Turbot Eggs Research Articles

Effects of different light spectra on embryo development and the performance of newly hatched turbot (Scophthalmus maximus) larvae

Light is a key environmental factor that synchronizes various life stages from embryo development to sexual maturation in fish. For turbot, light spectra have the most influence at the larval and juvenile stages. In the current study, differences in the development of embryos and the performance of newly hatched turbot larvae exposed to five different spectra: full spectrum (LDF), blue (LDB, peak at 450 nm), green (LDG, peak at 533 nm), orange (LDO, peak at 595 nm) and red (LDR, peak at 629 nm), were examined. At 62.8 h post fertilization, a higher number of embryos exposed to short-wavelengths (LDG and LDB) had developed a heartbeat in comparison with embryos exposed to other wavelengths. Larvae exposed to the green spectrum had higher malformation rates than larvae exposed to the other spectra, indicating that larvae exposed to green light may have significantly reduced survival rates. The results of non-specific immunity parameters showed that the mRNA expression levels of cathepsin D (CTSD), cathepsin F (CTSF), catalase (CAT) and metallothionein (MT) in larvae exposed to LDB were significantly higher than those exposed to other spectra, but CAT activity in larvae exposed to LDB was significantly lower than larvae exposed to the other spectra. There was no significant difference in MT activity in larvae exposed to the five different spectra. The mRNA expression level of lysozyme (LZM) in larvae exposed to LDR was significantly higher than other spectra, while there was no significant difference in LZM activity observed in larvae exposed to LDR, LDG, LDB and LDF. The difference of the enzyme activity of total superoxide dismutase (T-SOD) was not significant among larvae exposed to the five spectra. mRNA expression of the heat shock protein 70 (HSP70) was significantly higher in newly hatched larvae exposed to LDB, LDR and LDG, indicating that larvae exposed to LDB, LDG and LDR exhibited a stress response. The mRNA expression level of the insulin-like growth factor-1 (IGF-1) and growth parameters in the newly hatched larvae exposed to the different spectra were not significantly different. The results of the present study indicate that LDO and LDF should be used for embryo incubation and newly hatched larvae when rearing turbot. This study provides a theoretical basis for optimizing the incubation light environment for fertilized turbot eggs, promoting immunity and reducing stress responses in newly hatched larvae.

Water turbulence effect on egg survival and characteristics of hatched larvae of the Black Sea turbot Scophthalmus maeoticus (Pallas, 1814)

The impact of different level of turbulence on developing eggs and prelarvae of the Black Sea turbot Scophthalmus maeoticus obtained from the pair of wild spawners at the end of natural spawning season has been studied. The incubation was carried out at three different levels of turbulence generated by diffused aeration: without aeration (calm water), low aeration (34 ml per minute) and high aeration (75 ml per minute). In calm water hatching rate (HR) of turbot eggs was 20 % and the hatched prelarvae had the longest standard length (SL = 3.13 mm) and the biggest volume of the yolk sac (VYS = 0.37 mm³) as compared to those of low aeration (HR = 18 %; SL = 3.10 mm; VYS = 0.32 mm³) and high aeration conditions (HR = 14 %; SL = 3.10 mm; VYS = 0.29 mm³). One day after hatching the percentage of survived prelarvae from the number of hatched was the highest – 86 % at high aeration, compared with that of low aeration (72 %) and calm water (61 %). Application of turbulence leads to elimination of “poor” quality eggs during incubation, presumably enables hatching of more viable larvae and can reduce mortality of more advanced larvae.

Patterns of catabolic capacities and energy metabolism in developing embryos and yolk-sac larvae of turbot (Scophthalmus maximus L.)

