Fish Physiol Biochem DOI 10.1007/s10695-014-9928-5

Blood chemistry profile as indicator of nutritional status in European seabass (Dicentrarchus labrax) Helena Peres • Sara Santos • Aires Oliva-Teles

Received: 6 January 2014 / Accepted: 10 March 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract This study was carried out to establish biochemical parameters with potential diagnostic value to assess the nutritional status of healthy seabass. For that purpose, triplicate groups of seabass juveniles were submitted to different feeding protocols: fed for 14 days; fed for 7 days followed by 7 days of fasting or fasted for 14 days. At the end of the trial, body, liver and viscera were randomly sampled for proximate composition analysis. Blood was also collected and the following plasma parameters were analyzed by standard clinical methods: glucose; cholesterol; triglycerides; protein; inorganic phosphorus; calcium; magnesium; alkaline phosphatase (ALP); aspartate aminotransferase; lactate dehydrogenase; creatine phosphokinase and lipase. No major effect of feed deprivation on body composition, visceral index, perivisceral and hepatic lipid content were observed, whereas hepatosomatic index and hepatic glycogen were reduced. Previous feeding conditions strongly influenced the plasma parameters in seabass. Comparatively to the fed group, plasma glucose,

cholesterol and calcium levels were reduced after 2 weeks of fasting while plasma triglycerides, protein, inorganic phosphorus and ALP attained minimum levels after 1 week of fasting. Overall, enzymatic activity parameters showed higher variability than biochemistry parameters. In conclusion, during shortterm starvation (\14 days) hepatic energy depots were extensively mobilized while perivisceral and body lipids reserves were preserved. Among measured parameters, plasma protein, triglycerides, inorganic phosphorus and ALP seem to have potential as predicative diagnostic tools to assess the nutritional status of seabass and may be useful to monitor feeding practices in aquaculture. Further studies are, however, required to extend results of this study to other fish size classes. Keywords Blood  Plasma biochemistry  Plasma enzymes  Seabass  Starvation

Introduction H. Peres (&)  A. Oliva-Teles Centro de Investigac¸a˜o Marinha e Ambiental (CIIMAR/ CIMAR – Laborato´rio Associado), Universidade do Porto, Rua dos Bragas, 289, 4050 Porto, Portugal e-mail: [email protected] S. Santos  A. Oliva-Teles Departamento de Biologia, Faculdade de Cieˆncias, Universidade do Porto, Edifı´cio FC4 - Rua do Campo Alegre, 4169-007 Porto, Portugal

Optimum growth performance, immune competence and well-being of fish have long been recognized to be straightly conditioned by adequacy of feeds and feeding conditions (Maita 2007; Oliva-Teles 2012). Under intensive aquaculture, diagnostic of fish nutritional and health conditions is mainly based on morphological examination and disease and malnutrition status are only detected by evident visual signals

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or weight losses. For terrestrial animals, with the development of routine blood tests, evaluation of nutritional status became more practical, sophisticated and precise, allowing an earlier detection of impaired malnutrition and health. Indeed, for terrestrial animals, blood analysis is usually one of the routine evaluation tools, providing important diagnostic and prognostic information of pathological and metabolic disorders and of therapeutic approaches (Knox et al. 1998; Kerr 2008). In fish, although previous studies have shown that impair feeding, specific nutritional deficiencies, stress and disease induce changes in the blood constituents, the use of blood biochemistry as diagnostic tool is not usually done. This is due to the paucity of reliable information on physiological values of hematology and blood biochemistry parameters in well-nourished, healthy and stress-free fish. This issue is, however, very pertinent, as fish reared under intensive conditions are daily subjected to different stressors that compromise their nutritional and immune competence and an earlier and trustful diagnosis of such conditions would help to improve management practices. Up to now, clinical hematology and biochemical reference values were determined for only a few aquaculture fish species, including dojo loach; Amur sturgeon; skipjack, yellowfin and bluefin tuna (Svoboda et al. 2001; De Pedro et al. 2005; Coz-Rakovac et al. 2005; Knowles et al. 2006; Tavares-Dias and Moraes 2007; Zhou et al. 2009; Cao and Wang 2010; Di Marco et al. 2011). Available data on biochemistry parameters in fish were recently compiled by Peres et al. (2013), and data on hematology parameters were compiled by Hrubec and Smith (2010). However, for the majority of well-established aquaculture species, including seabass, such data are still missing. For clinical interpretation of blood data, besides the importance of the establishment of normal reference values, there is also a need to select the metabolites and/or enzymes that have specificity, sensitivity and predictive or diagnostic value for a specific situation (i.e., malnutrition, stress, infection/diseases) and for a specific species (Kerr 2008). Therefore, the present study aims to establish the most sensitivity plasma biochemical parameters to feed deprivation, i.e., the plasma biochemistry parameters with diagnostic potential to assess the nutritional condition of seabass under intensive aquaculture conditions.

