sm78n2-3965.html

Yearly growth and metabolic changes in earthen pond-cultured meagre Argyrosomus regius

Luis Vargas-Chacoff¹, Ignacio Ruiz-Jarabo², Inês Páscoa², Odete Gonçalves²,
Juan Miguel Mancera²

¹Instituto de Ciencias Marinas y Limnológicas, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile. E-mail: luis.vargas@uach.cl
²Departamento de Biología, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, Puerto Real 11510, Cádiz, Spain.

Summary: Metabolic modifications associated with natural environmental conditions were assessed in the meagre Argyrosomus regius cultured in earthen ponds under natural photoperiod and temperature. Juvenile specimens (90-100 g initial weight) were sampled (plasma, liver and muscle) every two months for 18 months (between December 2004 and May 2006). Specimens showed seasonal variations in growth rate, with the highest values in spring and summer. Plasmatic, hepatic and muscular metabolite levels and hepatic and muscular metabolic enzymes also showed significant variations throughout the year. Enzymatic activity related to carbohydrate metabolism in the liver (HK, FBPase and G6PDH) showed great modifications in summer, increasing glycogenogenic pathways, while amino acid metabolism (GDH and GOT activity) was enhanced in spring and summer. However lipid-related (G3PDH activity) metabolic enzymes did not show a clear seasonal pattern. In muscle, enzymatic activity related to amino acid, lipid and lactate metabolism (LDH-O activity), but not carbohydrate metabolism, showed seasonal changes in parallel with changes in growth rate. Thus A. regius specimens showed a trend to grow in summer months and mobilize their energy reserves in winter. Differences in the hepatic level were observed between the first and the second year of the study, suggesting the possible existence of metabolic changes related to specimen age or size. Our results indicate that growth and metabolic responses in A. regius are environmentally dependent and that this species is a very good candidate for diversification in aquaculture.

Keywords: Argyrosomus regius; energy; growth; metabolic parameters; ponds; seasons.

Crecimiento y cambios metabólicos anuales en corvina Argyrosomus regius cultivada en esteros

Resumen: Modificaciones metabólicas asociadas a condiciones ambientales temporales fueron evaluadas en la corvina Argyrosomus regius, cultivadas en esteros con fotoperiodo y temperatura natural. Ejemplares juveniles (90-100 g de peso inicial) fueron muestreados (plasma, hígado y músculo) cada dos meses durante 18 meses (entre diciembre de 2004 y mayo de 2006). Las muestras mostraron variaciones estacionales en la tasa de crecimiento, con valores más altos durante la primavera y el verano. Niveles de metabolitos plasmáticos, hepáticos y musculares, así como las actividades de enzimas metabólicas hepáticas y musculares también presentaron variaciones significativas a lo largo del año. La actividad enzimática relacionada con el metabolismo de carbohidratos en el hígado (HK, FBPasa y G6PDH) mostró altas modificaciones en el verano, el aumento de las vías glucogenogénicas, mientras el metabolismo de aminoácidos (actividades de GDH y GOT) se incrementó en temporadas de primavera y verano. Sin embargo la actividad de G3PDH (enzima metabólica relacionada con los lípidos) no mostró un claro patrón estacional. En el músculo, la actividad enzimática respecto a los aminoácidos, lípidos y el metabolismo del lactato (LDH-O) presentó cambios estacionales en paralelo con los cambios en la tasa de crecimiento, pero el metabolismo de los carbohidratos no presentó cambios estacionales. Así especímenes de A. regius mostraron una tendencia a crecer en los meses de verano y movilizar sus reservas de energía en invierno. Se observaron diferencias en el nivel hepático entre el primer y el segundo año de estudio, lo que sugiere la posible existencia de cambios metabólicos relacionados con la edad o el tamaño del espécimen. Nuestros resultados indican que el crecimiento y las respuestas metabólicas en A. regius dependen del medio ambiente, pero también indican que esta especie es una muy buena candidata para la diversificación de la acuicultura.

Palabras clave: Argyrosomus regius; energía; crecimiento; parámetros metabólicos; esteros; estaciones del año.

Citation/Como citar este artículo: Vargas-Chacoff L., Ruiz-Jarabo I., Páscoa I., Gonçalves O., Mancera J.M. 2014. Yearly growth and metabolic changes in earthen pond-cultured meagre Argyrosomus regius. Sci. Mar. 78(2): 000-000. doi: http://dx.doi.org/10.3989/scimar.03965.06B

Editor: S. Zanuy.

Received: October 4, 2013. Accepted: April 1, 2014. Published: June 6, 2014.

Contents

Summary
Resumen
Introduction
Materials and methods
Results
Discussion
Acknowledgements
References

IntroductionTop

These earthen ponds are quite shallow (Arias 1976Arias A. 1976. Sobre la biología de la dorada, Sparus aurata L., de los esteros de la provincia de Cádiz. Invest. Pesq. 40: 201-222.) and large variations in abiotic variables (such as salinity, temperature and photoperiod) have a great influence on the physiology of cultured species. Seasonal changes in those abiotic factors affect and modulate the physiological responses, which force fish to vary osmoregulatory, metabolic and growth rates for efficient environmental adaptation (Boeuf and Payan 2001Boeuf G., Payan P. 2001. How should salinity influence fish growth? Comp. Biochem. Physiol. 130 C: 411-423., Vargas-Chacoff et al. 2009aVargas-Chacoff L., Arjona F.J., Ruiz-Jarabo I., et al. 2009a. Seasonal variation in osmoregulatory and metabolic parameters in earthen pond cultured gilthead sea bream Sparus auratus. Aquac. Res. 40: 1279-1290.). Low water temperatures could induce fasting in cultured species, modifying their metabolic status and triggering an allostatic overload (Polakof et al. 2006Polakof S., Arjona F.J., Sangiao-Alvarellos S., et al. 2006. Food deprivation alters osmoregulatory and metabolic responses to salinity acclimation in gilthead sea bream Sparus auratus. J. Comp. Physiol. 176B: 441-452., Estévez et al. 2011Estévez A., Treviño L., Kotzamanis Y., et al. 2011. Effects of different levels of plant proteins on the ongrowing of meagre (Argyrosomus regius) juveniles at low temperatures. Aquacul. Nutr. 17: e572-e582.), as occurs in the gilthead seabream (Sparus aurata) (Ibarz et al. 2010Ibarz A., Padrós F., Gallardo M.A., et al. 2010. Low-temperature challenges to gilthead sea bream culture: review of cold-induce alterations and “Winter Syndrome”. Rev. Fish Biol. Fish. 20: 539-556.). On the other hand, short but intensive rainfall in autumn/winter (usual in southwestern Spain) can decrease water salinity in earthen ponds, inducing osmotic and consequently metabolic challenges in cultured fish (Vargas-Chacoff et al. 2009aVargas-Chacoff L., Arjona F.J., Ruiz-Jarabo I., et al. 2009a. Seasonal variation in osmoregulatory and metabolic parameters in earthen pond cultured gilthead sea bream Sparus auratus. Aquac. Res. 40: 1279-1290.). To the best of our knowledge, metabolic approaches have not been performed in A. regius.

The aim of this study was to provide key information on the metabolic changes in A. regius specimens cultured in earthen ponds under natural environmental conditions during an eighteen-month period (this period was chosen because it is the time expected commercial growth of this species). This information can be useful for optimizing its culture, as it may indicate seasonal metabolic behaviour that can be used to determine the best maintenance conditions as an alternative to aquaculture practice in tanks and cages.

