on macronutrient selection are still unknown. The aim of this study was to determine the effects of three salinity levels (25°, 7°, and 0°) on total food intake and encapsulated macronutrient selection in a euryhaline teleost, European sea bass. A total of 40 fish (five per tank) with an average body weight of 52.4 T 7.1 g were used. Lowering the salinity level from 25° to 7° and 0° reduced food intake by 27% and 42%, respectively. Regarding macronutrient selection, these salinity changes significantly decreased the percentage of CH intake by 31% and 27%, while increasing that of P by 30% and 25%, respectively. Fat selection remained unaltered, with an average value of 22% for all tested salinities. Specific growth rate (SGR) and feed conversion efficiency (FCE) were affected by macronutrient selection pattern, which in turn was salinity-dependent. These results indicate a strong influence of salinity on European sea bass food intake and macronutrient selection. D 2005 Elsevier Inc. All rights reserved. Keywords: Salinity; Food intake; Macronutrient selection; Sea bass; Dicentrarchus labrax

1. Introduction Growth and food intake are controlled by internal factors involving the central nervous system, endocrine system, and neuroendocrine system, and also by environmental factors such as temperature, photoperiod, salinity, oxygen level, ammonia, pH, etc. Salinity, like other environmental factors specific to aquatic habitats, has prompted many studies on its influence on fish growth. For the most part, these studies have demonstrated that water salinity affects the growth of both marine [1 –5] and freshwater species [6– 10]; in fact, it is unusual to find a species whose growth is not affected by salinity [11,12]. Salinity appears to modify a number of aspects related to growth performance including (a) standard metabolic rate [1,5,13], (b) total food intake [2,9,11,14,15], (c) the feed conversion efficiency [2,6,8,15,16], and (d) the balance of hormones involved in metabolism [17 – 21]. * Corresponding author. Tel.: +34 968 364 931; fax: +34 968 363 963. E-mail address: [email protected] (V.C. Rubio). 0031-9384/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2005.04.022

Nevertheless, depending on the species, developmental state, season, and acclimation period, some discrepancies have been found as regards the effects of salinity on food intake. It is generally accepted that the highest food intake occurs at intermediate salinity levels [22,23], although numerous exceptions can be found. Thus, for example, increasing salinity increases food intake in grass carp [24], sea bass [14], and flounder [25], whereas it has very little effect, if any, on bluefish [11], spotted wolfish [26], turbot [2], or rainbow trout [27], or even diminishes food intake in other species such as common carp [28], Atlantic cod [15], gilthead seabream [29], rainbow trout [30], and black bream [23]. Previous studies using self-feeders have shown that some fish species like goldfish [31], rainbow trout [32], European sea bass [33], common carp [34], and sharpsnout seabream [35] are able to select a balanced diet from unbalanced granulated diets with different macronutrient proportions (i.e., they can regulate their energy and macronutrient intake to put together a complete diet matching their specific nutritional habits; carnivorous or omnivorous). Recently, it

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V.C. Rubio et al. / Physiology & Behavior 85 (2005) 333 – 339

has been shown that fish can also select a complete diet from pure macronutrient sources packaged into gelatine capsules [36,37]—a method that prevents chemosensory properties at oropharyngeal level from interfering with macronutrient selection. However, despite the number of reports devoted to the influence of salinity on fish food intake, its possible effects on macronutrient selection have not been investigated. European sea bass (Dicentrarchus labrax L.) is a euryhaline and eurytherm fish that can live in different osmotic media, including the sea, estuaries, coastal lagoons, and rivers, which makes this species a good experimental model to study the influence of salinity on fish physiology. It appears that sea bass can endure a very wide range of salinities, being able to tolerate direct transfer from 37° to 3.9° salinity, and also levels as low as 0.5° if salinity is gradually reduced over 24 h [38]. The aim of this work was to study the influence of three salinity levels (25°, 7°, and 0°) on European sea bass food intake regulation and encapsulated macronutrient selection.

