Investigations on the metabolism of viable and nonviable gilthead sea bream (Sparus aurata) eggs

Aquaculture 223 (2003) 159 – 174 www.elsevier.com/locate/aqua-online

Investigations on the metabolism of viable and nonviable gilthead sea bream (Sparus aurata) eggs Franz Lahnsteiner a,*, Pierpaolo Patarnello b a

Institute for Zoology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria b Valle Ca’ Zuliani Sr., Via Timavo, 76, 34074 Monfalcone, Italy

Received 18 July 2002; received in revised form 30 January 2003; accepted 11 February 2003

Abstract The present study investigated selected biochemical parameters in viable and nonviable eggs of the gilthead sea bream, Sparus aurata. During embryogenesis, S. aurata eggs had a balanced and stable energy metabolism as the levels of adenosine nucleotides and acetyl-CoA, and the adenylate energy charge (EC), remained constant. Mg2 +-dependent ATPase, which is involved in membranedriven ion transport during oxidative phosphorylation, increased in activity. In nonviable eggs, the levels of ATP, acetyl-CoA, the adenylate energy charge, and the activities of malate dehydrogenase were significantly decreased in comparison to viable eggs. Viable eggs had high Na+/K+-ATPase activity which remained constant during embryogenesis while Ca2 +-ATPase activity increased. These enzymes were similarly high in nonviable eggs indicating that the ability for ion transport and for osmoregulation did not differ. However, nonviable eggs contained nonphysiological high levels of magnesium and calcium ions indicating ion influx from the seawater. As the phospholipid levels were significantly lower in nonviable eggs, this ion influx is thought to be related to changed composition of the oolemma. Activities of glucose-6-phosphate dehydrogenase, transaldolase, phosphofructokinase, and pyruvate kinase were constant in viable eggs of S. aurata during embryogenesis. Pyruvate carboxylase increased in activity in the embryonic stage. The occurrence of these enzymes indicated the presence of the enzymatic system for glycolysis for gluconeogenesis and for the pentose phosphate pathway. The monosaccharide levels (i.e. total amount, glucose, fructose, galactose) increased steadily during egg development. Monosaccharides are necessary for nucleic acid synthesis levels, which increased during embryogenesis, and may also play a role as osmotically active compounds. In nonviable eggs, levels of all assayed sugars as well as activities of pyruvate carboxylase and transaldolase were very significantly decreased. Enzymes involved in the catabolism of proteins and amino acids (proteases, aspartate aminotransferase, glutamate dehydrogenase) were constant in the viable eggs with the exception

* Corresponding author. Tel.: +43-662-8044-5630; fax: +43-662-8044-5698. E-mail address: [email protected] (F. Lahnsteiner). 0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0044-8486(03)00159-5

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of aspartate aminotransferase, which increased significantly in the embryonic stage. Nonviable eggs had lower activities of glutamate dehydrogenase than viable eggs, while the other enzyme activities were similar. Amino acid levels and inorganic phosphate levels were lower in nonviable than in viable eggs. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Sea bream; Sparus aurata; Egg viability; Egg quality; Biochemistry

1. Introduction The culture of marine fish is a new and continuously expanding industry, which is dependent on the steady improvement of technologies. Efficient production of high quality fry is one of the most important requirements to obtain independence from wild populations and to produce healthy and high quality fish stocks. One of the main marine species kept in mariculture is the gilthead sea bream (Sparus aurata). As in other sparid species (Sakai et al., 1985; Matsuura et al., 1988; Lin et al., 1990; Kjørsvik et al., 1990), brood stocks are able to produce high quantities of eggs, but the egg quality varies greatly and in a noncontrollable way. Freshly spawned eggs often contain a high proportion of nonviable eggs. Also, high percentages of viable eggs may cease development during embryogenesis. To obtain knowledge about the biochemical parameters limiting the viability of marine fish eggs, numerous biochemical investigations have been performed on egg composition and energy resources during embryogenesis (Dicentrarchus labrax— Rønnestad et al., 1998; S. aurata—Mourente and Odriozola, 1990; Koven et al., 1992; Pascual and Yu´fera, 1993; Rønnestad et al., 1994; Gadus morhua—Pickova et al., 1997; Carnevali et al., 1999) and on biochemical differences between viable and nonviable eggs (S. aurata—Mauro, 1990; Carnevali et al., 2001; Pagrus major—Seoka et al., 1996; Lates calcarifer—Nocillado et al., 2000; for reviews, see Bromage and Roberts, 1994; Brooks et al., 1997). However, many biochemical parameters remain unstudied. Their investigation may lead to increased knowledge about embryogenesis and parameters essential for egg viability. The present study investigates five selected metabolic parameters in viable and nonviable eggs of the gilthead sea bream (S. aurata) during embryogenesis (first cleavage to embryo stage before hatching). (1) The egg energy status was characterized by investigation of energy shuttles (creatinine kinase-ATP generation from creatine phosphate, adenylate kinase-ATP generation from ADP), energetic compounds (ADP, AMP ATP, adenylate energy charge), and coenzymes with a central position in metabolism (acetyl-CoA, NADH). Malate dehydrogenase, succinate dehydrogenase, and Mg2 + dependent ATPase were investigated as tricarboxylic acid cycle, and membrane-driven ion transport for oxidative phosphorylation are essential for developing eggs (Rombough, 1988; Finn et al., 1995). (2) Parameters related to osmoregulation were studied. Na+/K+- and Ca2 +-dependent ATPases were investigated as they are important for ion transport and subsequently for

