- Email: [email protected]
Egg quality criteria in common dentex (Dentex dentex)
Aquaculture 260 (2006) 232 – 243 www.elsevier.com/locate/aqua-online
Egg quality criteria in common dentex (Dentex dentex) G. Giménez a , A. Estévez a,⁎, F. Lahnsteiner b , B. Zecevic b , J.G. Bell c , R.J. Henderson c , J.A. Piñera d , J.A. Sanchez-Prado d a
d
Centro de Acuicultura-IRTA, Ctra. Poble Nou Km 6, 43540 Sant Carles de la Rápita, Tarragona, Spain b Insitute for Zoology, University of Salzburg, Hellbrunnerstrasse 34, A-5020, Salzburg, Austria c Laboratory of Nutrition, Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, Scotland, UK Departamento de Biología Funcional. Laboratorio de Genética Acuícola, Facultad de Medicina, IUBA. Universidad de Oviedo, Julián Clavería s/n, 33071 Oviedo, Spain Received 4 April 2006; accepted 20 June 2006
Abstract The spawning quality, in terms of hatching rate, larval mortality at 3 and 5 days post-hatching (dph) and day of total mortality of two broodstock groups of common dentex was evaluated for 1 month in 2005. Several biochemical parameters including total lipid content, lipid class and fatty acid composition, carbohydrate content and metabolic enzyme activities were analysed in all the egg batches collected. Comparison was carried out between low- (mortality at 3 dph higher than 35%) and high-quality (mortality at 3 dph lower than 10%) batches. No differences were observed in lipid content and/or lipid class and fatty acid composition although a slightly higher content of neutral lipids was detected in high-quality batches. However, significant differences were obtained regarding carbohydrate composition and the activity of enzymes such as alkaline phosphatase and pyruvate kinase being higher in low-quality egg batches. © 2006 Elsevier B.V. All rights reserved. Keywords: Egg quality; Lipids; Carbohydrates; Enzymes; Common dentex
1. Introduction Although common dentex (Dentex dentex) has been considered as a promising species for aquaculture in the Mediterranean coast, for a long time, due to its high market price and growth rate, its culture under intensive conditions is still a problem and not enough numbers of juveniles are produced for ongrowing in sea cages. Common dentex is a batch spawner that usually spawns at nightfall or early morning (Abellan, 2000) for a long period of up to 40–50 days (Giménez and Estévez, personal observation) and the number and size of the eggs ⁎ Corresponding author. Tel.: +34 977745427; fax: +34 977744138. E-mail address: [email protected] (A. Estévez). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.06.028
released in each batch varies over the spawning season as well as the quality between egg batches. One of the main problems with this very carnivorous and cannibalistic (Efthimiou et al., 1994) species is the high mortality rate during the larval phase, especially in the early feeding period with rotifers (Glamuzina et al., 1989). The need for a precise estimation of egg quality is therefore of paramount importance in order to clarify if the low survival rate during early larval rearing is due to the initial viability of the larvae or the quality of the eggs. Hatchery production can be optimized by starting the production cycle with high-quality eggs giving high egg survival and hatching rates and robust larvae with better growth, survival and stress resistance. Identification of a method to reliably discriminate between low- and high-quality egg batches
G. Giménez et al. / Aquaculture 260 (2006) 232–243
will be vital to reduce hatchery production costs by ensuring time and resources are not wasted on egg batches with low survival and/or performance potential. Several morphological (Kjorsvik et al., 1990; Thorsen et al., 2003) and biochemical parameters such as lipids (Sargent, 1995; Bell and Sargent, 2003; Tveiten et al., 2004), amino acids (Ronnestad and Fyhn, 1993) or vitamins (Ronnestad et al., 1997, 1999; Maeland et al., 2003) have been considered as indicators of egg quality. Recently, several compounds and enzymes involved in carbohydrate metabolism have been identified as good markers for egg quality in other Sparidae species such as Sparus aurata and Puntazzo puntazzo (Lahnsteiner and Patarnello, 2004a). Lipids and fatty acids have been also considered in order to compare egg quality from wild and captive broodstocks (Rodriguez et al., 2004; Salze et al., 2005). In this paper, we have measured total lipid content, lipid class and fatty acid composition, as well as enzymes and mono- and disaccharides of common dentex eggs taken at the same developmental stage from different batches of 2 broodstock groups. Both broodstock groups were maintained under the same environmental and feeding conditions as a tool to evaluate initial egg quality. Hatching and daily larval mortality rates, time to yolk resorption and time to 100% mortality were also assessed using 96-well EIA plates. 2. Materials and methods 2.1. Materials The daily spawnings of 2 broodstock groups of common dentex were assessed for a month during 2005. The broodstock groups were composed of 5 females:8 males and 4 females:6 males, respectively (Table 1). All the fish were conveniently tagged by the use of pit tags placed in the muscle near the dorsal fin and identified by the use of microsatellite DNA markers previously isolated and used for other Sparidae. The fish were stocked in two 4000-l circular tanks connected to a recirculation unit (Carbó et al., 2002) and fed semi-moist pellets in which fresh minced fish (Boops boops), fish meal (Skretting, Spain), a fish oil concentrate (TG0525, Croda, UK) and vitamin premix (Skretting, Spain) were mixed in a 40:40:15:2 ratio, respectively. Photoperiod was maintained at 14 h L:10 h D during the spawning season to allow natural egg release. Everyday, the eggs were removed from the outside collector (a cylindro-conical tank containing a 500 μm net immersed in a 100-l holding tank connected to the recirculation system), separated into floating and non-floating eggs in a measuring cylinder. Floating eggs were isolated, thoroughly washed and subsequently kept in UV-treated
233
Table 1 Description of common dentex broodstock used in the study Broodstock 1
Broodstock 2
Pit tag code
Sex
Weight (g) Pit tag code Sex
Weight (g)
37CD 1E43 5530 A4DF 1D38 EA2A 7FC2 108D 1AE3 340F 8337 1140 Ratio F:M Density (kg/m3)
M M M M M M F F F F F M 1:1.4 3.82
985 1376 1485 1300 735 965 1182 1430 1470 1150 2015 1180
1600 1920 1510 1700 2298 1560 1250 1395 1115 789
AE97 2F8E 118F 8D1D 0668 DB32 A953 ACA5 3F5E 22B3
M F F F M M M M F M
1:1.2 3.78
seawater, observed under a stereomicroscope to assess the morphology, fertilization rate and embryological stage and transferred to the laboratory where a sample of the eggs was plated onto a 96-well cell culture multi-well plate (Nunc) as in Shields et al. (1997). One egg was placed in each well using a Pasteur pipette and each well then filled with autoclaved seawater and incubated in darkness at 19 °C in a refrigerated incubator. Sub-samples of the eggs were rinsed in distilled water and placed in capped vials with chloroform/methanol (2:1, v/v), covered with nitrogen gas and kept at −20 °C for lipid analysis, and in Eppendorf tubes filled with either 0.6 ml 100 mM/l Tris buffer pH 7.5 and 0.6 ml 3 mol/l perchloric acid kept at −80 °C for the analysis of enzymes and acid-stable metabolites, respectively (Lahnsteiner and Patarnello, 2004b). Once per week, the eggs were incubated in a 35 l tank for 2–3 days, a sample of the larvae obtained were fixed in 100% ethanol and subjected to DNA extraction. Genomic DNA was extracted following the standard Chelex@100 (Biorad) resin protocol with Proteinase K (Walsh et al., 1991). PCRs (polymerase chain reaction) for the nine microsatellites mentioned above were carried out following the published conditions (Bernardo et al., 2004; De la Herrán et al., 2005; Piñera et al., 2006) and the amplified fragment sizes were determined using an OpenGene Sequencing System (BayerHealth, Inc). Once the microsatellite genotypes of all broodstocks and offsprings were profiled, each offspring was assigned to the most probable parental couple using Cervus software (Marshall et al., 1998). For the EIA plate samples, the eggs/larvae were inspected and counted on a daily basis in order to calculate the hatching rate, percentage survival after hatching, mortality rate at days 3 and 5 post-hatching (dph), percentage of
234
G. Giménez et al. / Aquaculture 260 (2006) 232–243
Fig. 1. Volume of sinking and floating eggs as well as parental female identified in some of the egg batches, and presumable spawning time for each identified female. G and B denote good and bad egg batches. Contribution of each female (%) in some of the genetically identified batches is also indicated.
deformities and time to 100% mortality. Having in mind that first feeding and point of no return occur in this species at days 3 and 7, respectively, we have considered, and analysed more closely, high-quality batches (good) of eggs being those with a mortality rate less than 10% between 3
and 5 dph and low-quality batches (bad) of eggs being those with mortality rates greater than 35% at 3 dph. Total lipids were extracted from samples by homogenisation in chloroform/methanol (2:1, v/v) containing 0.01% butylated hydroxytoluene (BHT) (Folch et al.,
G. Giménez et al. / Aquaculture 260 (2006) 232–243
235
Table 2 Hatching rate, survival, mortality at 3 and 5 dph, last day of survival, deformities and parental female of the eggs obtained from broodstock 1 and plated on 96-well EIA plates Broodstock 1 Spawning date
Hatching rate (%)
Hatching survival (%)
Mortality 3 dph (%)
Mortality 5 dph (%)
Day 100% mortality
Deformities (%)
27/04/2005
97.92
100.00
57.61
72.83
10
0
28/04/2005 29/04/2005 30/04/2005 02/05/2005 03/05/2005 04/05/2005 05/05/2005 06/05/2005
85.26 57.29 92.71 92.93 83.51 96.88 100.00 100.00
100.00 94.55 84.88 96.74 91.36 92.86 98.95 98.92
56.79 16.36 34.88 21.74 40.74 22.62 9.47 6.45
69.14 16.36 38.37 27.17 40.74 26.19 11.58 11.83
9 9 10 10 11 11 11 11
0 0.00 4.17 1.01 7.22 2.08 2.11 0.00
07/05/2005 08/05/2005
95.83 90.53
81.61 75.00
24.14 35.00
24.14 35.00
11 10
10.42 9.47
09/05/2005 11/05/2005 12/05/2005 13/05/2005 17/05/2005 23/05/2005 26/05/2005 Average S.D.
96.84 98.96 91.67 100.00 93.75 96.88 81.63 91.81 10.28
94.57 96.84 100.00 97.92 83.95 96.74 93.51 93.24 7.25
8.70 3.16 9.09 4.17 32.10 36.96 27.27 24.85 16.85
29.35 6.32 9.09 4.17 33.33 42.39 27.27 29.18 19.27
10 10 11 11 10 10 11 10.33 0.69
Female code and % contribution 7FC2 97%, 340F 8%
2.11 0.00 7.14 0.00 0.00 0.00 2.04 2.65 3.52
340F 63%, 8337 37% 7FC2 50%, 340F 50%
340F 100%
High-quality eggs are denoted by bold + italic letters, low-quality eggs by bold letters only.
Table 3 Hatching rate, survival, mortality at 3 and 5 dph, last day of survival, deformities and parental female of the eggs obtained from broodstock 2 and plated on 96-well EIA plates Broodstock 2 Spawning date
Hatching rate (%)
Hatching survival (%)
Mortality 3 dph (%)
Mortality 5 dph (%)
Day 100% mortality
Deformities (%)
28/04/2005 29/04/2005 30/04/2005 02/05/2005 03/05/2005 04/05/2005 05/05/2005 06/05/2005 07/05/2005 08/05/2005 09/05/2005 12/05/2005 13/05/2005 23/05/2005 26/05/2005 27/05/2005 Average S.D.
