Fish Physiol Biochem DOI 10.1007/s10695-014-9908-9
Biochemical composition and quality of turbot (Scophthalmus maximus) eggs throughout the reproductive season Yudong Jia • Zhen Meng • Xinfu Liu Jilin Lei
•
Received: 18 March 2013 / Accepted: 6 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The present study investigated the biochemical composition and quality of turbot (Scophthalmus maximus) eggs throughout the reproductive season. Results showed that the fertilization, hatching and egg floating rates were variable throughout the reproductive season, with the highest values recorded during the mid-season. Meanwhile, positive correlations were found between fertilization, hatching rate and floating rate. The composition of turbot eggs, including total lipid, protein, carbohydrate, moisture and dry weight showed no significant differences during the reproductive season. Furthermore, no correlations were found between egg compositions and viability parameters (VPs), including fertilization and hatching rates as well as larval deformity rate. However, egg diameter varied and correlated with fertilization, hatching and egg floating rates. The fatty acid in eggs at mid-season had significantly higher levels of C14:0, C16:0, C16:1n-7, C18:0, C18:1, C20:4n-6, C20:5n-3 and C22:6n-3. Moreover, significant relationships were found between fatty acids and
Y. Jia Z. Meng X. Liu J. Lei (&) Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, No. 106 Nanjing Road, Qingdao 266071, People’s Republic of China e-mail: [email protected] Y. Jia Z. Meng X. Liu J. Lei Qingdao Key Laboratory for Marine Fish Breeding and Biotechnology, Qingdao 266071, People’s Republic of China
VPs. Eggs of the middle season had significantly higher concentration of isoleucine, leucine, lysine, methionine, valine, alanine, aspartate, glutamate and serine, whereas no significant relationships were found between amino acids and VPs. These observations suggest that the biochemical profile of eggs may be useful in evaluating egg quality and improving broodstock management for turbot. Keywords Turbot (Scophthalmus maximus) Biochemical composition Egg quality Reproduction
Introduction Egg quality is one of the limiting factors of seed production as well as the main obstacle for aquaculture development for seawater and freshwater species. Knowledge of the processes that influence egg quality, which can have direct practical use in broodstock management, is important for the control of aquatic production. Fish egg quality can be defined as the ability of the egg to be fertilized and subsequently developed into a normal embryo (Bobe and Labbe 2010). In fish, egg quality is highly variable and is affected by nutritional status, stress, overripening, hormonal induction of spawning, genetics and age of broodstock, in addition to water quality and environmental factors (Lahnsteiner 2000; Finn 2007; Zakeri et al. 2009; Mylonas et al. 2010; Schreck 2010;
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Fish Physiol Biochem
Villamizar et al. 2011; Jerez et al. 2012; Lahnsteiner and Kletzl 2012). Numerous efforts have been undertaken to evaluate the criteria for fish egg quality. Although fertilization and hatching rates are the most widely used indicators, other parameters for egg morphology, such as buoyancy, egg size, distribution of lipid droplets and abnormal blastomere morphology, have been cited as reliable indicators of egg quality (Unuma et al. 2005; Mansour et al. 2008; Aristizabal et al. 2009; Kohn and Symonds 2012). The above indicators of egg quality could be species-specific. In addition, biochemical parameters [yolk protein, lipids, carbohydrates, amino acids, vitamins and enzymes] have been considered as viable indicators of egg quality (Gime´nez et al. 2006; Faulk and Holt 2008; Lubzens et al. 2010; Samaee et al. 2010; Lanes et al. 2012). Morphological criteria, as well as fertilization and hatching rates, may be indicative of egg quality, but tell us nothing about what factors determine egg quality. Therefore, investigating the biochemical composition of fish egg and identifying a method for the reliable discrimination between low- and high-quality egg batches are vital in reducing hatchery production costs. These approaches would prevent the unnecessary occupation of hatchery staff time and facilities on what may prove to be unproductive egg batches. Turbot (Scophthalmus maximus) is a widely cultured marine fish of considerable commercial value in Europe and China. In recent years, studies have been conducted on the reproduction (Mugnier et al. 2000; Gosz et al. 2011), immune response (Pereiro et al. 2012), environmental conditions (Imsland et al. 2003; Nissling et al. 2006; Foss et al. 2009; Silva et al. 2011) and nutritional requirements (Leknes et al. 2012; Qi et al. 2012) of turbot in captivity. However, information on the biochemical composition of spawned eggs and subsequent consequences on the egg/larval quality parameters is sparse, particularly, reports on the proportion of floating eggs, fertilization and hatch rates, larval survival and growth for this species. In addition, the relationship among morphological characteristics, biochemical composition and viability parameters (VPs: fertilization rate [FR], hatching rate [HR] and larval deformity rate [LD]) at various stages of the reproductive season has not been evaluated. Such information would be important for producing a steady supply of high-quality eggs and for ensuring that time and resources are not wasted on egg batches with low survival and/or performance potential.
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The present study aims to compare VPs and biochemical profiles (proteins, carbohydrates, lipids, fatty acids and amino acids) of turbot (S. maximus) eggs throughout the reproductive season in pursuit of better broodstock management. Meanwhile, relationship between changes observed in biochemical composition and egg quality is also evaluated.
Materials and methods Broodstock management and egg collection Four-year-old mature turbots used for the experiments were obtained from the broodstock of a commercial fish farm (Yantai Tianyuan Aquatic Limited Corporation, China). The fish (five females, two males) weighting 3,500–4,000 g were kept in round tanks of 8 m3 at a stocking of 4 kg/m3. The tanks were supplied with recirculating water at a rate of 25 L/min and exposed to a constant photoperiod (16 h light:8 h dark). Water salinity and oxygen were ranged from 20 to 25 g/L and 5 to 9 mg/L, respectively. Temperature was maintained between 12 and 13 °C. Fish were fed with a diet of frozen sardines, squid and shrimp. Females had spawned multiple times during the reproductive season. In this study, the spawning period commenced in mid-October and ended in midDecember in artificial broodstock. Ovulatory rhythms were determined based on the first ovulation date by using daily abdominal stripping at the start of the spawning season. This procedure was performed to ensure that fresh ovulated eggs were obtained and to prevent overripening (Mcevoy 1984). Due to their ovulatory cycles of 70–90 h, females were stripped every 4–5 days to avoid overripening. Each female could spawn 8–12 times at 3–5-day intervals throughout the spawning season (Mugnier et al. 2000). Three periods of 10–15 days length were considered, namely early, mid- and late seasons, to study the variation of egg quality parameters throughout the spawning season. In the present study, three females could spawn all along the spawning period and three batches of egg samples were collected from each female in each period. Thus, we collect a total of 9 batches of samples in each period and performed artificial fertilization on each spawn independently. The number of eggs per batch was estimated by counting the eggs contained in an aliquot of 500 lL and measuring
Fish Physiol Biochem Table 1 Number of egg batches, egg volume per batch and ovulatory rhythm of turbot during the reproductive season Early season
Mid-season
Late season
Number of egg batches
3 (3)
3 (3)
3 (3)
Batch volume (mL)
105.33 ± 1.50 (3)a
156.56 ± 1.80 (3)b
Ovulatory rhythm (days)
a
4.46 ± 0.11 (3)
b
3.50 ± 0.09 (3)
100.11 ± 1.32 (3)a 4.17 ± 0.06 (3)c
Values are mean ± SEM (number of fish). Means within a row with different superscripts are significantly different (P \ 0.05)
the total volume of the batch. The egg batches, egg volume per batch and ovulatory rhythm of turbot during the reproductive season are shown in Table 1. Eggs were fertilized in seawater by gentle mixing with newly obtained milt from two males, which was collected with a plastic syringe and kept at 4 °C. Samples contaminated with urine or seawater was discarded. Only those milt samples that showed a motility of over 80 % of spermatozoa were used for fertilization. Sperm motility was measured by activating 1 mL of sperm (pre-diluted 1:5 v/v in a Ringer solution) with 19 lL of seawater and scored immediately under light microscopy. The percentage of motile spermatozoa was scored according to a previous method (Cejko et al. 2010). The volume ratio sperm:eggs:water used was 0.5:100:100, for which fertilization rate is maximal in turbot (Mugnier et al. 2000). Egg and larval quality measurements After the eggs were collected, their diameter was measured using a digital caliper (Mitutoyo CD-20C). Turbot egg is a type of buoyant eggs. Floating eggs are viable and used to calculate the FR and HR (Mcevoy 1984; Kjørsvik et al. 2003). The floating rate was calculated as the number of floating eggs divided by the total number of eggs spawned. FR was evaluated as follows: Three samples of one hundred eggs from the floating fraction of each batch were placed into Petri dishes and observed under a stereoscopic microscope (Carl Zeiss Vision, Aalen, Germany) at least 4 h after egg fertilized. Eggs at the 8- to 32-cell stage were classified as fertilized (Kjørsvik et al. 2003). HR was calculated based on the number of fertilized eggs placed into the incubators and the number of larvae that appear after hatching. In addition, the percentage of body deformities in newly hatched larvae, including lordosis, kyphosis and scoliosis, was visually determined under a stereoscopic microscope (Carl Zeiss
