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- Solea senegalensis sperm cryopreservation: new insights on sperm qualityPublication . F RIESCO, MARTA; Oliveira, Catarina; Soares, Florbela; Gavaia, Paulo J.; Dinis, Maria Teresa; Cabrita, ElsaCryopreservation 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.
- Nutritional physiology during development of Senegalese sole (Solea senegalensis)Publication . Conceição, L. E. C.; Ribeiro, Laura; Engrola, S.; Aragão, C.; Morais, S.; Lacuisse, M.; Soares, Florbela; Dinis, Maria TeresaThe Senegalese sole, a species with a complex metamorphosis, difficulties in weaning and with occasional problems of malpigmentation and skeletal deformities, is a good model species to study larval nutritional physiology. In addition, the early metamorphosis and acquisition of a peculiar non-proactive bottom-feeding behaviour make early weaning an important issue in sole hatcheries. The present work reviews recent findings in different aspects of nutritional physiology during the development of Senegalese sole, in an attempt to optimize the composition of sole diets and to understand what are the limiting factors for weaning sole. Both digestive enzymes activity and tracer studies using 14C-Artemia show that sole larvae, even at young stages, have a high capacity for digesting live preys. This is reflected in a high growth potential and low mortality rates for this species during the larval stage compared to other marine fish species. Based on the observation of the digestive enzymes profile, early introduction of inert microdiets in co-feeding with Artemia does not seem to affect intestinal function. However, when co-feeding is not provided, intestinal activity may be depressed. Furthermore, early introduction of microdiets in co-feeding with Artemia may have a positive effect on survival rates, but at the expense of lower growth rates and higher size dispersal. This may reflect variation in the adaptation capacity of individual larvae to inert diets. High dietary neutral lipid (soybean oil) content results in reduced growth and accumulation of lipid droplets in the enterocytes and affects the capacity of Senegalese sole larvae to absorb and metabolise dietary fatty acids (FA) and amino acids (AA). Through tube feeding of different 14C-lipids and free FA it has been shown that FA absorption efficiency increases with unsaturation and that sole larvae spare DHA from catabolism. In addition, it was demonstrated that absorption efficiency varies according to molecular form, being highest for free FA, lowest for triacylglycerols and intermediate for phospholipids. Live preys commonly used in larviculture do not seem to have a balanced AA profile for sole larvae. Furthermore, the ideal dietary AA composition probably changes during development. Rotifers and Artemia metanauplii are apparently deficient in one or more of the following AA depending on the larval development stage: histidine, sulphur AA, lysine, aromatic AA, threonine and arginine. It has also been demonstrated that balancing the dietary AA profile with dipeptides inArtemia-fed larvae increases AA retention and reduces AA catabolism. When supplementing larval diets with limiting AA it should also be considered that sole larvae have different absorption, and retention efficiencies for individual AA, and that they have the capacity to spare indispensable AA. In addition, the absorption of free AA is faster and more efficient than that of complex proteins. Improvements in biochemical composition of inert microdiets for sole are likely to contribute to the reproducible weaning success of Senegalese sole.
- Growth, stress response and free amino acid levels in Senegalese sole (Solea senegalensis Kaup 1858) chronically exposed to exogenous ammoniaPublication . Pinto, W.; Aragão, C.; Soares, Florbela; Dinis, Maria Teresa; Conceição, L. E. C.Stressful husbandry conditions are likely to a¡ect growth and amino acid metabolism in ¢sh. In this study, chronic ammonia exposure was used to test the e¡ects of a stressor on growth and amino acid metabolism of Senegalese sole juveniles. The ¢sh were exposed for 52 days to 11.6 mg L 1 [low-TAN (L-TAN)] or 23.2 mg L 1 [high-TAN (H-TAN)] of total ammonia nitrogen (TAN), or to 0 mg L 1 (Control). Growth in L-TAN groups was slightly but signi¢- cantly di¡erent from the Control groups [relative growth rate (RGR50.35 0.13 and 0.52 0.23% day 1respectively)]. In H-TAN groups, growth was severely a¡ected (RGR50.01 0.13% day 1 ).
- Pigmentation abnormalities in juveniles sole Solea senegalensis Kaup, 1858 | Anomalías en la pigmentación de juveniles de lenguado Solea senegalensis Kaup, 1858Publication . Soares, Florbela; Engrola, S.; Dinis, Maria TeresaEl lenguado Solea senegalensis Kaup, 1858 es una especie de valor comercial elevado y está considerada de interés potencial para la acuicultura. En consecuencia, existe una creciente necesidad de desarrollar el cultivo larval de esta especie a escala industrial y producir juveniles para engorde en las piscifactorías. Se han realizado diversos trabajos con el objetivo de mejorar la metodología en la producción de S. senegalensis (Dinis, 1986, 1992; Dinis et al., 1999) y se señalan algunas limitaciones: entre ellas, las anomalías de pigmentación. Se describen aquí las principales anomalías en la pigmentación del lenguado a partir de puestas naturales de reproductores salvajes mantenidos en cautividad. El porcentaje total de alevines mal pigmentados fue del 11 %, lo que puede constituir un problema cuando se pretende la producción a gran escala, pues entraña un obstáculo para su comercialización, con el consiguiente descenso de la rentabilidad.
- Multivariate cluster analysis to study motility activation of Solea senegalensis spermatozoa: a model for marine teleostsPublication . Martínez-Pastor, Felipe; Cabrita, Elsa; Soares, Florbela; Anel-Lopez, Luis; Dinis, Maria TeresaComputer-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 CO and CL4 with time. Both CL3 and CL4 were higher in samples activated with SUC, at least in early post-activation. Proportions of CO and CL4 at 15 s were associated with higher quality at 60 sand 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.