MATERIALS AND METHODSTop

Abbreviations

BSA: Bovine serum albumin
FBPase: Fructose 1,6-bisphosphatase (EC 3.1.3.11)
G6PDH: Glucose 6-phosphate dehydrogenase (EC 1.1.1.49)
GDH: Glutamate dehydrogenase (EC 1.4.1.2)
G3PDH: Glycerol 3-phosphate dehydrogenase (EC 1.1.1.8)
GOT: Aspartate aminotransferase (EC 2.6.1.1)
HK: Hexokinase (EC 2.7.1.1)
LDH-O: Lactate dehydrogenase-oxidase (EC 1.1.1.27)

Animals and maintenance

Juveniles of meagre (A. regius, n=90 total, n=10 for each sampled period) were supplied by a local fish farm (ACUINOVA, San Fernando Cádiz, Spain). According to the fish farm procedure, specimens (n=6500, 12.0±0.5 g weight) were placed in natural earthen ponds (88 m length × 15 m width × 1.5 m depth, total volume: 1980 m³) in September 2004 at an initial density of 0.039 kg m^–3. Specimens were sampled approximately every two months for 18 months (2/12/2004, 21/2/2005, 13/4/2005, 30/5/2005, 27/7/2005, 26/9/2005, 22/11/2005, 8/3/2006 and 23/5/2006). In the first sample (2/12/2004) the fish weighed 90-100 g, while the final weight was 1100-1400 g at a final density of 4 kg m^–3. The mortality during the experimental period was 10%. Fish were maintained in earthen ponds under natural photoperiod, temperature and salinity regimens. Salinity was measured with an ATAGO S/MILL refractometer and temperature was measured with a digital thermometer. Both parameters were measured each day at the same time (12:00); Figure 1 shows the average variations of temperature and salinity over time (months). Fish were fed daily according to fish farm procedures (2% of body weight) with commercial dry pellets for sea bream (Dibaq-Diproteg SA, Segovia, Spain) (see Table 1 for nominal food composition). They were fasted 24 h before sampling. All experimental procedures described complied with the Guidelines of the European Union Council (86/609/EU) for the use of animals in research.

Full size image

Fig. 1. – Average values for water temperature (°C) and salinity (‰) during the experimental period. Data are shown as mean±SEM. Each factor was measured daily; the average is for number of days between sampling points.

Table 1. – Proximate composition of the diet used during the experiment (% dry weight).

Pellet	Protein	Fat	Carbohydrates	Fibre	Ash	Total P	Digestible energy (Mj/Kg)
Sea bream (60-200g)	46	21	11.07	1.4	9.1	1.2	19.7
Sea bream (190-600g)	44	22	11.47	1.5	8.5	1.1	19.8
Sea bream (>600g)	42	22	14.20	1.7	8.0	1.05	19.9

Sampling procedure

Fish were netted, submitted to lethal doses of 2-phenoxyethanol (1 mL L^–1) and completely euthanized by spinal section before tissues were removed, weighed and sampled (n=10 per time-point). The time between capture and sampling was always less than 3 minutes). Blood was collected from the caudal peduncle into 1-mL syringes rinsed with a solution containing 25000 units of ammonium heparin per 3 mL 0.6% NaCl. Plasma was separated from cells by centrifugation of whole blood (3 min, 10000 × g, 4°C), snap-frozen in liquid N2 and stored at –80°C until analysis. Liver and muscle were removed from each fish, the liver was weighed and both were freeze-clamped in liquid nitrogen and stored at –80°C until further assay.

To evaluate seasonal effects on fish performance, several biometric indices were calculated:

- Fulton’s condition factor (K) was calculated as K=100·W/L3, where W= fish weight (g) and L= total length (cm).
- Hepatosomatic index (HSI) = (liver weight/total weight)*100
- Specific growth rate (% daily SGR) was calculated as SGR=100(Ln W_f – Ln W_i)/T, where W_f = final body weight (g), W_i = initial body weight (g) and T is number of days between weighings.

Plasma determinations

Plasma glucose, triglycerides and lactate levels were measured using commercial kits from Spinreact (Glucose-HK Ref. 1001200; Triglycerides Ref. 1001311; Lactate Ref. 1001330) adapted for 96-well microplates. Plasma total proteins were determined in 1:50 (v/v) diluted plasma samples using the bicinchoninic acid BCA Protein Assay Kit (Pierce #23225). All assays were performed with a Bio Kinetics EL-340i Automated Microplate Reader (Bio-Tek Instruments) using DeltaSoft3 software for Macintosh (BioMetallics Inc.).

Tissue metabolites and enzymatic activities

Frozen liver and muscle were finely minced in an ice-cooled Petri dish, vigorously mixed and divided into two aliquots to assess enzyme activities and metabolite levels. The frozen tissue used for the assessment of metabolite concentration was homogenized by ultrasonic disruption with 7.5 volumes of ice-cooled 0.6 N perchloric acid, neutralized (using 1 mol L^–1 potassium bicarbonate) and centrifuged (30 min, 13000 g, 4°C, Eppendorf 5415R), and the supernatant was used to assay tissue metabolite levels. Tissue lactate and triglyceride levels were determined spectrophotometrically using commercial kits (Spinreact, see before). Tissue glycogen concentrations were assessed using the method of Keppler and Decker (1974)Keppler D., Decker K. 1974. Glycogen: Determination with amyloglucosidase. In: Bergmeyer H.U. (ed.) Methods of enzymatic analysis. Academic, New York, pp 1127-1131.. Glucose obtained after glycogen breakdown (after subtracting free glucose levels) was determined with a commercial kit (Spinreact, see above). Total α-amino acid (Total aa) levels were assessed colorimetrically using the ninhydrin method of Moore (1968)Moore S. 1968. Amino acid analysis: aqueous dimethyl sulfoxide as solvent for the ninhydrin reaction. J. Biol. Chem. 1242: 6281-6283. adapted to a microplate assay. The aliquots of tissues used for the assessment of enzyme activities were homogenized by ultrasonic disruption in 10 volumes of ice-cold stopping-buffer containing 50 mM imidazole-HCl (pH 8.5), 1 mM 2-mercaptoethanol, 50 mM NaF, 4 mM EDTA, 250 mM sucrose, and 0.5 mM p-methyl-sulphonylfluoride (Sigma Chemical Co., St. Louis, MO, USA), the last added as dry crystals immediately before homogenization. The homogenates were centrifuged (30 min, 13000 g, 4°C) and the supernatants were stored in different aliquots at −80°C until use in enzymatic assays. Enzyme activities were determined using a PowerWave^TM 340 microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA) with KCjunior Data Analysis Software for Microsoft® Windows XP. Reaction rates of enzymes were determined by changes in absorbance of NAD(P)H at 340 nm. The reactions were started by the addition of homogenates (15 μL) at a pre-established protein concentration, omitting the substrate in control wells (final volume of 275-295 μL) and allowing the reactions to proceed at 37°C (5-15 min). Protein levels were assayed in triplicate as in plasma samples. Enzymatic analyses were carried out at conditions meeting requirements for optimal velocities. The specific conditions for the assay of HK, FBPase, G6PDH, GDH, GOT, G3PDH, and LDH-O have been described previously (Laiz-Carrión et al. 2003Laiz-Carrión R., Martín del Río M.P., Miguez J., et al. 2003 Influence of cortisol on osmoregulation and energy metabolism in gilthead sea bream Sparus aratus. J. Exp. Zool. 298A: 105-118., Sangiao-Alvarellos et al. 2003Sangiao-Alvarellos S., Laiz-Carrión R., Guzmán J.M., et al. 2003. Acclimation of Sparus aurata to various salinities alters energy metabolism of osmoregulatory and nonosmoregulatory organs Am. J. Physiol. 285: R897-R907., 2005Sangiao-Alvarellos S., Arjona F.J., Martín del Río M.P., et al. 2005. Time course of osmoregulatory and metabolic changes during osmotic acclimation in Sparus auratus. J. Exp. Biol. 208: 4291-4304., Polakof et al. 2006Polakof S., Arjona F.J., Sangiao-Alvarellos S., et al. 2006. Food deprivation alters osmoregulatory and metabolic responses to salinity acclimation in gilthead sea bream Sparus auratus. J. Comp. Physiol. 176B: 441-452., Vargas-Chacoff et al. 2009bVargas-Chacoff L., Arjona F.J., Polakof S., et al. 2009b. Interactive effects of environmental salinity and temperature on metabolic responses of gilthead sea bream Sparus aurata. Comp. Biochem. Physiol. 154A: 417-424.).