Vision, Aalen, Germany). FR, HR, LD and floating rate were calculated using the following formulae: Floating rate ð100 %Þ ¼ 100 no: of buoyant eggs= no: of ovulated eggs Fertilization rate ð100 %Þ ¼ 100 no: of fertilized eggs=no: of buoyant eggs Hatching rate ð100 %Þ ¼ 100 no: of hatched larvae=no: of buoyant eggs Larval deformity rate ð100 %Þ ¼ 100 no: of deformed larvae=no: of hatched larvae Biochemical analysis From each egg batch, three sample eggs were collected and rinsed three times with 50 ml distilled water and immediately stored at -80 °C until analysis. The amount of samples used for each analysis was as follows: wet/dry weight (100 eggs), protein (8–10 mg dry weight, 150 eggs), carbohydrate (5–6 mg dry weight, 80 eggs), total lipid (6–10 mg dry weight, 150 eggs), fatty acids (500–600 mg dry weight, 800 eggs) and amino acids (100–200 mg dry weight, 200 eggs). Moisture content was determined according to the previous method (Faulk and Holt 2008). Briefly, after rinsing the eggs, external water was removed by placing the sample on top of paper towels. The sample was immediately transferred to a pre-weighed glass vial and weighed on microbalance (Shimadzu Corp., Kyoto, Japan). Samples were then lyophilized for 24 h and then reweighed to obtain their dry weight. Moisture content was calculated by subtracting the egg dry weight of eggs from their wet weight, dividing by wet weight and multiplying by 100. Protein was quantified using the Bradford protein assay kit (Beyotime Biotechnology, Haimen, China). A standard curve (0.5–5 mg) was generated using
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Fish Physiol Biochem
bovine serum albumin with two replicates per standard concentration, and the absorbance at 485 nm was measured. Carbohydrate content was extracted and measured according to a previous method (Guisande et al. 1998). Briefly, samples were homogenized in 2 mL of distilled water, and an aliquot of 400 lL was used in the assay. To sample volumes of 400 lL, 10 lL of 81 % phenol was added to the solution, and after gently shaking, 1 mL of concentrated sulfuric acid (18 M) was added. The samples were again shaken and maintained at room temperature for 30 min. Then, the absorbance was read at 485 nm. A standard curve (2.5–50 lg) was established by using reagent-grade glucose. Total lipids were cold extracted from lyophilized samples using chloroform/methanol (2:1 v/v) as described by Folch et al. (1957) with 0.01 % (w/v) butylated hydroxytoluene (BHT) as an antioxidant. Prior to homogenization, heptadecanoic acid (17:0, Sigma, St. Louis, MO, USA) was added as an internal standard for subsequent quantification of fatty acid. Fatty acids were saponified and methylated using the method described by Mansour et al. (2011). The fatty acid methyl esters were analyzed on a gas chromatograph (Agilent 6890N, Palo Alto, CA, USA) equipped with an Agilent 7,683 autosampler (Agilent Technologies), an Agilent capillary column (length, 20 m; internal diameter, 0.10 mm; film thickness, 0.10 lm; Agilent Technologies) and a flame ionization detector. Fatty acids were identified by comparing the retention times with standard values. The results were expressed as gram of each fatty acid per kilogram of tissue dry weight (g/kg dry weight). The amino acid compositions of eggs were determined using a reversed phase high-performance liquid chromatograph (Agilent 1100 Series, Santa Clara, CA, USA), equipped with a UV detector. Samples for amino acid analysis were hydrolyzed using 6 N HCl for 24 h at 115 °C. Cysteine was oxidized with 0.01 N sodium hydroxide and determined as cystine. Tryptophan could not be measured because of its degradation during acid hydrolysis. Hydrolysate amino acid concentrations of eggs were expressed as gram of each fatty acid per kilogram of tissue dry weight (g/kg dry weight). Statistical analysis Shapiro–Wilk normality test and Bartlett test were used to estimate the data complied or not with normal distribution
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and homogeneity of variance, respectively (Faraway 2005). The data were analyzed via one-way analysis of variance (ANOVA) and Duncan’s multiple-range tests using the SAS 8.0 software. Relationships between biochemical parameters of eggs and VPs were assessed by Pearson’s correlation coefficient and simple regression models. All data are presented as the mean ± standard error of the mean (SEM). In all statistical tests used, P \ 0.05 was considered significantly different.
Results The FR, HR and floating rate were varied throughout the reproductive season, with the highest values observed in the mid-season (Table 2, P \ 0.05); the early and late seasons showed no significant differences (Table 2, P [ 0.05). Meanwhile, the linear relationship between the FR and HR and the floating rate on the one hand and the floating rate on the other hand were significant during the spawning season (Fig. 1a, b). The lowest LD was obtained in the middle season (Table 2, P \ 0.05), the early and late seasons showed no significant differences (Table 2, P [ 0.05). The composition of turbot eggs, including total lipid, protein, carbohydrate, moisture and dry weight showed no significant differences during the reproductive seasons (Tables 2, 3, P [ 0.05). Egg diameter was significantly lower in the mid-season (Table 2, P \ 0.05). Moreover, a negative correlation was observed between egg diameter and the fertilization, hatching and floating rates during the reproductive season (Figs. 1c, d, 2). The fatty acid profile of egg lipids differed significantly between the early and middle reproductive seasons (Table 4, P \ 0.05). In general, eggs spawned of the mid-season had significantly higher concentrations of C14:0, C16:0, C16:1n-7, C18:0, C18:1, C20:4n-6, C20:5n-3, C22:6n-3, saturated fatty acid (SFA), polyunsaturated fatty acid (PUFA) and monounsaturated fatty acid (MUFA). However, the fatty acid composition of total lipid was not significantly different between the late and early or middle reproductive seasons (Table 4, P [ 0.05). Furthermore, significant relationships were found between VPs and concentrations of other fatty acids (Table 5, P \ 0.05). Significant correlations were observed between C14:0, C16:0, C16:1n-7, C20:4n-6, C20:5n-3, C22:6n-3, SFA, PUFA and LD (linear), and also between
Fish Physiol Biochem Table 2 Mean floating, fertilization, hatching and larval deformity rate (%) throughout the reproductive season of turbot Early season Egg diameter (mm) Floating rate (%)
Mid-season
Late season
1.17 ± 0.03a
1.01 ± 0.01b
1.16 ± 0.02a
a
b
48.81 ± 2.09a
46.12 ± 1.16
90.48 ± 0.51
Fertilization rate (%) Hatching rate (%)
a
37.42 ± 2.13 41.18 ± 3.43a
b
90.74 ± 1.01 76.49 ± 3.29b
35.67 ± 2.01a 39.43 ± 2.71a
Larval deformity rate (%)
12.69 ± 0.32a
9.24 ± 0.09b
12.38 ± 0.23a
Data are expressed as mean ± SEM (n = 9). Means within a row with different superscripts are significantly different (P \ 0.05)
Fig. 1 Relations between floating rate (a, b), egg diameter (c, d) and the fertilization, hatching rate in turbot. Early (cross), middle (triangle) and late (circle) seasons
C16:0, C16:1n-7, C20:4n-6, C20:5n-3, C22:6n-3, SFA and FR (quadratic) and HR (quadratic). The content of amino acid content (g/kg dry weight) was significantly higher in the mid-season
than in the early and late seasons (Table 6, P \ 0.05). Essential amino acids, including isoleucine, leucine, lysine, methionine and valine, were significantly higher in the mid-season (Table 6, P \ 0.05). Non-
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Fish Physiol Biochem Table 3 Mean egg diameter, dry weight, moisture content, total lipid, protein and carbohydrate throughout the reproductive season of turbot Early season
Mid-season
Late season
Dry weight (mg 100 egg)
6.85 ± 0.06
6.89 ± 0.03
6.42 ± 0.26
Moisture (% wet weight) Proteins (% dry weight)
90.17 ± 0.06
90.04 ± 0.06
90.11 ± 0.58
11.83 ± 0.68
12.77 ± 0.67
13.17 ± 0.75
Total lipid (% dry weight)
19.81 ± 0.35
20.57 ± 0.84
20.03 ± 0.67
Carbohydrate (% dry weight)
9.74 ± 0.75
9.36 ± 0.30
8.91 ± 0.28
Data are mean ± SEM (n = 9). No significant differences were found during reproductive season (P [ 0.05)
Fig. 2 Relationships between the egg diameter and the floating rate in turbot. Early (cross), middle (triangle) and late (circle) seasons
essential amino acids, including alanine, aspartate, glutamate and serine, were also significantly higher in the mid-season than in the early season (Table 6, P \ 0.05). However, no significant relationships were found between VPs and the composition of amino acid in turbot eggs (data not shown).