24.21 88.54 94.79 95.51 100.00 93.68 98.96 97.89 95.83 97.83 91.67 95.83 95.83 57.73 71.43 48.45 84.26 22.20
82.61 97.62 95.56 91.67 100.00 96.63 100.00 98.92 93.48 95.56 96.59 90.70 92.13 98.21 78.46 76.60 92.80 7.38
34.78 7.14 21.11 19.05 8.79 3.37 3.16 1.08 7.61 7.78 13.64 16.28 17.98 41.07 41.54 40.43 17.80 14.23
34.78 10.71 23.33 26.19 13.19 4.49 3.16 2.15 7.61 7.78 21.59 17.44 17.98 50.00 49.23 46.81 21.03 16.36
8 9 10 10 10 11 10 10 11 10 9 10 10 10 11 10 9.94 0.77
0.00 0.00 0.00 6.74 1.03 2.11 0.00 0.00 1.04 1.09 1.04 2.08 2.08 0.00 5.10 1.03 1.46 1.93
High-quality eggs are denoted by bold + italic letters, low-quality eggs by bold letters only.
Female code and % contribution 3F5E 80%, 118F 20%
3F5E 80%, 118F 20% 3F5E 100% 3F5E 70%, 118F 30%
3F5E 20%, 118F 20%, 8D1D 60%
236
G. Giménez et al. / Aquaculture 260 (2006) 232–243
1957). Lipid class composition was determined by highperformance thin-layer chromatography (HPTLC). Approximately 10 μg of lipid was applied as a 2 mm streak and the plate developed to two-thirds distance with methyl acetate/isopropanol/chloroform/methanol/0.25% aqueous KCl (25:25:25:10:9, by vol.), to separate polar lipid classes, and then fully developed with isohexane/ diethyl ether/acetic acid (85:15:1, by vol.). Lipid classes were visualised by charring at 160 °C for 15 min after spraying with 3% (w/v) aqueous cupric acetate containing 8% (v/v) phosphoric acid and quantified by densitometry using a Camag 3 TLC Scanner (Camag, Muttenz, Switzerland) and winCATS software (Henderson and Tocher, 1992). The identities of individual lipid classes were confirmed by comparison with authentic standards. Fatty acid methyl esters (FAME) were prepared from total lipid by acid-catalysed transesterification using 2 ml of 1% H2SO4 in methanol plus 1 ml toluene (Christie,
1982), and FAME were then extracted and purified (Tocher and Harvie, 1988). FAME were separated and quantified by gas–liquid chromatography (Fisons GC8600, Carlo Erba, Milan, Italy) using a 30 m × 0.32 mm capillary column (CP wax 52CB; Chrompak, London, U.K) utilizing on-column injection at 50 °C and flame ionization detection at 250 °C. Hydrogen was used as carrier gas and temperature programming was from 50 °C to 180 °C at 40 °C min− 1 and then to 225 °C at 2 °C min− 1. Individual methyl esters were identified by comparison to known standards (Ackman, 1980). Biochemical samples for enzyme and acid-stable metabolites analyses were thawed at room temperature and homogenized. The extracts were centrifuged at 1500×g for 10 min at 4 C°. The supernatants were used for analysis by colorimetry and UV spectrophotometry following the methods described by Lahnsteiner and Patarnello (2004b). For the determination of egg wet
Table 4 Biochemical compounds (content per egg in dry weight) analysed in common dentex eggs (N = 36) and simple regression models obtained with hatching rate and mortality at 3 and 5 dph Mean
S.D.
Enzymes (nmol/min/egg) Adenylate kinase Pyruvate kinase
0.59 2.72
0.41 1.94
Malate dehydrogenase Glucose-6-phosphate dehydrogenase Transaldolase Aspartate aminotransferase Succinate dehydrogenase Acid phosphatase Alkaline phosphatase
10.17 0.46 1.14 2.48 11.90 15.68 0.50
3.55 0.61 1.50 3.98 13.90 5.72 0.12
Glucose-6-phosphatase
0.18
0.20
Metabolites (nmol/egg) Free monosaccharides Total monosaccharides Free ketose Total ketose Sialic acid Free ribose Total ribose Free heptose Total heptose Free 6-deoxyhexose Total 6-deoxyhexose Glucose-6-phosphate Fructose-6-phosphate ATP Glucose
129.33 411.15 23.31 19.54 29.83 22.66 25.71 290.60 288.96 631.63
42.79 158.67 24.90 12.95 19.50 10.62 14.86 590.75 684.37 175.73
1225.24 14.65 4.26 9.61 7.51
605.51 13.87 4.87 7.23 7.39
H: hatching rate, M3: % mortality at 3 dph, M5: % mortality at 5 dph.
Regression
r2
P
H = 28.34x − 7.883x2 + 0.64x3 + 64.821 M3 = − 19.279x + 4.973x2 − 0.407x3 + 40.285 M5 = − 10.916x + 1.053x2 + 43.726
0.306 0.254 0.245
0.009 0.030 0.013
M3 = 60.933x − 9.671 M5 = 66.879x − 8.902
0.200 0.184
0.008 0.011
H = 1.90x − 0.01x2 + 0.000015x3 − 23.75
0.352
0.003
H = 5.196x − 0.139x2 + 0.001x3 + 32.716
0.253
0.027
M3 = 0.047x − 8.248 M5 = 0.065x − 15.7
0.273 0.395
0.002 0.000
H = −1.503x + 99.012 M3 = 1.063x + 18.525
0.490 0.214
0.001 0.053
G. Giménez et al. / Aquaculture 260 (2006) 232–243
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 °C for 24 h and re-weighed. 2.2. Statistical analysis Metabolite values and enzyme activities were expressed in units per egg dry weight. To define relationships between analysed parameters and egg viability and larval mortality the coefficient of Pearson and simple regression models were used. However, the explanatory effect of the enzymes and metabolites in simple regression was low and then a stepwise multiple regression model had to be formulated. Only those independent variables which increased r2 for >0.02 were considered to increase the explanatory effect of the models. Multiple regression models with a significance P < 0.001 are shown in the results. For lipid analysis, the data were expressed in mg fatty acid/g of lipids in the case of fatty acids and in relative percentage in the case of lipid classes. Results in egg lipid composition between high and low quality of egg batches were then analysed by Student's t-test (SigmaStat 3.1 package). 3. Results The volume of floating and nonfloating eggs collected from April 27 to May 27, 2005, together with the identified parental female, is shown in Fig. 1. High accuracy in parentage analyses were obtained as a result of high polymorphism detected for analysed microsatellite loci. We successfully assigned 100% of the
237
offspring analysed to a single parental couple, showing Tables 2 and 3 and Fig. 1 shows the females that contributed to each sample analysed and the proportion of the larvae coming from each female. Only 6 females (3 per stock) and 10 males (5 per stock) were involved in the overall spawning season (data not shown). The spawning season lasted for 3 months (beginning of April until the end of June) with a total production of 7.25 million eggs for broodstock 1 (56.96% floating) and 5.51 million eggs for broodstock 2 (65.7% floating). Although in Fig. 1, high-quality egg batches seem to have a higher proportion of floating eggs, no significant differences in the ratio floating: sinking eggs (Student's t-test, P = 0.083, N = 29) were found between good and bad egg batches. In all the batches examined, the eggs were between 25% epiboly (approx. 10 h post-fertilization at 19 °C) and beginning of neurulation (approx. 16 h postfertilization at 19 °C). Tables 2 and 3 show the results obtained from the 96 multi-well plates. Although the rates of hatching and survival after hatching were relatively high for all the batches, different mortality rates at 3 and 5 dph were obtained. Ten batches were considered of high quality (mortality at 3 dph < 10%), whereas 6 batches produced bad eggs (mortality at 3 dph > 35%). Regression analysis between hatching and larval mortality rates at 3 and 5 dph (square root transformed data) were very low (r2 = 0.245 and r2 = 0.217, respectively) and even lower when high- and low-quality egg batches were analysed separately. The means plus standard deviation for the enzymes and metabolites analysed are shown in Table 4. Based on the calculation of correlation coefficients of simple
Table 5 Multiple regression models using biochemical egg parameters as independent variables and % hatching and % mortality at 3 and 5 dph as dependent variables to predict egg quality in common dentex Analysed parameters (in dry weight) Hatching rate (%) x1 glucose (nmol/egg), x2 monosaccharides (nmol/egg), x3 pyruvate kinase (nmol/min/egg), x4 ribose (nmol/egg), x5 ketose (nmol/egg)
Mortality at 3 dph (%) x1 free 6-deoxyhexose (nmol/egg), x2 pyruvate kinase (nmol/min/egg), x3 glucose (nmol/egg), x4 alkaline phosphatase (nmol/min/egg)
Mortality at 5 dph (%) x1 free 6-deoxyhexose (nmol/egg), x2 pyruvate kinase (nmol/min/egg), x3 alkaline phosphatase (nmol/min/egg), x4 glucose (nmol/egg)
Multiple regression model
R2
F
P
y = −1.527x1 + 0.101x2 + 0.002x22 − 0.0000081x32 − 18.159x3 + 9.829x23 − 1.360x33 − 1.515x4 + 0.133x24 − 0.002x34 + 0.289x5 − 0.003x25 + 75.681
0.948
9.186
0.006
y = 0.033x1 − 14.051x2 + 2.989x22 − 0.149x32 + 0.349x3 + 41.069x4 − 3.482
0.761
5.851
0.006
y = 0.045x1 − 14.599x2 + 2.148x22 + 38.283x3 + 0.336x4 − 3.255
0.780
8.496
0.001
238
G. Giménez et al. / Aquaculture 260 (2006) 232–243
Fig. 2. Significant differences (Student's t-test, P ≤ 0.05) found between good (N= 10, grey bars) and bad (N = 6, black bars) egg batches in terms of carbohydrate composition, hatching rate and mortality at days 3 and 5 post-hatching.
regression models, it was found that only pyruvate kinase, glucose, ribose and monosaccharides were related to hatching rate whereas pyruvate kinase, alkaline phosphatase, monosaccharides, 6-deoxihexose, ketose, glucose-6 phosphate and glucose were related to larval mortality.
However, in all the cases, r2 was very low and multiple regression models were then formulated. Multiple regression models were formulated using the biochemical parameters which were correlated with hatching rate and larval mortality as independent variables.
Fig. 3. Total lipid content (mg DW) and fatty acid composition (% total fatty acids, TFA), in terms of total saturates, monoenes and PUFA as well as important fatty acids, of high- (grey bars) and low (black bars)-quality batches of eggs. No significant differences were detected in terms of lipid and fatty acid composition (Student's t-test, P ≤ 0.05). Sat: total saturated, Mono: total monounsaturated, PUFA: total polyunsaturated, ARA: arachidonic acid, EPA: eicosapentaenoic acid, DHA: docosahexaenoic acid.