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osmoregulation (the Ca2 + dependent ATPase additionally for mobilizing and demobilizing intracellular calcium). Egg internal levels of calcium and magnesium were determined, as their accumulation could be indicative for increased permeability of the oolemma; phospholipids were measured, as these are the main compounds of the membranes. Further sialic acids were measured as they occur in the cortical vesicles and are involved in the formation of the perivitelline space. (3) The carbohydrates (total levels of carbohydrates, monosaccharides) were analyzed as well as the enzymes related to their metabolism (glycolysis: lactate dehydrogenase, pyruvate kinase, phosphofructokinase; gluconeogenesis: pyruvate carboxylase, glucose-6-phosphate dehydrogenase; pentose phosphate way: transaldolase). Carbohydrates may be energy delivering compounds during egg development (Terner, 1979; Mommsen and Walsh, 1988), but are also necessary for synthesis of nucleic acids. (4) Concerning the protein and amino acid catabolism, protease activities, free amino acids and free phosphate, and enzymes involved in catabolism of amino acids (aspartate aminotransferase: transamination; glutamate dehydrogenase: deamination) were studied. (5) Finally, also enzymes related to autolytic processes (acid phosphatase, h-D-glucuronidase) were investigated.

2. Material and methods Experiments were performed in the commercial hatchery Valle Ca’ Zuliani Sr in Monfalcone, Italy, with gilthead sea bream eggs. The brood stock were kept under artificial temperature and day light rhythms, which produced fry from September to June. Brood stock fish were F2 or F3 generations from captured wild populations and matured and spawned without hormonal treatment. Eggs were sampled from seven brood stock groups on two consecutive days to obtain a total of 14 different egg batches. Each brood stock group consisted out of 50 – 60 females and 20– 23 males and was kept in spawning tanks with running seawater (fish density: 5 kg/m3). The fish were fed with a mixture of commercial fish pellets, shrimp, and squid. 2.1. Collection of eggs Eggs originating from natural spawning were collected via an overflow tube at the outflow end of the spawning tank with a commercial collector system. As samples of early and homogenous embryonic stages were required, eggs were collected every 2 h, i.e. until about 20 g of eggs were in the collectors. For the different brood stock groups, sampling was done during maximum spawning activity, which was known from preliminary sampling trials. Eggs were taken out of the collectors with fine-meshed nets. They were separated into floating and nonfloating samples. Floating eggs were considered viable; nonfloating eggs sank to the bottom of the incubator and were considered nonviable. Eggs from both groups were sampled for biochemical analysis. The rest of the nonfloating eggs was discarded. From the floating eggs, a 10-g sample was taken, disinfected with 0.2x

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iodine, and incubated in a cylindrical 10-l vessel containing 17 jC seawater (salinity of 37x ) with constant, gentle aeration. Egg stocking density, salinity, temperature, and aeration were similar to routine hatchery incubation conditions. At 10, 30, and 90 h after incubation, eggs were removed from the tanks with a fine-meshed net and separated into floating and nonfloating. The required subsamples were taken from floating and nonfloating eggs. Nonfloating eggs were discarded. Floating eggs were returned to incubation vessels after water renewal. 2.2. Sample preparation For biochemical analyses, five subsamples were taken from each sample. Seawater was drained away with filter paper and amounts of 50 – 100 mg were weighed in Eppendorf tubes with an analytical balance to obtain a reference unit for the metabolic measurements. Five different extractions were performed. Enzymes were extracted in 0.6 ml 100 mM/l Tris buffer supplemented with 0.1% Triton X-100 (pH 7.5). Acid-stable metabolites were extracted in 0.6 ml 3 mol/l perchloric acid. Alkali stable metabolites were extracted in 0.6 ml 0.5 mol/l alcoholic (50% ethanol) potassium hydroxide solution. Lipids were extracted in 2 ml of chloroform, methanol mixture (2:1, v/v). For measurement of egg internal ion levels, a subsample was placed in a small finemeshed sieve and washed with five rinses of distilled water. The remaining water was allowed to drain away; the eggs were weighed into Eppendorf tubes and 0.6 ml of distilled water was added. Additionally, eggs were fixed in 5% glutaraldehyde in 0.1 mol/l cacodylate buffer containing 5% sucrose (osmolality of buffer was 125 mosMol/kg) at 10 jC. From these subsamples, digitized micrographs were taken in a stereomicroscope at five times magnification for determining embryonic stages and for measurement of egg dimensions. For determination of egg wet weight, about 20 – 30 mg eggs were weighed to the nearest 0.1 mg and the number of eggs counted. For the determination of egg dry weight, samples were placed in an incubator at 100 jC for 24 h and reweighed. All biochemical samples were immediately frozen at 25 jC. After transport into the lab, they were stored at 70 jC. Biochemical samples were thawed at room temperature, homogenized, and kept in extraction solution for at least 30 min under constant agitation. The Tris extract, perchloric acid extract, alkaline extract, and distilled water extract were centrifuged at 1500  g for 10 min at 4 jC. The supernatant from the perchloric acid extract was neutralized with 2 mol/l KOH. The alkaline extract was neutralized with triethanolamine/phosphate buffer mixture (TEA, 0.5 mol/l; KH2PO4, 0.4 mol/l; K2HPO4, 0.1 mol/l) to pH 7.4. The solvent layer was collected from the chloroform, methanol extract. All extractions were performed at 5– 10 jC. 2.3. Enzymatic measurements Routine enzymatic assays were optimized for S. aurata to obtain highest reaction velocity. The assay conditions for the enzymes are shown in Table 1. Adequate blanks and