2. Materials and methods 2.1. Animal housing A total of 40 European sea bass (D. labrax) with initial and final body weights of 52.4 T 7.1 g and 78.0 T 14.2 g (mean T S.D.), respectively, were distributed among eight 75-l tanks (five fish per tank) with a flow-through and water turnover of 200% per hour. Fish were kept at 23 T 0.5 -C, under 12:12 LD (350 lx: total darkness, lights onset at 08:00 h). The tanks were placed in isolated chambers, and each one of them fitted with a closed water circuit that recycled artificial seawater through biological and mechanical filters. Marine water was prepared by adding synthetic sea salt (SERA Premium Sea Salt, Heinsberg, Germany) to aerated tap water until the appropriate density was obtained. Three salinity levels used were: 25°, 7°, and 0°, achieved by gradually replacing filtered seawater with freshwater in order to reach the target salinity in 72 h, which represents the period of acclimatization for each salinity. Water salinity levels were recorded using a manual refractometer (Leica Microsystems, Inc., Buffalo, USA) and adjusted on a daily basis. Sea bass were allowed to acclimatize to laboratory conditions for several weeks. During this period, fish were fed ad libitum once a day using a complete diet (CD) containing pure macronutrients (protein (P), carbohydrate (CH), and fat (F)) mixed inside gelatine capsules (Table 1). 2.2. Diet manufacture The encapsulated feed was prepared by filling gelatine capsules (no. 4, 0.2 ml; Roig Farma, SA, Barcelona,

Table 1 Ingredients and proximate analysis calculated from the assay of the macronutrients of the four kinds of capsules, expressed as mean (n = 4) CD Ingredients of capsule content Casein Gelatin Cod liver oil Soya oil Dextrin Vitamin and mineral mix CaCO3/CaPO4

P

(g/kg dry weight) 430 783 90 157 158 – 52 – 210 – 20 20 40 40

Proximate analysis of the capsules (% dry matter) Dry matter 92.6 90.2 Gross protein 57.3 90.8 Ether extract 16.7 0.6 NFE 22.0 3.3 Ash 4.0 5.3 Gross energy (kJ/g)a 23.7 22.2

F

CH

– – 705 235 – 20 40

– – – – 940 20 40

98.8 17.1 80.7 1.1 1.1 35.6

90.5 18.8 0.7 77.4 3.1 17.6

a

Calculated from the macronutrient percentage mean using the following energy coefficients: 23.6 kJ/g for P; 38.9 kJ/g for F; and 16.7 kJ/g for CH [60].

Spain) with powdered macronutrients using a semiautomatic encapsulator (Tecnyfarma, Miranda de Ebro, Spain). Depending on the experimental phase, adaptation, or macronutrient self-selection phase, fish were fed either a complete diet or pure macronutrient capsules, respectively. During the experimental period, a previously described [36,37] three-choice macronutrient paradigm was used. Briefly, fish had to self-select from three types of capsules, each one containing a single macronutrient source (P, CH, or F) supplemented with vitamin and mineral complexes, sodium alginate as binder, and cellulose as filler (Table 1). The F source used was a mixture of cod liver oil and soya oil (3:1). Protein was provided by vitamin-free casein and gelatine (5:1), and CH by dextrin. The capsules’ analytical compositions are given in Table 1. Proximate composition was determined using the following techniques: moisture, by drying the samples at 110 -C for 24 h to constant weight; proteins, by the Kjeldahl method (N  6.25); fats, by diethyl ether extraction; ash, by heating at 450 -C for 24 h; and nitrogen-free extract (NFE), by subtraction of protein + fats + ash. The capsules supplied to a given tank were stored in a single bag for at least 10 days before being used. This prevented the fish from distinguishing the capsules content based on the chemical properties of any potential external contamination. 2.3. Experimental design Once salinity level was adjusted at 25°, to evaluate the influence of salinity on total food intake, fish were self-fed gelatine capsules filled with a complete diet (52% P, 21% CH, and 21% F) for 20 days and switched to pure macronutrients for 35 days, with periods of acclimatization