Patterns of catabolic capacities and energy metabolism in turbot eggs during embryogenesis and yolk-sac larvae were investigated. The catabolization of energy fuels were evaluated by specific enzymes including trypsin-like proteases (TRY) for protein and triglyceride lipase (LIP) for lipids. Meanwhile, citrate synthase (CS) and lactate dehydrogenase (LDH) were measured to assess energy production pathway. Regarding contribution to energy production, the enzymes of pyruvate kinase (PK), aspartate aminotransferase (AAT) and hydroxyacyl CoA dehydrogenase (HOAD) which was indicative of the mobilizations of carbohydrates, amino acids and fatty acids respectively and their ratios were detected.Results showed the metabolic enzymes and catabolic enzymes were present and involved in the embryogenesis of turbot. LIP activity exhibited a pattern of ‘low-high-low-high’. TRY activity dropped to the minimal value at blastula stage, and then increased significantly to the maximal value at 2days after hatching (DAH). The enzymatic activities of HOAD, AAT, LDH and CS all showed similar upward tendency and increased significantly as development went on. PK activity increased to the peak at cleavage stage, and then decreased to the lowest point at hatching day.Concerning enzymatic ratios, both AAT/HOAD and PK/HOAD presented similar “high-low” patterns, showing the maximal value 3.18 and 45.27 respectively at cleavage stage and the minimal value 0.46 and 0.84 respectively at 2 DAH. PK/AAT ratio declined from fertilized eggs (14.31) to 2 DAH (1.82). PK/CS ratio decreased significantly from cleavage stage (1.63) to 2 DAH (0.16). In contrast, a rising tendency of HOAD/CS ratio appeared as growth went on. AAT/CS ratio peaked at gastrula stage (0.13) and declined thereafter. LDH/CS ratio increased from fertilized eggs (1.42) to cleavage stage (1.85), and then decreased until 2 DAH (0.55). LDH/PK ratio reached the peak of 3.85 at hatching day.Combined with patterns of enzymatic ratios, it can be concluded that carbohydrates are predominantly mobilized before cleavage stage and are gradually substituted by amino acids from cleavage stage to gastrula stage and fatty acids during the following days, revealing their importance for morphogenesis in turbot. Regarding energy production pathway, the dominant mode was anaerobic pathway before cleavage stage and aerobic pathway during subsequent periods.

Using early life stages of marine animals to screen the toxicity of priority hazardous and noxious substances.

This study provides toxicity values for early life stages (ELS) of two phylogenetically distinct marine animal taxa, the sea urchin (Paracentrotus lividus), a deuterostome invertebrate, and the turbot (Scophthalmus maximus), a vertebrate (teleost), when challenged by six hazardous and noxious substances (HNS): aniline, butyl acrylate, m-cresol, cyclohexylbenzene, hexane and trichloroethylene. The aim of the study was to provide preliminary information on toxic effects of representative and relevant priority HNS to assess the risk posed by spills to marine habitats and therefore improve preparedness and the response at the operational level. Selection criteria to include each compound in the study were (1) inclusion in the HASREP (2005) list; (2) presence on the priority list established by Neuparth et al. (2011); (3) paucity of toxicological data (TOXnet and ECOTOX) for marine organisms; (4) behaviour in the water according to the categories defined by the European Behaviour classification system (GESAMP 2002), by selecting compounds with different behaviours in water; and (5) physicochemical and toxicological properties, where available, in order to anticipate the most toxic compounds. Aniline and m-cresol were the most toxic compounds with no observed apical effect concentration (NOAEC) values for sea urchin ranging between 0.01 and 0.1mg/L, followed by butyl acrylate and cyclohexylbenzene with NOAECs ranging between 0.1 and 1.0mg/L and trichloroethylene with NOAEC values that were in the range between 1 and 10mg/L, reflecting their behaviour in water, mostly vapour pressure, but also solubility and log Kow. Hexane was toxic only for turbot embryos, due to its neurotoxic effects, and not for sea urchin larvae, at concentrations in the range between 1 and 10mg/L. The concentrations tested were of the same order of magnitude for both species, and it was observed that sea urchin embryos (length of the longest arm) are more sensitive than turbot eggs larvae (hatching and cumulative mortality rates) to the HNS tested (except hexane). For this specific compound, concentrations up to 70mg/L were tested in sea urchin larvae and no effects were observed on the length of the larvae. Both tests were found to be complementary depending on behaviour in water and toxicity target of the compounds analysed.