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Table 1 Setup for enzymatic assays in S. aurata Enzyme and assay type

Substrate in mmol/l

Additives and co-substrates, mmol/l

pH

Dt

Acid phosphatase, colorimetrica Adenylate kinase, UVa Allanine aminotransferase, UVa Aspartate aminotransferase, UVa Ca2 +-ATPase, phosphate determinationb NA+/K+-ATPase, phosphate determinationc Mg2 +-ATPase, UVa Creatinine kinase, UVa

p-nitrophenyl phosphate, 11; Km = 3.9 AMP, 2.6; Km = 2 2-oxoglutarate, 5; Km = 2.7 2-oxoglutarate, 3.3; Km = 1 ATP, 5; Km = 2.75

–

4.4

30

ATP 1; MgSO4, 13; KCl, 43 allanine, 500; pyridoxal phosphate, 100 aspartate, 240; pyridoxal phosphate, 100 CaCl2, 5

7.6 7.4

5 5

7.6

2

7.6

20

MgCl2, 3; NaCl, 130; KCl, 20; histidine, 30; ouabin, 1 MgSO4, 4 –

7.6

20

7.6

3 5

MgCl2, 6; maleinimide 5

7.6

5

–

4.5

60

NH4 acetate, 100; ADP, 1

7.8

5

NaCl, 200

7.6

5

MgCl2, 0.05

9.4

3

ATP, 1; MgSO4, 5; KCl 0.05

7.4

5

ATP, 3.3; MgSO4, 6.7; acetyl-CoA, 0.16 EDTA, 2

7.8

5

7.4

20

CaCl2, 0.6

7.4

20

–

7.6

20

MgCl2, 10; KCl, 100

8.0

5

sodium azide, 5; dichloroindophenol, 2.5 erythrose 4-phosphate, 0.18

7.4

10

7.6

5

Glucose-6-phosphate dehydrogenase, UVa h-D-glucuronidase, colorimetrica Glutamate dehydrogenase, UV Lactate dehydrogenase, UVa Malate dehydrogenase, UVa Phosphofructokinase, UVd Pyruvate carboxylasee Calcium-independent protease, colorimetricf Metalloprotease, colorimetricf Total protease, UV-amino acid determinationf Pyruvate kinase, UVa Succinate dehydrogenase, colorimetricg Transaldolasea

ATP, 2.5; Km = 1.43 ATP, 0.55; Km = 0.25 creatinine phosphate, 17.5; Km = 2 G-6-P, 3.3; Km = n.d. phenolphthalein glucuronide, 1.5; Km = 0.4 2-oxoglutarate, 3.5; Km = 2 pyruvate, 0.20; Km = 0.15 malate, 0.27; Km = 0.18 fructose-6-phosphate, 1; Km = n.d. pyruvate, 10; Km = 5.5 N-a-benzoyl-L-arginine ethyl ester (BAEE), 0.88; Km = 1.4 BAEE 0.88; Km = 1.4 Casein, 0.25%; Km = n.d. phosphoenolpyruvate, 1; Km = 0.45 succinate, 4.55; Km = 2.3 fructose-6-phosphate, 2.7; Km = n.d.

Dt = incubation period in minutes, Km = Michaelis constant determined for three floating egg samples in the embryo stage. a Bergmeyer (1985). b Rorive and Kleinzeller (1974). c Jørgense (1974). d Mansour (1966). e Young et al. (1969). f Beynon and Bond (1989). g Clark and Switzer (1977).