G. Giménez et al. / Aquaculture 260 (2006) 232–243
239
Fig. 4. Lipid class composition of high- (grey bars) and low (black bars)-quality batches of eggs. No significant differences were detected (Student's t-test, P ≤ 0.05). LPC: lysophosphatidylcholine, SM: sphingomyeline, PC: phosphatidylcholine, PS+ PI: phosphatidylserine and phosphatidylinositol, PE: phosphatidylethanolamine, MAG: monoacylglycerols, CHO: cholesterol, FFA: free fatty acids, TAG: triacylglycerols, SE + W: sterol esters and waxes.
Metabolites such as monosaccharides, glucose, ketose and ribose and enzymes such as pyruvate kinase as independent variables, the hatching rate (%) could be predicted with high statistical significance (Table 5). Whereas, in order to predict larval viability (larval mortality rates at 3 and 5 dph), multiple regression models must include 6-deoxyhexose, glucose, pyruvate kinase and alkaline phosphatase (Table 5). The same parameters were used in order to compare high- (N =10) and low (N= 6)-quality batches of eggs. In this case only the content of glucose, ketose, glucose-6phosphate and 6-deoxyhexose showed significant differences (Student's t-test) between good and bad eggs (Fig. 2). However, no significant differences could be found in total lipid content that varied between 167.8–225.7 mg/g in the high-quality egg batches and 166.9–223.7 mg/g in those considered as bad (Fig. 3). Relative fatty acid composition of good-quality eggs was saturates 26.6– 33.9%, monoenes 28.3–34.4%, polyunsaturates 29.8– 46.4%, and n −3 PUFA 23.6–37.5%, whereas bad-quality eggs contained 25.8–35.3% saturates, 28.5–34.2% monoenes, 30.4–45.7% PUFA of which n − 3 PUFA represented
24.3–36.9%, showing no significant differences between both groups of eggs. ARA, EPA and DHA were 1.6–2.5%, 3.4–4.9% and 17.8–29.1%, respectively, for good egg batches and 1.6–2.4%, 3.5–5.5% and 18.5–28.6%, respectively, for bad egg batches, showing no significant differences (Fig. 3). Neutral lipid classes predominated (65.0± 2.6% in low-quality eggs and 67.3 ± 6.5% in highquality eggs), with sterol esters+ waxes (SE + W) and triglycerides (TAG) as the main neutral lipid classes (25.1 ± 2.3 and 34.1 ± 4.7 for good eggs and 24.9 ± 1.7 and 32.4 ± 1.7 for bad-quality eggs, respectively). Among the polar lipids, phosphatidyl choline (PC) was the most abundant lipid class, being 16.8 ± 3.2 for good and 17.9± 1.1 for badquality eggs. No significant differences were obtained when comparing both groups of eggs although slightly higher amounts of SE + W as well as total neutral lipids were observed in high-quality egg batches (Fig. 4). 4. Discussion In most hatchery strains, an unequal contribution of broodstock to the next generation seems to be typical. The
240
G. Giménez et al. / Aquaculture 260 (2006) 232–243
use of microsatellite loci to aid in paternity inference and pedigree analysis in fish species has arisen in recent years (Bernardo et al., 2004; Borrell et al., 2003, 2004; Piñera et al., 2004, 2006). In this work microsatellite loci have made possible retrospective assignment to parental origin of individuals from different family groups reared communally and have allowed us to determine the females (and males) involved in the overall spawning and also to evaluate their contribution to egg production (Fig. 1 and Tables 2 and 3). According to the results shown in Fig. 1 and Tables 2 and 3, high-quality eggs were obtained in the middle of the spawning period of each female (i.e., eggs released by females 7FC2 from broodstock 1 and 3F5E and 118F from broodstock 2 at the beginning of May), whereas lowquality eggs were produced at the end and/or the beginning of the spawning period (i.e., eggs produced by 340F female on May 23rd and 7FC2 female on April 27th– beginning and May 8th–end). Similar results have been obtained for cod (Kjorsvik, 1994) with the highest egg viability during peak spawning, becoming more variable in the later part of the season. Several parameters related to egg morphology, such as egg size, egg dry weight and especially abnormal blastomere morphology have been cited as reliable indicators of hatching success (Kjorsvik et al., 1990; Shields et al., 1997) and negative correlations between this last factor and egg survival and hatching rates have been observed. Other authors (Vallin and Nissling, 1998; Rani, 2005) concluded that abnormal blastomere morphology was not correlated to embryonic development and hatching success because aberrations at early stages of development can be repaired and the larva was able to hatch normally. In the case when abnormal blastomere morphology is low (Penney et al., 2006) or the eggs are assessed at later developmental stages, as it is in our case, any mortality of aberrant eggs may have been masked by other factors. Egg morphology parameters may have some use as coarse screening tools to identify very poor egg batches but cannot be of use in the case of natural fertilization in tanks (as is the case of most Sparidae) when the earlier stages of embryonic development cannot be assessed and/or in the case that broodstock groups are kept in good health and good husbandry conditions and the overall egg quality is good enough to produce very low rates of morphologically aberrant eggs. On the other hand, hatching rate cannot be considered as the only indicator of good egg quality. In the present study, hatching rate was very high in all the egg batches considered (around 80–100% with rare exceptions) and it was found to be unrelated to the mortality rates obtained at days 3 and 5 post-hatching. Mortality rates of larvae that have no access to external food later in
development, such as at day 3 (mouth opening) and day 5 post-hatching (closest day to the point of no return) in the case of common dentex and/or other Sparidae, can be more useful as indicators of quality. This is because they indicate the quality of the endogenous reserves contained in the yolk sac and the intrinsic survival potential of the larva. In terms of biochemical parameters, lipids and free amino acids have been considered as good indicators of egg quality since these compounds are the main sources of energy and membrane constituents for the developing embryo (Sargent, 1995; Ronnestad and Fyhn, 1993). The composition of broodstock diet is believed to have profound effects on the reproduction and egg quality of marine fish (Watanabe, 1985; Izquierdo et al., 2001) and the use of diets with different levels of essential fatty acids, especially arachidonic acid (ARA) and the n − 3 HUFA, EPA and DHA, had a significant effect on hatching and fertilization rates, and survival in the early larval stages of sea bream (Fernandez-Palacios et al., 1995; Rodriguez et al., 1998) and sea bass (Bruce et al., 1999). Furthermore, n − 3 HUFA, DHA/EPA ratio and ARA content have been cited to be related to blastomere morphology, hatching rate (Pickova et al., 1997) and larval survival (in terms of survival activity index, SAI, Furuita et al., 2000). However, in the present study, SAI was similar between high- and low-quality batches of eggs and no relationship could be found with any of the compounds analysed (data not shown). In the case of lipid classes, triacylglycerol (TAG) is the most common form of energy storage in eggs, together with sterol esters and waxes in the case of pelagic eggs with oil globules, whereas phosphatidylcholine (PC) and total phospholipids are the major constituents of membranes, a source of phosphorus and choline and an important source of energy during embryonic development (Tocher et al., 1985; Fraser et al., 1988). Lipid composition of common dentex eggs used in this study was similar to that described previously (Mourente et al., 1999) with a slightly higher content of ARA in the eggs of the present study (1.5% in Mourente et al., 1999 and 1.6–2.5% in this study) probably due to the use of moist pellets in which B. boops (rich in ARA) was included. Total lipid content, and lipid class and fatty acid composition of the eggs showed no significant differences between high- and low-quality batches of eggs although a slightly higher content of neutral lipids (mainly sterol esters + waxes) in high-quality eggs was observed, perhaps indicating a higher lipid storage for the developing larvae and a subsequently superior source of energy. Similar results were found by Salze et al. (2005) in an attempt to associate the levels of n − 3 HUFA, eicosapentaenoic acid (EPA) and
G. Giménez et al. / Aquaculture 260 (2006) 232–243
docosahexaenoic acid (DHA) with egg quality and performance in cod. These same authors found a positive correlation between phosphatidyl inositol (and ARA) levels with performance parameters that were not observed in our study. Larval mortality at the beginning of the exogenous feeding period may be a function not only of lipid reserves but rather of the combined energy available from lipids plus proteins, bearing in mind that proteins are used when limiting food concentrations occur since a reduction in larval protein content is usually observed during the time of yolk exhaustion and the initial stages of exogenous feeding (Riveiro et al., 2000). Unfortunately, in the present study neither total protein content nor free amino acids were analysed. Carbohydrate composition and metabolism of common dentex eggs was similar to that of other Sparidae (Lahnsteiner and Patarnello, 2004a,b) and plays an important role during embryonic development. For each metabolite and enzyme a high deviation was obtained and subsequently the application of single regression curves was not possible and multiple regression analysis had to be utilised in order to relate the composition of the eggs with egg quality variables such as hatching rate and larval survival. Multiple regressions obtained for common dentex were different to that observed previously for gilthead seabream and sharpsnout seabream (Lahnsteiner and Patarnello, 2004a,b). In the case of common dentex a minimal activity of pyruvate kinase and minimal concentrations of monosaccharides and ribose are needed for normal embryonic development, whereas high concentration of 6-deoxyhexose and glucose resulted in decreased viability parameters of the larvae due to the interruption or low activity of the pathways of carbohydrate metabolism. For the embryos that showed a high alkaline phosphatase activity, hatching rate was reduced and larval mortality increased. If good and bad batches of eggs were compared, no differences in enzyme activity could be found but significant differences were obtained in terms of carbohydrate content. Low-quality eggs were found to contain higher amounts of ketose, glucose-6-phosphate, glucose and 6deoxyhexose which might indicate impaired catabolism (Glycolysis) for energy production and consequently the accumulation of glycolysis metabolites. Considering that carbohydrates are synthesized during embryonic development for use in the final stages of energy production and for mechanical and osmoregulatory function, it might be the case that the embryos of low-quality eggs were not utilising carbohydrates for energy production and other substrates, most probably lipids, were used instead. Alkaline phosphatase (ALP) is involved in the catabolism of phospholipids and in dephosphorylation of phosvitin in the yolk (Sire et al., 1994). In the present study high levels of this
241
enzyme were found to be correlated with larval mortality which might indicate that the yolk was consumed very fast in the eggs showing high ALP activity and as a consequence, the resulting larvae had lower amounts of yolk sac reserves before exogenous feeding. In comparison to other studies on S. aurata and P. puntazzo (Lahnsteiner and Patarnello, 2004a,b), higher hatching rates were obtained in the case of common dentex, and more advanced developmental stages were used for the analysis. Consequently, no correlation could be found between hatching rate and acid phosphatase, also involved in the catabolism of phospholipids, adenylate kinase, glucose-6-phosphatase, transaldolase and sialic acid which are involved in gluconeogenesis and in the pentose pathway. The differences observed in the composition of highand low-quality batches of eggs cannot be related to the developmental stage (all the eggs collected were in midepiboly and/or beginning of neurulation) or the quality of the broodstock. Both broodstock groups were cultured using the same photoperiod and water quality conditions and were fed the same semi-moist pellets and presumably egg quality was fairly high in both groups. Finally, in communal broodstock groups of batchspawning species the egg batches obtained everyday may include eggs from more than one female and differences in egg quality parameters due to parental variability may be high. Microsatellite identification of egg batches or newly hatched larvae is a very practical tool for the identification of parentage, maternal contribution to egg quality and composition, and for discrimination between egg batches. Acknowledgements GG thanks the financial support provided by the Ministry of Science and Education (INIA fellowship). Funding was partially provided to AE by the Spanish Ministries of Agriculture Fisheries and Food (Jacumar) and Science and Education (INIA project ACU02-006). Thanks are also due to J. Canoura, N. Gras and O. Bellot (CA-IRTA) for their help during common dentex rearing season and to Martina Radner (Salzourg University) for her help in the carbohydrate and enzyme analysis. References Abellan, E., 2000. Culture of common dentex (Dentex dentex L.) present knowledge, problems and perspectives. Cah. Options Mediterr. 47, 157–168. Ackman, R.G., 1980. Fish lipids. Part 1. In: Connell, J.J. (Ed.), Advances in Fish Science and Technology. Fishing News (Books) Ltd., Farnham, Surrey, UK.