Discussion Egg and larval quality are key factors to the successful production of viable offspring in marine teleosts. Egg
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quality is influenced by several parameters that frequently change during the reproductive season. Among these parameters, egg morphology as well as FR and HR are the most widely used indicators of egg quality. The buoyancy of eggs has often been used as an indicator in the assessment of egg quality in marine fish. It was reported that the ratio of buoyant eggs to total eggs spawned correlates positively with egg hatchability (Seoka et al. 2003; Unuma et al. 2005; Furuita et al. 2007). In addition, the size of the eggs is sometimes considered to be beneficial for the future development of the embryo. Mansour et al. (2008) found that low-quality (low FR) eggs of Salvelinus alpinus tend to have large egg diameter than good- and fair-quality eggs. In addition, the egg diameter of Melanogrammus aeglefinus declines over the spawning season and positively correlates with FR (Trippel and Neil 2004). Similar observations have been reported in other marine fish species (Lahnsteiner 2000; Aegerter and Jalabert 2004). Likewise, turbot egg diameter becomes significantly smaller as the season progresses (Lavens et al. 1999). Therefore, egg diameter is often cited as an important criterion for the assessment of egg quality and reproductive performance in many fish species. In the present study, the FR, HR and floating rate of eggs were found to be variable throughout the reproductive season, with the highest values observed in the mid-season, whereas low values was seen in the early and late season. Furthermore, positive correlations were found between FR, HR and the egg floating rate. However, egg diameter was significantly the lowest in the middle of the productive season and negative correlation was detected between egg diameter and the FR, HR, floating rate in turbot eggs. These results indicated that high-quality egg batches were observed in the middle of the reproductive season. Furthermore, our results show that buoyancy and diameter of eggs may be useful indicators for assessing turbot egg quality during the reproductive season. Embryogenesis and larval development are long processes that require a high input of nutrients and energy from egg reserves in most fish species (Boulekbache 1981; Dayal et al. 2003; Cejas et al. 2004; Kamler 2005). In principle, fish eggs contain all the nutrients to support both homeostasis and development of larvae during the lecithotrophic phase (Lubzens et al. 2010). Moreover, the biochemical composition of fish eggs is species-specific, and the
Fish Physiol Biochem Table 4 Fatty acid composition (g/kg dry weight) of turbot eggs throughout the reproductive season Fatty acid
Early season
Mid-season
Late season
C14:0
1.63 ± 0.35b
2.72 ± 0.12a
2.30 ± 0.18ab
C16:0
b
a
17.06 ± 2.11ab
12.69 ± 2.59
22.06 ± 0.68
b
5.35 ± 0.99 2.82 ± 0.55b
a
10.08 ± 0.71 4.50 ± 0.25a
7.45 ± 0.71ab 3.96 ± 0.50ab
C18:1
13.91 ± 2.53b
25.34 ± 1.52a
21.56 ± 2.92ab
C18:2
1.32 ± 0.22
1.97 ± 0.13
1.90 ± 0.23
C18:3
0.49 ± 0.12
0.78 ± 0.04
0.70 ± 0.04
C20:4n-6(AA)
1.49 ± 0.28a
2.58 ± 0.08b
2.02 ± 0.23ab
C20:5n-3(EPA)
4.25 ± 0.96a
7.62 ± 0.55b
5.63 ± 0.51ab
C16:1n-7 C18:0
C22:5n-3
2.38 ± 0.50
3.65 ± 0.15
3.21 ± 0.42
C22:6n-3(DHA)
16.78 ± 3.42a
29.21 ± 0.62b
22.16 ± 2.38ab
SFA
17.73 ± 3.61a
30.50 ± 0.78b
24.20 ± 2.82ab
b
a
37.11 ± 4.06ab
PUFA
25.97 ± 5.00
47.31 ± 1.12
MUFA
20.05 ± 3.70b
36.61 ± 2.19a
29.76 ± 3.73ab
DHA:EPA
4.01 ± 0.16
3.87 ± 0.28
3.93 ± 0.12
EPA:ARA
2.82 ± 0.35
2.96 ± 0.20
2.80 ± 0.07
Data are expressed as mean ± SEM (n = 9). Means within a row with different superscripts are significantly different (P \ 0.05) SFA saturated fatty acid, PUFA polyunsaturated fatty acid and MUFA monounsaturated fatty acid
precise sequence of consumption (proteins and amino acids, lipids and fatty acids and carbohydrates) varies both qualitatively and quantitatively. Embryos of turbot (S. maximus) have been reported to catabolize proteins and carbohydrates exclusively (Planas et al. 1989), whereas embryos of trout (Oncorhynchus mykiss) use proteins, lipids and carbohydrates to satisfy their energy requirements (Boulekbache 1981). In addition, the biochemical composition profile of fish eggs varies among species and has been shown to increase, decrease or remain constant during the spawning season (Aegerter and Jalabert 2004; Faulk and Holt 2008; Fuiman and Ojanguren 2011). The current work found that the dry weight, moisture, protein, total lipid and carbohydrate were fairly consistent and no significant difference was observed throughout the reproductive season in turbot eggs. Similar results have been described for other marine fish species (Nocillado et al. 2000; Dayal et al. 2003; Gime´nez et al. 2006; Faulk and Holt 2008). The total lipid content of marine fish eggs frequently ranges from 15 to 35 % dry weight (Vetter et al. 1983; Ostrowski and Divakaran 1991; Sargent 1995; Dayal et al. 2003; Samaee et al. 2009), which is similar to our study here for turbot eggs. In addition, the total lipid content of fish eggs is generally more conserved than
other fish tissues during spawning season (Sheridan 1988; Tocher 2003; Salze et al. 2005). We found no significant difference in the total lipid content of turbot eggs during reproductive season. However, lipid content of turbot eggs was higher than the ones reported by Silversand et al. (1996) for this species. This difference may be on account of different reared condition (the temperature was maintained 10 ± 1 °C) and nutritional regime (they fed three times a week with a formulated moist diet). Proteins in eggs are extremely important for the functionality of the female gamete. Amino acids are basic unit of protein. In the current study, we found the composition of amino acid content variable during the spawning season (Table 6), whereas the total amino acid content showed no significant change in turbot egg (data not shown). This is consistent with our observation that the protein contents of turbot eggs did not change during the spawning season. In the nutritional profile of fish eggs, lipid and fatty acids provide metabolic substrates for embryonic and larval development, and they serve as structural components in the biogenesis of membranes. These are particularly important in regulating numerous physiological processes including steroidogenesis, embryonic development, hatching and early larval
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Fish Physiol Biochem Table 5 Simple regression equations, explanatory effect (R2), F and P values of the significant relationships found between the turbot egg fatty acid content and the VPs (FR, HR, LD) considered in current study Variables Dependent (fatty acids)
Equations
R2
F
P
Independent (VPs)
1 C14:0
LD
y = -2.010x ? 15.889
0.498
6.956
0.034
2 C16:0
FR
y = 0.447x2 - 11.380x ? 98.128
0.700
6.990
0.027
3 C16:0
HR
y = 0.354x2 - 8.786x ? 90.684
0.680
6.362
0.033
4 C16:0
LD
y = -0.252x ? 15.778
0.498
6.956
0.034
5 C16:1n-7
FR
y = 1.449x2 - 12.988x ? 61.985
0.704
7.140
0.026
2
6 C16:1n-7
HR
y = 1.124x - 10.998x ? 65.122
0.707
7.241
0.025
7 C16:1n-7
LD
y = -0.252x ? 15.778
0.557
8.809
0.021
8 C20:4n-6
FR
y = 39.528x2 - 115.826x ? 115.364
0.711
7.383
0.024
9 C20:4n-6
HR
y = 28.669x2 - 86.305x ? 101.096
0.691
6.705
0.030
10 C20:4n-6
LD
y = -2.186x ? 15.872
0.543
8.319
0.024
FR HR
2
y = 2.692x - 18.745x ? 64.565 y = 2.088x2 - 15.906x ? 68.047
0.692 0.684
6.751 6.485
0.029 0.032 0.024
11 C20:5n-3 12 C20:5n-3 13 C20:5n-3
LD
y = -2.186x ? 15.872
0.543
8.319
14 C22:6n-3
FR
y = 0.317x2-10.200x ? 110.718
0.801
12.055
0.008
15 C22:6n-3
HR
y = 0.234x2-7.793x ? 99.928
0.782
10.760
0.010
16 C22:6n-3
LD
y = -0.012x2 ? 0.281x ? 11.486
0.714
7.498
0.023 0.031
2
17 SFA
FR
y = 0.263x -8.827x ? 103.357
0.687
6.600
18 SFA
HR
y = 0.200x2-6.991x ? 96.503
0.685
6.519
0.031
19 SFA
LD
y = -0.182x ? 15.819
0.538
8.165
0.024
20 PUFA
FR
y = 0.114x2-5.973x ? 108.801
0.719
7.684
0.022
21 PUFA
HR
y = 0.087x2-4.79x ? 101.707
0.717
7.584
0.023
2
22 PUFA
LD
y = -0.004x ? 0.125x ? 12.087
0.652
5.633
0.042
23 MUFA
LD
y = -0.135x ? 15.322
0.485
6.584
0.037
R2 [ 0.49 and P \ 0.05 are considered significance levels for regression analysis SFA saturated fatty acid, PUFA polyunsaturated fatty acid, MUFA monounsaturated fatty acid, FR fertilization rate, HR hatching rate and LD larval deformity rate
performance (Cejas et al. 2004; Mansour et al. 2011). In general, marine fish egg lipids are rich in highly unsaturated fatty acids including docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and arachidonic acid (AA). DHA plays an important role as a structural component of cell membranes, especially in the processes of synaptogenesis and retinogenesis, during embryonic development in fish (Sargent et al. 2002; Tocher 2010). EPA is also a structural component of membrane phospholipids, and both fatty acids compete for enzymes that esterify them into phospholipids (Sargent et al. 2002). AA is known as a primary precursor of physiologically active molecules such as prostaglandins and leukotrienes, which are involved in the regulation of embryonic development, hatching and early larval performance (Bell and Sargent 2003).
