Florbela Soares

A review on the cultivation potential of Solea senegalensis in Spain and in Portugal

Abstract The sole, Solea senegalensis , is a common high-value flatfish in Southern Europe, commonly reared in extensive aquacultural production in Portugal and Spain. Research in recent years has focused on the production of larvae and juveniles of good quality. Natural spawning of broodstock in captivity has been accomplished, and is the only way viable eggs have been obtained to date. The broodstock feeding regime is based on squid ( Loligo vulgaris ), being supplemented with polychaetes ( Hediste diversicolor ) during final maturation. Temperature plays a very important role in the onset and duration of the spawning period, with egg emission stopping below 16°C. Observed duration of the spawning period has ranged from 4 to 6 months. The total daily egg collection during the spawning season ranged from 0 to 180 g for a broodstock of 15 fish. Egg fertilization rates varied between 20% and 100%, and the percentage of viable eggs had a mean of 72.1±26.5%. Variations in egg size between batches were detected, with egg size tending to decline during the spawning season. Larvae hatch with an average size of 2.4±0.1 mm total length, but this varies widely by batch. Larvae accept Artemia nauplii as first prey and reach a size of 8 mm by day 15. Metamorphosis starts on 11 days after hatching (DAH) and is completed by 19 DAH. Survival at 19 DAH range from 29% to 87%. From 19 DAH onwards fish are fed live Artemia metanauplii, and reach 16±0.8 mm on 40 DAH. Ponds stocked with unweaned juveniles produced after one year fish with 16.6±2.1 cm total length and 40.3±2.5 g wet weight, with a survival of 20%. Unweaned juveniles stocked together with Sparus aurata reached a size of 35.3±1.8 cm total length and 456.1±3.6 g wet weight after one year fish, with a survival of 8%. Epizootic mortalities due to pasteurellosis have been observed in juvenile S. senegalensis . As in other flatfish, pigmentation abnormalities as well as some malformations associated with the migration of the eye have also been observed. The optimization of the weaning and growout procedures, including the development of appropriate feeding regimes, and further studies into the causes of pigmentation abnormalities are necessary before this species can be reared intensively on a large scale.

Effect of dietary non-protein energy levels on condition and oxidative status of Senegalese sole (Solea senegalensis) juveniles

The effects of dietary non-protein energy levels on growth, oxidative status and condition were studied in juveniles of Senegalese sole (Solea senegalensis). Four isonitrogenous diets with four energy levels were used: A-low (11% lipid, raw carbohydrate); B-intermediate (11% lipid, digestible carbohydrate); C-intermediate (21% lipid, raw carbohydrate); and D-high (21% lipid, digestible carbohydrate). Survival, relative growth rate (RGR) and feed conversion rate were not significantly affected by the dietary treatments. The fatty acid composition of the fish muscle varied little among the treatments, but trans 18:2n - 6, total n - 6 and EPA/DHA ratio, tended to be higher in fish fed diets with low lipid level. Cellular energy allocation (CEA) results (indicative of metabolic status and net energy budgets) showed significant differences in liver, but not in muscle samples. Livers of fish fed diet C contained the lowest carbohydrate, protein and CEA values, but the highest cellular energy consumption. Fish fed diet A had the highest CEA for growth, followed by fish fed diets B and D and then diet C. The liver and muscle peroxidation and antioxidant activity were measured by thiobarbituric acid reactive substances (TBARS) test and the enzymatic activities levels of catalase (CAT) and superoxide dismutase (SOD). TBARS values were higher for fish fed diets with high lipid content. However, no clear relation was found between HUFA level and TBARS value. The activity levels of the antioxidant enzymes CAT and SOD were higher in livers of fish fed diets with a high lipid level. Furthermore, CAT and SOD activity and TBARS values were influenced by the type of dietary starch in the diet. Higher oxidation rates were observed in fish fed diets containing raw carbohydrate. These data suggest that lipid and carbohydrate energy sources affect the oxidative status of Senegalese sole. Diets containing low levels of lipid and digestible starch reduce the susceptibility of the fish to oxidation and may enhance growth rate

Multivariate cluster analysis to study motility activation of Solea senegalensis spermatozoa: a model for marine teleosts.