242
G. Giménez et al. / Aquaculture 260 (2006) 232–243
Bell, J.G., Sargent, J.R., 2003. Arachidonic acid in aquaculture feeds: current status and future opportunities. Aquaculture 218, 491–499. Bernardo, D., Piñera, J.A., Blanco, G., Vázquez, E., Sánchez, J.A., 2004. Development of microsatellite markers in Pagellus bogaraveo and their usefulness in other five species with interest in aquaculture. Special Publication, vol. 34. European Aquaculture Society, pp. 160–161. Borrell, Y., Blanco, G., Vázquez, E., Álvarez, J., Pineda, H., Espinosa, G., Fernández-Pato, C., Martínez, C. y Sánchez, J.A. (2003). La variabilidad microsatélite y su aplicación en Acuicultura. CIVA2003 (http://www.civa2003.org), 1042–1056. Borrell, Y.J., Álvarez, J., Vázquez, E., Fernández Pato, C., Martínez Tapia, C., Sánchez, J.A., Blanco, G., 2004. Applying microsatellites to the management of farmed turbot stocks (Scophtalmus maximus L) in hatcheries. Aquaculture 241, 133–150. Bruce, M., Oyen, F., Bell, J.G., Asturiano, J.F., Carrillo, M., Zanuy, S., Ramos, J., Bromage, N., 1999. Development of broodstock diets for the European sea bass (Dicentrarchus labrax) with special emphasis on the importance of n − 3 and n − 6 highly unsaturated fatty acids to reproductive performance. Aquaculture 177, 85–97. Carbó, R., Estévez, A., Furones, M.D., 2002. Intelligent and multifunctional recirculation system. Its application in research at CA-IRTA. EAS Spec. Publ., vol. 32, pp. 171–172. Christie, W.W., 1982. Lipid Analysis. Robert Maxwell, M.C., Oxford. De la Herrán, R., Magoulas, A., Garrido-Ramos, M.A., Ruiz-Rejón, C., Ruiz-Rejón, M., Zouros, E., 2005.Desarrollo de microsatélies en tres especies de espáridos con interés en acuicultura. In: La acuicultura como actividad económica de las zonas costeras. IX Congreso Nacional de Acuicultura pp133–136. Ed. Consejería de Agricultura y Pesca. Junta de Andalucia. Sevilla. Spain. Efthimiou, S., Divanach, P., Rosenthal, H., 1994. Growth, food conversion and agonistic behaviour in common dentex (Dentex dentex) juveniles fed on pelleted moist and dry diets. Aquat. Living Resour. 7, 267–275. Fernandez-Palacios, H., Izquierdo, M.S., Robaina, L., Valencia, A., Salí, M., Vergara, J.M., 1995. Effect of n− 3 HUFA level in broodstock diets on egg quality of gilthead sea bream (Sparus aurata L.). Aquaculture 132, 325–337. Folch, J.M., Lees, M., Sloane Standley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509. Fraser, A.J., Gamble, J.C., Sargent, J.R., 1988. Changes in lipid content, lipid class composition and fatty acid composition in developing eggs and unfed larvae of cod (Gadus morhua). Mar. Biol. 99, 307–313. Furuita, H., Tanaka, H., Yamamoto, T., Shiraishi, M., Takeuchi, T., 2000. Effects of n − 3 HUFA levels in broodstock diet on the reproductive performance and egg and larval quality of the Japanese flounder, Paralichthys olivaceus. Aquaculture 187, 387–398. Glamuzina, B., Jug-Dujakovic, J., Katavic, I., 1989. Preliminary studies on reproduction and larval rearing of common dentex, Dentex dentex (Linnaeus, 1758). Aquaculture 77, 75–84. Henderson, R.J., Tocher, D.R., 1992. Thin-layer chromatography. In: Hamilton, R.J., Hamilton, S. (Eds.), Lipid Analysis. A Practical Approach. IRL Press, Oxford, pp. 65–111. Izquierdo, M.S., Fernández-Palacios, H., Tacon, A.G.J., 2001. Effect of broodstock nutrition on reproductive performance of fish. Aquaculture 197, 25–42. Kjorsvik, E., 1994. Egg quality in wild and broodstock cod L. J. World Aquac. Soc. 25, 22–29. Kjorsvik, E., Mangor Jensen, A., Holmefjord, I., 1990. Egg quality in fishes. Adv. Mar. Biol. 26, 71–113. Lahnsteiner, F., Patarnello, P., 2004a. Biochemical egg quality determination in the gilthead seabream, Sparus aurata: reproduc-
ibility of the method and its application for sharpsnout seabream, Puntazzo puntazzo. Aquaculture 237, 433–442. Lahnsteiner, F., Patarnello, P., 2004b. Egg quality determination in the gilthead seabream, Sparus aurata, with biochemical parameters. Aquaculture 237, 443–459. Maeland, A., Ronnestad, I., Waagbo, R., 2003. Folate in eggs and developing larvae of Atlantic halibut, Hippoglossus hippoglossus. Aquac. Nutr. 9, 185–188. Marshall, T.C., Slate, J., Kruuk, L.E.B., Pemberton, J., 1998. Statistical confidence for likelihood-based paternity inference in natural populations. Mol. Ecol. 7, 639–655. Mourente, G., Rodríguez, A., Grau, A., Pastor, E., 1999. Utilization of lipids by Dentex dentex L. (Osteichthyes, Sparidae) larvae during lecitotrophia and subsequent starvation. Fish Physiol. Biochem. 21, 45–58. Penney, R.W., Lush, P.L., Wade, J., Brown, J.A., Parrish, C.C., Burton, M.P.M., 2006. Comparative utility of egg blastomere morphology and lipid biochemistry for prediction of hatching success in Atlantic cod, Gadus morhua L. Aquac. Res. 37, 272–283. Pickova, J., Dutta, P.C., Larsson, P.O., Kiessling, A., 1997. Early embryonic cleavage pattern, hatching success, and egg-lipid fatty acid composition: comparison between two cod (Gadus morhua) stocks. Can. J. Fish. Aquat. Sci. 54, 2410–2416. Piñera, J.A., Bernardo, D., Blanco, G., Vázquez, E., Sánchez, J.A., 2004. Finding my parents: how many genetic markers are needed? Special Publication, vol. 34. European Aquaculture Society, pp. 661–662. Piñera, J.A., Bernardo, D., Vázquez, E., Blanco, G., Sánchez, J.A., 2006. Isolation and characterization of polymorphic microsatellite markers in Pagellus bogaraveo, and cross-species amplification in Sparus aurata and Dicentrarchus labrax. Mol. Ecol. 33–35. Rani, M.S., 2005. Prediction of larval viability based on egg quality parameters and early cleavage patterns in the experiments of triploidy induction in Atlantic cod, Gadus morhua L. M. Sc. Thesis, Univ. Tromso, Norway, 64 pp. Riveiro, I., Guisande, C., Lloves, M., Maneiro, I., Cabanas, J.M., 2000. Importante of parental effects on larval survival in Sardina pilchardus. Mar. Ecol., Prog. Ser. 205, 249–258. Rodriguez, C., Cejas, J.R., Martin, M.V., Badia, P., Samper, M., Lorenzo, A., 1998. Influence of n − 3 highly unsaturated fatty acid deficiency on the lipid composition of broodstock gilthead seabream (Sparus aurata L.) and on egg quality. Fish Physiol. Biochem. 18, 177–187. Rodriguez, C., Acosta, C., Badia, P., Cejas, J.R., Santamaria, F.J., Lorenzo, A., 2004. Assessment of lipid and essential fatty acids requirements of black seabream (Spondyliosoma cantharus) by comparison of lipid composition in muscle and liver of wild and captive adult fish. Comp. Biochem. Physiol., Part B Biochem. Mol. Biol. 139, 619–629. Ronnestad, I., Fyhn, H.J., 1993. Metabolic aspects of free amino acids in developing marine fish eggs and larvae. Rev. Fish Sci. 1, 239–259. Ronnestad, I., Lie, O., Waagbo, R., 1997. Vitamin B6 in Atlantic halibut, Hippoglossus hippoglossus—endogenous utilization and retention in larvae fed natural zooplankton. Aquaculture 157, 337–345. Ronnestad, I., Thorsen, A., Finn, R.N., 1999. Fish larval nutrition: a review of recent advances in the roles of amino acids. Aquaculture 177, 201–216. Salze, G., Tocher, D.R., Roy, W.J., Robertson, D.A., 2005. Egg quality determinants in cod (Gadus morhua L.): egg performance and lipids in eggs from farmed and wild broodstock. Aquac. Res. 36, 1488–1499. Sargent, J.R., 1995. Origins and functions of egg lipids: nutritional implications. In: Bromage, N., Roberts, R.J. (Eds.), Broodstock Management and Egg and Larval Quality. Blackwell Science, Cambridge, UK, pp. 353–372.
G. Giménez et al. / Aquaculture 260 (2006) 232–243 Shields, R.J., Brown, N.P., Bromage, N.R., 1997. Blastomere morphology as a predictive measure of fish egg viability. Aquaculture 155, 1–12. Sire, M.F., Babin, P.J., Vernier, J.M., 1994. Involvement of the lysosomal system in yolk protein deposit and degradation during vitellogenesis and embryonic development in trout. J. Exp. Zool. 269, 69–83. Thorsen, A., Trippel, E.A., Lambert, Y., 2003. Experimental methods to monitor the production and quality of eggs of captive marine fish. J. Northwest Atl. Fish. Sci. 33, 55–70. Tocher, D.R., Harvie, D.G., 1988. Fatty acid compositions of the major phosphoglycerides from fish neural tissues: (n − 3) and (n − 6) polyunsaturated fatty acids in rainbow trout (Salmo gairdneri) and cod (Gadus morhua) brains and retinas. Fish Physiol. Biochem. 5, 229–239. Tocher, D.R., Fraser, A.J., Sargent, J.R., Gamble, J.C., 1985. Lipid class composition during embryonic and early larval development in Atlantic herring (Clupea harengus L.). Lipids 20, 84–89.
243
Tveiten, H., Jobling, M., Andreassen, I., 2004. Influence of egg lipids and fatty acids on egg viability and their utilization during embryonic development of spotted wolfish Anahichas minor Olafsen. Aquac. Res. 35, 152–161. Vallin, L., Nissling, A., 1998. Cell morphology as an indicator of viability of cod eggs—results from an experimental study. Fish. Res. 38, 247–255. Walsh, P.S., Metzger, D.A., Higuchi, R., 1991. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques 10, 506–513. Watanabe, T., 1985. Importance of the study of broodstock nutrition for further development of aquaculture. In: Cowey, C.B, Mackie, A.M., Bell, J.K. (Eds.), Nutrition and Feeding in Fish. Academic Press, London, UK, pp. 395–414.