Statistics

Data were checked for normality, independence and homogeneity of variance before one-way analysis of variance was conducted using months (time) as a factor. Tukey’s a posteriori test was used to identify significantly different groups. Logarithmic transformations of the data were performed when necessary to fulfil the conditions of the parametric analysis of variance. Statistical significance was accepted at P<0.05.

RESULTSTop

Water conditions

The abiotic water parameters temperature and salinity varied throughout the year, showing relevant temperature differences (maximum of 25°C in summer and minimum of 11°C in winter), while salinity showed sudden fluctuations between 36% and 39% (Fig. 1).

Biometrics

Length and weight showed a similar trend, with a strong linear relationship (R²=0.94) between these parameters (Fig. 2). Condition factor (K), hepatosomatic index (HSI) and specific growth rate (% daily SGR) of specimens in each sampling point are shown in Table 2.

Full size image

Fig. 2. – Changes in body weight (A) and total length (B) over 18 months in A. regius specimens cultured in an earthen pond. Data points represents mean±SEM (n=10 per group at each sampling time). Different letters indicate significant differences among sampling points (P<0.05, one-way ANOVA, Tukey test).

Table 2. – Seasonal growth performance indices in A. regius specimens cultured in earthen ponds. Further details as in legend of Figure 2.

	Dec-04	Feb-05	Apr-05	May-05	Jul-05	Sep-05	Nov-05	Mar-06	May-06
Hepatosomatic index (%)	1.63±0.01a	1.39±0.02b	1.00±0.01c	1.85±0.03d	1.81±0.01d	1.01±0.01c	1.14±0.03b	1.09±0.03b	1.18±0.01b
Condition factor	1.23±0.03a	1.11±0.02b	1.16±0.02b	1.09±0.02b	1.08±0.02b	1.14±0.03b	1.08±0.03b	1.05±0.02b	1.07±0.02b
Specific growth rate (% daily)	0.00	0.18	0.78	0.40	1.76	0.41	0.48	0.00	0.37

Plasma

Plasma metabolite levels are given in Table 3. All parameters showed significant modifications due to seasonal effects. Glucose concentrations showed the lowest values in summer (P<0.05) and lactate during winter (P<0.05). However, proteins showed no clear pattern of change, but were at maximum levels in late winter and spring. Maximum levels of triglycerides were observed in late winter and early spring (P<0.05).

Table 3. – Seasonal changes in plasmatic metabolite levels in A. regius specimens cultured in earthen ponds. Further details as in legend of Figure 2.

	Dec-04	Feb-05	Apr-05	May-05	Jul-05	Sep-05	Nov-05	Mar-06	May-06
Glucose (mM)	6.01±0.77a	5.43±0.46a	5.82±0.60a	3.80±0.27b	2.67±0.19c	5.65±0.58a	6.51±0.39a	6.38±0.35a	3.66±0.30b
Lactate (mM)	1.33±0.08a	2.74±0.23bc	3.01±0.39bc	1.77±0.17b	3.72±0.46c	3.71±0.44c	2.0±0.24b	2.63±0.18bc	3.25±0.42c
Protein (mg/mL)	35.61±1.32ab	37.10±1.44b	33.06±1.49ab	36.67±0.67b	32.54±1.23ab	33.54±1.08ab	29.94±1.58a	35.20±1.71b	35.88±0.94b
Triglycerides (mM)	3.03±0.17a	4.32±0.33b	6.93±0.17c	3.40±0.17ab	2.43±0.40a	2.89±0.10a	1.37±0.2d	3.67±0.35ab	2.97±0.25a

Liver

Time-related metabolic changes are shown in Table 4 (metabolite levels) and Figure 3 (enzymatic activities). Liver glycogen and glucose levels showed an inverse relationship, with glycogen stores increasing while free glucose levels decreased. Liver proteins and amino acid levels showed different patterns of change, with the highest protein values in spring and the highest amino acid levels in winter (P<0.05). Liver triglyceride content showed no clear seasonal pattern. Enzymatic activities related to carbohydrate metabolism (HK, FBPase and G6PDH) showed statistical differences between seasons; HK activity decreased in summer (Fig. 3A) while FBPase and G6PDH increased in summer and autumn, respectively (Fig. 3B, C). However, amino acid-related enzymes (GDH and GOT activities) were enhanced in spring and summer, while lipid-related (G3PDH activity) metabolic enzymes showed no clear seasonal pattern (P<0.05) (Fig. 3D-F).

Table 4. – Seasonal changes in hepatic metabolite levels in A. regius specimens cultured in earthen ponds. Further details as in legend of Figure 2.

	Dec-04	Feb-05	Apr-05	May-05	Jul-05
Glucose (μmol/hepatic unit)	20.99±2.97a	17.78±1.25a	23.84±2.02a	23.16±2.07a	78.01±4.85b
Glycogen (μmol glucidic units/hepatic unit)	55.09±3.39ab	43.74±4.39ab	65.64±6.82a	34.09±6.48b	139.54±14.11cde
Protein (μmol/hepatic unit)	37.89±6.19ab	27.90±2.73a	47.13±4.92b	39.42±3.12b	113.13±9.11c
Total Aa (μmol/hepatic unit)	219.83±18.64a	101.43±7.71b	114.31±11.53b	83.16±14.61b	252.17±47.56a
Triglycerides (μmol/hepatic unit)	5.14±0.82a	2.29±0.33b	3.91±0.77ab	5.19±0.77a	15.98±2.68c
	Sep-05	Nov-05	Mar-06	May-06
Glucose (μmol/hepatic unit)	152.20±7.82c	196.11±16.83cd	170.10±12.02cd	206.76±14.77d
Glycogen (μmol glucidic units/hepatic unit)	144.13±16.71cde	153.12±10.47cde	110.49±15.20d	181.976±15.24e
Protein (μmol/hepatic unit)	176.80±15.26d	231.35±22.80d	173.30±13.18d	228.79±14.99d
Total Aa (μmol/hepatic unit)	240.78±27.11a	673.54±118.85c	599.54±42.28c	3 79.27±29.08d
Triglycerides (μmol/hepatic unit)	32.61±4.88d	30.01±2.51d	24.21±0.89d	15.83±2.12c

Full size image

Fig. 3. – Seasonal changes in liver HK (A), FBP (B), G6PDH (C), GDH (D), GOT (E) and G3PDH (F) activities in A. regius specimens cultured in earthen ponds. Further details as in legend of Figure 2.

Muscle

Seasonal changes of metabolic parameters in muscle are shown in Table 5 (metabolite levels) and Figure 4 (enzymatic activities). Muscle glycogen content increased in mid- to late spring (P<0.05), while glucose levels showed no clear pattern of change; lactate levels were elevated after summer. Protein content showed significant variations between seasons (P<0.05), while amino acid values increased in mid- to late spring. Triglycerides showed no statistical differences throughout the year. Carbohydrate metabolic enzymes (HK, FBPase and G6PDH) showed statistical differences between seasons: i) HK had high activity in autumn and winter, ii) FBPase decreased in spring and fall, and iii) G6PDH decreased mainly in spring but values were maintained high throughout the year (Fig. 4A-C). Amino acid–related metabolic enzyme (GDH activity) was lowest in summer (P<0.05, Fig. 4D). No pattern of variation was distinguishable for GOT activity (Fig. 4D, E). Lipid-related metabolic enzyme (G3PDH activity) showed seasonal variation, with the highest activity occurring in spring (P<0.05) (Fig. 4F). Lactic-related metabolic enzyme activity (LDH-O activity) was highest in spring and summer (P<0.05) (Fig. 4G).