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Materials and methods The experiment was conducted at the Marine Zoological Station, University of Porto, Portugal, according to the European Economic Community animal experimentation guidelines (Directive of 24 November 1986; 86/609/EEC) by accredited researchers (following FELASA category C recommendations). Immature 0? seabass obtained from a local commercial fish farm (Timar, Algarve, Portugal) were used in this study. Experimental facilities consisted of a thermoregulated recirculation water system equipped with 18 cylindrical fiberglass tanks of 300-l capacity, supplied with continuous flow of filtered seawater at 3–3.5 l min-1. During the trial, a natural photoperiod was adopted, water temperature was maintained at 25 ± 0.5 °C, salinity averaged 35 ± 1 %, oxygen averaged 7–7.5 mg l-1 and ammonia and nitrite levels were kept around zero mg l-1. Before the beginning of the trial, fish were acclimatized for 2 weeks to the experimental conditions. During this period, fish were controlled for external disease signals, skin and fins lesions, feeding, territorial and aggression behavior, frantic swimming or increased opercular movements, to confirm health and welfare conditions. Then, fish were unfed for 24 h and thereafter 9 homogeneous groups of 18 seabass (average body weight of 65 g; weight range 60–70 g) were established and randomly distributed for each tank. Triplicate groups of fish were then fed by hand, twice a day, to apparent satiety, for 14 days (group A); for 7 days followed by 7 days of starvation (group B); starved for 14 days (group C). During both acclimatization and experimental period, fish were fed a fishmeal-based diet (Table 1). At the end of the trial, 24 h after the last meal (for group A), 3 fish per tank (9 fish per treatment) were randomly sampled for blood collection, body, liver and viscera composition. In order to reduce stress induced by handling, the 3 fish sampled in each tank were quickly removed from the water and blood immediately collected from the caudal vein by function with a heparinized syringe, without prior anesthesia, according to the protocol proposed by Marino et al. (2001) for seabass. Time elapsing from capture to blood collection was \3 min, and all blood samples were collected between 10 and 12 a.m. in order to reduce the potential influence of circadian rhythms on

Fish Physiol Biochem Table 1 Composition and proximate analyses of the experimental diet Ingredients (% dry weight) Fish meala

66.6

Dextrin

21.1

Cod liver oil Vitamin premix

8.8 b

1.0

Choline chloride (50 %)

0.5

Mineral premixc

1.0

Binderd

1.0

Proximate analyses (% dry weight) Dry matter

91.1

Crude protein Crude fat

50.8 15.7

Ash

13.7

Gross energy (kJ g-1 DM)

21.8

aspartate aminotransferase (AST); lactate dehydrogenase (LDH); creatine phosphokinase (CPK); and lipase, alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT) and creatinine. Chemical analyses of the experimental diet, fish and viscera were done according to Peres et al. (2011). Data on plasma parameters was analyzed by oneway analyses of variance (ANOVA), after testing the homogeneity of variances with the Levene test. Significant differences between group A (starved for 24 h) against each treatment (group B; starved for 1 week and C starved for 2 weeks) were determined by the Dunnett test. All statistical analysis were done using the SPSS 21 software package for Windows.