Egg quality criteria in common dentex (Dentex dentex) G. Giménez a , A. Estévez a,⁎, F. Lahnsteiner b , B. Zecevic b , J.G. Bell c , R.J. Henderson c , J.A. Piñera d , J.A. Sanchez-Prado d a
d
Centro de Acuicultura-IRTA, Ctra. Poble Nou Km 6, 43540 Sant Carles de la Rápita, Tarragona, Spain b Insitute for Zoology, University of Salzburg, Hellbrunnerstrasse 34, A-5020, Salzburg, Austria c Laboratory of Nutrition, Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, Scotland, UK Departamento de Biología Funcional. Laboratorio de Genética Acuícola, Facultad de Medicina, IUBA. Universidad de Oviedo, Julián Clavería s/n, 33071 Oviedo, Spain Received 4 April 2006; accepted 20 June 2006
Abstract The spawning quality, in terms of hatching rate, larval mortality at 3 and 5 days post-hatching (dph) and day of total mortality of two broodstock groups of common dentex was evaluated for 1 month in 2005. Several biochemical parameters including total lipid content, lipid class and fatty acid composition, carbohydrate content and metabolic enzyme activities were analysed in all the egg batches collected. Comparison was carried out between low- (mortality at 3 dph higher than 35%) and high-quality (mortality at 3 dph lower than 10%) batches. No differences were observed in lipid content and/or lipid class and fatty acid composition although a slightly higher content of neutral lipids was detected in high-quality batches. However, significant differences were obtained regarding carbohydrate composition and the activity of enzymes such as alkaline phosphatase and pyruvate kinase being higher in low-quality egg batches. © 2006 Elsevier B.V. All rights reserved. Keywords: Egg quality; Lipids; Carbohydrates; Enzymes; Common dentex
1. Introduction Although common dentex (Dentex dentex) has been considered as a promising species for aquaculture in the Mediterranean coast, for a long time, due to its high market price and growth rate, its culture under intensive conditions is still a problem and not enough numbers of juveniles are produced for ongrowing in sea cages. Common dentex is a batch spawner that usually spawns at nightfall or early morning (Abellan, 2000) for a long period of up to 40–50 days (Giménez and Estévez, personal observation) and the number and size of the eggs ⁎ Corresponding author. Tel.: +34 977745427; fax: +34 977744138. E-mail address: [email protected] (A. Estévez). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.06.028
released in each batch varies over the spawning season as well as the quality between egg batches. One of the main problems with this very carnivorous and cannibalistic (Efthimiou et al., 1994) species is the high mortality rate during the larval phase, especially in the early feeding period with rotifers (Glamuzina et al., 1989). The need for a precise estimation of egg quality is therefore of paramount importance in order to clarify if the low survival rate during early larval rearing is due to the initial viability of the larvae or the quality of the eggs. Hatchery production can be optimized by starting the production cycle with high-quality eggs giving high egg survival and hatching rates and robust larvae with better growth, survival and stress resistance. Identification of a method to reliably discriminate between low- and high-quality egg batches
G. Giménez et al. / Aquaculture 260 (2006) 232–243
will be vital to reduce hatchery production costs by ensuring time and resources are not wasted on egg batches with low survival and/or performance potential. Several morphological (Kjorsvik et al., 1990; Thorsen et al., 2003) and biochemical parameters such as lipids (Sargent, 1995; Bell and Sargent, 2003; Tveiten et al., 2004), amino acids (Ronnestad and Fyhn, 1993) or vitamins (Ronnestad et al., 1997, 1999; Maeland et al., 2003) have been considered as indicators of egg quality. Recently, several compounds and enzymes involved in carbohydrate metabolism have been identified as good markers for egg quality in other Sparidae species such as Sparus aurata and Puntazzo puntazzo (Lahnsteiner and Patarnello, 2004a). Lipids and fatty acids have been also considered in order to compare egg quality from wild and captive broodstocks (Rodriguez et al., 2004; Salze et al., 2005). In this paper, we have measured total lipid content, lipid class and fatty acid composition, as well as enzymes and mono- and disaccharides of common dentex eggs taken at the same developmental stage from different batches of 2 broodstock groups. Both broodstock groups were maintained under the same environmental and feeding conditions as a tool to evaluate initial egg quality. Hatching and daily larval mortality rates, time to yolk resorption and time to 100% mortality were also assessed using 96-well EIA plates. 2. Materials and methods 2.1. Materials The daily spawnings of 2 broodstock groups of common dentex were assessed for a month during 2005. The broodstock groups were composed of 5 females:8 males and 4 females:6 males, respectively (Table 1). All the fish were conveniently tagged by the use of pit tags placed in the muscle near the dorsal fin and identified by the use of microsatellite DNA markers previously isolated and used for other Sparidae. The fish were stocked in two 4000-l circular tanks connected to a recirculation unit (Carbó et al., 2002) and fed semi-moist pellets in which fresh minced fish (Boops boops), fish meal (Skretting, Spain), a fish oil concentrate (TG0525, Croda, UK) and vitamin premix (Skretting, Spain) were mixed in a 40:40:15:2 ratio, respectively. Photoperiod was maintained at 14 h L:10 h D during the spawning season to allow natural egg release. Everyday, the eggs were removed from the outside collector (a cylindro-conical tank containing a 500 μm net immersed in a 100-l holding tank connected to the recirculation system), separated into floating and non-floating eggs in a measuring cylinder. Floating eggs were isolated, thoroughly washed and subsequently kept in UV-treated
233
Table 1 Description of common dentex broodstock used in the study Broodstock 1
Broodstock 2
Pit tag code
Sex
Weight (g) Pit tag code Sex
Weight (g)
37CD 1E43 5530 A4DF 1D38 EA2A 7FC2 108D 1AE3 340F 8337 1140 Ratio F:M Density (kg/m3)
M M M M M M F F F F F M 1:1.4 3.82
985 1376 1485 1300 735 965 1182 1430 1470 1150 2015 1180
1600 1920 1510 1700 2298 1560 1250 1395 1115 789
AE97 2F8E 118F 8D1D 0668 DB32 A953 ACA5 3F5E 22B3
M F F F M M M M F M
1:1.2 3.78
seawater, observed under a stereomicroscope to assess the morphology, fertilization rate and embryological stage and transferred to the laboratory where a sample of the eggs was plated onto a 96-well cell culture multi-well plate (Nunc) as in Shields et al. (1997). One egg was placed in each well using a Pasteur pipette and each well then filled with autoclaved seawater and incubated in darkness at 19 °C in a refrigerated incubator. Sub-samples of the eggs were rinsed in distilled water and placed in capped vials with chloroform/methanol (2:1, v/v), covered with nitrogen gas and kept at −20 °C for lipid analysis, and in Eppendorf tubes filled with either 0.6 ml 100 mM/l Tris buffer pH 7.5 and 0.6 ml 3 mol/l perchloric acid kept at −80 °C for the analysis of enzymes and acid-stable metabolites, respectively (Lahnsteiner and Patarnello, 2004b). Once per week, the eggs were incubated in a 35 l tank for 2–3 days, a sample of the larvae obtained were fixed in 100% ethanol and subjected to DNA extraction. Genomic DNA was extracted following the standard Chelex@100 (Biorad) resin protocol with Proteinase K (Walsh et al., 1991). PCRs (polymerase chain reaction) for the nine microsatellites mentioned above were carried out following the published conditions (Bernardo et al., 2004; De la Herrán et al., 2005; Piñera et al., 2006) and the amplified fragment sizes were determined using an OpenGene Sequencing System (BayerHealth, Inc). Once the microsatellite genotypes of all broodstocks and offsprings were profiled, each offspring was assigned to the most probable parental couple using Cervus software (Marshall et al., 1998). For the EIA plate samples, the eggs/larvae were inspected and counted on a daily basis in order to calculate the hatching rate, percentage survival after hatching, mortality rate at days 3 and 5 post-hatching (dph), percentage of
234
G. Giménez et al. / Aquaculture 260 (2006) 232–243
Fig. 1. Volume of sinking and floating eggs as well as parental female identified in some of the egg batches, and presumable spawning time for each identified female. G and B denote good and bad egg batches. Contribution of each female (%) in some of the genetically identified batches is also indicated.
deformities and time to 100% mortality. Having in mind that first feeding and point of no return occur in this species at days 3 and 7, respectively, we have considered, and analysed more closely, high-quality batches (good) of eggs being those with a mortality rate less than 10% between 3
and 5 dph and low-quality batches (bad) of eggs being those with mortality rates greater than 35% at 3 dph. Total lipids were extracted from samples by homogenisation in chloroform/methanol (2:1, v/v) containing 0.01% butylated hydroxytoluene (BHT) (Folch et al.,
G. Giménez et al. / Aquaculture 260 (2006) 232–243
235
Table 2 Hatching rate, survival, mortality at 3 and 5 dph, last day of survival, deformities and parental female of the eggs obtained from broodstock 1 and plated on 96-well EIA plates Broodstock 1 Spawning date
Hatching rate (%)
Hatching survival (%)
Mortality 3 dph (%)
Mortality 5 dph (%)
Day 100% mortality
Deformities (%)
27/04/2005
97.92
100.00
57.61
72.83
10
0
28/04/2005 29/04/2005 30/04/2005 02/05/2005 03/05/2005 04/05/2005 05/05/2005 06/05/2005
85.26 57.29 92.71 92.93 83.51 96.88 100.00 100.00
100.00 94.55 84.88 96.74 91.36 92.86 98.95 98.92
56.79 16.36 34.88 21.74 40.74 22.62 9.47 6.45
69.14 16.36 38.37 27.17 40.74 26.19 11.58 11.83
9 9 10 10 11 11 11 11
0 0.00 4.17 1.01 7.22 2.08 2.11 0.00
07/05/2005 08/05/2005
95.83 90.53
81.61 75.00
24.14 35.00
24.14 35.00
11 10
10.42 9.47
09/05/2005 11/05/2005 12/05/2005 13/05/2005 17/05/2005 23/05/2005 26/05/2005 Average S.D.
96.84 98.96 91.67 100.00 93.75 96.88 81.63 91.81 10.28
94.57 96.84 100.00 97.92 83.95 96.74 93.51 93.24 7.25
8.70 3.16 9.09 4.17 32.10 36.96 27.27 24.85 16.85
29.35 6.32 9.09 4.17 33.33 42.39 27.27 29.18 19.27
10 10 11 11 10 10 11 10.33 0.69
Female code and % contribution 7FC2 97%, 340F 8%
2.11 0.00 7.14 0.00 0.00 0.00 2.04 2.65 3.52
340F 63%, 8337 37% 7FC2 50%, 340F 50%
340F 100%
High-quality eggs are denoted by bold + italic letters, low-quality eggs by bold letters only.
Table 3 Hatching rate, survival, mortality at 3 and 5 dph, last day of survival, deformities and parental female of the eggs obtained from broodstock 2 and plated on 96-well EIA plates Broodstock 2 Spawning date
Hatching rate (%)
Hatching survival (%)
Mortality 3 dph (%)
Mortality 5 dph (%)
Day 100% mortality
Deformities (%)
28/04/2005 29/04/2005 30/04/2005 02/05/2005 03/05/2005 04/05/2005 05/05/2005 06/05/2005 07/05/2005 08/05/2005 09/05/2005 12/05/2005 13/05/2005 23/05/2005 26/05/2005 27/05/2005 Average S.D.