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standards were run for each assay to exclude interactions from other enzymes in the crude extract. All enzymatic assays were performed at 22 F 1 jC. Protein was measured with the method of Lowry et al. (1951). 2.4. Measurement of metabolites The perchloric acid extraction method yielded measurements for acetyl-CoA, ATP, ADP, AMP, glucose, fructose, galactose, glucose-6-phosphate, lactate with routine UVspectrophotometric assays described in Bergmeyer (1985), total free monosaccharides (hexoses and pentoses) with the phenol – sulphuric acid reagent, and total carbohydrates with the phenol – sulphuric acid reagent after sample treatment with 50% H2SO4 for 4 h at 120 jC to liberate bound monosaccharides, DNA levels with diphenylamine, free inorganic phosphorus with ascorbic acid and ammonium molybdate, and the free amino acids with the Folin – Ciocalteu phenol reagent according to methods described in Holtzhauer (1988). Sialic acids were measured with the ferric orcinol assay (Fukuda and Kobata, 1993). In the alkaline extract, NADH was measured by a routine UV-spectrophotometric assay described in Bergmeyer (1985). In the chloroform methanol extract, total lipids and phospholipids were measured with colorimetric assays described in Chaudhry (1989). In the distilled water extract, calcium was determined colorimetrically with the o-cresolphthalein complexone method and magnesium with calmagite (Tietz, 1990). For all assays, sample volumes were adjusted to fall in the linear range of the assay and standards used. 2.5. Statistics Metabolites and enzyme activities of eggs were expressed as unit per gram tissue. Enzyme activities were expressed additionally as unit per gram protein. For statistical analysis, data were normalized by logarithmic transformation. Adenylate energy charge (EC) was calculated according to the formula EC=[(ATP) + 1/2(ADP)]/ [(ATP)+(ADP)+(AMP)]. Analysis of variance (ANOVA) with subsequent Tukey’s b-test was used to compare the means of the different parameters of different embryonic stages and between floating and nonfloating eggs.

3. Results 3.1. General parameters of the eggs The samples collected out of the spawning tanks contained eggs in the first cleavage stages (2 – 16 cell stage). Eggs in later ontogenetic stages occurred in frequencies < 5%. The morula stage was visible after 10 h and blastopore closure after 30 h. Organogenesis started after 40 h and hatching occurred after 110 h. Immediately after collection, 51.6 F 36.6% (mean F S.D., n = 14) of the eggs were floating. Of these floating eggs, 42.8 F 31.6% survived just before hatching. The eggs

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had a wet weight of 702 F 94 mg, a dry weight of 7.34 F 2.57 mg, a diameter of 1.07 F 0.02 mm, and a lipid vesicle diameter of 0.27 F 0.02 mm. These parameters were constant during egg development and did not differ significantly between viable and nonviable eggs. 3.2. Biochemical changes during embryonic development From the first cleavage stages (4– 32 cell) to the embryo stage, acid phosphatase activity increased constantly (Table 2). Activities of aspartate aminotransferase, Ca2 +ATPase, Mg2 +-ATPase, and pyruvate carboxylase increased from the stage of blastopore closure to the embryo stage (Table 2). Creatinine kinase was not detectable before blastopore closure (Table 2). Activities of adenylate kinase, allanine aminotransferase, h-D-glucuronidase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, Na+/K+-ATPase, proteases, phosphofructokinase, pyruvate kinase, succinate dehydrogenase, and transaldolase were constant throughout the investigated period (Table 3). Levels of DNA, glucose, galactose, and of free and total monosaccharides increased constantly (Table 4). Levels of fructose increased and levels of NADH decreased from first

Table 2 Changes in enzyme activities of eggs of S. aurata during embryogenesis Enzymes

4 – 32 cell

Morula

Blastopore closure

Embryos before hatching

Acid phosphatase nmol/min/egg nmol/min/mg protein

0.27 F 0.09a 2.84 F 0.72a

0.40 F 0.10a,b 3.33 F 0.79a,b

0.39 F 0.18a,b 3.70 F 1.77a,b

0.43 F 0.18b 4.03 F 1.56b

Aspartate aminotransferase nmol/min/egg 2.24 F 1.69a nmol/min/mg protein 23.4 F 2.7a

2.89 F 2.31a 25.3 F 4.8a

2.52 F 2.17a 25.5 F 7.3a

6.87 F 2.32b 219.7 F 27.7b

Creatinine kinase nmol/min/egg nmol/min/mg protein

0.00 F 0.00a 0.00 F 0.00a

0.00 F 0.00a 0.00 F 0.00a

0.99 F 0.18b 7.04 F 1.22b

1.04 F 0.31b 6.48 F 0.81b

Ca2 + -ATPase nmol/min/egg nmol/min/mg protein

24.5 F 7.5a 78.6 F 17.8a

31.9 F 6.9a 73.9 F 10.6a

47.8 F 8.7a 188.0 F 13.2a

218.2 F 10.1b 510.6 F 43.6b

Mg2 + -ATPase nmol/min/egg nmol/min/mg protein

3.8 F 1.4a 36.0 F 5.6a

3.8 F 2.2a,b 37.2 F 2.7a,b

4.0 F 1.9a,b 35.1 F 2.0a,b

7.6 F 2.6b 44.5 F 4.8b

Pyruvate carboxylase nmol/min/egg nmol/min/mg protein

0.38 F 0.15a 3.66 F 1.41a

0.35 F 0.24a 3.29 F 2.25a

0.36 F 0.21a 3.41 F 2.24a

1.39 F 0.78b 13.33 F 8.25b

Data are mean F S.D., n = 14. For each parameter, data were subjected to ANOVA with subsequent Tukey’s btest; values superscripted by the same letter are not significantly different, p>0.05.