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The DHA/EPA ratio can lead to an inadequate balance in the structural composition of cells and can affect the developing embryo as well as the subsequent larval survival and growth (Furuita et al. 2002). The level of essential fatty acids—DHA, EPA and AA—in eggs as well as the balance among them must be considered simultaneously (Sargent et al. 2002; Bell and Sargent 2003). Moreover, the composition of fatty acid and DHA/EPA in eggs is species-specific. Other fatty acids in eggs of marine fish vary and correlate with egg quality during spawning season. Samaee et al. (2009) reported that myristic acid (C14:0) level was higher in high-quality eggs (high FR and HR) than in other egg categories in Dentex dentex, and a statistical significance was found between myristic acid (C14:0) and LD during spawning season. However, palmitic acid
Fish Physiol Biochem Table 6 Amino acid composition (g kg-1 dry weight) of turbot eggs throughout the reproductive season Amino acid
Early season
Mid-season
Late season
Essential Arginine
37.73 ± 0.66
39.63 ± 0.50
39.00 ± 0.80
Histidine Isoleucine
17.27 ± 0.47 37.97 ± 0.70b
18.33 ± 0.32 42.40 ± 0.50a
17.80 ± 0.45 39.87 ± 0.92b
Leucine
58.57 ± 1.07b
65.47 ± 0.91a
61.67 ± 1.24b
48.20 ± 1.01
c
a
51.63 ± 0.83b
Methionine
14.87 ± 0.29
b
a
17.57 ± 0.28
16.67 ± 0.47a
Phenylalanine
33.10 ± 0.74
34.57 ± 0.35
34.23 ± 0.72
Threonine
29.80 ± 0.90
31.07 ± 0.61
31.00 ± 0.30
Valine
44.17 ± 0.83b
49.67 ± 0.55a
46.97 ± 0.93a
48.27 ± 1.23b
55.77 ± 1.70a
53.07 ± 0.38a
b
a
Lysine
54.87 ± 0.84
Non-essential Alanine Aspartate
45.07 ± 0.84
Cysteine
4.33 ± 0.19
Glutamate
86.97 ± 0.77
b
48.00 ± 0.81
46.97 ± 0.78ab
4.73 ± 0.23
4.80 ± 0.25
a
97.13 ± 1.78
90.93 ± 1.82b
Glycine
20.43 ± 0.27
21.60 ± 0.26
21.17 ± 0.44
Proline
42.77 ± 2.05
46.30 ± 1.91
46.27 ± 0.73
31.80 ± 1.29a
32.60 ± 0.61a
29.27 ± 0.41
29.53 ± 0.57
Serine Tyrosine
27.1 ± 0.20b 27.76 ± 0.58
Data are expressed as mean ± SEM(n = 9). Means within a row with different superscripts are significantly different (P \ 0.05)
(C16:0) and palmitoleic acid (C16:1n-7) content of fish eggs and their correlations with VPs are speciesspecific during spawning season (Samaee et al. 2009; Jerez et al. 2012). In the present study, the fatty acid compositions of eggs were found to be significantly different with variable changes throughout the reproductive season, with the highest value of DHA, EPA, AA, C14:0, C16:0 and C16:1n-7 observed in the midseason. Significantly, relationships were also found between VPs and the concentrations of DHA, EPA, AA and C14:0, C16:0, C16:1n-7 in turbot eggs (Table 5). This might indicate the existence of a series of synergisms (positive or negative) among the egg fatty acids and effects of the synergisms on fish biological processes. These statistical findings suggest the need for more suitable experimental approaches to find the biological grounds for these interrelations among different fatty acids. Moreover, in other marine fish species, significant correlations have been reported between fatty acids and VPs (Salze et al. 2005; Mansour et al. 2011; Jerez et al. 2012; Lanes et al. 2012). Meanwhile, DHA/EPA ratios ranged between 4:1 and 3:1. Similar values have been described by Silversand et al. (1996). These results
indicated that fatty acid of turbot eggs has a higher direct effect on VPs during the reproductive season. Therefore, fatty acid composition and the concentration of eggs may be considered as potential biochemical markers to predict turbot egg quality. Amino acids are the main energy resources during egg development and are also osmotic active compounds that regulate egg hydration and, subsequently, egg buoyancy during the final maturation of oocytes (Rønnestad et al. 1992, 1996). Several studies have demonstrated that amino acids are lower in nonviable than in viable eggs (Nocillado et al. 2000; Samaee et al. 2010; Lanes et al. 2012). High levels of amino acids could provide more energy resource for embryonic and larval development after fertilization. Samaee et al. (2010) showed that egg amino acid contents were significantly correlated with VPs in common dentex (Dentex dentex). Furthermore, the amino acid composition profile of fish eggs varies among species and has been shown to increase, decrease or remain constant during the spawning season (Rønnestad and Fyhn 1993; Rønnestad et al. 1996; Faulk and Holt 2008; Samaee et al. 2010). The present study demonstrated that the levels of amino
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Fish Physiol Biochem
acids were variable during the reproductive season, with the highest value in the mid-season. In addition, the profile generated here for turbot was similar to that reported for a number of marine fish eggs during the spawning season (Rønnestad et al. 1996; Faulk and Holt 2008; Lanes et al. 2012). However, significant relationships were not found between VPs and the profile of amino acids in turbot eggs. In summary, the results of this study indicated that egg quality was highly variable throughout the reproductive season, whereas high-quality turbot eggs were observed in the middle of the reproductive season. The diameter and buoyancy may be used as predictors of turbot egg quality during reproductive season due to correlation with VPs. Meanwhile, the fatty acid and amino acid profiles of eggs were variable throughout the reproductive season, possibly promoting the synthesis of vitellogenin and consequently affecting embryo and larval viability. These findings suggest that information on the biochemical profile of eggs may be useful in evaluating egg quality and improving broodstock management for turbot. In addition, further studies should seek to understand the relationship between the biochemical profiles of eggs and VPs over different female ages and spawning seasons, which will aid in the identification of new biomarkers of egg quality. Acknowledgments This study was supported by National Natural Science Foundation of China (31302205), Natural Science Foundation of Shandong Province (ZR2012CQ024, BS2013SW004) and the China Postdoctoral Science Foundation (2012M511559, 2013T60690). We thank Huaxin Niu and Peng Hu (Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences) for help in the experiment.