Computer-assisted sperm analysis (CASA) and clustering analysis have enabled to study sperm subpopulations in mammals, but their use in fish sperm has been limited. We have used spermatozoa from Senegalese sole (Solea senegalensis) as a model for subpopulation analysis in teleostei using two different activating solutions. Semen from six males was activated using 1100 mOsm/kg solutions: artificial seawater (ASW) or sucrose solution (SUC). Motility was acquired at 15, 30, 45, and 60 s post-activation. CASA parameters were combined into two principal components, which were used in a non-hierarchical clustering analysis, obtaining four subpopulations (CL): CL1 (slow/non-linear), CL2 (slow/linear), CL3 (fast/non-linear), and CL4 (fast/linear). We detected spermatozoa lysis, especially in ASW. Sperm motility was higher for SUC and decreased with time. The subpopulation proportions varied with time and activating treatment, showing both an increase in CL1 and CL2 and a decrease in CL3 and CL4 with time. Both CL3 and CL4 were higher in samples activated with SUC, at least in early post-activation. Proportions of CL3 and CL4 at 15 s were associated with higher quality at 60 s and with lower lysis. A second clustering analysis was conducted, classifying the males accordingly to their motility subpopulations. This analysis showed a high heterogeneity between samples. Subpopulation analysis of CASA data can be applied to Solea spermatozoa, allowing identification of potentially interesting sperm subpopulations. Future studies might benefit from these techniques to establish the relationship of these subpopulations with fish sperm quality and fertility, helping to characterize males according to their reproductive potential.

Sperm quality evaluation in Solea senegalensis during the reproductive season at cellular level.

Sperm quality seems to be one of the reasons for the reproduction constraints faced by Senegalese sole (Solea senegalensis) aquaculturists. Previous studies in this species indicated that the sperm quality of individuals kept in culture varies throughout the year and that different sperm subpopulations can be identified in ejaculates according to the motility pattern of spermatozoa. Aiming to better understand factors affecting sole sperm quality in captivity, sperm of 11 males was assessed during the reproductive season using different parameters: motility characteristics using CASA analysis; cell plasma membrane resistance to seawater hyperosmolarity; DNA fragmentation with single-cell gel electrophoresis; and early apoptosis, labeled with Annexin-V FITC. Computer-assisted sperm analyses motility data were treated using multivariate analysis to identify the presence of different spermatozoa subpopulations according to their motility pattern. Four distinct sperm subpopulations were obtained: Subpop1, which includes fast linear spermatozoa; Subpop2, made up of fast nonlinear spermatozoa; Subpop3, which includes slow linear spermatozoa; and Subpop4, which contains slow nonlinear spermatozoa. The sperm subpopulation structure varied with time after activation and with male. Low cell resistance to the seawater hyperosmotic conditions was noticed. The Annexin-V assay allowed the identification of an apoptotic population ranging from 6% to 20%. A high percentage of cells (64.1%) showed a DNA fragmentation level below 30%, but these values varied significantly between males. DNA fragmentation appears to be related to cell membrane resistance to hyperosmotic conditions faced by the cells when in contact with seawater. This condition seems to modulate the composition of the motile sperm population and performance after activation. This phenomenon could be related to the spermatozoa maturation process.

Changes in Solea senegalensis sperm quality throughout the year

Some of the Senegalese sole (Solea senegalensis) broodstock reproductive constraints are related to sperm quality. Although they present two defined spawning season (spring and autumn), males gave semen during all the year thus an exhaustive annual sperm analysis is important to determine the seasonal changes in semen quality. Sampling was performed monthly during one year, analyzing different cellular parameters to better understand sperm quality limitations obstructing sole mass production. The percentage of progressive motile cells and their linear velocity showed a decrease from March (beginning of the first spawning season) to July (when the highest temperatures were observed), followed by a slight increase in August and October (second spawning season). DNA fragmentation values showed highest values between the two spawning seasons and decreased to the end of the year. The percentage of apoptotic cells was lowest in March (beginning of the first spawning season) and the highest in November. The percentage of cells resistant to seawater exposure presented two peaks related with both spawning seasons. There was a tendency for the semen to attain a quality peak between the beginning and the middle of the first spawning season (March-May), followed by a pronounced decrease, achieving the lowest values during the months with the highest temperature. Also, the different males present in the broodstocks reach their sperm quality peak at different times, which will result in an unequal contribution for the next generation.