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Table 3 Enzymatic activities of eggs of S. aurata, which were unchanged in the investigated stages of embryogenesis Enzymes

nmol/min/mg protein

nmol/min/egg

Adenylate kinase Allanine aminotransferase Glucose-6-phosphate dehydrogenase h-D-glucuronidase Glutamate dehydrogenase Lactate dehydrogenase Na+, K+-ATPase Malate dehydrogenase Phosphofructokinase Pyruvate kinase Succinate dehydrogenase Proteases (BAEE substrate, without CaCl2) Proteases (BAEE substrate, 0.6 mM CaCl2) Proteases, casein substrate Transaldolase

8.90 F 3.46 0.252 F 0.356 0.35 F 0.49 1.21 F 0.88 9.76 F 5.82 25.4 F 18.6 108.0 F 58.5 343.3 F 127.7 15.19 F 6.12 2.05 F 2.00 0.044 F 0.036 0.065 F 0.026 0.054 F 0.053 8.82 F 5.62 4.98 F 1.16

0.93 F 0.74 0.044 F 0.063 0.049 F 0.040 0.77 F 0.45 1.02 F 0.62 3.09 F 2.62 20.97 F 13.16 37.38 F 12.67 0.15 F 0.19 0.35 F 0.34 0.006 F 0.005 0.009 F 0.010 0.009 F 0.008 2.30 F 1.91 2.10 F 0.53

Values are mean F S.D., n = 56.

cleavage stages to the morula stage. Acetyl-CoA, amino acids, protein, ATP, ADP, AMP, adenylate energy charge, glucose-6-phosphate, free inorganic phosphate, lactate, and sialic acid remained constant (Table 5). 3.3. Changes between viable and nonviable eggs Eggs were defined as nonviable when they lost buoyancy and sank to the bottom of the incubator. Those eggs stopped development, and in some cases, they also showed signs of abnormal development. Viable eggs had significantly higher activities of malate dehydrogenase, glutamate dehydrogenase, pyruvate carboxylase and transaldolase (Table 6), and higher levels of amino acids, DNA, fructose, total monosaccharides and a higher adenylate energy charge than nonviable eggs during the whole investigated period (Table 7). Floating eggs had significantly lower activities of acid

Table 4 Changes in metabolite levels of eggs of S. aurata during embryogenesis Metabolites

4 – 32 cell

Morula

Blastopore closure

Embryos before hatching

DNA, ng/egg Fructose, nmol/egg Galactose, nmol/egg Glucose, nmol/egg NADH, nmol/egg Free monosaccharides, nmol/egg Total amount of monosaccharides, nmol/egg

21.1 F 2.7a 0.74 F 0.38a 1.37 F 0.33a 4.80 F 0.28a 1.27 F 0.56a 2.33 F 0.20a 224 F 86a

24.2 F 5.5a,b 3.37 F 0.50b 8.84 F 1.41b 6.43 F 1.53a,b 0.42 F 0.22b 4.10 F 0.88a,b 453 F 283b

27.6 F 4.4b 4.10 F 1.46b 9.30 F 1.57b,c 7.70 F 1.54a,b 0.22 F 0.34b 6.05 F 0.84b,c 500 F 57b

32.6 F 4.9c 3.06 F 0.84b 14.18 F 1.32c 13.3 F 2.96b 0.26 F 0.30b 7.31 F 1.71c 1103 F 694c

Data are mean F S.D., n = 14. Values superscripted by the same letter are not significantly different, p>0.05.

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Table 5 Metabolite levels in eggs of S. aurata, which were unchanged in the investigated stages of embryogenesis Metabolites Acetyl-CoA, nmol/egg Amino acids, nmol/egg ATP, nmol/egg ADP, nmol/egg AMP, nmol/egg Glucose-6-phosphate, nmol/egg Inorganic phosphate, nmol/egg Lactate, nmol/egg Sialic acid, ng/egg Adenylate energy charge Protein, ng/egg

10.86 F 5.32 36.5 F 18.0 5.38 F 1.70 5.05 F 3.36 0.41 F 0.22 0.66 F 0.31 57.29 F 24.93 4.06 F 3.09 60.9 F 46.5 0.55 F 0.11 94.57 F 20.72

Values are mean F S.D., n = 56.

phosphatase and higher levels of acetyl-CoA, glucose, galactose, free monosaccharides, and inorganic phosphate than nonfloating eggs in all investigated stages with exception of the embryo stage (Table 7). ATP levels did not significantly differ between floating and nonfloating eggs but were highly variable in nonfloating eggs. Phospholipid, total lipid, calcium, and magnesium were measured only for the first cleavage stages and the morula stage. Phospholipid levels were significantly higher while calcium and magne-

Table 6 Differences in enzyme activities between floating and nonfloating eggs of S. aurata Enzymes