Table 5. – Seasonal changes in muscle metabolite levels in A. regius specimens cultured in earthen ponds. Further details as in legend of Figure 2.

	Dec-04	Feb-05	Apr-05	May-05	Jul-05
Glucose (μmol/g wet weight)	6.84±0.33a	5.18±0.20b	6.63±0.20a	6.18±0.08a	6.46±0.44a
Glycogen (μmol glucidic units /g wet weight)	0.31±0.05a	0.37±0.05ab	1.33±0.09c	1.07±0.09c	0.46±0.09b
Lactate (μmol/g wet weight)	0.91±0.05a	0.95±0.07a	0.84±0.06a	0.74±0.06a	1.36±0.07b
Protein (μmol/g wet weight)	8.17±0.32a	7.66±0.46a	7.90±0.37a	7.55±0.37a	6.47±0.21b
Total Aa (μmol/g wet weight)	34.99±3.87a	47.76±3.49ab	44.36±3.28ab	50.68±3.44b	37.22±4.02a
Triglycerides (μmol/g wet weight)	0.13±0.03	0.05±0.01	0.08±0.02	0.08±0.01	0.23±0.13
	Sep-05	Nov-05	Mar-06	May-06
Glucose (μmol/g wet weight)	5.70±0.14b	5.45±0.11b	5.68±0.21b	5.55±0.41b
Glycogen (μmol glucidic units /g wet weight)	0.58±0.13b	0.52±0.16b	0.58±0.16b	0.27±0.07a
Lactate (μmol/g wet weight)	1.37±0.11b	1.24±0.06b	1.05±0.08ab	1.57±0.07b
Protein (μmol/g wet weight)	6.03±0.25b	6.59±0.23b	6.53±0.30b	6.56±0.23b
Total Aa (μmol/g wet weight)	34.52±3.49a	43.63±3.35ab	42.53±2.39ab	48.35±2.80b
Triglycerides (μmol/g wet weight)	0.21±0.01	0.17±0.05	0.08±0.01	0.12±0.02

Full size image

Fig. 4. – Seasonal changes in muscle HK (A), FBP (B), G6PDH (C), GDH (D), GOT (E), G3PDH (F) and LDH-O (G) activities in A. regius specimens cultured in earthen ponds. Further details as in legend of Figure 2.

DISCUSSIONTop

The meagre (Argyrosomus regius) showed variations in growth rate that correlated well with the seasonal metabolic changes observed. Specimens showed the highest growth rates during spring and summer, when environmental temperatures are highest (20-25°C), while growth was lower in the cold winter months. It is interesting that the growth enhancement was greater in the second spring (from March/06 to May/06) than in the first spring (from April/05 to May/05). This difference could be attributed to a greater temperature increase in the second spring (from 16°C to 22°C) than in the first (from 16°C to 20°C). However, this better capacity to recover from metabolic stress associated with the winter season could also be attributed to two factors: i) the greater size of specimens in the second spring, and/or ii) the possibility that the fish population contained stronger specimens, survivors from the first year of culture (Estévez et al. 2011Estévez A., Treviño L., Kotzamanis Y., et al. 2011. Effects of different levels of plant proteins on the ongrowing of meagre (Argyrosomus regius) juveniles at low temperatures. Aquacul. Nutr. 17: e572-e582.). Many studies have been performed with A. regius using different culture systems (cages, tanks or earthen ponds), so the data available to compare the growth rates between them should be treated with care (Cárdenas 2010Cárdenas S. 2010. Crianza de la Corvina (Argyrosomus regius). Serie Cuardenos de Acuicultura nº 3. Fundación OESA y CSIC, Madrid, 100 pp.). The growth of A. regius appears to be higher in cages than in tanks and earthen ponds. In cages, juveniles of 110 g reached 1850 g in 8 months (a growth rate of 217.5 g per month) (Pastor et al. 2002Pastor E., Grau A., Massutí E., et al. 2002. Preliminary results on growth of meagre, Argyrosomus regius (Asso, 1801) in sea cages and indoor tanks. EAS Especial Publication 32: 422-423.); while in tanks, specimens of 230 g reached 380 g in 3.5 months (a growth rate of 42.8 g per month) (Chatzifotis et al. 2010Chatzifotis S., Panagiotidou M., Papaioannou N., et al. 2010. Effect of dietary lipid levels on growth, feed utilization, body composition and serum metabolites of meagre (Argyrosomus regius) juveniles. Aquaculture 307: 65-70.). These contrasts could be attributed to many different reasons such as food and water quality and presence/absence of parasites, so no clear conclusions can be drawn from those studies. Our results showed that specimens of 90 g kept in natural earthen ponds reached 1231.3 g in 18 months (a growth rate of 63.3 g per month), indicating that A. regius cultured in this culture system had good growth rates and condition factor indexes (Cárdenas et al. 2008Cárdenas S., Duncan N., Pastor E., et al. 2008. Meagre (Argyrosomus regius) broodstock management in the Spanish R&D project PLANACOR (JACUMAR). Aquaculture Europe, Krakow, Poland.). Our results also indicate that metabolism showed temporal variation, with plasmatic, hepatic and muscular metabolites as well as hepatic and muscular metabolic enzymes showing significant variation throughout the seasons of the year.