24.21 88.54 94.79 95.51 100.00 93.68 98.96 97.89 95.83 97.83 91.67 95.83 95.83 57.73 71.43 48.45 84.26 22.20
82.61 97.62 95.56 91.67 100.00 96.63 100.00 98.92 93.48 95.56 96.59 90.70 92.13 98.21 78.46 76.60 92.80 7.38
34.78 7.14 21.11 19.05 8.79 3.37 3.16 1.08 7.61 7.78 13.64 16.28 17.98 41.07 41.54 40.43 17.80 14.23
34.78 10.71 23.33 26.19 13.19 4.49 3.16 2.15 7.61 7.78 21.59 17.44 17.98 50.00 49.23 46.81 21.03 16.36
8 9 10 10 10 11 10 10 11 10 9 10 10 10 11 10 9.94 0.77
0.00 0.00 0.00 6.74 1.03 2.11 0.00 0.00 1.04 1.09 1.04 2.08 2.08 0.00 5.10 1.03 1.46 1.93
High-quality eggs are denoted by bold + italic letters, low-quality eggs by bold letters only.
Female code and % contribution 3F5E 80%, 118F 20%
3F5E 80%, 118F 20% 3F5E 100% 3F5E 70%, 118F 30%
3F5E 20%, 118F 20%, 8D1D 60%
236
G. Giménez et al. / Aquaculture 260 (2006) 232–243
1957). Lipid class composition was determined by highperformance thin-layer chromatography (HPTLC). Approximately 10 μg of lipid was applied as a 2 mm streak and the plate developed to two-thirds distance with methyl acetate/isopropanol/chloroform/methanol/0.25% aqueous KCl (25:25:25:10:9, by vol.), to separate polar lipid classes, and then fully developed with isohexane/ diethyl ether/acetic acid (85:15:1, by vol.). Lipid classes were visualised by charring at 160 °C for 15 min after spraying with 3% (w/v) aqueous cupric acetate containing 8% (v/v) phosphoric acid and quantified by densitometry using a Camag 3 TLC Scanner (Camag, Muttenz, Switzerland) and winCATS software (Henderson and Tocher, 1992). The identities of individual lipid classes were confirmed by comparison with authentic standards. Fatty acid methyl esters (FAME) were prepared from total lipid by acid-catalysed transesterification using 2 ml of 1% H2SO4 in methanol plus 1 ml toluene (Christie,
1982), and FAME were then extracted and purified (Tocher and Harvie, 1988). FAME were separated and quantified by gas–liquid chromatography (Fisons GC8600, Carlo Erba, Milan, Italy) using a 30 m × 0.32 mm capillary column (CP wax 52CB; Chrompak, London, U.K) utilizing on-column injection at 50 °C and flame ionization detection at 250 °C. Hydrogen was used as carrier gas and temperature programming was from 50 °C to 180 °C at 40 °C min− 1 and then to 225 °C at 2 °C min− 1. Individual methyl esters were identified by comparison to known standards (Ackman, 1980). Biochemical samples for enzyme and acid-stable metabolites analyses were thawed at room temperature and homogenized. The extracts were centrifuged at 1500×g for 10 min at 4 C°. The supernatants were used for analysis by colorimetry and UV spectrophotometry following the methods described by Lahnsteiner and Patarnello (2004b). For the determination of egg wet
Table 4 Biochemical compounds (content per egg in dry weight) analysed in common dentex eggs (N = 36) and simple regression models obtained with hatching rate and mortality at 3 and 5 dph Mean
S.D.
Enzymes (nmol/min/egg) Adenylate kinase Pyruvate kinase
0.59 2.72
0.41 1.94
Malate dehydrogenase Glucose-6-phosphate dehydrogenase Transaldolase Aspartate aminotransferase Succinate dehydrogenase Acid phosphatase Alkaline phosphatase
10.17 0.46 1.14 2.48 11.90 15.68 0.50
3.55 0.61 1.50 3.98 13.90 5.72 0.12
Glucose-6-phosphatase
0.18
0.20
Metabolites (nmol/egg) Free monosaccharides Total monosaccharides Free ketose Total ketose Sialic acid Free ribose Total ribose Free heptose Total heptose Free 6-deoxyhexose Total 6-deoxyhexose Glucose-6-phosphate Fructose-6-phosphate ATP Glucose
129.33 411.15 23.31 19.54 29.83 22.66 25.71 290.60 288.96 631.63
42.79 158.67 24.90 12.95 19.50 10.62 14.86 590.75 684.37 175.73
1225.24 14.65 4.26 9.61 7.51
605.51 13.87 4.87 7.23 7.39
H: hatching rate, M3: % mortality at 3 dph, M5: % mortality at 5 dph.
Regression
r2
P
H = 28.34x − 7.883x2 + 0.64x3 + 64.821 M3 = − 19.279x + 4.973x2 − 0.407x3 + 40.285 M5 = − 10.916x + 1.053x2 + 43.726
0.306 0.254 0.245
0.009 0.030 0.013
M3 = 60.933x − 9.671 M5 = 66.879x − 8.902
0.200 0.184
0.008 0.011
H = 1.90x − 0.01x2 + 0.000015x3 − 23.75
0.352
0.003
H = 5.196x − 0.139x2 + 0.001x3 + 32.716
0.253
0.027
M3 = 0.047x − 8.248 M5 = 0.065x − 15.7
0.273 0.395
0.002 0.000
H = −1.503x + 99.012 M3 = 1.063x + 18.525
0.490 0.214
0.001 0.053
G. Giménez et al. / Aquaculture 260 (2006) 232–243
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 °C for 24 h and re-weighed. 2.2. Statistical analysis Metabolite values and enzyme activities were expressed in units per egg dry weight. To define relationships between analysed parameters and egg viability and larval mortality the coefficient of Pearson and simple regression models were used. However, the explanatory effect of the enzymes and metabolites in simple regression was low and then a stepwise multiple regression model had to be formulated. Only those independent variables which increased r2 for >0.02 were considered to increase the explanatory effect of the models. Multiple regression models with a significance P < 0.001 are shown in the results. For lipid analysis, the data were expressed in mg fatty acid/g of lipids in the case of fatty acids and in relative percentage in the case of lipid classes. Results in egg lipid composition between high and low quality of egg batches were then analysed by Student's t-test (SigmaStat 3.1 package). 3. Results The volume of floating and nonfloating eggs collected from April 27 to May 27, 2005, together with the identified parental female, is shown in Fig. 1. High accuracy in parentage analyses were obtained as a result of high polymorphism detected for analysed microsatellite loci. We successfully assigned 100% of the
237
offspring analysed to a single parental couple, showing Tables 2 and 3 and Fig. 1 shows the females that contributed to each sample analysed and the proportion of the larvae coming from each female. Only 6 females (3 per stock) and 10 males (5 per stock) were involved in the overall spawning season (data not shown). The spawning season lasted for 3 months (beginning of April until the end of June) with a total production of 7.25 million eggs for broodstock 1 (56.96% floating) and 5.51 million eggs for broodstock 2 (65.7% floating). Although in Fig. 1, high-quality egg batches seem to have a higher proportion of floating eggs, no significant differences in the ratio floating: sinking eggs (Student's t-test, P = 0.083, N = 29) were found between good and bad egg batches. In all the batches examined, the eggs were between 25% epiboly (approx. 10 h post-fertilization at 19 °C) and beginning of neurulation (approx. 16 h postfertilization at 19 °C). Tables 2 and 3 show the results obtained from the 96 multi-well plates. Although the rates of hatching and survival after hatching were relatively high for all the batches, different mortality rates at 3 and 5 dph were obtained. Ten batches were considered of high quality (mortality at 3 dph < 10%), whereas 6 batches produced bad eggs (mortality at 3 dph > 35%). Regression analysis between hatching and larval mortality rates at 3 and 5 dph (square root transformed data) were very low (r2 = 0.245 and r2 = 0.217, respectively) and even lower when high- and low-quality egg batches were analysed separately. The means plus standard deviation for the enzymes and metabolites analysed are shown in Table 4. Based on the calculation of correlation coefficients of simple
Table 5 Multiple regression models using biochemical egg parameters as independent variables and % hatching and % mortality at 3 and 5 dph as dependent variables to predict egg quality in common dentex Analysed parameters (in dry weight) Hatching rate (%) x1 glucose (nmol/egg), x2 monosaccharides (nmol/egg), x3 pyruvate kinase (nmol/min/egg), x4 ribose (nmol/egg), x5 ketose (nmol/egg)
Mortality at 3 dph (%) x1 free 6-deoxyhexose (nmol/egg), x2 pyruvate kinase (nmol/min/egg), x3 glucose (nmol/egg), x4 alkaline phosphatase (nmol/min/egg)
Mortality at 5 dph (%) x1 free 6-deoxyhexose (nmol/egg), x2 pyruvate kinase (nmol/min/egg), x3 alkaline phosphatase (nmol/min/egg), x4 glucose (nmol/egg)
Multiple regression model
R2
F
P
y = −1.527x1 + 0.101x2 + 0.002x22 − 0.0000081x32 − 18.159x3 + 9.829x23 − 1.360x33 − 1.515x4 + 0.133x24 − 0.002x34 + 0.289x5 − 0.003x25 + 75.681
0.948
9.186
0.006
y = 0.033x1 − 14.051x2 + 2.989x22 − 0.149x32 + 0.349x3 + 41.069x4 − 3.482
0.761
5.851
0.006
y = 0.045x1 − 14.599x2 + 2.148x22 + 38.283x3 + 0.336x4 − 3.255
0.780
8.496
0.001
238
G. Giménez et al. / Aquaculture 260 (2006) 232–243
Fig. 2. Significant differences (Student's t-test, P ≤ 0.05) found between good (N= 10, grey bars) and bad (N = 6, black bars) egg batches in terms of carbohydrate composition, hatching rate and mortality at days 3 and 5 post-hatching.
regression models, it was found that only pyruvate kinase, glucose, ribose and monosaccharides were related to hatching rate whereas pyruvate kinase, alkaline phosphatase, monosaccharides, 6-deoxihexose, ketose, glucose-6 phosphate and glucose were related to larval mortality.
However, in all the cases, r2 was very low and multiple regression models were then formulated. Multiple regression models were formulated using the biochemical parameters which were correlated with hatching rate and larval mortality as independent variables.
Fig. 3. Total lipid content (mg DW) and fatty acid composition (% total fatty acids, TFA), in terms of total saturates, monoenes and PUFA as well as important fatty acids, of high- (grey bars) and low (black bars)-quality batches of eggs. No significant differences were detected in terms of lipid and fatty acid composition (Student's t-test, P ≤ 0.05). Sat: total saturated, Mono: total monounsaturated, PUFA: total polyunsaturated, ARA: arachidonic acid, EPA: eicosapentaenoic acid, DHA: docosahexaenoic acid.
G. Giménez et al. / Aquaculture 260 (2006) 232–243
239
Fig. 4. Lipid class composition of high- (grey bars) and low (black bars)-quality batches of eggs. No significant differences were detected (Student's t-test, P ≤ 0.05). LPC: lysophosphatidylcholine, SM: sphingomyeline, PC: phosphatidylcholine, PS+ PI: phosphatidylserine and phosphatidylinositol, PE: phosphatidylethanolamine, MAG: monoacylglycerols, CHO: cholesterol, FFA: free fatty acids, TAG: triacylglycerols, SE + W: sterol esters and waxes.