Induction of mitotic gynogenesis in turbot Scophthalmus maximus

All-female production will maximize the profit potential for the aquaculture of the turbot Scophthalmus maximus, which exhibits significant sexually dimorphic growth. However, the basis for the technology of all-female production, i.e. the genetic mechanism of sex determination in turbot has been obscure so far, and might be ascertained by analysis of the sex ratio of its diploid mitotic gynogens. In this study, artificial mitotic gynogenesis in turbot was induced by hydrostatic pressure shock of its eggs activated by ultraviolet (UV)-irradiated sperm of red sea bream Pagrus major. Before the activation of turbot eggs, cryopreserved sperm of red sea bream were thawed, diluted 1:20 with Hank's solution, and UV-irradiated at a dosage of 6480–7200erg/mm2. Under water temperature of 14.5±0.5°C, treatments were tested by shocking the activated eggs at designed protocols: timings of 70–95maf (min after fertilization) with an intensity of 70MPa for 6min; intensities of 60–85MPa with a timing of 90maf for 6min; durations of 2–10min with an intensity of 75MPa initiated at 90maf. The optimal protocol of the induction was determined to be pressure-shocking activated eggs initiated at 85–90maf with 75MPa for 6min. A mitotic gynogen of 124 juveniles survived by 150days after hatching. The ploidy of these juveniles was confirmed to be diploid by flow cytometry and their sex ratio was 1:1 female to male. The sex ratio of these juveniles supported the assumption of a genetic mechanism of sex determination of female heterogametes (ZW/ZZ) in turbot. This protocol can be used to create homozygous clones of double haploids that are very useful for researches on both the sex determination mechanism and selective breeding of this important farmed fish species.

Biochemical composition and quality of turbot (Scophthalmus maximus) eggs throughout the reproductive season

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 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.

Effect of iodine treatment on the hatching rate of black sea turbot, (Psetta maxima Linnaeus, 1758), eggs

A study evaluating the effects of iodine treatment on the hatching success of turbot eggs was carried out. A hundred, 500, 1500 and 3000 ppm iodine treatment were performed for 10 minutes. The treatment with 3000 ppm iodine presented lower hatching percentage (70.0 ±6.08%). The control group (97.6 ±1.42%) and disinfected with 100 ppm iodine (91.3 ±4.04%) resulted in slightly higher hatching percentage, and significantly different from the other disinfected groups (P < 0.05). The control group and disinfected eggs with 1500 and 3000 ppm iodine were statistically significant in abnormality (P < 0.05). Zero-day-old eggs, 1-day-old eggs and 3-day-old eggs were treated with a dose of 100 ppm iodine for ten minutes. Differences in abnormalities between 0-day-old eggs (early morula stage) and 1 and 3 day-old eggs were statistically significant (P < 0.05). In conclusion, 100 ppm iodine appeared suitable for disinfecting turbot eggs if treatment is applied at the early morula stage.

Induction of diploid gynogenesis in turbot Scophthalmus maximus with left-eyed flounder Paralichthys olivaceus sperm

Turbot Scophthalmus maximus exhibits sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. In this paper, gynogenetic diploids of turbot were induced by activating egg development with ultraviolet (UV)-irradiated left-eyed flounder Paralichthys olivaceus sperm combined with cold shock to prevent extrusion of the second polar body. The results of UV irradiation experiments showed that survival, motility, and duration of activity of P. olivaceus sperm generally decreased with increase in UV dose. The typical Hertwig's effect was observed after fertilized turbot eggs with UV-irradiated P. olivaceus sperm and the optimal UV dose for gynogenetic haploid production was 36,000 erg mm(-2). At 15 degrees C, appropriate timing of cold shock for retention of the second polar body in turbot eggs was at 6 min after fertilization. Results of different combinations of two shock temperatures (1 or 3 degrees C) and four shock durations (15, 25, 35 or 45 min) at 6 min after fertilization demonstrated that shock of 25 min at 1 degrees C gave the highest production of diploid gynogens (39.58% relative to its diploid control). The results of this study reveal that the use of UV-irradiated P. olivaceus sperm for activation of turbot eggs and cold shock for polar body retention is an effective method to produce gynogenetic offspring.