In plasma, glucose showed its lowest values in spring and summer, while lactate showed its lowest levels in spring and autumn. These results are different from those described for other species, such as S. aurata (Gómez-Milán et al. 2007Gómez-Millán E., Sánchez-Muros M.J. 2007. Daily and annual variations of the hepatic glucose-6 phosphate dehydrogenase activity and seasonal changes in the body fats of the gilthead sea bream Sparus aurata. J. Exp. Zool. 307A: 516-526., Vargas-Chacoff et al. 2009aVargas-Chacoff L., Arjona F.J., Ruiz-Jarabo I., et al. 2009a. Seasonal variation in osmoregulatory and metabolic parameters in earthen pond cultured gilthead sea bream Sparus auratus. Aquac. Res. 40: 1279-1290.) and Oreochromis mossambicus (Fiess et al. 2007Fiess J., Kunkel-Patterson A., Mathias L., et al. 2007. Effects of environmental salinity and temperature on osmorregulatory ability, organic osmolytes, and plasma hormone profiles in the Mozambique tilapia (Oreochromis mossambicus). Comp. Biochem. Physiol. 146A: 252-264.). In the present study, the lowest glucose or lactate concentrations in these months are associated with changes in hepatic parameters related to glucose or lactic acid metabolism (see below), and could indicate a reduction in the hepatic capacity to produce these metabolites, focusing these resources into somatic growth (Foster and Moon 1991Foster F.D., Moon T.W. 1991. Hypometabolism with fasting in the yellow perch (Perca flavescens): a study of enzymes, hepatocyte metabolism, and tissue size. Physiol. Zool. 64: 259-275., Sala-Rabanal et al. 2003Sala-Rabanal M., Sánchez J., Ibarz A., et al. 2003. Effects of low temperature and fasting on haematology and plasma composition on gilthead sea bream (Sparus aurata). Fish Physiol. Biochem. 29: 105-115.). In addition, in spring (April) plasmatic levels of proteins, triglycerides (TAG) and liver glycogen increased, while HSI decreased (probably due to hepatic TAG mobilization), followed by a depletion in plasma glucose, proteins and TAG as well as liver glycogen (May-July), which may also be related to an acceleration in the growth of the animals (Gallardo et al. 2003Gallardo M.A., Sala-Rabanal M., Ibarz A., et al. 2003. Functional alterations associated with “Winter syndrome” in gilthead sea bream (Sparus aurata). Aquaculture 223: 15-27., Sala-Rabanal et al. 2003Sala-Rabanal M., Sánchez J., Ibarz A., et al. 2003. Effects of low temperature and fasting on haematology and plasma composition on gilthead sea bream (Sparus aurata). Fish Physiol. Biochem. 29: 105-115., Ibarz et al. 2005Ibarz A., Blasco J., Beltrán M., et al. 2005. Cold-Induced alterations on proximate composition and fatty acid profiles of several tissues in gilthead sea bream (Sparus aurata). Aquaculture 249: 477-486., 2007Ibarz A., Fernández-Borràs J., Gallardo M.A., et al. 2007. Alterations in lipid metabolism and use of energy depots of gilthead sea bream (Sparus aurata) at low temperature. Aquaculture 262: 470-480., Vargas-Chacoff et al. 2009aVargas-Chacoff L., Arjona F.J., Ruiz-Jarabo I., et al. 2009a. Seasonal variation in osmoregulatory and metabolic parameters in earthen pond cultured gilthead sea bream Sparus auratus. Aquac. Res. 40: 1279-1290., bVargas-Chacoff L., Arjona F.J., Polakof S., et al. 2009b. Interactive effects of environmental salinity and temperature on metabolic responses of gilthead sea bream Sparus aurata. Comp. Biochem. Physiol. 154A: 417-424.). In winter (November), metabolic pathways have to confront a critical situation due to lower temperatures (Somero 2004Somero G. 2004. Adaptation of enzymes to temperature: searching for basic strategies. Comp. Biochem. Physiol. 139B: 321-333.). Plasmatic levels of lactate, TAG and proteins dropped and maintained a proper homeostasis by conversion to glucose, as indicated by the increased gluconeogenesis pathways such as liver FBPase in autumn and winter. Under these conditions there is a tendency in A. regius specimens to mobilize fat deposits, which may explain the observed enhancement in plasma triglyceride values. This situation agrees with previous results reported in S. aurata in winter (Tort et al. 1998Tort L., Padròs F., Rotland J., et al. 1998. Winter syndrome in the gilthead sea bream Spatus aurata. Immunological and histopathological features. Fish Shellfish Immun. 8: 37-47., 2004Tort L., Rotllant J., Liarte C., et al. 2004. Effects of temperature decrease on feeding rate, immune indicators and histopathological changes of gilthead sea bream Sparus aurata fed with an experimental diet. Aquaculture 229: 55-65., Gallardo et al. 2003Gallardo M.A., Sala-Rabanal M., Ibarz A., et al. 2003. Functional alterations associated with “Winter syndrome” in gilthead sea bream (Sparus aurata). Aquaculture 223: 15-27., Vargas-Chacoff et al. 2009aVargas-Chacoff L., Arjona F.J., Ruiz-Jarabo I., et al. 2009a. Seasonal variation in osmoregulatory and metabolic parameters in earthen pond cultured gilthead sea bream Sparus auratus. Aquac. Res. 40: 1279-1290.). Finally, plasma proteins levels increased previous to each important growth increase period (spring), and decreased during summer months, supporting muscle (somatic) growth. A similar situation has been reported for S. aurata (Chaves-Pozo et al. 2008Chaves-Pozo E., Arjona F.J., García-López A., et al. 2008. Sex steroids and metabolic parameter levels in a seasonal breeding fish (Sparus auratus L.). Gen. Comp. Endocr. 156: 531-536., Vargas-Chacoff et al. 2009aVargas-Chacoff L., Arjona F.J., Ruiz-Jarabo I., et al. 2009a. Seasonal variation in osmoregulatory and metabolic parameters in earthen pond cultured gilthead sea bream Sparus auratus. Aquac. Res. 40: 1279-1290.). Gómez-Millán et al. (2007)Gómez-Millán E., Cardenete G., Sánchez-Muros M.J. 2007. Annual variations in the specific activity of fructose 1,6-bisphosphatase, alanine aminotransferase and pyruvate kinase in the Sparus aurata liver. Comp. Biochem. Physiol. 147B: 49-55. observed in specimens of S. aurata an inverse pattern of variation in plasma amino acids levels with respect to environmental temperature (with the lowest values in cool months and the highest in warm months). This pattern has been related to a higher growth rate during spring and summer months, where available plasma amino acids are necessary to synthesize structural proteins (Vargas-Chacoff et al. 2009aVargas-Chacoff L., Arjona F.J., Ruiz-Jarabo I., et al. 2009a. Seasonal variation in osmoregulatory and metabolic parameters in earthen pond cultured gilthead sea bream Sparus auratus. Aquac. Res. 40: 1279-1290.).

Hepatic energy metabolism processes (i.e. glycogen/glucose turnover, ammoniagenesis, fatty acid synthesis and gluconeogenesis) were affected by temporal changes, and could induce physiological changes associated with differences on growth. Variations in metabolite levels and enzymatic activities related to different abiotic (i.e. salinity, temperature) or biotic (i.e. reproduction, growth) factors have been reported in teleost species (Gómez-Millán and Sánchez-Muros 2007Gómez-Millán E., Sánchez-Muros M.J. 2007. Daily and annual variations of the hepatic glucose-6 phosphate dehydrogenase activity and seasonal changes in the body fats of the gilthead sea bream Sparus aurata. J. Exp. Zool. 307A: 516-526., Gómez-Millán et al. 2007Gómez-Millán E., Cardenete G., Sánchez-Muros M.J. 2007. Annual variations in the specific activity of fructose 1,6-bisphosphatase, alanine aminotransferase and pyruvate kinase in the Sparus aurata liver. Comp. Biochem. Physiol. 147B: 49-55., Arjona et al. 2009Arjona F.J., Vargas-Chacoff L., Ruiz-Jarabo I., et al. 2009. Tertiary stress responses in Senegalese sole (Solea senegalensis Kaup, 1858) to osmotic acclimation: Implications for osmoregulation, energy metabolism and growth. Aquaculture 287: 419-426., Herrera et al. 2009Herrera M., Vargas-Chacoff L., Hachero I., et al. 2009. Osmoregulatory changes in wedge sole (Dicologoglossa cuneata Moreau, 1881) after acclimation to different environmental salinities. Aquac. Res. 40: 762-771., Arjona et al. 2010Arjona F.J., Vargas-Chacoff L., Ruiz-Jarabo I., et al. 2010. Acclimation of Solea senegalensis to different ambient temperatures: implications for thyroidal status and osmoregulation. Mar. Biol. 157: 1325-1335.).

Glycogen and triglyceride content as well as HSI presented a clear pattern depending on the developmental stage of specimens and the season. The glycogen consumed in autumn is related to the increase in HK activity, glucose being its major export product, as observed in plasma glucose levels. However, in the winter moths (February/05-march/06) the lowest values of glycogen were observed, in agreement with the idea of decreased food intake due to low water temperatures and the use of this compound as an energy source (Navarro et al. 1997Navarro I., Blasco J., Baños N., et al. 1997. Effects of fasting and feeding on plasma amino acid levels in brown trout. Fish Physiol. Biochem.16: 303-309., Sala-Rabanal et al. 2003Sala-Rabanal M., Sánchez J., Ibarz A., et al. 2003. Effects of low temperature and fasting on haematology and plasma composition on gilthead sea bream (Sparus aurata). Fish Physiol. Biochem. 29: 105-115.). This differential behaviour of hepatic glycogen stores depending on the age and/or size of the specimens deserves future research. FBPase is a key gluconeogenic enzyme that enhances liver glucose production. The results showed an increased FBPase activity at the beginning of the autumn in the second year. Taking all this evidence together, the increased total FBPase activity may indicate an upregulation of gluconeogenesis at this time, which would explain the high hepatic glucose levels observed in A. regius during this period. A similar FBPase activity profile has been reported in S. aurata (Gómez-Millán et al. 2007Gómez-Millán E., Cardenete G., Sánchez-Muros M.J. 2007. Annual variations in the specific activity of fructose 1,6-bisphosphatase, alanine aminotransferase and pyruvate kinase in the Sparus aurata liver. Comp. Biochem. Physiol. 147B: 49-55.).