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response induced by intramuscular DNA vaccine encoding for viral haemorrhagic septicaemia virus (VHSV) G glycoprotein in turbot (Scophthalmus maximus). Fish Shellfish Immunol 32:1088–1094 Planas M, Ferreiro MJ, Fernandez-Reiriz MJ, Labarta U (1989) Evolucio´n de la composicio´n bioquı´mı´ca y actividades enzima´ticas en huevos de rodaballo (Scophthalmus maximus.) durante la embrioge´nesis. In: Yufera M (ed) Acuicultura intermareal. Andalucı´a, Cadiz, pp 215–227 Qi GS, Ai QH, Mai KS, Xu W, Liufu ZG, Yun B, Zhou HH (2012) Effects of dietary taurine supplementation to a casein-based diet on growth performance and taurine distribution in two sizes of juvenile turbot (Scophthalmus maximus L.). Aquaculture 358:122–128 Rønnestad I, Fyhn HJ (1993) Metabolic aspects of free amino acids in developing marine fish eggs and larvae. Rev Fish Sci 1:239–259 Rønnestad I, Fyhn HJ, Gravningen K (1992) The importance of free amino acids to the energy metabolism of eggs and larvae of turbot (Scophthalmus maximus). Mar Biol 114:517–525 Rønnestad I, Robertson RR, Fyhn HJ (1996) Free amino acids and protein content in pelagic and demersal eggs of tropical marine fishes. In: MacKinlay DD, Eldridge M (eds) The fish egg. American Fisheries Society, Vancouver, pp 81–84. ISBN 0-9698631-0-7 Salze G, Tocher DR, Roy WJ, Robertson DA (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 Samaee SM, Estevez A, Gimenez G, Lahnsteiner F (2009) Evaluation of quantitative importance of egg lipids and fatty acids during embryos and larvae development in marine pelagophil teleosts: with an emphasis on Dentex dentex. J Exp Zool 311A:735–751 Samaee SM, Mente E, Estevez A, Gimenez G, Lahnsteiner F (2010) Embryo and larva development in common dentex (Dentex dentex), a pelagophil teleost: the quantitative composition of egg-free amino acids and their interrelations. Theriogenology 73:909–919 Sargent JR (1995) Origins and functions of lipids in fish eggs: nutritional implications. In: Bromage NR, Roberts RR (eds) Broodstock management and egg and larva quality. Blackwell, Oxford Sargent JR, Tocher DR, Bell JG (2002) The lipids. In: Halver JE, Hardy RW (eds) Fish nutrition, 3rd edn. Academic Press, New York, pp 181–257 Schreck CB (2010) Stress and fish reproduction: the roles of allostasis and hormesis. Gen Comp Endocrinol 165:549– 556 Seoka M, Yamada S, Iwata Y, Yanagisawa T, Nakagawa T, Kumai H (2003) Differences in the biochemical content of buoyant and non-buoyant eggs of the Japanese eel, Anguilla japonica. Aquaculture 216:355–362 Sheridan MA (1988) Lipid dynamics in fish: aspects of absorption, transportation, deposition and mobilization. Comp Biochem Physiol B 90:679–690 Silva J, Laranjeira A, Serradeiro R, Santos MA, Pacheco M (2011) Ozonated seawater induces genotoxicity and hematological alterations in turbot (Scophthalmus maximus)—implications for management of recirculation aquaculture systems. Aquaculture 318:180–184
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Biochemical composition and quality of turbot (Scophthalmus maximus) eggs throughout the reproductive season Yudong Jia • Zhen Meng • Xinfu Liu Jilin Lei
•
Received: 18 March 2013 / Accepted: 6 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The present study investigated the biochemical composition and quality of turbot (Scophthalmus maximus) eggs throughout the reproductive season. Results showed that the fertilization, hatching and egg floating rates were variable throughout the reproductive season, with the highest values recorded during the mid-season. Meanwhile, positive correlations were found between fertilization, hatching rate and floating rate. The composition of turbot eggs, including total lipid, protein, carbohydrate, moisture and dry weight showed no significant differences during the reproductive season. Furthermore, no correlations were found between egg compositions and viability parameters (VPs), including fertilization and hatching rates as well as larval deformity rate. However, egg diameter varied and correlated with fertilization, hatching and egg floating rates. The fatty acid in eggs at mid-season had significantly higher levels of C14:0, C16:0, C16:1n-7, C18:0, C18:1, C20:4n-6, C20:5n-3 and C22:6n-3. Moreover, significant relationships were found between fatty acids and
Y. Jia Z. Meng X. Liu J. Lei (&) Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, No. 106 Nanjing Road, Qingdao 266071, People’s Republic of China e-mail: [email protected] Y. Jia Z. Meng X. Liu J. Lei Qingdao Key Laboratory for Marine Fish Breeding and Biotechnology, Qingdao 266071, People’s Republic of China
VPs. Eggs of the middle season had significantly higher concentration of isoleucine, leucine, lysine, methionine, valine, alanine, aspartate, glutamate and serine, whereas no significant relationships were found between amino acids and VPs. These observations suggest that the biochemical profile of eggs may be useful in evaluating egg quality and improving broodstock management for turbot. Keywords Turbot (Scophthalmus maximus) Biochemical composition Egg quality Reproduction
Introduction Egg quality is one of the limiting factors of seed production as well as the main obstacle for aquaculture development for seawater and freshwater species. Knowledge of the processes that influence egg quality, which can have direct practical use in broodstock management, is important for the control of aquatic production. Fish egg quality can be defined as the ability of the egg to be fertilized and subsequently developed into a normal embryo (Bobe and Labbe 2010). In fish, egg quality is highly variable and is affected by nutritional status, stress, overripening, hormonal induction of spawning, genetics and age of broodstock, in addition to water quality and environmental factors (Lahnsteiner 2000; Finn 2007; Zakeri et al. 2009; Mylonas et al. 2010; Schreck 2010;
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Villamizar et al. 2011; Jerez et al. 2012; Lahnsteiner and Kletzl 2012). Numerous efforts have been undertaken to evaluate the criteria for fish egg quality. Although fertilization and hatching rates are the most widely used indicators, other parameters for egg morphology, such as buoyancy, egg size, distribution of lipid droplets and abnormal blastomere morphology, have been cited as reliable indicators of egg quality (Unuma et al. 2005; Mansour et al. 2008; Aristizabal et al. 2009; Kohn and Symonds 2012). The above indicators of egg quality could be species-specific. In addition, biochemical parameters [yolk protein, lipids, carbohydrates, amino acids, vitamins and enzymes] have been considered as viable indicators of egg quality (Gime´nez et al. 2006; Faulk and Holt 2008; Lubzens et al. 2010; Samaee et al. 2010; Lanes et al. 2012). Morphological criteria, as well as fertilization and hatching rates, may be indicative of egg quality, but tell us nothing about what factors determine egg quality. Therefore, investigating the biochemical composition of fish egg and identifying a method for the reliable discrimination between low- and high-quality egg batches are vital in reducing hatchery production costs. These approaches would prevent the unnecessary occupation of hatchery staff time and facilities on what may prove to be unproductive egg batches. Turbot (Scophthalmus maximus) is a widely cultured marine fish of considerable commercial value in Europe and China. In recent years, studies have been conducted on the reproduction (Mugnier et al. 2000; Gosz et al. 2011), immune response (Pereiro et al. 2012), environmental conditions (Imsland et al. 2003; Nissling et al. 2006; Foss et al. 2009; Silva et al. 2011) and nutritional requirements (Leknes et al. 2012; Qi et al. 2012) of turbot in captivity. However, information on the biochemical composition of spawned eggs and subsequent consequences on the egg/larval quality parameters is sparse, particularly, reports on the proportion of floating eggs, fertilization and hatch rates, larval survival and growth for this species. In addition, the relationship among morphological characteristics, biochemical composition and viability parameters (VPs: fertilization rate [FR], hatching rate [HR] and larval deformity rate [LD]) at various stages of the reproductive season has not been evaluated. Such information would be important for producing a steady supply of high-quality eggs and for ensuring that time and resources are not wasted on egg batches with low survival and/or performance potential.
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The present study aims to compare VPs and biochemical profiles (proteins, carbohydrates, lipids, fatty acids and amino acids) of turbot (S. maximus) eggs throughout the reproductive season in pursuit of better broodstock management. Meanwhile, relationship between changes observed in biochemical composition and egg quality is also evaluated.