V.C. Rubio et al. / Physiology & Behavior 85 (2005) 333 – 339

2.4. Data analysis Food intake is expressed as grams per 100 g BW per day. Results are presented as mean T S.E. values. Specific growth rate was calculated using the following formula: SGR (% BW/day) = 100(lnW f lnWi) / days, where W f and W i are the final and initial body weight (g), respectively. The feed conversion ratio was calculated as: FCE (%) = 100(W f Wi) / R, where R is total food intake (g). Mean daily food intake differences for the capsules filled with either a complete diet vs. pure macronutrient were statistically compared by an independent samples t test. The effects of salinity on total food intake and macronutrient selection were analyzed using a one-way analysis of variance (ANOVA), with salinity and macronutrient content as factors. Arcsine (¾) transformations of macronutrient intake percentages were performed to achieve homogeneity of variance. Prior to ANOVA, normality and homogeneity of variances were checked by Kolmogorov – Smirnov and F max tests, respectively. Significant main effects were further analysed using Tukey’s multiple range test to make pairwise comparisons between means. Correlations between macronutrient selection and SGR or FCE were analyzed using the Pearson correlation test, with the regression lines for those correlations that were found to be statistically significant being calculated. A probability level of P < 0.05 was used in order to consider the observed effects as statistically significant. All statistics were conducted with tank mean as observation.

3. Results

(1.52 T 0.10 vs. 1.54 T 0.08 g/100 g BW/day, P = 0.13). In contrast and irrespective of the salinity level, statistically significant differences were found between the numbers of capsules of each macronutrient (P: 3.54 T 0.21; CH: 2.78 T 0.29; and F: 1.79 T 0.14 capsules/100 g BW/day) ingested by the animals [ F(2,15) = 15.25, P < 0.001]. Total food intake significantly dropped when salinity was reduced from 25° (1.52 T 0.08) to 7° (1.12 T 0.03), and then again to 0° (0.89 T 0.06 g/100 g BW/day) [ F(2,23) = 19.41, P < 0.001], although this last difference was not statistically significant (Fig. 1). Macronutrient intake was also affected by salinity. As shown in Fig. 2a, the total intake of all three macronutrients decreased along with salinity, with the differences between the initial (25°) and final (0°) salinity levels being statistically significant for all macronutrients. However, if the results are expressed as macronutrient intake percentages, CH intake decreased and P intake increased in response to decreasing salinity (Fig. 2b), whereas the percentage of F remained unaltered at an average value of 22%. Growth performance (Fig. 3) and feeding efficiency showed a tendency to decrease at 0° compared to 25° salinity (SGR was reduced by 68% and FCR by 58%); however, these effects were not statistically significant [ F(2,23) = 1.652, P = 0.216 and F(2,23) = 0.993, P = 387, respectively]. SGR and FCE depended on the macronutrient selection pattern, which was in turn modified by salinity. Fig. 4 shows the relationship between the different macronutrient intake percentages vs. FCE at all three salinity levels. A significant positive correlation between P selection and SGR [ y = 0.02x 0.85, R 2 = 0.51 at 0°; y = 0.03x 1.35, R 2 = 0.55 at 7°] or FCE (Fig. 4a) [ y = 1.85x 80.56, R 2 = 0.59 at 0°; y = 2.90x 117.92, R 2 = 0.72 at 7°] was found at 0° and 7° salinity levels, respectively. In contrast, a negative correlation that was also statistically significant was observed between SGR [ y = 0.03x + 0.75, R 2 = 0.64 at 0°; y = 0.04x + 1.06, R 2 = 0.67 at 25°] or FCE (Fig. 4b) [ y = 3.04x + 70.50, R 2 = 0.64 at 0°; y = 3.12x + 86.26,