Hatchability of eggs from Atlantic cod, turbot and Atlantic halibut after disinfection with ozonated seawater

Abstract In aquaculture the risk of transmission of fish pathogens via eggs is reduced by disinfection in ozonated seawater, but this treatment may delay or reduce hatching. The objective of this study was to investigate the tolerance of Atlantic cod, turbot and Atlantic halibut eggs to ozonated seawater. Groups of eggs were treated with different concentrations and exposure times 2 days before hatching, and the effects on hatchability were observed. The groups of eggs of all three species that had been exposed to 2 mg O 3 /l for 2 min or less showed normal hatching. In the groups with high total exposure (4 mg O 3 /l for 1 min or higher), a clearly lower percentage of hatching was found. Interspecies differences in tolerance were observed, with turbot eggs displaying higher tolerance than eggs of either halibut or cod. Due to the interspecies differences, the tolerance of eggs to ozonated seawater should be carefully evaluated in controlled laboratory-scale experiments in order to establish a basis for disinfection protocols. Two milligrams O 3 per liter for 2 min and lower exposures ought to be sufficient to ensure an excess of oxidants for efficient inactivation of fish pathogens while avoiding negative effects on the hatchability of halibut, cod and turbot eggs.

Reproduction, gamete supply and larval rearing of New Zealand turbot Colistium nudipinnis (Waite 1910) and brill Colistium guntheri (Hutton 1873): a potential new aquaculture species

New Zealand turbot Colistium nudipinnis (Waite 1910) and brill Colistium guntheri (Hutton 1873) were studied to assess their potential for aquaculture development. The reproductive cycle of wild fish showed a long spawning season from winter to summer, during which it is possible to obtain gametes. Both species have a diurnal ovulatory cycle, and gamete collection, by stripping and fertilizing at sea, was most successful within 2–3 h before and after sunset. Male reproductive anatomy suggests that these flatfish spawn in close proximity and that pair formation is highly likely. The eggs of both species have multiple oil droplets, turbot eggs being slightly larger (0.99 mm diameter) with more droplets (18–55) than brill eggs (0.97 mm, 13–26 droplets). Hatching occurred approximately 84 h after fertilization at 14 °C. Newly hatched turbot averaged 2.2 mm in length, and brill averaged 2.1 mm. First feeding began 4 days post-hatch (DPH). During larval rearing, rotifers were replaced by Artemia nauplii at 10 DPH. Metamorphosis commenced at 12–15 DPH and was completed and the larvae settled by 45 DPH. Weaning to inert foods began at 20–22 DPH (50 mg weight) and was completed by 57 DPH. Survival of turbot was 22.8% from fertilized egg to hatching, 7.3% through incubation to 22 DPH and 2.1% through incubation to fully weaned juveniles. Weaning success for turbot from metamorphosis to 57 DPH was 31.5%.

Induction of triploidy in the turbot ( Scophthalmus maximus) : I. Ploidy determination and the effects of cold shocks

The basis for induction of triploidy in the turbot by applying cold shocks shortly after fertilization (AF) was studied. Since this species exhibits a polymorphism in a number of nucleolar organizing regions (NOR), determination of the ploidy level through NOR analysis was first validated. Results showed that NOR analysis could discriminate well between diploid and triploid individuals whose ploidy level was verified karyotypically ( n=44 chromosomes in diploids; n=66 in triploids). In diploids, the mean number of NOR per cell ranged from 1.10 to 1.85, whereas in triploids, it ranged from 1.50 to 2.35. However, histogram distribution of data on mean number of NOR per cell showed that the number of fish in the overlapping region (NOR number between 1.50 and 1.85) was very low. Cold-shocked fish with a NOR value >1.735 were considered triploids. The error in ploidy assessment using NOR analysis in the turbot was found to be consistently around 3% and always <5%. In this way, NOR analysis could be safely applied to monitor the effects of cold shocks on triploidy induction. Cold shocks were applied 5 min AF for 5, 10, 20 or 40 min at either 0°C, 2°C, or 4°C. Results showed that the number of triploids increased with lower shock temperatures and longer shock duration in the range from 5 to 20 min. In particular, cold shocks of 0°C applied during 20 min consistently resulted in ∼90% triploid turbot ( P<0.001). Shocks longer than 20 min (40 min) did not increase the number of triploid turbot in contrast to what has been found in other flatfish species. This is probably related to the higher pre-shock temperature at which turbot eggs are incubated. Survival, 1 day after hatching, was ∼80% of the untreated controls and not different ( P>0.05) from appropriate sham controls, indicating that lower survival is due to the effects of mechanical handling and stress during triploidy induction rather than the triploid condition per se. The highest triploid yield obtained in this study, ∼70%, is higher than the triploid yield obtained in other flatfishes.