Materials and methods Broodstock management and egg collection Four-year-old mature turbots used for the experiments were obtained from the broodstock of a commercial fish farm (Yantai Tianyuan Aquatic Limited Corporation, China). The fish (five females, two males) weighting 3,500–4,000 g were kept in round tanks of 8 m3 at a stocking of 4 kg/m3. The tanks were supplied with recirculating water at a rate of 25 L/min and exposed to a constant photoperiod (16 h light:8 h dark). Water salinity and oxygen were ranged from 20 to 25 g/L and 5 to 9 mg/L, respectively. Temperature was maintained between 12 and 13 °C. Fish were fed with a diet of frozen sardines, squid and shrimp. Females had spawned multiple times during the reproductive season. In this study, the spawning period commenced in mid-October and ended in midDecember in artificial broodstock. Ovulatory rhythms were determined based on the first ovulation date by using daily abdominal stripping at the start of the spawning season. This procedure was performed to ensure that fresh ovulated eggs were obtained and to prevent overripening (Mcevoy 1984). Due to their ovulatory cycles of 70–90 h, females were stripped every 4–5 days to avoid overripening. Each female could spawn 8–12 times at 3–5-day intervals throughout the spawning season (Mugnier et al. 2000). Three periods of 10–15 days length were considered, namely early, mid- and late seasons, to study the variation of egg quality parameters throughout the spawning season. In the present study, three females could spawn all along the spawning period and three batches of egg samples were collected from each female in each period. Thus, we collect a total of 9 batches of samples in each period and performed artificial fertilization on each spawn independently. The number of eggs per batch was estimated by counting the eggs contained in an aliquot of 500 lL and measuring
Fish Physiol Biochem Table 1 Number of egg batches, egg volume per batch and ovulatory rhythm of turbot during the reproductive season Early season
Mid-season
Late season
Number of egg batches
3 (3)
3 (3)
3 (3)
Batch volume (mL)
105.33 ± 1.50 (3)a
156.56 ± 1.80 (3)b
Ovulatory rhythm (days)
a
4.46 ± 0.11 (3)
b
3.50 ± 0.09 (3)
100.11 ± 1.32 (3)a 4.17 ± 0.06 (3)c
Values are mean ± SEM (number of fish). Means within a row with different superscripts are significantly different (P \ 0.05)
the total volume of the batch. The egg batches, egg volume per batch and ovulatory rhythm of turbot during the reproductive season are shown in Table 1. Eggs were fertilized in seawater by gentle mixing with newly obtained milt from two males, which was collected with a plastic syringe and kept at 4 °C. Samples contaminated with urine or seawater was discarded. Only those milt samples that showed a motility of over 80 % of spermatozoa were used for fertilization. Sperm motility was measured by activating 1 mL of sperm (pre-diluted 1:5 v/v in a Ringer solution) with 19 lL of seawater and scored immediately under light microscopy. The percentage of motile spermatozoa was scored according to a previous method (Cejko et al. 2010). The volume ratio sperm:eggs:water used was 0.5:100:100, for which fertilization rate is maximal in turbot (Mugnier et al. 2000). Egg and larval quality measurements After the eggs were collected, their diameter was measured using a digital caliper (Mitutoyo CD-20C). Turbot egg is a type of buoyant eggs. Floating eggs are viable and used to calculate the FR and HR (Mcevoy 1984; Kjørsvik et al. 2003). The floating rate was calculated as the number of floating eggs divided by the total number of eggs spawned. FR was evaluated as follows: Three samples of one hundred eggs from the floating fraction of each batch were placed into Petri dishes and observed under a stereoscopic microscope (Carl Zeiss Vision, Aalen, Germany) at least 4 h after egg fertilized. Eggs at the 8- to 32-cell stage were classified as fertilized (Kjørsvik et al. 2003). HR was calculated based on the number of fertilized eggs placed into the incubators and the number of larvae that appear after hatching. In addition, the percentage of body deformities in newly hatched larvae, including lordosis, kyphosis and scoliosis, was visually determined under a stereoscopic microscope (Carl Zeiss
Vision, Aalen, Germany). FR, HR, LD and floating rate were calculated using the following formulae: Floating rate ð100 %Þ ¼ 100 no: of buoyant eggs= no: of ovulated eggs Fertilization rate ð100 %Þ ¼ 100 no: of fertilized eggs=no: of buoyant eggs Hatching rate ð100 %Þ ¼ 100 no: of hatched larvae=no: of buoyant eggs Larval deformity rate ð100 %Þ ¼ 100 no: of deformed larvae=no: of hatched larvae Biochemical analysis From each egg batch, three sample eggs were collected and rinsed three times with 50 ml distilled water and immediately stored at -80 °C until analysis. The amount of samples used for each analysis was as follows: wet/dry weight (100 eggs), protein (8–10 mg dry weight, 150 eggs), carbohydrate (5–6 mg dry weight, 80 eggs), total lipid (6–10 mg dry weight, 150 eggs), fatty acids (500–600 mg dry weight, 800 eggs) and amino acids (100–200 mg dry weight, 200 eggs). Moisture content was determined according to the previous method (Faulk and Holt 2008). Briefly, after rinsing the eggs, external water was removed by placing the sample on top of paper towels. The sample was immediately transferred to a pre-weighed glass vial and weighed on microbalance (Shimadzu Corp., Kyoto, Japan). Samples were then lyophilized for 24 h and then reweighed to obtain their dry weight. Moisture content was calculated by subtracting the egg dry weight of eggs from their wet weight, dividing by wet weight and multiplying by 100. Protein was quantified using the Bradford protein assay kit (Beyotime Biotechnology, Haimen, China). A standard curve (0.5–5 mg) was generated using
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bovine serum albumin with two replicates per standard concentration, and the absorbance at 485 nm was measured. Carbohydrate content was extracted and measured according to a previous method (Guisande et al. 1998). Briefly, samples were homogenized in 2 mL of distilled water, and an aliquot of 400 lL was used in the assay. To sample volumes of 400 lL, 10 lL of 81 % phenol was added to the solution, and after gently shaking, 1 mL of concentrated sulfuric acid (18 M) was added. The samples were again shaken and maintained at room temperature for 30 min. Then, the absorbance was read at 485 nm. A standard curve (2.5–50 lg) was established by using reagent-grade glucose. Total lipids were cold extracted from lyophilized samples using chloroform/methanol (2:1 v/v) as described by Folch et al. (1957) with 0.01 % (w/v) butylated hydroxytoluene (BHT) as an antioxidant. Prior to homogenization, heptadecanoic acid (17:0, Sigma, St. Louis, MO, USA) was added as an internal standard for subsequent quantification of fatty acid. Fatty acids were saponified and methylated using the method described by Mansour et al. (2011). The fatty acid methyl esters were analyzed on a gas chromatograph (Agilent 6890N, Palo Alto, CA, USA) equipped with an Agilent 7,683 autosampler (Agilent Technologies), an Agilent capillary column (length, 20 m; internal diameter, 0.10 mm; film thickness, 0.10 lm; Agilent Technologies) and a flame ionization detector. Fatty acids were identified by comparing the retention times with standard values. The results were expressed as gram of each fatty acid per kilogram of tissue dry weight (g/kg dry weight). The amino acid compositions of eggs were determined using a reversed phase high-performance liquid chromatograph (Agilent 1100 Series, Santa Clara, CA, USA), equipped with a UV detector. Samples for amino acid analysis were hydrolyzed using 6 N HCl for 24 h at 115 °C. Cysteine was oxidized with 0.01 N sodium hydroxide and determined as cystine. Tryptophan could not be measured because of its degradation during acid hydrolysis. Hydrolysate amino acid concentrations of eggs were expressed as gram of each fatty acid per kilogram of tissue dry weight (g/kg dry weight). Statistical analysis Shapiro–Wilk normality test and Bartlett test were used to estimate the data complied or not with normal distribution
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and homogeneity of variance, respectively (Faraway 2005). The data were analyzed via one-way analysis of variance (ANOVA) and Duncan’s multiple-range tests using the SAS 8.0 software. Relationships between biochemical parameters of eggs and VPs were assessed by Pearson’s correlation coefficient and simple regression models. All data are presented as the mean ± standard error of the mean (SEM). In all statistical tests used, P \ 0.05 was considered significantly different.
Results The FR, HR and floating rate were varied throughout the reproductive season, with the highest values observed in the mid-season (Table 2, P \ 0.05); the early and late seasons showed no significant differences (Table 2, P [ 0.05). Meanwhile, the linear relationship between the FR and HR and the floating rate on the one hand and the floating rate on the other hand were significant during the spawning season (Fig. 1a, b). The lowest LD was obtained in the middle season (Table 2, P \ 0.05), the early and late seasons showed no significant differences (Table 2, P [ 0.05). The composition of turbot eggs, including total lipid, protein, carbohydrate, moisture and dry weight showed no significant differences during the reproductive seasons (Tables 2, 3, P [ 0.05). Egg diameter was significantly lower in the mid-season (Table 2, P \ 0.05). Moreover, a negative correlation was observed between egg diameter and the fertilization, hatching and floating rates during the reproductive season (Figs. 1c, d, 2). The fatty acid profile of egg lipids differed significantly between the early and middle reproductive seasons (Table 4, P \ 0.05). In general, eggs spawned of the mid-season had significantly higher concentrations of C14:0, C16:0, C16:1n-7, C18:0, C18:1, C20:4n-6, C20:5n-3, C22:6n-3, saturated fatty acid (SFA), polyunsaturated fatty acid (PUFA) and monounsaturated fatty acid (MUFA). However, the fatty acid composition of total lipid was not significantly different between the late and early or middle reproductive seasons (Table 4, P [ 0.05). Furthermore, significant relationships were found between VPs and concentrations of other fatty acids (Table 5, P \ 0.05). Significant correlations were observed between C14:0, C16:0, C16:1n-7, C20:4n-6, C20:5n-3, C22:6n-3, SFA, PUFA and LD (linear), and also between
Fish Physiol Biochem Table 2 Mean floating, fertilization, hatching and larval deformity rate (%) throughout the reproductive season of turbot Early season Egg diameter (mm) Floating rate (%)
Mid-season
Late season
1.17 ± 0.03a
1.01 ± 0.01b
1.16 ± 0.02a
a
b
48.81 ± 2.09a
46.12 ± 1.16
90.48 ± 0.51
Fertilization rate (%) Hatching rate (%)
a
37.42 ± 2.13 41.18 ± 3.43a
b
90.74 ± 1.01 76.49 ± 3.29b
35.67 ± 2.01a 39.43 ± 2.71a
Larval deformity rate (%)
12.69 ± 0.32a
9.24 ± 0.09b
12.38 ± 0.23a
Data are expressed as mean ± SEM (n = 9). Means within a row with different superscripts are significantly different (P \ 0.05)
Fig. 1 Relations between floating rate (a, b), egg diameter (c, d) and the fertilization, hatching rate in turbot. Early (cross), middle (triangle) and late (circle) seasons
C16:0, C16:1n-7, C20:4n-6, C20:5n-3, C22:6n-3, SFA and FR (quadratic) and HR (quadratic). The content of amino acid content (g/kg dry weight) was significantly higher in the mid-season
than in the early and late seasons (Table 6, P \ 0.05). Essential amino acids, including isoleucine, leucine, lysine, methionine and valine, were significantly higher in the mid-season (Table 6, P \ 0.05). Non-
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Fish Physiol Biochem Table 3 Mean egg diameter, dry weight, moisture content, total lipid, protein and carbohydrate throughout the reproductive season of turbot Early season
Mid-season
Late season
Dry weight (mg 100 egg)
6.85 ± 0.06
6.89 ± 0.03
6.42 ± 0.26
Moisture (% wet weight) Proteins (% dry weight)
90.17 ± 0.06
90.04 ± 0.06
90.11 ± 0.58
11.83 ± 0.68
12.77 ± 0.67
13.17 ± 0.75
Total lipid (% dry weight)
19.81 ± 0.35
20.57 ± 0.84
20.03 ± 0.67
Carbohydrate (% dry weight)
9.74 ± 0.75
9.36 ± 0.30
8.91 ± 0.28
Data are mean ± SEM (n = 9). No significant differences were found during reproductive season (P [ 0.05)
Fig. 2 Relationships between the egg diameter and the floating rate in turbot. Early (cross), middle (triangle) and late (circle) seasons
essential amino acids, including alanine, aspartate, glutamate and serine, were also significantly higher in the mid-season than in the early season (Table 6, P \ 0.05). However, no significant relationships were found between VPs and the composition of amino acid in turbot eggs (data not shown).