Results

a

Steam dried LT fish meal, Pesquera Diamante, Spain (CP 72.1 % DM; GL 9.3 % DM)

b

Vitamins (mg kg-1 diet): retinol, 18,000 (IU kg-1 diet); calciferol, 2,000 (IU kg-1 diet); alpha tocopherol, 35; menadion sodium bis., 10; thiamin, 15; riboflavin, 25; Ca pantothenate, 50; nicotinic acid, 200; pyridoxine, 5; folic acid, 10; cyanocobalamin, 0.02; biotin, 1.5; ascorbyl monophosphate, 50; inositol, 400

c

Minerals (mg kg-1 diet): cobalt sulfate, 1.91; copper sulfate, 19.6; iron sulfate, 200; sodium fluoride, 2.21; potassium iodide, 0.78; magnesium oxide, 830; manganese oxide, 26; sodium selenite, 0.66; zinc oxide, 37.5; potassium chloride, 1.15 (g kg-1 diet); sodium chloride, 0.40 (g kg-1 diet); dibasic calcium phosphate, 5.9 (g kg-1 diet)

d

Aquacube, Agil, England

the analyzed parameters. Blood was then centrifuged at 3.0009g for 5 min, and two plasma aliquots from each fish were obtained and frozen at -80 °C until analysis. Total weigh of these fish, liver and viscera were record and then were frozen at -80 °C for subsequent composition analysis. Plasma analyses were performed within 48 h after sampling, in a certified Veterinary Clinical Laboratory (NP EN ISO 9001-2000 by Bureau Veritas), using standard clinical methods in an auto-analyzer (Architect ci8200; Abbot Diagnostics, Canada), stringently monitored with appropriate quality control procedures to insure accuracy and reproducibility. The following parameters were selected, based on its potential clinical relevance, reproducibility and cost-effectiveness to diagnose the nutritional status of the fish: glucose; protein; triglycerides; cholesterol; calcium; magnesium; inorganic phosphorus; alkaline phosphatase (ALP);

No mortality or signs of disease or stress were observed during the experiment including the acclimatization period. Due to the imposed feeding protocol, growth performance, feed intake, nitrogen and energy retention decreased with the duration of starvation (Table 2). Eviscerated body composition, perivisceral and liver lipid content were not affected by duration of starvation. Contrarily, hepatosomatic index and liver glycogen content were significantly reduced by starvation. Mean values, range and coefficient of variation of the measured plasma biochemical parameters of seabass kept under the different feeding protocols are presented in Table 3. Alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT) and creatinine values were below the limit of detection for the majority of the fish analyzed and therefore are not reported in the table. A general tendency for a reduction in the measured plasma parameters after 1 week of starvation was observed for some parameters but it was statistically significant only for triglycerides, protein, phosphorus and ALP. Thus, after 1 week of starvation, plasma triglycerides levels were reduced from 405 to 241 mg dl-1; plasma total protein was reduced from 4.9 to 4.2 g dl-1 and inorganic phosphorus was reduced from 8.4 to 7.0 mg dl-1. From the first to the second week of starvation, a trend for further reduction in these parameters was also noticed but it was not statistically significant. Comparatively to the fed group, after 2 weeks of starvation, there was a significant reduction in plasma

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Fish Physiol Biochem Table 2 Growth, feed utilization and body composition of seabass kept under the different feeding protocols Groupsa

Initial

A

B

C

SEM

Initial body weight (g)



65.0

65.0

65.0

0.02

Final body weight (g)



84.1

70.3*

60.8*

3.40

Daily growth indexb Feed intake (g fish-1)

– –

2.6 20.0

-0.63* 0.0

0.47 3.04

N retention (g kg-1 day-1)



E retention (kJ kg-1 day-1)



0.74* 9.46

0.90

0.45*

0.15*

149.4

21.6*

-55.0*

0.11 30.3

Eviscerated body composition (% wet weight) Dry matter

27.7

31.5

30.7

30.9

0.41

Protein

16.9

20.0

19.4

19.4

0.24

Lipids

8.8

7.5

6.9

7.3

0.15

Energy (kJ g-1)

7.3

7.5

7.1

7.0

0.20

Ash

4.8

5.3

5.5

5.7

0.10

HSIc



1.5

1.0*

0.6*

0.14

Liver glycogen



0.13

0.05*

0.005*

0.02

Liver lipid



0.14

0.16

0.10

0.01

VId



8.8

7.3

7.0

0.35

Perivisceral lipids



5.3

3.8

4.0

0.32

Visceral composition (% body weight)

Asterisks denotes significant difference between group A against group B or C (Dunnett test; P \ 0.05; n = 3) a

Group A: seabass unfed for 24 h; B: seabass unfed for 1 week; C: seabass unfed for 2 weeks

b

DGI: (final body weight1/3 - initial body weight1/3)/(time in days)) 9 100

c

Hepatosomatic index: (liver weight/body weight) 9 100

d

Visceral index: (viscera weight/body weight) 9 100

glucose levels (from 130 to 95 mg dl-1), cholesterol (from 272 to 214 mg dl-1) and calcium (from 16 to 13 mg dl-1). Magnesium, AST, LDH, CPK and lipase were not significantly affected by starvation, though a trend for reduction in mean values was also observed for most of these parameters, which was quite expressive for AST and LDH. The activity of these enzymes decreased, from a mean value of 84–26 U l-1 and from 127 to 65 U l-1, respectively, after 2 weeks of starvation. However, due to the high degree of variation among individuals, these differences were proved to be not statistically significant.