Does Carnobacterium divergens isolated from Atlantic salmon, Salmo salar L., colonize the gut of early developing turbot, Scophthalmus maximus L., larvae?

It is well established from endothermic animals that the adhesion of lactic acid bacteria involves host speci®city (Fuller 1986). In juvenile ®sh, JoEborn, Olsson, Westerdahl, Conway & Kjellberg (1997) reported by in vitro tests that a Carnobacterium sp. isolated from Atlantic salmon, Salmo salar L., colonized the gastrointestinal tract of rainbow trout, Onchorynchus mykiss (Walbaum). However, no information from ®sh larvae is available on whether the adhesion of lactic acid bacteria involves host speci®city. This is relevant in the context of whether or not it is possible to colonize the gastrointestinal tract of larvae at early stages with probiotics, notably Gram-positive bacteria, such as lactic acid bacteria, or by a resident micro ora, which may contribute to nutrition. The following bacteria were used in the present study: Vibrio pelagius isolated from copepod-fed turbot, Scophthalmus maximus L., larvae (Munro, Barbour & Birkbeck 1994), which is unusual in that it contains the polyunsaturated fatty acid eicosapentaenoic acid (20:5 n-3) (Ringu, Sinclair, Birkbeck & Barbour 1992), and a lactic acid bacterium, Carnobacterium divergens, isolated from Atlantic salmon. The main reason for using this species was that it produced a substance that inhibited the growth of both Vibrio anguillarum and Proteus vulgaris (Strum 1988), and Aeromonas salmonicida (Ringu 1999). Recently, colonization of different bacterial strains in larval turbot was determined using an enzyme-linked immunosorbent assay (ELISA) (Ringu, Birkbeck, Munro, Vadstein & Hjelmeland 1996; Ringu & Vadstein 1998). The purpose of the current study, was to evaluate whether a C. divergens, orginally isolated from Atlantic salmon, was able to colonize the gut of turbot larvae by adding this bacterial species to the rearing water and to compare these results with those for larvae exposed to V. pelagius, by using the ELISA method. Furthermore, a previous study (Ringu et al. 1996) demonstrated that antisera used in ELISA showed high sensitivity against homologous bacterial antigens and very little crossreaction with heterologous bacteria. Turbot eggs were obtained from Tinfos Aqua, Norway, and 1000 eggs were incubated in 80-L circular tanks with at bottoms in static water. A detailed description of larval rearing conditions and cultivation of live feed (Brachionus plicatilis SINTEFstrain) is given by Ringu et al. (1996). The following bacterial strains were used: C. divergens, originally isolated from young Atlantic salmon juveniles (Strum 1988); and Vibrio pelagius, isolated from copepod-fed turbot larvae (Munro et al. 1994). Vibrio pelagius was identi®ed as described by Munro et al. (1994) using the `Bacterial Identi®er' program (Bryant, Lee, West & Colwell 1986). Carnobacterium divergens was identi®ed using carboR

Effect of Dietary Essential Fatty Acids and Vitamins on Egg Quality in Turbot Broodstocks

Varying egg quality is one of the main factors interfering with the reliable performance of marine fish hatcheries. As larval performance during the first period largely depends on the availability of essential nutrients, the endogenous provision of these nutrients through the egg stages, and possibly parental diet, might be an important tool in improving hatchery output. Therefore, several feeding experiments were conducted on turbot (Scophthalmus maximus) broodstock reared under production conditions at two commercial facilities in France: France Turbot (FT) and Sepia Conseil (SC). Diets varied mainly in essential fatty acids (ω-3 HUFA), Vitamins C and E, and were fed for 2–3 months prior to the reproductive season. Egg quality parameters, i.e. fertilization and hatching rate, egg and oil droplet diameter, and biochemical composition, were monitored for each female during its total reproductive phase. Egg size during the reproductive cycle showed a low variability among females receiving the same treatment, but became significantly smaller as the season progressed. The same occurred for the oil globule in the non-vitamin supplemented groups, whereas it significantly increased in the vitamin-supplemented groups. However, these observations could not be correlated with any of the hatching or fertilization characteristics. The egg dimensions also varied as a function of the diet supplied, i.e. high HUFA levels in the broodstock diet resulted in a significant increase of egg diameter, oil globule diameter as well as fertilization rate. Interestingly, the control groups, with the lowest fertilization rate, had the highest hatching percentage, significantly higher than the HUFA/non-vitamin enriched groups. Using non-selected sources of trash fish as a food source at SC resulted in low levels of ascorbic acid (AA) in the eggs compared to the administration of an optimal quality batch of mackerel at FT. Enrichment of the trash fish with ascorbate-2–polyphosphate (ApP) resulted in a tripling of the AA content. Extra enrichment of the FT control diet did not further increase the levels in the eggs, suggesting that a saturation level was obtained. The major fatty acids in turbot eggs as well as freshly hatched larva are 16:0, 18:1ω-9, 20:5ω-3 and 22:6ω-3, but no obvious changes in their pattern could be detected for the different broodstock treatments. However, the level of 20:4ω-6 was significantly higher in the control group of FT, and showed a high correlation with the hatching percentage of the eggs obtained from the various broodstock treatments.