Discussion Egg and larval quality are key factors to the successful production of viable offspring in marine teleosts. Egg
123
quality is influenced by several parameters that frequently change during the reproductive season. Among these parameters, egg morphology as well as FR and HR are the most widely used indicators of egg quality. The buoyancy of eggs has often been used as an indicator in the assessment of egg quality in marine fish. It was reported that the ratio of buoyant eggs to total eggs spawned correlates positively with egg hatchability (Seoka et al. 2003; Unuma et al. 2005; Furuita et al. 2007). In addition, the size of the eggs is sometimes considered to be beneficial for the future development of the embryo. Mansour et al. (2008) found that low-quality (low FR) eggs of Salvelinus alpinus tend to have large egg diameter than good- and fair-quality eggs. In addition, the egg diameter of Melanogrammus aeglefinus declines over the spawning season and positively correlates with FR (Trippel and Neil 2004). Similar observations have been reported in other marine fish species (Lahnsteiner 2000; Aegerter and Jalabert 2004). Likewise, turbot egg diameter becomes significantly smaller as the season progresses (Lavens et al. 1999). Therefore, egg diameter is often cited as an important criterion for the assessment of egg quality and reproductive performance in many fish species. In the present study, the FR, HR and floating rate of eggs were found to be variable throughout the reproductive season, with the highest values observed in the mid-season, whereas low values was seen in the early and late season. Furthermore, positive correlations were found between FR, HR and the egg floating rate. However, egg diameter was significantly the lowest in the middle of the productive season and negative correlation was detected between egg diameter and the FR, HR, floating rate in turbot eggs. These results indicated that high-quality egg batches were observed in the middle of the reproductive season. Furthermore, our results show that buoyancy and diameter of eggs may be useful indicators for assessing turbot egg quality during the reproductive season. Embryogenesis and larval development are long processes that require a high input of nutrients and energy from egg reserves in most fish species (Boulekbache 1981; Dayal et al. 2003; Cejas et al. 2004; Kamler 2005). In principle, fish eggs contain all the nutrients to support both homeostasis and development of larvae during the lecithotrophic phase (Lubzens et al. 2010). Moreover, the biochemical composition of fish eggs is species-specific, and the
Fish Physiol Biochem Table 4 Fatty acid composition (g/kg dry weight) of turbot eggs throughout the reproductive season Fatty acid
Early season
Mid-season
Late season
C14:0
1.63 ± 0.35b
2.72 ± 0.12a
2.30 ± 0.18ab
C16:0
b
a
17.06 ± 2.11ab
12.69 ± 2.59
22.06 ± 0.68
b
5.35 ± 0.99 2.82 ± 0.55b
a
10.08 ± 0.71 4.50 ± 0.25a
7.45 ± 0.71ab 3.96 ± 0.50ab
C18:1
13.91 ± 2.53b
25.34 ± 1.52a
21.56 ± 2.92ab
C18:2
1.32 ± 0.22
1.97 ± 0.13
1.90 ± 0.23
C18:3
0.49 ± 0.12
0.78 ± 0.04
0.70 ± 0.04
C20:4n-6(AA)
1.49 ± 0.28a
2.58 ± 0.08b
2.02 ± 0.23ab
C20:5n-3(EPA)
4.25 ± 0.96a
7.62 ± 0.55b
5.63 ± 0.51ab
C16:1n-7 C18:0
C22:5n-3
2.38 ± 0.50
3.65 ± 0.15
3.21 ± 0.42
C22:6n-3(DHA)
16.78 ± 3.42a
29.21 ± 0.62b
22.16 ± 2.38ab
SFA
17.73 ± 3.61a
30.50 ± 0.78b
24.20 ± 2.82ab
b
a
37.11 ± 4.06ab
PUFA
25.97 ± 5.00
47.31 ± 1.12
MUFA
20.05 ± 3.70b
36.61 ± 2.19a
29.76 ± 3.73ab
DHA:EPA
4.01 ± 0.16
3.87 ± 0.28
3.93 ± 0.12
EPA:ARA
2.82 ± 0.35
2.96 ± 0.20
2.80 ± 0.07
Data are expressed as mean ± SEM (n = 9). Means within a row with different superscripts are significantly different (P \ 0.05) SFA saturated fatty acid, PUFA polyunsaturated fatty acid and MUFA monounsaturated fatty acid
precise sequence of consumption (proteins and amino acids, lipids and fatty acids and carbohydrates) varies both qualitatively and quantitatively. Embryos of turbot (S. maximus) have been reported to catabolize proteins and carbohydrates exclusively (Planas et al. 1989), whereas embryos of trout (Oncorhynchus mykiss) use proteins, lipids and carbohydrates to satisfy their energy requirements (Boulekbache 1981). In addition, the biochemical composition profile of fish eggs varies among species and has been shown to increase, decrease or remain constant during the spawning season (Aegerter and Jalabert 2004; Faulk and Holt 2008; Fuiman and Ojanguren 2011). The current work found that the dry weight, moisture, protein, total lipid and carbohydrate were fairly consistent and no significant difference was observed throughout the reproductive season in turbot eggs. Similar results have been described for other marine fish species (Nocillado et al. 2000; Dayal et al. 2003; Gime´nez et al. 2006; Faulk and Holt 2008). The total lipid content of marine fish eggs frequently ranges from 15 to 35 % dry weight (Vetter et al. 1983; Ostrowski and Divakaran 1991; Sargent 1995; Dayal et al. 2003; Samaee et al. 2009), which is similar to our study here for turbot eggs. In addition, the total lipid content of fish eggs is generally more conserved than
other fish tissues during spawning season (Sheridan 1988; Tocher 2003; Salze et al. 2005). We found no significant difference in the total lipid content of turbot eggs during reproductive season. However, lipid content of turbot eggs was higher than the ones reported by Silversand et al. (1996) for this species. This difference may be on account of different reared condition (the temperature was maintained 10 ± 1 °C) and nutritional regime (they fed three times a week with a formulated moist diet). Proteins in eggs are extremely important for the functionality of the female gamete. Amino acids are basic unit of protein. In the current study, we found the composition of amino acid content variable during the spawning season (Table 6), whereas the total amino acid content showed no significant change in turbot egg (data not shown). This is consistent with our observation that the protein contents of turbot eggs did not change during the spawning season. In the nutritional profile of fish eggs, lipid and fatty acids provide metabolic substrates for embryonic and larval development, and they serve as structural components in the biogenesis of membranes. These are particularly important in regulating numerous physiological processes including steroidogenesis, embryonic development, hatching and early larval
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Fish Physiol Biochem Table 5 Simple regression equations, explanatory effect (R2), F and P values of the significant relationships found between the turbot egg fatty acid content and the VPs (FR, HR, LD) considered in current study Variables Dependent (fatty acids)
Equations
R2
F
P
Independent (VPs)
1 C14:0
LD
y = -2.010x ? 15.889
0.498
6.956
0.034
2 C16:0
FR
y = 0.447x2 - 11.380x ? 98.128
0.700
6.990
0.027
3 C16:0
HR
y = 0.354x2 - 8.786x ? 90.684
0.680
6.362
0.033
4 C16:0
LD
y = -0.252x ? 15.778
0.498
6.956
0.034
5 C16:1n-7
FR
y = 1.449x2 - 12.988x ? 61.985
0.704
7.140
0.026
2
6 C16:1n-7
HR
y = 1.124x - 10.998x ? 65.122
0.707
7.241
0.025
7 C16:1n-7
LD
y = -0.252x ? 15.778
0.557
8.809
0.021
8 C20:4n-6
FR
y = 39.528x2 - 115.826x ? 115.364
0.711
7.383
0.024
9 C20:4n-6
HR
y = 28.669x2 - 86.305x ? 101.096
0.691
6.705
0.030
10 C20:4n-6
LD
y = -2.186x ? 15.872
0.543
8.319
0.024
FR HR
2
y = 2.692x - 18.745x ? 64.565 y = 2.088x2 - 15.906x ? 68.047
0.692 0.684
6.751 6.485
0.029 0.032 0.024
11 C20:5n-3 12 C20:5n-3 13 C20:5n-3
LD
y = -2.186x ? 15.872
0.543
8.319
14 C22:6n-3
FR
y = 0.317x2-10.200x ? 110.718
0.801
12.055
0.008
15 C22:6n-3
HR
y = 0.234x2-7.793x ? 99.928
0.782
10.760
0.010
16 C22:6n-3
LD
y = -0.012x2 ? 0.281x ? 11.486
0.714
7.498
0.023 0.031
2
17 SFA
FR
y = 0.263x -8.827x ? 103.357
0.687
6.600
18 SFA
HR
y = 0.200x2-6.991x ? 96.503
0.685
6.519
0.031
19 SFA
LD
y = -0.182x ? 15.819
0.538
8.165
0.024
20 PUFA
FR
y = 0.114x2-5.973x ? 108.801
0.719
7.684
0.022
21 PUFA
HR
y = 0.087x2-4.79x ? 101.707
0.717
7.584
0.023
2
22 PUFA
LD
y = -0.004x ? 0.125x ? 12.087
0.652
5.633
0.042
23 MUFA
LD
y = -0.135x ? 15.322
0.485
6.584
0.037
R2 [ 0.49 and P \ 0.05 are considered significance levels for regression analysis SFA saturated fatty acid, PUFA polyunsaturated fatty acid, MUFA monounsaturated fatty acid, FR fertilization rate, HR hatching rate and LD larval deformity rate
performance (Cejas et al. 2004; Mansour et al. 2011). In general, marine fish egg lipids are rich in highly unsaturated fatty acids including docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and arachidonic acid (AA). DHA plays an important role as a structural component of cell membranes, especially in the processes of synaptogenesis and retinogenesis, during embryonic development in fish (Sargent et al. 2002; Tocher 2010). EPA is also a structural component of membrane phospholipids, and both fatty acids compete for enzymes that esterify them into phospholipids (Sargent et al. 2002). AA is known as a primary precursor of physiologically active molecules such as prostaglandins and leukotrienes, which are involved in the regulation of embryonic development, hatching and early larval performance (Bell and Sargent 2003).