Antemortem versus postmortem methods for detection of betanodavirus in Senegalese sole (Solea senegalensis)

The suitability of nested reverse transcription polymerase chain reaction (nRT-PCR) to detect betanodavirus in blood samples from naturally infected Senegalese sole (Solea senegalensis) was evaluated in comparison with other diagnostic methods. Results indicated that histologic examination of brain lesions could be regarded as the most consistent indicator of nodavirus infection in this species. The nRT-PCR showed low to moderate levels of detection; the best values were obtained in brain samples followed by blood samples. Inoculation of SSN-1 and SAF-1 cells with fish samples did not cause cytopathic effect, although virus was detected by reverse transcription polymerase chain reaction in approximately 25% of the SSN-1 inoculated wells. The efficiency of detection of the viral genome was dramatically increased by the use of nRT-PCR, reaching 90.6% of positives in brain samples and 84.4% in blood samples. The sensitivity and the negative predictive value of nRT-PCR in blood samples were slightly lower than those obtained using brain samples. Nevertheless, it is suggested that the advantage of being able to perform diagnosis on live fish adequately counterbalances the slightly lower sensitivity of nRT-PCR on blood samples. This technique is proposed as a useful tool, not only for the selection of nodavirus-free breeders but also to check the fish status during ongrowing.

Effect of two sulfur-containing amino acids, taurine and hypotaurine in European sea bass (Dicentrarchus labrax) sperm cryopreservation

In the present work, taurine and hypotaurine were evaluated as potential additives to improve European sea bass (Dicentrarchus labrax) sperm quality after cryopreservation. For cryopreservation, three different extenders were used: control extender (NAM), supplemented with 1mM taurine or supplemented with 1mM hypotaurine, all of them containing 10% Me₂SO as cryoprotectant. To evaluate sperm quality of fresh and thawed sperm, motility (CASA: computer assisted sperm analysis), viability (SYBR Green/propidium iodide), lipid peroxidation (malondialdehyde level), protein oxidation (carbonyl content), glutathione peroxidase, glutathione reductase and superoxide dismutase activities and DNA fragmentation (comet assay) were quantified. The result demonstrated that 1 mM hypotaurine supplemented extender increased total motility (30.1 ± 3.2%), and that 1 mM taurine extender produced higher velocity (18.1 ± 2.6 μm/s) and linearity (46.0 ± 4.8%) than the control extender (21.8 ± 3.2%, 15.5 ± 1.3 μm/s, 41.8 ± 2.4%, respectively). Cell viability, lipid peroxidation and protein oxidation were not statistically different between treatments. Similar results were obtained for glutathione peroxidase and superoxide dismutase activities. Only glutathione reductase showed differential activity before and after freezing, increasing its activity in thawed sperm. Regarding the comet assay results, taurine and hypotaurine significantly reduced DNA fragmentation (52.8 ± 0.9% and 51.8 ± 0.9%, respectively) in comparison to the control (55.7 ± 0.8%). In conclusion, for European sea bass sperm cryopreservation, extenders supplemented with 1 mM taurine and 1 mM hypotaurine improved some parameters of sperm quality after thawing, resulting in better motility and lower DNA damage than the control, two very important factors related to fertilization success.