Metabolites such as monosaccharides, glucose, ketose and ribose and enzymes such as pyruvate kinase as independent variables, the hatching rate (%) could be predicted with high statistical significance (Table 5). Whereas, in order to predict larval viability (larval mortality rates at 3 and 5 dph), multiple regression models must include 6-deoxyhexose, glucose, pyruvate kinase and alkaline phosphatase (Table 5). The same parameters were used in order to compare high- (N =10) and low (N= 6)-quality batches of eggs. In this case only the content of glucose, ketose, glucose-6phosphate and 6-deoxyhexose showed significant differences (Student's t-test) between good and bad eggs (Fig. 2). However, no significant differences could be found in total lipid content that varied between 167.8–225.7 mg/g in the high-quality egg batches and 166.9–223.7 mg/g in those considered as bad (Fig. 3). Relative fatty acid composition of good-quality eggs was saturates 26.6– 33.9%, monoenes 28.3–34.4%, polyunsaturates 29.8– 46.4%, and n −3 PUFA 23.6–37.5%, whereas bad-quality eggs contained 25.8–35.3% saturates, 28.5–34.2% monoenes, 30.4–45.7% PUFA of which n − 3 PUFA represented
24.3–36.9%, showing no significant differences between both groups of eggs. ARA, EPA and DHA were 1.6–2.5%, 3.4–4.9% and 17.8–29.1%, respectively, for good egg batches and 1.6–2.4%, 3.5–5.5% and 18.5–28.6%, respectively, for bad egg batches, showing no significant differences (Fig. 3). Neutral lipid classes predominated (65.0± 2.6% in low-quality eggs and 67.3 ± 6.5% in highquality eggs), with sterol esters+ waxes (SE + W) and triglycerides (TAG) as the main neutral lipid classes (25.1 ± 2.3 and 34.1 ± 4.7 for good eggs and 24.9 ± 1.7 and 32.4 ± 1.7 for bad-quality eggs, respectively). Among the polar lipids, phosphatidyl choline (PC) was the most abundant lipid class, being 16.8 ± 3.2 for good and 17.9± 1.1 for badquality eggs. No significant differences were obtained when comparing both groups of eggs although slightly higher amounts of SE + W as well as total neutral lipids were observed in high-quality egg batches (Fig. 4). 4. Discussion In most hatchery strains, an unequal contribution of broodstock to the next generation seems to be typical. The
240
G. Giménez et al. / Aquaculture 260 (2006) 232–243
use of microsatellite loci to aid in paternity inference and pedigree analysis in fish species has arisen in recent years (Bernardo et al., 2004; Borrell et al., 2003, 2004; Piñera et al., 2004, 2006). In this work microsatellite loci have made possible retrospective assignment to parental origin of individuals from different family groups reared communally and have allowed us to determine the females (and males) involved in the overall spawning and also to evaluate their contribution to egg production (Fig. 1 and Tables 2 and 3). According to the results shown in Fig. 1 and Tables 2 and 3, high-quality eggs were obtained in the middle of the spawning period of each female (i.e., eggs released by females 7FC2 from broodstock 1 and 3F5E and 118F from broodstock 2 at the beginning of May), whereas lowquality eggs were produced at the end and/or the beginning of the spawning period (i.e., eggs produced by 340F female on May 23rd and 7FC2 female on April 27th– beginning and May 8th–end). Similar results have been obtained for cod (Kjorsvik, 1994) with the highest egg viability during peak spawning, becoming more variable in the later part of the season. Several parameters related to egg morphology, such as egg size, egg dry weight and especially abnormal blastomere morphology have been cited as reliable indicators of hatching success (Kjorsvik et al., 1990; Shields et al., 1997) and negative correlations between this last factor and egg survival and hatching rates have been observed. Other authors (Vallin and Nissling, 1998; Rani, 2005) concluded that abnormal blastomere morphology was not correlated to embryonic development and hatching success because aberrations at early stages of development can be repaired and the larva was able to hatch normally. In the case when abnormal blastomere morphology is low (Penney et al., 2006) or the eggs are assessed at later developmental stages, as it is in our case, any mortality of aberrant eggs may have been masked by other factors. Egg morphology parameters may have some use as coarse screening tools to identify very poor egg batches but cannot be of use in the case of natural fertilization in tanks (as is the case of most Sparidae) when the earlier stages of embryonic development cannot be assessed and/or in the case that broodstock groups are kept in good health and good husbandry conditions and the overall egg quality is good enough to produce very low rates of morphologically aberrant eggs. On the other hand, hatching rate cannot be considered as the only indicator of good egg quality. In the present study, hatching rate was very high in all the egg batches considered (around 80–100% with rare exceptions) and it was found to be unrelated to the mortality rates obtained at days 3 and 5 post-hatching. Mortality rates of larvae that have no access to external food later in
development, such as at day 3 (mouth opening) and day 5 post-hatching (closest day to the point of no return) in the case of common dentex and/or other Sparidae, can be more useful as indicators of quality. This is because they indicate the quality of the endogenous reserves contained in the yolk sac and the intrinsic survival potential of the larva. In terms of biochemical parameters, lipids and free amino acids have been considered as good indicators of egg quality since these compounds are the main sources of energy and membrane constituents for the developing embryo (Sargent, 1995; Ronnestad and Fyhn, 1993). The composition of broodstock diet is believed to have profound effects on the reproduction and egg quality of marine fish (Watanabe, 1985; Izquierdo et al., 2001) and the use of diets with different levels of essential fatty acids, especially arachidonic acid (ARA) and the n − 3 HUFA, EPA and DHA, had a significant effect on hatching and fertilization rates, and survival in the early larval stages of sea bream (Fernandez-Palacios et al., 1995; Rodriguez et al., 1998) and sea bass (Bruce et al., 1999). Furthermore, n − 3 HUFA, DHA/EPA ratio and ARA content have been cited to be related to blastomere morphology, hatching rate (Pickova et al., 1997) and larval survival (in terms of survival activity index, SAI, Furuita et al., 2000). However, in the present study, SAI was similar between high- and low-quality batches of eggs and no relationship could be found with any of the compounds analysed (data not shown). In the case of lipid classes, triacylglycerol (TAG) is the most common form of energy storage in eggs, together with sterol esters and waxes in the case of pelagic eggs with oil globules, whereas phosphatidylcholine (PC) and total phospholipids are the major constituents of membranes, a source of phosphorus and choline and an important source of energy during embryonic development (Tocher et al., 1985; Fraser et al., 1988). Lipid composition of common dentex eggs used in this study was similar to that described previously (Mourente et al., 1999) with a slightly higher content of ARA in the eggs of the present study (1.5% in Mourente et al., 1999 and 1.6–2.5% in this study) probably due to the use of moist pellets in which B. boops (rich in ARA) was included. Total lipid content, and lipid class and fatty acid composition of the eggs showed no significant differences between high- and low-quality batches of eggs although a slightly higher content of neutral lipids (mainly sterol esters + waxes) in high-quality eggs was observed, perhaps indicating a higher lipid storage for the developing larvae and a subsequently superior source of energy. Similar results were found by Salze et al. (2005) in an attempt to associate the levels of n − 3 HUFA, eicosapentaenoic acid (EPA) and
G. Giménez et al. / Aquaculture 260 (2006) 232–243
docosahexaenoic acid (DHA) with egg quality and performance in cod. These same authors found a positive correlation between phosphatidyl inositol (and ARA) levels with performance parameters that were not observed in our study. Larval mortality at the beginning of the exogenous feeding period may be a function not only of lipid reserves but rather of the combined energy available from lipids plus proteins, bearing in mind that proteins are used when limiting food concentrations occur since a reduction in larval protein content is usually observed during the time of yolk exhaustion and the initial stages of exogenous feeding (Riveiro et al., 2000). Unfortunately, in the present study neither total protein content nor free amino acids were analysed. Carbohydrate composition and metabolism of common dentex eggs was similar to that of other Sparidae (Lahnsteiner and Patarnello, 2004a,b) and plays an important role during embryonic development. For each metabolite and enzyme a high deviation was obtained and subsequently the application of single regression curves was not possible and multiple regression analysis had to be utilised in order to relate the composition of the eggs with egg quality variables such as hatching rate and larval survival. Multiple regressions obtained for common dentex were different to that observed previously for gilthead seabream and sharpsnout seabream (Lahnsteiner and Patarnello, 2004a,b). In the case of common dentex a minimal activity of pyruvate kinase and minimal concentrations of monosaccharides and ribose are needed for normal embryonic development, whereas high concentration of 6-deoxyhexose and glucose resulted in decreased viability parameters of the larvae due to the interruption or low activity of the pathways of carbohydrate metabolism. For the embryos that showed a high alkaline phosphatase activity, hatching rate was reduced and larval mortality increased. If good and bad batches of eggs were compared, no differences in enzyme activity could be found but significant differences were obtained in terms of carbohydrate content. Low-quality eggs were found to contain higher amounts of ketose, glucose-6-phosphate, glucose and 6deoxyhexose which might indicate impaired catabolism (Glycolysis) for energy production and consequently the accumulation of glycolysis metabolites. Considering that carbohydrates are synthesized during embryonic development for use in the final stages of energy production and for mechanical and osmoregulatory function, it might be the case that the embryos of low-quality eggs were not utilising carbohydrates for energy production and other substrates, most probably lipids, were used instead. Alkaline phosphatase (ALP) is involved in the catabolism of phospholipids and in dephosphorylation of phosvitin in the yolk (Sire et al., 1994). In the present study high levels of this
241
enzyme were found to be correlated with larval mortality which might indicate that the yolk was consumed very fast in the eggs showing high ALP activity and as a consequence, the resulting larvae had lower amounts of yolk sac reserves before exogenous feeding. In comparison to other studies on S. aurata and P. puntazzo (Lahnsteiner and Patarnello, 2004a,b), higher hatching rates were obtained in the case of common dentex, and more advanced developmental stages were used for the analysis. Consequently, no correlation could be found between hatching rate and acid phosphatase, also involved in the catabolism of phospholipids, adenylate kinase, glucose-6-phosphatase, transaldolase and sialic acid which are involved in gluconeogenesis and in the pentose pathway. The differences observed in the composition of highand low-quality batches of eggs cannot be related to the developmental stage (all the eggs collected were in midepiboly and/or beginning of neurulation) or the quality of the broodstock. Both broodstock groups were cultured using the same photoperiod and water quality conditions and were fed the same semi-moist pellets and presumably egg quality was fairly high in both groups. Finally, in communal broodstock groups of batchspawning species the egg batches obtained everyday may include eggs from more than one female and differences in egg quality parameters due to parental variability may be high. Microsatellite identification of egg batches or newly hatched larvae is a very practical tool for the identification of parentage, maternal contribution to egg quality and composition, and for discrimination between egg batches. Acknowledgements GG thanks the financial support provided by the Ministry of Science and Education (INIA fellowship). Funding was partially provided to AE by the Spanish Ministries of Agriculture Fisheries and Food (Jacumar) and Science and Education (INIA project ACU02-006). Thanks are also due to J. Canoura, N. Gras and O. Bellot (CA-IRTA) for their help during common dentex rearing season and to Martina Radner (Salzourg University) for her help in the carbohydrate and enzyme analysis. References Abellan, E., 2000. Culture of common dentex (Dentex dentex L.) present knowledge, problems and perspectives. Cah. Options Mediterr. 47, 157–168. Ackman, R.G., 1980. Fish lipids. Part 1. In: Connell, J.J. (Ed.), Advances in Fish Science and Technology. Fishing News (Books) Ltd., Farnham, Surrey, UK.