123
The DHA/EPA ratio can lead to an inadequate balance in the structural composition of cells and can affect the developing embryo as well as the subsequent larval survival and growth (Furuita et al. 2002). The level of essential fatty acids—DHA, EPA and AA—in eggs as well as the balance among them must be considered simultaneously (Sargent et al. 2002; Bell and Sargent 2003). Moreover, the composition of fatty acid and DHA/EPA in eggs is species-specific. Other fatty acids in eggs of marine fish vary and correlate with egg quality during spawning season. Samaee et al. (2009) reported that myristic acid (C14:0) level was higher in high-quality eggs (high FR and HR) than in other egg categories in Dentex dentex, and a statistical significance was found between myristic acid (C14:0) and LD during spawning season. However, palmitic acid
Fish Physiol Biochem Table 6 Amino acid composition (g kg-1 dry weight) of turbot eggs throughout the reproductive season Amino acid
Early season
Mid-season
Late season
Essential Arginine
37.73 ± 0.66
39.63 ± 0.50
39.00 ± 0.80
Histidine Isoleucine
17.27 ± 0.47 37.97 ± 0.70b
18.33 ± 0.32 42.40 ± 0.50a
17.80 ± 0.45 39.87 ± 0.92b
Leucine
58.57 ± 1.07b
65.47 ± 0.91a
61.67 ± 1.24b
48.20 ± 1.01
c
a
51.63 ± 0.83b
Methionine
14.87 ± 0.29
b
a
17.57 ± 0.28
16.67 ± 0.47a
Phenylalanine
33.10 ± 0.74
34.57 ± 0.35
34.23 ± 0.72
Threonine
29.80 ± 0.90
31.07 ± 0.61
31.00 ± 0.30
Valine
44.17 ± 0.83b
49.67 ± 0.55a
46.97 ± 0.93a
48.27 ± 1.23b
55.77 ± 1.70a
53.07 ± 0.38a
b
a
Lysine
54.87 ± 0.84
Non-essential Alanine Aspartate
45.07 ± 0.84
Cysteine
4.33 ± 0.19
Glutamate
86.97 ± 0.77
b
48.00 ± 0.81
46.97 ± 0.78ab
4.73 ± 0.23
4.80 ± 0.25
a
97.13 ± 1.78
90.93 ± 1.82b
Glycine
20.43 ± 0.27
21.60 ± 0.26
21.17 ± 0.44
Proline
42.77 ± 2.05
46.30 ± 1.91
46.27 ± 0.73
31.80 ± 1.29a
32.60 ± 0.61a
29.27 ± 0.41
29.53 ± 0.57
Serine Tyrosine
27.1 ± 0.20b 27.76 ± 0.58
Data are expressed as mean ± SEM(n = 9). Means within a row with different superscripts are significantly different (P \ 0.05)
(C16:0) and palmitoleic acid (C16:1n-7) content of fish eggs and their correlations with VPs are speciesspecific during spawning season (Samaee et al. 2009; Jerez et al. 2012). In the present study, the fatty acid compositions of eggs were found to be significantly different with variable changes throughout the reproductive season, with the highest value of DHA, EPA, AA, C14:0, C16:0 and C16:1n-7 observed in the midseason. Significantly, relationships were also found between VPs and the concentrations of DHA, EPA, AA and C14:0, C16:0, C16:1n-7 in turbot eggs (Table 5). This might indicate the existence of a series of synergisms (positive or negative) among the egg fatty acids and effects of the synergisms on fish biological processes. These statistical findings suggest the need for more suitable experimental approaches to find the biological grounds for these interrelations among different fatty acids. Moreover, in other marine fish species, significant correlations have been reported between fatty acids and VPs (Salze et al. 2005; Mansour et al. 2011; Jerez et al. 2012; Lanes et al. 2012). Meanwhile, DHA/EPA ratios ranged between 4:1 and 3:1. Similar values have been described by Silversand et al. (1996). These results
indicated that fatty acid of turbot eggs has a higher direct effect on VPs during the reproductive season. Therefore, fatty acid composition and the concentration of eggs may be considered as potential biochemical markers to predict turbot egg quality. Amino acids are the main energy resources during egg development and are also osmotic active compounds that regulate egg hydration and, subsequently, egg buoyancy during the final maturation of oocytes (Rønnestad et al. 1992, 1996). Several studies have demonstrated that amino acids are lower in nonviable than in viable eggs (Nocillado et al. 2000; Samaee et al. 2010; Lanes et al. 2012). High levels of amino acids could provide more energy resource for embryonic and larval development after fertilization. Samaee et al. (2010) showed that egg amino acid contents were significantly correlated with VPs in common dentex (Dentex dentex). Furthermore, the amino acid composition profile of fish eggs varies among species and has been shown to increase, decrease or remain constant during the spawning season (Rønnestad and Fyhn 1993; Rønnestad et al. 1996; Faulk and Holt 2008; Samaee et al. 2010). The present study demonstrated that the levels of amino
123
Fish Physiol Biochem
acids were variable during the reproductive season, with the highest value in the mid-season. In addition, the profile generated here for turbot was similar to that reported for a number of marine fish eggs during the spawning season (Rønnestad et al. 1996; Faulk and Holt 2008; Lanes et al. 2012). However, significant relationships were not found between VPs and the profile of amino acids in turbot eggs. In summary, the results of this study indicated that egg quality was highly variable throughout the reproductive season, whereas high-quality turbot eggs were observed in the middle of the reproductive season. The diameter and buoyancy may be used as predictors of turbot egg quality during reproductive season due to correlation with VPs. Meanwhile, the fatty acid and amino acid profiles of eggs were variable throughout the reproductive season, possibly promoting the synthesis of vitellogenin and consequently affecting embryo and larval viability. These findings suggest that information on the biochemical profile of eggs may be useful in evaluating egg quality and improving broodstock management for turbot. In addition, further studies should seek to understand the relationship between the biochemical profiles of eggs and VPs over different female ages and spawning seasons, which will aid in the identification of new biomarkers of egg quality. Acknowledgments This study was supported by National Natural Science Foundation of China (31302205), Natural Science Foundation of Shandong Province (ZR2012CQ024, BS2013SW004) and the China Postdoctoral Science Foundation (2012M511559, 2013T60690). We thank Huaxin Niu and Peng Hu (Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences) for help in the experiment.
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