4 – 32 cell

Morula

Blastopore closure

Embryos before hatching

Acid phosphatase, nmol/min/egg Floating 0.27 F 0.09a Nonfloating 0.43 F 0.13b

0.28 F 0.09a 1.00 F 0.28b

0.40 F 0.18a 0.71 F 0.37b

0.42 F 0.18a 0.61 F 0.15a

Glutamate dehydrogenase, nmol/min/egg Floating 1.29 F 1.57a Nonfloating 0.20 F 0.13b

1.46 F 0.41a 0.17 F 0.28b

1.28 F 0.54a 0.69 F 0.54b

1.66 F 0.19a 1.14 F 0.16b

Malate dehydrognase, nmol/min/egg Floating 42.2 F 13.8a Nonfloating 28.3 F 7.8b

46.7 F 10.9a 26.0 F 8.6b

41.5 F 10.6a 24.1 F 9.39b

43.3 F 12.7a 24.1 F 8.4b

Transaldolase, nmol/min/mg protein Floating 2.75 F 2.78a Nonfloating 0.34 F 0.31b

2.67 F 1.23a 0.43 F 0.72b

2.16 F 1.28a 0.78 F 0.58b

2.26 F 1.12a 0.51 F 0.63b

Pyruvate carboxylase, nmol/min/egg Floating 0.38 F 0.15a Nonfloating 0.05 F 0.07b

0.35 F 0.24a 0.08 F 0.11b

0.36 F 0.21a 0.18 F 0.12b

1.39 F 0.78a 0.20 F 0.28b

Values for floating and nonfloating eggs from the same development stage were compared by student’s t-test. Values superscripted by the same letter are not significantly different.

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Table 7 Differences in metabolite levels between floating and nonfloating eggs of S. aurata Metabolites

4 – 32 cell

Morula

Blastopore closure

Embryos before hatching

Acetyl-CoA, nmol/egg Floating 19.6 F 5.6a Nonfloating 12.3 F 3.5a

11.0 F 3.9a 5.6 F 2.7b

16.2 F 7.2a 5.5 F 2.6b

17.2 F 4.0a 15.5 F 5.1a

Adenylate energy charge Floating 0.75 F 0.20a Nonfloating 0.41 F 0.19b

0.87 F 0.11a 0.54 F 0.14b

0.72 F 0.09a 0.60 F 0.11b

0.73 F 0.07a 0.51 F 0.14b

Amino acids, nmol/egg Floating 19.8 F 6.7a Nonfloating 8.88 F 5.2b

37.2 F 10.9a 11.3 F 4.1b

41.9 F 16.8a 15.1 F 10.5b

31.7 F 9.7a 20.6 F 8.7b

DNA, lg/egg Floating Nonfloating

21.1 F 2.7a 2.4 F 0.3b

24.2 F 5.5a 6.9 F 0.9b

27.6 F 4.4a 6.9 F 3.9b

32.6 F 4.9a 12.8 F 5.3b

Free monosaccharides, nmol/egg Floating 2.3 F 0.2a Nonfloating 1.2 F 1.0b

4.1 F 0.8a 2.4 F 0.6b

6.0 F 0.8a 2.9 F 1.1b

7.3 F 0.7a 5.5 F 0.9a

Total monosaccharides, nmol/egg Floating 224 F 86a Nonfloating 196 F 98b

453 F 283a 361 F 265b

500 F 57a 365 F 120b

1103 F 694a 950 F 5125b

Fructose, nmol/egg Floating Nonfloating

0.74 F 0.38a 0.22 F 0.10b

3.37 F 0.50a 1.08 F 0.50b

4.10 F 1.46a 0.36 F 0.25b

3.06 F 0.84a 1.57 F 0.89b

Glucose, nmol/egg Floating Nonfloating

4.80 F 0.28a 0.32 F 0.55b

6.43 F 1.53a 1.59 F 0.99b

7.70 F 1.54a 1.58 F 0.11b

13.3 F 2.96a 3.16 F 1.90a

Inorganic phosphate, nmol/egg Floating 63.5 F 19.2a Nonfloating 16.9 F 14.8b

60.9 F 25.0a 23.3 F 9.45b

71.0 F 29.8a 26.7 F 9.2b

80.2 F 21.1a 71.5 F 21.3a

Galactose, nmol/egg Floating Nonfloating

1.38 F 0.73a 0.31 F 1.01b

8.84 F 4.41a 3.80 F 3.04b

8.31 F 3.57a 5.25 F 0.74b

9.18 F 4.32a 9.41 F 0.74a

Phospholipid, ng/egg Floating 3.80 F 2.94a Nonfloating 0.84 F 1.20b

4.53 F 5.05a 0.72 F 1.32b

n.i. n.i.

n.i. n.i.

Calcium, nmol/egg Floating Nonfloating

1.6 F 1.9a 12.9 F 13.0b

n.i. n.i.

n.i. n.i.

1.6 F 1.6a 13.6 F 12.1b

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Table 7 (continued ) Metabolites

4 – 32 cell

Magnesium, nmol/egg Floating 2.2 F 2.4a Nonfloating 27.9 F 14.8b

Morula

Blastopore closure

Embryos before hatching

3.29 F 4.0a 24.01 F 13.0b

n.i. n.i.

n.i. n.i.