Discussion Prompt clinical diagnostic of malnutrition due to inadequacy of feed and feeding practices, stress factors or disease is of major importance for

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aquaculture. Blood analysis is a practical, fast and cost-effective procedure for evaluation of such situations with good diagnostic potential, as selected blood parameters provide an overall internal picture of the physiological process and health of the animal. Changes in the adequacy of feed, feeding practices, water quality and the presence of acute or chronic stressors and diseases have been reported to be reflected on plasma biochemistry profile (Maita 2007; Peres et al. 2013). Although not yet widely used in aquaculture, it is to expect that, similar to what happens in land animals, blood analysis will became a useful tool to assess the nutritional, well-being and health status of fish as long as data accumulate for the different species. Also, limiting the application of blood analysis to fish production is the lack of information on the diagnostic potential of the different plasma constituents, as well as the inexistence of reference biochemistry values for the main aquaculture species, including European seabass.

Fish Physiol Biochem Table 3 Plasma biochemistry values for seabass kept under different feeding protocols Groupa

A

B

Mean

Range

CV

C

Mean

Range

CV

Mean

Range

CV

Glucose (mg dl-1)

129.7

87–195

27.6

123.4

80–170

24.1

94.6*

60–122

21.6

Cholesterol (mg dl-1)

272.4

231–361

19.8

233.4

176–363

23.9

214.4*

176–248

11.4

Triglycerides (mg dl-1)

26.4

405.5

235–963

60.3

241.4*

177–375

23.3

189.5*

127–253

Protein (g dl-1)

4.9

3.9–6.2

14.6

4.2*

3.2–5.0

16.2

3.6*

31–4.1

9.5

Inorganic phosphorus (mg dl-1)

8.4

6.7–10.3

13.8

7.0*

5.9–8.0

10.7

6.3*

4.9–8.9

17.9

15.9

12.5–20.9

13.8

10.6–16.8

16.3

12.6*

10.4–15.1

14.5

2.1–5.4

27.1

3.41

2.9–4.2

12.8

3.12

1.7–4.2

22.4

36–74

23.4

37.0*

26–46

19.3

34.0*

26–44

16.6

Calcium (mg dl-1) Magnesium (mg dl-1) ALP (U l-1) -1

3.71 51.1

13.9

AST (U l )

83.6

19–257

108.2

41.8

12–119

91.6

26.0

14–44

36.3

LDH (U l-1)

126.6

31–282

69.2

57.3

24–156

80.3

65.2

30–99

37.6

CPK (U l-1)

689.3

123–1,566

78.8

430.3

84–1,113

84.3

645.3

101–1,229

70.7

Lipase (U l-1)

116.8

38–168

108.7

86.0

4–146

154.0

114.2

90–159

79.1

Asterisks denotes significant difference between group A against group B or C (Dunnett test; P \ 0.05; n = 9) ALP alkaline phosphatase, AST aspartate aminotransferase, LDH lactate dehydrogenase, CPK creatine phosphokinase CV: coefficient of variation: (standard deviation/mean) 9 100 a

Group A: seabass unfed for 24 h; B: seabass unfed for 1 week; C: seabass unfed for 2 weeks

