Metabolic modifications associated with natural environmental conditions were assessed in the meagre
Modificaciones metabólicas asociadas a condiciones ambientales temporales fueron evaluadas en la corvina
The meagre
Several studies have been conducted recently on meagre, mainly focusing on the reproduction of this species using hormone stimulation (
These earthen ponds are quite shallow (
The aim of this study was to provide key information on the metabolic changes in
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)
Juveniles of meagre (
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 |
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 Wf – Ln Wi)/T, where Wf = final body weight (g), Wi = initial body weight (g) and T is number of days between weighings.
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.).
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
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.
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% (
Length and weight showed a similar trend, with a strong linear relationship (R2=0.94) between these parameters (
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 metabolite levels are given in
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 |
Time-related metabolic changes are shown in
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 |
Seasonal changes of metabolic parameters in muscle are shown in
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 |
The meagre (
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
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 (
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 (
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 (
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 (
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 (
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 (
In summary, the results of this study indicate that growth and metabolic responses in
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).