Another enzymatic activity related to carbohydrate metabolism, G6PDH, increased in level during spring months of the first year, fuelling the hepatic pentose shunt with glucose, which was enhanced in that season. An activated pentose phosphate pathway suggests an increased reducing capacity of liver, which may be related to a rise in lipid synthesis in agreement with reports for other species (Laiz-Carrión et al. 2005Laiz-Carrión R., Sangiao-Alvarellos S., Guzmán J.M., et al. 2005. Growth performance on gilthead sea bream Sparus aurata in different osmotic conditions: implications on osmoregulation and energy metabolism. Aquaculture 250: 849-861., Sangiao-Alvarellos et al. 2005Sangiao-Alvarellos S., Arjona F.J., Martín del Río M.P., et al. 2005. Time course of osmoregulatory and metabolic changes during osmotic acclimation in Sparus auratus. J. Exp. Biol. 208: 4291-4304., Arjona et al. 2009Arjona F.J., Vargas-Chacoff L., Ruiz-Jarabo I., et al. 2009. Tertiary stress responses in Senegalese sole (Solea senegalensis Kaup, 1858) to osmotic acclimation: Implications for osmoregulation, energy metabolism and growth. Aquaculture 287: 419-426.) and with the highest plasma triglyceride levels observed in spring of the first year in specimens of A. regius (present results). In summer the free amino acids in the liver decreased, probably because they are transported into the muscle, where they are deposited as muscle structural proteins. As the animal grows in summer, active routes are formation (anabolism: high activity of GDH and GOT) and not degradation (catabolism), in agreement with similar studies in other species such as S. aurata, where high anabolic-related enzymatic activities and plasma amino acid levels were also found in summer (Gómez-Millán et al. 2007Gómez-Millán E., Cardenete G., Sánchez-Muros M.J. 2007. Annual variations in the specific activity of fructose 1,6-bisphosphatase, alanine aminotransferase and pyruvate kinase in the Sparus aurata liver. Comp. Biochem. Physiol. 147B: 49-55., Vargas-Chacoff et al. 2009aVargas-Chacoff L., Arjona F.J., Ruiz-Jarabo I., et al. 2009a. Seasonal variation in osmoregulatory and metabolic parameters in earthen pond cultured gilthead sea bream Sparus auratus. Aquac. Res. 40: 1279-1290.).

The muscle metabolism showed changes in metabolite values and enzymatic activities, but without a clear seasonal pattern. The G6PDH enzyme increased its activity in winter, when the animal does not eat or reduces its rate of intake and is supported from body reserves. Thus, in winter fish consume muscle reserves and energy is obtained via gluconeogenesis (as indicated by the high levels of FBPase in February 2004) from amino acids (Mommsen et al. 1999Mommsen T.P., Vijayan M.M., Moon T.W. 1999. Cortisol in teleosts: dynamics, mechanisms of action, and metabolic regulation. Rev. Fish Biol. Fish. 9: 211-268.). This coincides with muscle HK activity, which is higher in winter months (producing glucose-6-phosphate, which would go to the pentose phosphate pathway and glycolysis), or GOT activity, which increased in this case and in February of the first year, possibly indicating a muscle catabolism that obtains basic metabolites capable of entering these energy production routes.

The TAG are used in part by the muscle to produce glycerol, and subsequently energy and reducing power via glycolysis. In this study, muscle TAG showed no variations throughout the year, but G3PDH activity increased in spring. This could be due to a better use of fats from the food. By comparison with other studies addressing the muscle levels of TAG in red porgy (P. pagrus) (Vargas-Chacoff et al. 2011Vargas-Chacoff L., Calvo A., Ruiz-Jarabo I., et al. 2011. Growth performance, osmoregulatory and metabolic modifications in red porgy fry, Pagrus pagrus, under different environmental salinities and stocking densities. Aquac. Res. 42: 1269-1278.), it can be inferred that their amount would not be related to the period of the year or even to a stressful situation.

The LDH-O in muscle showed its highest values in spring, when temperatures became warmer. This result points to an increase in lactate oxidation rates by those tissues involved in thermal accommodation that are able to use lactate as fuel (Vargas-Chacoff et al. 2009aVargas-Chacoff L., Arjona F.J., Ruiz-Jarabo I., et al. 2009a. Seasonal variation in osmoregulatory and metabolic parameters in earthen pond cultured gilthead sea bream Sparus auratus. Aquac. Res. 40: 1279-1290., bVargas-Chacoff L., Arjona F.J., Polakof S., et al. 2009b. Interactive effects of environmental salinity and temperature on metabolic responses of gilthead sea bream Sparus aurata. Comp. Biochem. Physiol. 154A: 417-424.). A very large fraction of the energy stored as glycogen in white muscle can be provided in the form of lactate, which besides being re-converted to glycogen in situ (Schulte et al. 1992Schulte P.M., Moyes C.D., Hochachka P.W. 1992. Integrating metabolic pathways in post-exercise recovery of white muscle. J. Exp. Biol. 166: 181-195.) can also be sent to oxidative tissues via the bloodstream (Weber 1992Weber J-M. 1992. Pathway for oxidative fuel provision to working muscles: ecological consequences of maximal supply limitations. Experientia 48: 557-564.).

In summary, the results of this study indicate that growth and metabolic responses in A. regius are time-dependent and position this species as a very good candidate for diversification in aquaculture. The growth of this species decreased in the coldest months, so it would be advisable to concentrate efforts on warmer growing environments or warmer months, so animals would grow faster and be more cost effective. The use of earthen ponds as a culture system for meagre appears to be a good alternative for countries that have the conditions to use them, because they are much easier to manage than offshore cultivation systems.

ACKNOWLEDGEMENTSTop

This study was partly supported by grants AGL2007-61211/ACU (Ministerio de Educación y Ciencia and FEDER, Spain) and Proyecto de Excelencia PO7-RNM-02843 (Junta de Andalucía) to J.M.M. The authors wish to thank ACUINOVA (San Fernando, Cádiz) for providing experimental fish. L.V.C. is funded by the programme Beca Presidente de la República de Chile and Comisión de Ciencia y Tecnología de Chile (CONICYT). We thank Dr. Lafayette Eaton for his help checking this manuscript and the Dirección de Investigación of the Universidad Austral de Chile (DID).

REFERENCESTop

Arias A. 1976. Sobre la biología de la dorada, Sparus aurata L., de los esteros de la provincia de Cádiz. Invest. Pesq. 40: 201-222.

Arjona F.J., Vargas-Chacoff L., Ruiz-Jarabo I., et al. 2009. Tertiary stress responses in Senegalese sole (Solea senegalensis Kaup, 1858) to osmotic acclimation: Implications for osmoregulation, energy metabolism and growth. Aquaculture 287: 419-426.
http://dx.doi.org/10.1016/j.aquaculture.2008.10.047

Arjona F.J., Vargas-Chacoff L., Ruiz-Jarabo I., et al. 2010. Acclimation of Solea senegalensis to different ambient temperatures: implications for thyroidal status and osmoregulation. Mar. Biol. 157: 1325-1335.
http://dx.doi.org/10.1007/s00227-010-1412-x

Boeuf G., Payan P. 2001. How should salinity influence fish growth? Comp. Biochem. Physiol. 130 C: 411-423.