Endocrine and milt response of Senegalese sole, Solea senegalensis, males maintained in captivity

Improving fertilization success in captive Senegalese sole broodstocks has been a challenge in the last years. Recent reports suggest that low sperm volume and quality could be one of the reasons leading to poor fertilization rates, although further studies are needed to reach a conclusive explanation. Here, we report on several experiments focused on this issue. Seasonal profiles of plasma androgen levels (testosterone and 11-ketotestosterone) and sperm production and quality parameters were assessed, although no statistical correlations among them were identified. The response of males to female presence/absence was also analyzed. Long-term isolation from females decreased male androgen levels at the peak of the reproductive period, suggesting some kind of disrupting effects on the endocrine system. On the other hand, short-term exposure of previously isolated males to ripe females decreased androgen levels, possibly reflecting a rapid steroidogenic shift promoting final maturation of spermatozoa, and increased sperm viability, motility and velocity, thus, supporting the concept of positive effects of female contact on male sole performance. Further evidence sustaining the relevant female-to-male communication in sole reproduction was obtained after treating the females with progestagen 17α,20β-dihydroxy-4-pregnen-3-one (regarded as pre-ovulatory pheromone in fish) and registering a significant increase in sperm viability, velocity and motility in surrounding males. Finally, we found that a single administration of a 20 μg/kg GnRH analogue in males was effective in stimulating androgen release and sperm quality, although the effects were transient and thus, the use of sustained hormone delivery methods were suggested for improving efficiency. Our results point to velocity, viability, and motility as the most sensitive parameters in sole sperm, although further studies will have to evaluate whether these parameters have any relation with fertilization success in captive broodstocks of this important aquaculture species.

Molecular Taxonomic Profiling of Bacterial Communities in a Gilthead Seabream (Sparus aurata) Hatchery

As wild fish stocks decline worldwide, land-based fish rearing is likely to be of increasing relevance to feeding future human generations. Little is known about the structure and role of microbial communities in fish aquaculture, particularly at larval developmental stages where the fish microbiome develops and host animals are most susceptible to disease. We employed next-generation sequencing (NGS) of 16S rRNA gene reads amplified from total community DNA to reveal the structure of bacterial communities in a gilthead seabream (Sparus aurata) larviculture system. Early- (2 days after hatching) and late-stage (34 days after hatching) fish larvae presented remarkably divergent bacterial consortia, with the genera Pseudoalteromonas, Marinomonas, Acinetobacter, and Acidocella (besides several unclassified Alphaproteobacteria) dominating the former, and Actinobacillus, Streptococcus, Massilia, Paracoccus, and Pseudomonas being prevalent in the latter. A significant reduction in rearing-water bacterial diversity was observed during the larviculture trial, characterized by higher abundance of the Cryomorphaceae family (Bacteroidetes), known to populate microniches with high organic load, in late-stage rearing water in comparison with early-stage rearing-water. Furthermore, we observed the recruitment, into host tissues, of several bacterial phylotypes—including putative pathogens as well as mutualists—that were detected at negligible densities in rearing-water or in the live feed (i.e., rotifers and artemia). These results suggest that, besides host-driven selective forces, both the live feed and the surrounding rearing environment contribute to shaping the microbiome of farmed gilthead sea-bream larvae, and that a differential establishment of host-associated bacteria takes place during larval development.

Solea senegalensis sperm cryopreservation: New insights on sperm quality

Cryopreservation of Senegalese sole sperm can represent an alternative to overcome some reproductive problems of this species. However, it is important to guarantee the safe use of cryopreserved sperm by selecting an appropriate protocol according to a high demand quality need to be ensured. It has been demonstrated that traditional assays such as motility and viability do not provide enough information to identify specific damage caused by cryopreservation process (freezing and thawing). Specific tests, including lipid peroxidation and DNA damage, should be performed. In the present study, motility and lipid peroxidation were performed as specific tests allowing us to discard cryopreservation conditions such as methanol as internal cryoprotectant and bovine serum albumin as external cryoprotectant. In addition, a caspase 3/7 detection by flow cytometry was performed to analyze apoptosis activity in the best selected conditions. Moreover, new highly sensitive tests based on transcript number detection have recently been described in fish sperm cryopreservation. For this reason, a transcript level detection assay was performed on certain oxidative and chaperone genes related to fertilization ability and embryo development (hsp70, hsp90BB, hsp90AA, gpx) to select the best cryopreservation conditions. DMSO+ egg yolk proved to be the best cryoprotectant combination in terms of transcript level. This study describes an optimized cryopreservation protocol for Solea senegalensis sperm demonstrating for the first time that transcript degradation is the most sensitive predictor of cell status in this species after cryopreservation.