Although starvation induced a significant reduction on growth performance and nutrient retention, body composition and perivisceral lipid content were not affected. On the contrary, liver weight was greatly reduced due to an intense mobilization of glycogen. These results suggest that during short-term starvation, energy needs are met essentially by ready mobilization of liver energy reserves while perivisceral and body lipids are preserved. Thus, hepatic energy reserves provide a good indication of the overall nutritional condition status of seabass. Similar results were previously described in this species (Peres and Oliva-Teles 2005; Perez-Jimenez et al. 2007) and in sea bream (Peres et al. 2011). Previous feeding conditions also lead to significant changes in some plasma biochemistry parameters, which may become useful indicators of the nutritional status of seabass. Basal blood glucose levels are known to differ considerably among fish species (Polakof et al. 2012). In this study, glycaemia levels 24 h after feeding averaged 130 mg dl-1, which is within previously reported values for seabass (96–191 mg dl-1; Peres et al. 1999; Coz-Rakovac et al. 2005; Maricchiolo et al. 2008; Roque et al. 2010; Chatzifotis et al. 2011; Enes et al. 2011), and decreased circa 4 and 27 %, respectively, during the

first and second weeks of fasting. This confirms the capacity of seabass to maintain basal blood glucose levels during short-term starvation periods as previously reported (Perez-Jimenez et al. 2007; Caruso et al. 2011; Chatzifotis et al. 2011). It also confirms that, in seabass, liver has a pivotal role in the maintenance of glucose homeostasis. Indeed, hepatic glycogen was reduced by 50 and 100 %, at the end of the first and second week of starvation, respectively, whereas hepatic and perivisceral lipid contents were nearly unchanged. Contrary to seabass, gilthead seabream does not seem to regulate as well blood glucose levels during short starvation periods. Indeed, in a study identical to the present one, seabream blood glucose decreased by 24 and 54 % during the first and second week of starvation (Peres et al. 2013). The relative constancy of plasma glucose levels in seabass, even during long periods of fasting, reduced the potential diagnostic value of this parameter to assess nutritional status. Plasma protein levels are often associated with fish nutritional and physiological status (Rehulka et al. 2005; Maita 2007). Basal plasma protein values averaged 4.9 g dl-1, which is comparable with basal values found for other fish species (De Pedro et al. 2005; Knowles et al. 2006; Tavares-Dias and Moraes

123

Fish Physiol Biochem

2007; Di Marco et al. 2011; Coeurdacier et al. 2011; Maricchiolo et al. 2008; Peres et al. 2013). Starvation induced a significant reduction in plasma protein levels, similarly to what was also observed in gilthead seabream (Peres et al. 2013). As plasma protein level is usually very stable in well-nourished animals but decreases under fasting conditions, it seems to have good potential for nutritional status prediction of fish (Coeurdacier et al. 2011). Indeed, under malnutrition or stress conditions, altered plasma total protein levels often occur as consequence of amino acid oxidation or peripheral proteolysis (Mommsen et al. 1999; Di Marco et al. 2008). Basal plasma triglycerides and cholesterol levels averaged 406 and 272 mg dl-1, respectively, values which are in agreement with previously reported values for this species (Echevarria et al. 1997; Peres et al. 1999; Perez-Jimenez et al. 2007; Chatzifotis et al. 2011), but higher than those reported by Roncarati et al. (2006). Starvation induced a significant reduction in both metabolites, with triglycerides being reduced faster and more exhaustively than cholesterol. Triglycerides levels were reduced by 41 and 14 % and cholesterol by 21 and 8 %, respectively, during the first and the second week of starvation. Also in seabass, Perez-Jimenez et al. (2007) observed an analogous mobilization pattern of these metabolites during fasting. The high reduction in plasma triglycerides levels during starvation is probably due to its use as energetic substrate and to the reduction in lipogenesis (Perez-Jimenez et al. 2007), which may have contributed for the maintenance of plasma glucose levels during the first days of fasting. The extensive mobilization of plasma triglycerides during fasting gives it some potential as a marker of nutritional condition in seabass. On the contrary, in seabream maintained under the same experimental protocol, plasma triglycerides mobilization during fasting was less exhaustive while the opposite was true for plasma cholesterol (Peres et al. 2013). Further, in seabass, plasma triglycerides levels were higher than cholesterol levels, while the inverse was true for seabream. Thus, differences between the two species regarding plasma metabolites mobilization during fasting may be related to differences in lipid metabolism between species. In an earlier study, Peres et al. (1999) also observed different metabolic strategies between these two species following an intramuscular load of glucose. While in seabream, it was observed a