Long-term effects of the cryopreservation of turbot (Psetta maxima) spermatozoa

The survival of turbot eggs and the rearing capacities of larvae stemmed from artificial fertilization practices using frozen-thawed spermatozoa were evaluated. Furthermore, the viability of sperm samples stored during a 9 month period in liquid nitrogen was assessed. No significant difference in the fertilization rate, hatching rate, survival and wet weight of 10-day old larvae were observed using fresh or frozen-thawed spermatozoa. The motility recorded at 10 s and 60 s post-activation and the fertilization capacity of frozen-thawed spermatozoa were not significantly decreased during a 9 month storage period in liquid nitrogen. These results confirm the high quality of the turbot spermatozoa stemmed from the cryopreservation process, allowing their use for routine aquaculture practices.

Surface disinfection of Atlantic halibut and turbot eggs with glutaraldehyde: evaluation of concentrations and contact times

To evaluate the effects of glutaraldehyde treatment at different disinfection temperatures, Atlantic halibut, Hippoglossus hippoglossus (L.), and turbot, Scophthalmus maximus (L.), eggs, incubated at 5 and 12°C, respectively, were disinfected with 400–1200 mg glutaraldehyde l-1 at three different contact times (2.5, 5 and 10 min). Egg batches of both poor and good quality were tested for halibut. Positive effects were more pronounced in poor than in good-quality batches at hatching. Egg disinfection had a highly positive effect on the viability of yolk-sac larvae in both types of batches. A level between 400 and 800 mg l-1 at a contact time of 5–10 min was optimal for halibut: at lower levels, the bactericidal effect was reduced, and at higher levels, there were indications of toxic effects. Halibut eggs disinfected with optimal doses of glutaraldehyde had furthermore a reduced hatching time and more synchronous hatching as compared with untreated eggs. Turbot was more sensitive to higher doses than halibut, and the best larval performance was obtained for 400–800 mg glutaraldehyde l-1 at a contact time of 2.5 min. A further evaluation should, however, be performed before recommendations are given for species incubated at temperatures higher than 5°C.

Microbially matured water: a technique for selection of a non-opportunistic bacterial flora in water that may improve performance of marine larvae

Before transfer to larval incubators, water was membrane filtered to remove >95% of the bacteria and then transiently maintained in a biofilter that promoted recolonization of the water by non-opportunistic bacteria. The process is termed microbial maturation of the water. Hypothetically the bacterial flora in the matured water should protect the marine larvae from colonization and proliferation by opportunistic bacteria. Testing of the hypothesis demonstrated 76% higher survival of yolk sac larvae of Atlantic halibut (Hippoglossus hippoglossus) in matured than in membrane filtered water. Proliferation of opportunistic bacteria was observed in the rearing water after hatching of turbot eggs (Scophthalmus maximus), but to a less extent in the microbially matured water. In the early phase of first feeding of turbot larvae, the matured water induced qualitative differences in the gut microflora. Significantly higher initial growth rate of the turbot larvae in the matured water affected 51% higher average weight of 13 days old larvae than in membrane filtered water. Algal addition to the matured water enhanced the larval growth further. The experiments conducted supported the proposed hypothesis that microbial maturation selects for non-opportunistic bacteria, which protects the marine larvae from proliferation of detrimental opportunistic bacteria.