1.8

Intake (g/100g BW/day)

of 72 h. For the effect of salinity on the macronutrient selection pattern, fish were fed color-coded capsules containing P, F, or CH for 114 days divided in 35-day periods for each salinity level and 3 days of acclimatization for each salinity change. The same color – macronutrient relationship was maintained in a given tank throughout the study, but this relationship was balanced between tanks to prevent a possible color effect. Each fish was provided an equal number of capsules of each macronutrient, with the quantity offered being always greater than what the fish could eat (30 –60 capsules/ macronutrient) to allow ad libitum intake of each macronutrient (i.e., one meal in excess per day). After 30 min (from 12:00 to 12:30 h), uneaten capsules were removed and counted to determine each macronutrient real intake. Throughout the experimental period, each fish was weighed at the beginning and at the end of each experimental phase.

a

1.5

b

1.2

b

0.9 0.6 0.3 0.0 25

Sea bass was able to regulate food intake regardless of the capsules composition, since no differences were observed in total food intake when capsules were filled with either a complete diet or pure macronutrients

335

7

0

Salinity (‰) Fig. 1. Effect of decreasing salinity levels on total food intake. Values represent the mean T S.E.M. of eight fish groups. Different superscripts denote statistically significant differences: P < 0.05 (ANOVA).

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V.C. Rubio et al. / Physiology & Behavior 85 (2005) 333 – 339

(a)

(a)

40

a a

ab

25‰

0.6

7‰

b

0.5

20

0‰

0.4

a

b b

0.3

ab

b

y = 1.85x - 80.56 R2 = 0.59

0 35

40

45

50

55

60

35

40

-10

0.1

-20

P

CH

Protein Intake (%)

F

Macronutrient

25%°

(b)

(b)

7%°

0%°

40 b

60

b

25‰ a

30

7‰

a

40

ab

b

30 20

y = -3.12x + 86.26 R2 = 0.77

20

0‰

FCE (%)

50

Intake (%)

10

0.2

0.0

10 0 15

10 -10

0 P

CH

F

R 2 = 0.77 at 25°] and the percentage of F selected by the animals at 0° and 25° salinity, respectively.

4. Discussion Pure macronutrient encapsulation has proved to be a valuable tool for macronutrient selection studies. This 0.6 0.5 0.4 0.3 0.2 0.1 0.0 7

25

30

Fat Intake (%)

Fig. 2. Effect of decreasing salinity levels on the macronutrient selection pattern expressed as: (a) macronutrient intake (in grams) and (b) macronutrient intake percentage. Values represent the mean T S.E.M. of eight fish groups. Different superscripts denote statistically significant differences inside each macronutrient group in macronutrient intake regarding the 25° phase: P < 0.05 (ANOVA).

25

20

y = -3.04x + 70.50 R2 = 0.64

-20

Macronutrient

SGR (% /day)

y = 2.90x - 117.92 R2 = 0.72

30

FCE (%)

Intake (g/100g BW/day)

0.7

0

Salinity level (‰) Fig. 3. Influence of decreasing salinity levels on specific growth rate (SGR). Values represent the mean T S.E.M. of eight fish groups. No significant differences were found.

25%°

7%°

0%°

Fig. 4. Relationship between protein (a) or fat selection (b) and FCE at different salinity levels. Values represent the mean of eight fish groups. Solid regression lines are displayed for those correlations that were found to be statistically significant. Dotted line represents a not statistically significant general trend.

technique allows sea bass to select a complete diet, in line with its carnivorous habits, from separate pure macronutrients sources contained inside colored gelatine capsules. One advantage of this method is that of preventing interference from the chemosensory properties inherent to granulated diets. In the present study, sea bass total daily food intake was the same regardless of the capsule content (complete diet or separate pure macronutrients); however, the numbers of capsules consumed of each macronutrient were statistically different irrespective of the salinity level, indicating that sea bass is able to develop an associative learning between the color and macronutrient content of the capsules through postingestive processes and/or postabsorptive mechanisms. Similar results have been previously described for this specie [36,37]. Total food intake progressively decreased when salinity was lowered from 25° to 7° and then to 0°, with the drop between the first two salinity levels being statistically significant. Previous studies [14] have reported a food intake increase with salinity levels over 28°; however, no studies have been published regarding the effects of salinity on fish fed dissociated macronutrient diets. Our results indicate that salinity has a differential effect on the macro-