Calderón J.A., Esteban J.C., Carrascosa M.A., et al. 1997. Estabulación y crecimiento en cautividad de un lote de reproductores de corvina (Argyrosomus regius (A.). VI Congreso Nacional de Acuicultura. MAPA. Cartagena, Murcia, España.

Cárdenas S. 2010. Crianza de la Corvina (Argyrosomus regius). Serie Cuardenos de Acuicultura nº 3. Fundación OESA y CSIC, Madrid, 100 pp.

Cárdenas S., Duncan N., Pastor E., et al. 2008. Meagre (Argyrosomus regius) broodstock management in the Spanish R&D project PLANACOR (JACUMAR). Aquaculture Europe, Krakow, Poland.

Chao L.N. 1986. Sciaenidae. Fishes of the North-eastern Atlantic and the Mediterranean. Unesco, Paris, pp. 865-874.

Chatzifotis S., Panagiotidou M., Papaioannou N., et al. 2010. Effect of dietary lipid levels on growth, feed utilization, body composition and serum metabolites of meagre (Argyrosomus regius) juveniles. Aquaculture 307: 65-70.
http://dx.doi.org/10.1016/j.aquaculture.2010.07.002

Chatzifotis S., Panagiotidou M., Divanach P. 2012. Effect of protein and lipid dietary levels on the growth of juvenile meagre (Argyrosomus regius). Aquacult. Int. 20: 91-98.
http://dx.doi.org/10.1007/s10499-011-9443-y

Chaves-Pozo E., Arjona F.J., García-López A., et al. 2008. Sex steroids and metabolic parameter levels in a seasonal breeding fish (Sparus auratus L.). Gen. Comp. Endocr. 156: 531-536.
http://dx.doi.org/10.1016/j.ygcen.2008.03.004

Dinis M.T., Ribeiro L., Soares F., et al. 1999. A review on the cultivation potential of Solea senegalensis in Spain and in Portugal. Aquaculture 176: 27-38.
http://dx.doi.org/10.1016/S0044-8486(99)00047-2

Duncan N., Estévez A., Porta J., et al. 2012. Reproductive development, GnRHa-induced spawning and egg quality of wild meagre (Argyrosomus regius) acclimatised to captivity. Fish. Physiol. Biochem. 38: 1273-1286.
http://dx.doi.org/10.1007/s10695-012-9615-3

El-Shebly A.A, El-Kady M.A.H., Hussin, A.B., et al. 2007. Preliminary observations on the pond culture of meagre, Argyrosomus regius (Asso, 1801) (Sciaenidae) in Egypt. J. Fish. Aquat. Sci. 2: 345-352.

Estévez A., Treviño L., Kotzamanis Y., et al. 2011. Effects of different levels of plant proteins on the ongrowing of meagre (Argyrosomus regius) juveniles at low temperatures. Aquacul. Nutr. 17: e572-e582.
http://dx.doi.org/10.1111/j.1365-2095.2010.00798.x

Fiess J., Kunkel-Patterson A., Mathias L., et al. 2007. Effects of environmental salinity and temperature on osmorregulatory ability, organic osmolytes, and plasma hormone profiles in the Mozambique tilapia (Oreochromis mossambicus). Comp. Biochem. Physiol. 146A: 252-264.

Foster F.D., Moon T.W. 1991. Hypometabolism with fasting in the yellow perch (Perca flavescens): a study of enzymes, hepatocyte metabolism, and tissue size. Physiol. Zool. 64: 259-275.

Gallardo M.A., Sala-Rabanal M., Ibarz A., et al. 2003. Functional alterations associated with “Winter syndrome” in gilthead sea bream (Sparus aurata). Aquaculture 223: 15-27.
http://dx.doi.org/10.1016/S0044-8486(03)00164-9

García-Mesa S., González G., Menchón A., et al. 2009. Cambios morfométricos y de composición durante el primer año de cultivo de la corvina (Argyrosomus regius). pp. 562-563 In: Beaz D., Villarroel M., Cárdenas S. (eds) XII Congreso Nacional de Acuicultura, Madrid, España.

Gil M.M., Grau A., Basilone G., et al. 2013. Reproductive strategy and fecundity of meagre Argyrosomus regius asso, 1801 (Pisces: Sciaenidae): Implications for restocking programs. Sci. Mar. 77: 105-118 .
http://dx.doi.org/10.3989/scimar.03688.28A

Giogios I., Grigorakis K., Kalogeropoulos N. 2013. Organoleptic and chemical quality of farmed meagre (Argyrosomus regius) as affected by size. Food. Chem. 141: 3153-3159.
http://dx.doi.org/10.1016/j.foodchem.2013.05.154

Gómez-Millán E., Sánchez-Muros M.J. 2007. Daily and annual variations of the hepatic glucose-6 phosphate dehydrogenase activity and seasonal changes in the body fats of the gilthead sea bream Sparus aurata. J. Exp. Zool. 307A: 516-526.
http://dx.doi.org/10.1002/jez.406

Gómez-Millán E., Cardenete G., Sánchez-Muros M.J. 2007. Annual variations in the specific activity of fructose 1,6-bisphosphatase, alanine aminotransferase and pyruvate kinase in the Sparus aurata liver. Comp. Biochem. Physiol. 147B: 49-55.
http://dx.doi.org/10.1016/j.cbpb.2006.12.013

Grau A., Rodríguez-Rúa A., Massuti-Pascual E., et al. 2007. Spawning of meagre Argyrosomus regius (Asso, 1801) using GnRHa. European Aquaculture Society, Istanbul, Turkey

Grigorakis K., Fountoulaki E., Vasilaki A., et al. 2011. Lipid quality and filleting yield of reared meagre (Argyrosomus regius). Int. J. Food Sci. Technol. 46: 711-716.
http://dx.doi.org/10.1111/j.1365-2621.2010.02537.x

Herrera M., Vargas-Chacoff L., Hachero I., et al. 2009. Osmoregulatory changes in wedge sole (Dicologoglossa cuneata Moreau, 1881) after acclimation to different environmental salinities. Aquac. Res. 40: 762-771.
http://dx.doi.org/10.1111/j.1365-2109.2008.02147.x

Ibarz A., Blasco J., Beltrán M., et al. 2005. Cold-Induced alterations on proximate composition and fatty acid profiles of several tissues in gilthead sea bream (Sparus aurata). Aquaculture 249: 477-486.
http://dx.doi.org/10.1016/j.aquaculture.2005.02.056

Ibarz A., Fernández-Borràs J., Gallardo M.A., et al. 2007. Alterations in lipid metabolism and use of energy depots of gilthead sea bream (Sparus aurata) at low temperature. Aquaculture 262: 470-480.
http://dx.doi.org/10.1016/j.aquaculture.2006.11.008

Ibarz A., Padrós F., Gallardo M.A., et al. 2010. Low-temperature challenges to gilthead sea bream culture: review of cold-induce alterations and “Winter Syndrome”. Rev. Fish Biol. Fish. 20: 539-556.
http://dx.doi.org/10.1007/s11160-010-9159-5

Jiménez M.T., Pastor E., Grau A., et al. 2005. Revisión sobre el cultivo de esciénidos en el mundo, con especial atención a la corvina (Argyrosomus regius). Bol. Inst. Esp. Oceanogr. 21: 169-176.

Keppler D., Decker K. 1974. Glycogen: Determination with amyloglucosidase. In: Bergmeyer H.U. (ed.) Methods of enzymatic analysis. Academic, New York, pp 1127-1131.