Values for floating and nonfloating eggs from the same development stage were compared by student’s t-test. Values superscripted by the same letter are not significantly different. n.i. = not investigated.

sium levels were lower in the floating eggs compared with nonfloating eggs (Table 7). Total lipid levels (31.94 F 14.47 ng/egg) were similar in floating and nonfloating eggs. All other analyzed compounds did not significantly differ between floating and nonfloating eggs.

4. Discussion Egg samples collected out of the spawning tanks during natural spawning were at similar stages of development, as most eggs were in the first cleavage stages. This indicates that spawning occurred simultaneously. Generally, during mass spawning, unspawned fish are stimulated by the spawning due to pheromones in milt, eggs, or urine (Scott and Vermeirssen, 1994). The eggs used for analysis were also a mixture from different individuals. When using such an egg pool for repeated subsampling, there exists the risk that eggs from various individuals are sampled in different frequencies. As biochemical composition of eggs differs generally between individual batches, this could reduce the accuracy of the analyses as individual effects might override those of the general population. However, the clear changes found in egg biochemistry in relation to the development stages and to the egg viability indicated that the influence of this latter possibility was low or negligible. The present data are similar to other studies for aspartate aminotransferase, free amino acids, phospholipids (P. major—Seoka et al., 1996), protein, and DNA (S. aurata—Pascual and Yu´fera, 1993; Carnevali et al., 2001). Therefore, the egg composition of the used S. aurata brood stock was comparable to other S. aurata brood stocks already investigated and to the egg composition of closely related species. 4.1. Energy metabolism During development, the viable eggs of S. aurata had a balanced and stable metabolism. In all stages of development, energy compounds were present at sufficient high levels and did not drop to critical low levels. This was demonstrated by the constant levels of adenosine nucleotides and acetyl-CoA, and the constant adenylate energy charge. The adenylate energy charge decreased in the eggs of the red drum (Sciaenops ocellata), possibly due to rapid hatching in only about 24 h (Vetter et al., 1983). Mg2 +-dependent ATPase, which catalyses the terminal transphosphorylation reaction by coupling the membrane-driven electrochemical gradient to ATP synthesis, increased. Generally, respiratory activity increases in developing fish eggs (Rombough, 1988; Rønnestad et al.,

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1998). Also, the decrease in NADH levels from the first cleavage stages to the morula stage may be related to the activity increase of tricarboxylic acid cycle and respiration. Contrary, in nonviable eggs, the energy metabolism became unstable as the adenylate energy charge, the levels of acetyl-CoA, and the activities of malate dehydrogenase all decreased. This might be due to inadequate levels of primary energy resources (e.g. amino acids, see below) or to a low metabolic rate of the energy producing pathways. 4.2. Osmoregulation When fish eggs are released into water, the oolemma reduces its permeability to avoid water fluxes following the osmotic gradients (Alderdice, 1988). The present data revealed that during development, the eggs of S. aurata maintained their ability for osmoregulation as the activity of Na+/K+-ATPase remained constant and the activity of Ca2 +-ATPase increased. The Ca2 +-ATPase has additional importance in transporting intracellular calcium between the endoplasmic reticulum and cytoplasm, and therefore also plays a role in cell regulatory processes, possibly to control complex embryonic differentiation processes. Nonviable eggs contained nonphysiological high levels of magnesium and calcium ions. As the activities of Na+/K+-dependent and Ca2 +-dependent ATPases were similar to viable eggs, it is suggested that they diffused into the eggs from the seawater. Therefore, ion influx should be related to changed composition and permeability of the oolemma. This hypothesis was supported by the fact that levels of phospholipids also decreased in nonviable eggs. S. aurata eggs contain mainly three types of phospholipids, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin (Mourente and Odriozola, 1990), which all are membrane components. The decreased phospholipid levels in nonviable eggs may be related to the increased acid phosphatase activity, as this enzyme is involved in the degeneration of phospholipids during lytic processes (Rawn, 1983). Seoka et al. (1996) also found low phospholipid levels as typical for nonviable P. major eggs. 4.3. Carbohydrate metabolism Developing eggs of S. aurata have the enzymatic capabilities for glycolysis (as indicated by activities of phosphofructokinase and pyruvate kinase), for gluconeogenesis (pyruvate carboxylase, glucose-6-phosphate dehydrogenase), and for the pentose phosphate path (transaldolase, transformation of monosaccharides in each other). The latter pathway has particular importance for delivering (desoxy) ribose for nucleic acid synthesis (Rawn, 1983). The present study and Carnevali et al. (2001) have shown a steady increase in nucleic acids during embryogenesis. In the present study, the rate of gluconeogenesis was high. The levels of free and of total monosaccharides (after acid treatment to split polysaccharide bonds) and the activity of pyruvate carboxylase and aspartate aminotransferase increased during egg development. Pyruvate carboxylase catalyses the intra-mitochondrial conversion of pyruvate to oxalacetate. Oxalacetate is transaminated into aspartate by aspartate aminotransferase, transported out of the mitochondria, and re-transaminated into oxalacetate for further glucogenic reactions (Rawn, 1983). Therefore, monosaccharides were not liberated from