V.C. Rubio et al. / Physiology & Behavior 85 (2005) 333 – 339

nutrient selection pattern, with the percentage of P selection increasing and that of CH decreasing with decreasing salinity. Although the reason why salinity modifies dietary selection is not known, a number of tentative explanations can be advanced: (a) The percentage of P would increase to compensate protein intake reduction, which is supported by some evidence indicating that, within certain limits, fish tend to maintain a stable protein intake [39,40]. CH reduction would be a passive consequence of the P increase. (b) Since encapsulated macronutrient intake is regulated through postingestive mechanisms, an alteration of the macronutrient selection pattern could be due to changes at the digestive, metabolic, and/or neurohumoral levels. To this effect, a number of studies have reported alterations of digestive enzymes in response to salinity changes [5,41,42], which could in turn alter the digestibility [43,44] and, consequently, metabolic availability of specific macronutrients. Salinity also affects the transport of nutrients [45], which might selectively affect the availability of some macronutrients, and thus the fish appetite for them, although this fact has not been described. Salinity-induced changes of key metabolic enzymes have also been reported, particularly for enzymes involved in CH metabolism [5,46 –48]. In addition, the use of 2-deoxy-dglucose and 2-mercaptoacetate in mammals has been reported to modify macronutrient selection, producing glucoprivation and lipoprivation, respectively [49 – 52]. Finally, macronutrient intake is modulated by several hormones and/or neurotransmitters acting at brain areas involved in feeding behavior regulation; consequently, modifications in any of these factors could alter the macronutrient selection pattern. In this sense, it has been described that salinity changes modify cortisol and GH levels in seawater fish, and also prolactin in freshwater fish [17,53 –55]. Furthermore, it is known that these hormones are involved in the regulation of macronutrient metabolism in fish [56 –59]. Considered overall and in agreement with our results, neither FCE nor SGR values are significantly affected by salinity. Yet, regression analysis between macronutrient selection and these two parameters provides some interesting results. Irrespective of salinity, FCE and SGR increase when protein selection increases and fat decreases. Although there appears to be no statistically significant relationship between FCE and protein intake at 25°, nor between FCE and fat intake at 7°, a general trend for FCE to increase along with protein intake was observed. Nevertheless, protein intake is highly preserved independently of the FCE, with values between 38% and 42% of selection at 25°. Probably, this lack of statistical significance at 25° could be attributed to the great heterogeneity in the protein

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selection between groups. When the salinity diminishes, animals probably try to compensate the physiologic changes by reduction, modification of the composition of the selected diet, and disappearance of the highly preserved protein intake. Another aspect to be considered is the fact that fish self-select a complete diet from independently encapsulated macronutrients; therefore, each fish selects a different diet, according to its nutritional preferences or physiological requirements. This variability in the selfselected diet could be a consequence of the metabolic changes produced by the diminution of the salinity, which induce a high variability in SGR and FCE. These results would appear to contradict the current tendency of increasing the lipid content of carnivorous fish diets in order to reduce the costs of using protein, presumably without any effect on feed efficiency. Yet, it must be pointed out that allowing fish to choose their diet is very different from forcing them to consume a diet with fixed proportions of previously established macronutrients. In summary, the present study reveals the important influence of salinity on food intake and macronutrient selection in sea bass. However, given that this species can be cultivated under different salinity levels and even in fresh water, further studies are required to determine the optimal composition of the diet depending on the salinity.

Acknowledgements This research was supported by grants from the ‘‘Ministerio de Ciencia y Tecnologı´a (MCYT)’’ project AGL2001-0698 to J.A. Madrid, EU (Concerted Action Q5CA-2001-989 to F.J. Sa´nchez-Va´zquez) and by the ‘‘Consejerı´a de Trabajo y Polı´tica Social, Fondo Social Europeo’’ and ‘‘Fundacio´n Se´neca’’ (Integral Operative Program for CARM 2000– 2006 of FSE to V.C. Rubio). The authors wish to thank M. Carmen Marı´n Jime´nez and other staff of CULMAREX, SA, for supplying the sea bass and for their kind support and assistance during the experiment.

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