Fatty-acid composition of ovulated eggs from wild and cultured turbot (Scophthalmus maximus) in relation to yolk and oil globule lipids

The fatty-acid composition of lipids from ovulated eggs of wild and cultured turbot was investigated in order to estimate the nutritional requirements during embryonic and early larval development. Lipid comprised 13.8±0.5% (n=5) and 13.2±0.7% (n=7) of the egg dry weight in wild and cultured turbot, respectively. Polyunsaturated fatty acids (PUFA) of the (n-3) series accounted for 39% of total fatty acids in total lipid of both wild and cultured fish. The predominant (n-3) FUFA was docosahexaenoic acid (22:6 n-3), which also was the most abundant fatty acid in turbot eggs and comprised 24 and 23% of the total egg fatty acids in wild and cultured fish, respectively. Phospholipids, triacylglycerols and cholesterol-wax esters of turbot eggs all exhibited a specific fatty-acid profile distinctly different from that of total lipid. The general pattern of the fatty-acid distribution in lipids of eggs from wild and cultured turbot was similar, but the relative amount of 18:2(n-6) was considerably higher and 20:1(n-9) slightly higher in cultured fish. These differences were extended to all lipid classes and probably reflect the dietary intake of certain vegetable and marine fish oils. Calculations based on light microscopical studies showed that 55 to 60% of the total lipids in cultured turbot eggs are confined to the oil globule. The size of the oil globule remained constant during embryogenesis, and a reduction in size occurred first after hatching and mainly after yolk depletion. This implies that the total amount of lipids utilised during the embryonic development is considerably less than the total lipids present in ovulated turbot eggs. Comparison of the fatty-acid composition of total lipids from eggs and vitellogenin of wild turbot reveals that egg lipids contained a lower level of saturated and a higher level of monounsaturated fatty acids. Eggs also contained wax esters, which were not detected in vitellogenin, suggesting that vitellogenin is not the only source of lipids for turbot eggs.

Artificial insemination in turbot ( Scophthalmus maximus): determination of the optimal sperm to egg ratio and time of gamete contact

In high-quality batches of eggs (mean egg viability rate 89.6%), fertilization success (FS = number of 4-cell-stage eggs/number of viable eggs) was maximal when the ratio of spermatozoa to egg was above 6 × 10 3. For lower ratios, FS decreased and became highly variable. In lower-quality batches of eggs (mean egg viability rates 72.0 and 75.9%), FS was variable even for the highest sperm to egg ratios. For the ratio of 1.5 × 10 3 spermatozoa per egg, the occurrence of maximum FS in relation to increasing contact time between gametes was scattered and mainly recorded after 2 and 3 min. For the ratio of 6 × 10 3 spermatozoa per egg, maximum FS was mainly observed after a contact of 1 min between gametes. In commercial production, a minimum ratio of 6 × 10 3 spermatozoa per egg and a contact time between gametes of 3 min is recommended for the artificial insemination of turbot eggs.

Pathways of bacterial contamination during egg incubation and larval rearing of turbot, Scophthalmus maximus

Summary Overall microbial levels in the water system of a turbot farm were similar to those found in regular sea-water. At the end of an incubation period, however, the numbers of colony-forming units (CFU) in Artemiu cultures and turbot eg incubation jars were up to four orders of magnitude higher than sea-water level. Rinsing of the foot organisms (rotifers) prior to feeding them to turbot larvae, however, significantly reduced bacterial numbers, thus reducing the number of cross-contaminations. Several species of Aeromonas, Pseudomonas and Vibrio among others, were identified in the eggs and larvae, or in the water in which these were incubated. Scanning electron microscop shows that the surface of unfertilized turbot eggs is a breeding ground for bacteria; the removal of these eggs from incubation jars is therefore recommended. The epidermis of turbot larvae was virtually free ofmicroorganisms.

The influence of temperature, salinity and external calcium on diffusional permeability of the eggs of turbot ( Scophthalmus maximus)

Turbot eggs, following activation, are relatively impermeable to tritiated water. Thirty degree-days after fertilisation, the permeability of turbot eggs increased with age. In general permeability increased with temperature, Q 10 ranging from 2.15 to 3.84, and decreased with age. Acute change in salinity had no significant effect on permeability. The effect of alterations in external calcium had no effect on permeability in 15.5 degree-day eggs, but at 31 degree-days permeability appeared to increase as external calcium concentration fell.