Laiz-Carrión R., Martín del Río M.P., Miguez J., et al. 2003 Influence of cortisol on osmoregulation and energy metabolism in gilthead sea bream Sparus aratus. J. Exp. Zool. 298A: 105-118.

Laiz-Carrión R., Sangiao-Alvarellos S., Guzmán J.M., et al. 2005. Growth performance on gilthead sea bream Sparus aurata in different osmotic conditions: implications on osmoregulation and energy metabolism. Aquaculture 250: 849-861.
http://dx.doi.org/10.1016/j.aquaculture.2005.05.021

Lavié A., Rodríguez-Rúa A., Ruiz-Jarabo I., et al. 2008. Physiological responses of juvenile of meagre, Argyrosomus regius (Asso, 1801), to density and temperature. Aquaculture Europe, Congress, Krakow, Poland.

Márquez P., Tinoco A.B., Ruiz-Jarabo I., et al. 2010. Osmoregulatory and metabolic responses of fry and juvenile meagre (Argyrosomus regius) to different environmental salinities. 9th International Congress on the Biology of Fishes, Barcelona, Spain.

Mommsen T.P., Vijayan M.M., Moon T.W. 1999. Cortisol in teleosts: dynamics, mechanisms of action, and metabolic regulation. Rev. Fish Biol. Fish. 9: 211-268.
http://dx.doi.org/10.1023/A:1008924418720

Moore S. 1968. Amino acid analysis: aqueous dimethyl sulfoxide as solvent for the ninhydrin reaction. J. Biol. Chem. 1242: 6281-6283.

Muñoz J.L., Rodríguez-Rúa A., Bustillos P., et al. 2008. Crecimiento de corvina Argyrosomus regius (Asso, 1801) en estanques de tierra a distintas salinidades. IV Jornadas de Acuicultura en el Litoral Suratlántico. Nuevos retos. Cartaya, Huelva, Spain.

Navarro I., Blasco J., Baños N., et al. 1997. Effects of fasting and feeding on plasma amino acid levels in brown trout. Fish Physiol. Biochem.16: 303-309.
http://dx.doi.org/10.1023/A:1007759316656

Papadakis I.E., Kentouri M., Divanach P., et al. 2013. Ontogeny of the digestive system of meagre Argyrosomus regius reared in a mesocosm, and quantitative changes of lipids in the liver from hatching to juvenile. Aquaculture 388: 76-88.
http://dx.doi.org/10.1016/j.aquaculture.2013.01.012

Pastor E., Grau A., Massutí E., et al. 2002. Preliminary results on growth of meagre, Argyrosomus regius (Asso, 1801) in sea cages and indoor tanks. EAS Especial Publication 32: 422-423.

Piccolo G., Bovera F., De Riu N., et al. 2008. Effect of two different protein/fat ratios of the diet on meagre (Argyrosomus regius) traits. Ital. J. Anim. Sci. 7: 363-371.

Poli B.M., Parisi G., Zampacavallo G., et al. 2003. Preliminary results on quality and quality changes in reared meagre (Argyrosomus regius): body and fillet traits and freshness changes in refrigerated commercial-size fish. Aquac. Int. 11: 301-311.
http://dx.doi.org/10.1023/A:1024840804303

Polakof S., Arjona F.J., Sangiao-Alvarellos S., et al. 2006. Food deprivation alters osmoregulatory and metabolic responses to salinity acclimation in gilthead sea bream Sparus auratus. J. Comp. Physiol. 176B: 441-452.

Quéméner L. 2002. Le maigre común (Argyrosomus regius). Biologie, pèche, marché et potentiel aquacole. Editions IFREMER, Plouzane, France, 31 pp.

Roo J., Hernández-Cruz C.M., Borrero C., et al. 2010. Effect of larval density and feeding sequence on meagre (Argyrosomus regius; Asso, 1801) larval rearing. Aquaculture 302: 82-88.
http://dx.doi.org/10.1016/j.aquaculture.2010.02.015

Sáenz de Rodrigáñez M.A.S., Fuentes J., Moyano F.J., et al. 2013. In vitro evaluation of the effect of a high plant protein diet and nucleotide supplementation on intestinal integrity in meagre (Argyrosomus regius). Fish. Physiol. Biochem. 39: 1365-1370.
http://dx.doi.org/10.1007/s10695-013-9790-x

Sala-Rabanal M., Sánchez J., Ibarz A., et al. 2003. Effects of low temperature and fasting on haematology and plasma composition on gilthead sea bream (Sparus aurata). Fish Physiol. Biochem. 29: 105-115.
http://dx.doi.org/10.1023/B:FISH.0000035904.16686.b6

Sangiao-Alvarellos S., Laiz-Carrión R., Guzmán J.M., et al. 2003. Acclimation of Sparus aurata to various salinities alters energy metabolism of osmoregulatory and nonosmoregulatory organs Am. J. Physiol. 285: R897-R907.

Sangiao-Alvarellos S., Arjona F.J., Martín del Río M.P., et al. 2005. Time course of osmoregulatory and metabolic changes during osmotic acclimation in Sparus auratus. J. Exp. Biol. 208: 4291-4304.
http://dx.doi.org/10.1242/jeb.01900

Schulte P.M., Moyes C.D., Hochachka P.W. 1992. Integrating metabolic pathways in post-exercise recovery of white muscle. J. Exp. Biol. 166: 181-195.

Somero G. 2004. Adaptation of enzymes to temperature: searching for basic strategies. Comp. Biochem. Physiol. 139B: 321-333.

Suzer C., Kamaci H.O., Çoban D., et al. 2013. Functional changes in digestive enzyme activities of meagre (Argyrosomus regius; Asso, 1801) during early ontogeny. Fish Physiol. Biochem. 39: 967-977.
http://dx.doi.org/10.1007/s10695-012-9755-5

Tort L., Padròs F., Rotland J., et al. 1998. Winter syndrome in the gilthead sea bream Spatus aurata. Immunological and histopathological features. Fish Shellfish Immun. 8: 37-47.
http://dx.doi.org/10.1006/fsim.1997.0120

Tort L., Rotllant J., Liarte C., et al. 2004. Effects of temperature decrease on feeding rate, immune indicators and histopathological changes of gilthead sea bream Sparus aurata fed with an experimental diet. Aquaculture 229: 55-65.
http://dx.doi.org/10.1016/S0044-8486(03)00403-4

Vallés R., Estévez A. 2013. Light conditions for larval rearing of meagre (Argyrosomus regius). Aquaculture 376: 15-19.
http://dx.doi.org/10.1016/j.aquaculture.2012.11.011

Vargas-Chacoff L., Arjona F.J., Ruiz-Jarabo I., et al. 2009a. Seasonal variation in osmoregulatory and metabolic parameters in earthen pond cultured gilthead sea bream Sparus auratus. Aquac. Res. 40: 1279-1290.
http://dx.doi.org/10.1111/j.1365-2109.2009.02226.x

Vargas-Chacoff L., Arjona F.J., Polakof S., et al. 2009b. Interactive effects of environmental salinity and temperature on metabolic responses of gilthead sea bream Sparus aurata. Comp. Biochem. Physiol. 154A: 417-424.

Vargas-Chacoff L., Calvo A., Ruiz-Jarabo I., et al. 2011. Growth performance, osmoregulatory and metabolic modifications in red porgy fry, Pagrus pagrus, under different environmental salinities and stocking densities. Aquac. Res. 42: 1269-1278.
http://dx.doi.org/10.1111/j.1365-2109.2010.02715.x

Weber J-M. 1992. Pathway for oxidative fuel provision to working muscles: ecological consequences of maximal supply limitations. Experientia 48: 557-564.
http://dx.doi.org/10.1007/BF01920239