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shift in metabolism toward an enhanced catabolism of stored body reserves; in seabass, an enhancement of anabolism was observed. Further studies are therefore required to elucidate the physiological basis for the differences between the two species and the potential value of these blood parameters as predictors of nutritional status. Although fish may absorb some inorganic nutrients from the aquatic environment, starvation induced a significant reduction in plasma phosphorus and calcium levels, confirming the essentiality of these mineral in the diet (Lall 2002). Starvation and malnutrition also induce severe drops of blood calcium and phosphorus level in different species (Peres et al. 2013). However, calcium to phosphorus ratio in plasma was conserved throughout the starvation period at around 2:1, which is consistent with earlier observations in this species (Roque et al. 2010). Ca/P ratio was, however, considerably higher in seabass than in seabream (1.4:1; Peres et al. 2013) or rainbow trout (1:1; Hemin and Paleczny 1987; Lee et al. 2010). Plasma calcium and phosphorus have been identified as good indicators of secondary phase of stress response in fish, being used as indirect indicators of altered plasma cortisol levels. Also, stress (Rehulka and Minarı´k 2007; Dobsikova et al. 2009; Roque et al. 2010) and pathological situations (Ziskowski et al. 2008) may alter the plasma levels of these minerals. On the contrary, plasma magnesium levels were kept constant during starvation, as previously reported for seabream (Peres et al. 2013), confirming the low diagnostic value of this parameter for evaluation of the nutritional status. Maintenance of plasma magnesium levels are probably related to its uptake from the water, as previously reported for some marine species (Ye et al. 2010). Plasma enzyme activity may also provide important information on the functional state of different organs, as intracellular and plasma enzymatic concentrations are usually proportional. An overall decline of plasma enzymes activities during fasting was previously reported for several species (McCue 2010; Peres et al. 2013). In this study, plasma non-specific enzyme activity was responsive to 1 week of fasting being reduced circa 50 % (AST and LDH) and 30 % (ALP, CPK and lipase). This level of activity was maintained during the second week of starvation, except for AST which activity was further reduced to circa 70 % of the

Fish Physiol Biochem

basal value. However, there was a very high variation in the activity measured in the different fish and consequently it precluded the detection of significant differences among groups, except for ALP activity which presented a CV much lower than for the other enzymes activity. This suggests that this enzyme may have potential for nutritional status prediction. The stabilization of enzymatic activity levels during the second week of starvation (except for AST) is in accordance with the overall pattern of plasma nutrients mobilization, which was more pronounced during the first week than during the second week of starvation. Besides being responsive to starvation or malnutrition, non-specific serum enzymes activity is also affected by disease, chemical exposure to toxics or stress conditions. Alterations of AST and ALT activities have be associated with liver damage, induced by pathological or stress situations (Lemarie et al. 1991; De La Torre et al. 2000; Tahmasebi-Kohyani et al. 2012) as well as with feeding nutrients with hepatic protective effects (Panigrahi et al. 2010; Harikrishnan et al. 2011). Increased activity of LHD, the enzyme responsible to conversion of lactate to pyruvate, may indicate hypoxia conditions, limiting water temperature or toxins (Perez-Jimenez et al. 2013; Harikrishnan et al. 2011). CPK, which is particularly active in heart and skeletal muscle, may indicate damage to these tissues. Pollutants and stress conditions were reported to increase CPK activity, probably due to muscle injury (Almeida et al. 2002; Rehulka and Minarı´k 2007). In conclusion, during short-term starvation periods (\14 days), growth and nutrient retention in juveniles seabass were reduced and hepatic glycogen depots were extensively mobilized, while perivisceral and body lipids reserves were preserved. Previous feeding condition also strongly influenced some of the measured plasma parameters in seabass juveniles. Among measured parameters, plasma protein, triglycerides, inorganic phosphorus and ALP activity seem to have potential as predicative diagnostic tools for evaluation seabass nutritional status. Further studies are, however, required to confirm and extend these results to fish submitted to other fish size classes. Acknowledgments This work was partially funded by the Project AQUAIMPROV (reference NORTE-07-0124-FEDER000038), co-financed by the North Portugal Regional Operational Programme (ON.2—O Novo Norte), under the

National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF).

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Ziskowski J, Mercaldo-Allen R, Pereira JJ, Kuropat C, Goldberg R (2008) The effects of fin rot disease and sampling method on blood chemistry and hematocrit measurements of winter flounder, Pseudopleuronectes americanus from New Haven Harbor (1987–1990). Mar Pollut Bull 56:740–750

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Blood chemistry profile as indicator of nutritional status in European seabass (Dicentrarchus labrax).

This study was carried out to establish biochemical parameters with potential diagnostic value to assess the nutritional status of healthy seabass. Fo...
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