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storage depots (e.g. glycogen) but synthesized de novo. Aside from their importance for nucleic acid synthesis, monosaccharides also could have importance as osmotic active compounds. Generally, for eggs, there are two mechanisms for osmotic regulation. Protein cleavage and liberation of free amino acids during oocyte final maturation regulates the rate of water uptake and specific gravity of the ovulated egg (Craik and Harvey, 1987). Also, monosaccharides can contribute to oocyte hydration. The cortical reaction and subsequent water influx between the environment and perivitelline space are osmotic regulatory processes, which occur after the egg has been released into water (Alderdice, 1988). The cortical vesicles contain mainly carbohydrates (Alderdice, 1988). Immediately after eggs have been released, water can flow into the egg as the permeability of the oolemma is still high. Aside from amino acids, monosaccharides could be the osmotic active compounds in the eggs. None of the analyzed carbohydrates decreased, indicating that they did not play a role as an energy source during egg development. In the red drum, the only species where carbohydrate metabolism was studied in detail, glycogen decreased during egg development. Glycolysis has been described as the most important energy resource for fish eggs during early development (Terner, 1979). Nonviable eggs revealed very significant changes in carbohydrate metabolism in comparison with viable eggs. Levels of all assayed sugars as well as activities of pyruvate carboxylase and transaldolase decreased. Therefore, gluconeogenesis and the pentose phosphate path were reduced. In many cases, the levels of monosaccharides reached viability limiting levels as they dropped towards zero. Levels of monosaccharides that were too low led to a reduced nucleic acid synthesis rate, which has been established by the data on DNA levels in this paper. Low rate of nucleic acid synthesis rate also was described in several other studies (Carnevali et al., 2001). Furthermore, low monosaccharide levels could lead to reduced osmoregulation ability and to a loss of egg buoyancy. 4.4. Protein and amino acid metabolism The constant protein levels measured during egg development are in agreement with other studies on D. labrax (Rønnestad et al., 1998), P. major (Seoka et al., 1996), and S. aurata (Carnevali et al., 2001). Also, constant proteolytic activities are conform to earlier studies (Carnevali et al., 2001). An increase in egg internal cathepsin L activity was observed immediately after fertilization (Carnevali et al., 2001). In the present study, free amino acids showed only a slight and nonsignificant tendency to decrease, while a clear decrease was observed for the same species by Rønnestad et al. (1994) and also for several other species (e.g. D. labrax—Rønnestad et al., 1998). Possibly, the more specific and sensitive analyses methods used by Rønnestad et al. (1994, 1998) were better suited to detect changes. Generally, free amino acids are energy resources during egg development (Rønnestad et al., 1994, 1998), and as osmotic active compounds, they also regulate egg buoyancy (Thorsen and Fyhn, 1996). In the present study, amino acid catabolism by transamination and oxidative desamination was demonstrated for S. aurata eggs by the occurrence of aspartate aminotransferase and glutamate dehydrogenase activities. The sharp increase in aspartate aminotransferase activity in the embryonic stages could be indicative of the onset of intensive amino acid catabolism. As stated above, it is also related to gluconeogenesis.

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The levels of free amino acids decreased in nonviable eggs in agreement with other studies (Seoka et al., 1996; Nocillado et al., 2000). These reduced levels of amino acids affect egg buoyancy, protein synthesis, and energetic status. The levels of free phosphate also were lower in nonviable eggs. This could indicate a low cleavage rate of phosphoprotein during oocyte final maturation (Craik and Harvey, 1987). Inorganic phosphate is required for synthesis of nucleic acids, glycolysis, and ATP synthesis (Rawn, 1983). Also, the glutamate dehydrogenase activity was lower in nonviable then in viable eggs, indicating that oxidative desamination was reduced. 4.5. Lytic enzymes Acid phosphatase and h-D-glucuronidase are lytic enzymes, which occur in lysosomes (Rawn, 1983). While h-D-glucuronidase remained constant, acid phosphatase increased constantly during egg development and was also higher in nonviable eggs. However, for nonviable eggs, it could indicate autolysis, for viable eggs, the cause is unclear.

5. Conclusions While the floating and nonfloating eggs could not be distinguished on base of morphological parameters, there existed very clear differences in the biochemistry and metabolism. Nonviable eggs had an altered biochemical composition, and activities of several important enzymes were reduced. Egg overmaturation, the retention of ovulated eggs in the ovary, is a factor that leads to changes in egg biochemistry and metabolism (Lahnsteiner, 2000). However, this parameter does not play a role for naturally spawning brood stocks, and therefore, the alterations must arise during oogenesis or during the final maturation of the oocyte. This indicates that in S. aurata, not all eggs reach a full grade of ‘‘biochemical’’ maturity. The reasons are unknown and may depend on genetic deficits or some suboptimal parameters under hatchery conditions.

Acknowledgements ¨ sterreichischer Forschungsfonds’’ (FWF), grant no. This work was supported by ‘‘O P15029.

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