242
G. Giménez et al. / Aquaculture 260 (2006) 232–243
Bell, J.G., Sargent, J.R., 2003. Arachidonic acid in aquaculture feeds: current status and future opportunities. Aquaculture 218, 491–499. Bernardo, D., Piñera, J.A., Blanco, G., Vázquez, E., Sánchez, J.A., 2004. Development of microsatellite markers in Pagellus bogaraveo and their usefulness in other five species with interest in aquaculture. Special Publication, vol. 34. European Aquaculture Society, pp. 160–161. Borrell, Y., Blanco, G., Vázquez, E., Álvarez, J., Pineda, H., Espinosa, G., Fernández-Pato, C., Martínez, C. y Sánchez, J.A. (2003). La variabilidad microsatélite y su aplicación en Acuicultura. CIVA2003 (http://www.civa2003.org), 1042–1056. Borrell, Y.J., Álvarez, J., Vázquez, E., Fernández Pato, C., Martínez Tapia, C., Sánchez, J.A., Blanco, G., 2004. Applying microsatellites to the management of farmed turbot stocks (Scophtalmus maximus L) in hatcheries. Aquaculture 241, 133–150. Bruce, M., Oyen, F., Bell, J.G., Asturiano, J.F., Carrillo, M., Zanuy, S., Ramos, J., Bromage, N., 1999. Development of broodstock diets for the European sea bass (Dicentrarchus labrax) with special emphasis on the importance of n − 3 and n − 6 highly unsaturated fatty acids to reproductive performance. Aquaculture 177, 85–97. Carbó, R., Estévez, A., Furones, M.D., 2002. Intelligent and multifunctional recirculation system. Its application in research at CA-IRTA. EAS Spec. Publ., vol. 32, pp. 171–172. Christie, W.W., 1982. Lipid Analysis. Robert Maxwell, M.C., Oxford. De la Herrán, R., Magoulas, A., Garrido-Ramos, M.A., Ruiz-Rejón, C., Ruiz-Rejón, M., Zouros, E., 2005.Desarrollo de microsatélies en tres especies de espáridos con interés en acuicultura. In: La acuicultura como actividad económica de las zonas costeras. IX Congreso Nacional de Acuicultura pp133–136. Ed. Consejería de Agricultura y Pesca. Junta de Andalucia. Sevilla. Spain. Efthimiou, S., Divanach, P., Rosenthal, H., 1994. Growth, food conversion and agonistic behaviour in common dentex (Dentex dentex) juveniles fed on pelleted moist and dry diets. Aquat. Living Resour. 7, 267–275. Fernandez-Palacios, H., Izquierdo, M.S., Robaina, L., Valencia, A., Salí, M., Vergara, J.M., 1995. Effect of n− 3 HUFA level in broodstock diets on egg quality of gilthead sea bream (Sparus aurata L.). Aquaculture 132, 325–337. Folch, J.M., Lees, M., Sloane Standley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509. Fraser, A.J., Gamble, J.C., Sargent, J.R., 1988. Changes in lipid content, lipid class composition and fatty acid composition in developing eggs and unfed larvae of cod (Gadus morhua). Mar. Biol. 99, 307–313. Furuita, H., Tanaka, H., Yamamoto, T., Shiraishi, M., Takeuchi, T., 2000. Effects of n − 3 HUFA levels in broodstock diet on the reproductive performance and egg and larval quality of the Japanese flounder, Paralichthys olivaceus. Aquaculture 187, 387–398. Glamuzina, B., Jug-Dujakovic, J., Katavic, I., 1989. Preliminary studies on reproduction and larval rearing of common dentex, Dentex dentex (Linnaeus, 1758). Aquaculture 77, 75–84. Henderson, R.J., Tocher, D.R., 1992. Thin-layer chromatography. In: Hamilton, R.J., Hamilton, S. (Eds.), Lipid Analysis. A Practical Approach. IRL Press, Oxford, pp. 65–111. Izquierdo, M.S., Fernández-Palacios, H., Tacon, A.G.J., 2001. Effect of broodstock nutrition on reproductive performance of fish. Aquaculture 197, 25–42. Kjorsvik, E., 1994. Egg quality in wild and broodstock cod L. J. World Aquac. Soc. 25, 22–29. Kjorsvik, E., Mangor Jensen, A., Holmefjord, I., 1990. Egg quality in fishes. Adv. Mar. Biol. 26, 71–113. Lahnsteiner, F., Patarnello, P., 2004a. Biochemical egg quality determination in the gilthead seabream, Sparus aurata: reproduc-
ibility of the method and its application for sharpsnout seabream, Puntazzo puntazzo. Aquaculture 237, 433–442. Lahnsteiner, F., Patarnello, P., 2004b. Egg quality determination in the gilthead seabream, Sparus aurata, with biochemical parameters. Aquaculture 237, 443–459. Maeland, A., Ronnestad, I., Waagbo, R., 2003. Folate in eggs and developing larvae of Atlantic halibut, Hippoglossus hippoglossus. Aquac. Nutr. 9, 185–188. Marshall, T.C., Slate, J., Kruuk, L.E.B., Pemberton, J., 1998. Statistical confidence for likelihood-based paternity inference in natural populations. Mol. Ecol. 7, 639–655. Mourente, G., Rodríguez, A., Grau, A., Pastor, E., 1999. Utilization of lipids by Dentex dentex L. (Osteichthyes, Sparidae) larvae during lecitotrophia and subsequent starvation. Fish Physiol. Biochem. 21, 45–58. Penney, R.W., Lush, P.L., Wade, J., Brown, J.A., Parrish, C.C., Burton, M.P.M., 2006. Comparative utility of egg blastomere morphology and lipid biochemistry for prediction of hatching success in Atlantic cod, Gadus morhua L. Aquac. Res. 37, 272–283. Pickova, J., Dutta, P.C., Larsson, P.O., Kiessling, A., 1997. Early embryonic cleavage pattern, hatching success, and egg-lipid fatty acid composition: comparison between two cod (Gadus morhua) stocks. Can. J. Fish. Aquat. Sci. 54, 2410–2416. Piñera, J.A., Bernardo, D., Blanco, G., Vázquez, E., Sánchez, J.A., 2004. Finding my parents: how many genetic markers are needed? Special Publication, vol. 34. European Aquaculture Society, pp. 661–662. Piñera, J.A., Bernardo, D., Vázquez, E., Blanco, G., Sánchez, J.A., 2006. Isolation and characterization of polymorphic microsatellite markers in Pagellus bogaraveo, and cross-species amplification in Sparus aurata and Dicentrarchus labrax. Mol. Ecol. 33–35. Rani, M.S., 2005. Prediction of larval viability based on egg quality parameters and early cleavage patterns in the experiments of triploidy induction in Atlantic cod, Gadus morhua L. M. Sc. Thesis, Univ. Tromso, Norway, 64 pp. Riveiro, I., Guisande, C., Lloves, M., Maneiro, I., Cabanas, J.M., 2000. Importante of parental effects on larval survival in Sardina pilchardus. Mar. Ecol., Prog. Ser. 205, 249–258. Rodriguez, C., Cejas, J.R., Martin, M.V., Badia, P., Samper, M., Lorenzo, A., 1998. Influence of n − 3 highly unsaturated fatty acid deficiency on the lipid composition of broodstock gilthead seabream (Sparus aurata L.) and on egg quality. Fish Physiol. Biochem. 18, 177–187. Rodriguez, C., Acosta, C., Badia, P., Cejas, J.R., Santamaria, F.J., Lorenzo, A., 2004. Assessment of lipid and essential fatty acids requirements of black seabream (Spondyliosoma cantharus) by comparison of lipid composition in muscle and liver of wild and captive adult fish. Comp. Biochem. Physiol., Part B Biochem. Mol. Biol. 139, 619–629. Ronnestad, I., Fyhn, H.J., 1993. Metabolic aspects of free amino acids in developing marine fish eggs and larvae. Rev. Fish Sci. 1, 239–259. Ronnestad, I., Lie, O., Waagbo, R., 1997. Vitamin B6 in Atlantic halibut, Hippoglossus hippoglossus—endogenous utilization and retention in larvae fed natural zooplankton. Aquaculture 157, 337–345. Ronnestad, I., Thorsen, A., Finn, R.N., 1999. Fish larval nutrition: a review of recent advances in the roles of amino acids. Aquaculture 177, 201–216. Salze, G., Tocher, D.R., Roy, W.J., Robertson, D.A., 2005. Egg quality determinants in cod (Gadus morhua L.): egg performance and lipids in eggs from farmed and wild broodstock. Aquac. Res. 36, 1488–1499. Sargent, J.R., 1995. Origins and functions of egg lipids: nutritional implications. In: Bromage, N., Roberts, R.J. (Eds.), Broodstock Management and Egg and Larval Quality. Blackwell Science, Cambridge, UK, pp. 353–372.
G. Giménez et al. / Aquaculture 260 (2006) 232–243 Shields, R.J., Brown, N.P., Bromage, N.R., 1997. Blastomere morphology as a predictive measure of fish egg viability. Aquaculture 155, 1–12. Sire, M.F., Babin, P.J., Vernier, J.M., 1994. Involvement of the lysosomal system in yolk protein deposit and degradation during vitellogenesis and embryonic development in trout. J. Exp. Zool. 269, 69–83. Thorsen, A., Trippel, E.A., Lambert, Y., 2003. Experimental methods to monitor the production and quality of eggs of captive marine fish. J. Northwest Atl. Fish. Sci. 33, 55–70. Tocher, D.R., Harvie, D.G., 1988. Fatty acid compositions of the major phosphoglycerides from fish neural tissues: (n − 3) and (n − 6) polyunsaturated fatty acids in rainbow trout (Salmo gairdneri) and cod (Gadus morhua) brains and retinas. Fish Physiol. Biochem. 5, 229–239. Tocher, D.R., Fraser, A.J., Sargent, J.R., Gamble, J.C., 1985. Lipid class composition during embryonic and early larval development in Atlantic herring (Clupea harengus L.). Lipids 20, 84–89.
243
Tveiten, H., Jobling, M., Andreassen, I., 2004. Influence of egg lipids and fatty acids on egg viability and their utilization during embryonic development of spotted wolfish Anahichas minor Olafsen. Aquac. Res. 35, 152–161. Vallin, L., Nissling, A., 1998. Cell morphology as an indicator of viability of cod eggs—results from an experimental study. Fish. Res. 38, 247–255. Walsh, P.S., Metzger, D.A., Higuchi, R., 1991. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques 10, 506–513. Watanabe, T., 1985. Importance of the study of broodstock nutrition for further development of aquaculture. In: Cowey, C.B, Mackie, A.M., Bell, J.K. (Eds.), Nutrition and Feeding in Fish. Academic Press, London, UK, pp. 395–414.