• Título:  SPERM QUALITY IN MARINE TELEOSTS: APPLICATIONS TO BROODSTOCK MANAGEMENT AND SPERM STORAGE IN TWO FARMED SPECIES, SOLEA SENEGALENSIS AND SPARUS AURATA
• Autor:  Beirão Santos, Jose
• Universidad:  Universidad de León
• Departamento:  Biología molecular
• Fecha de Lectura:  15/06/2011
• Mención Europeo / Mención Internacional: concedido
• Programa de doctorado:  BIOLOGÍA MOLECULAR Y BIOTECNOLOGÍA
• Dirección: 
  • HERRAEZ ORTEGA, PAZ (DIRECTOR)
  • Martins Silva Cabrita, Elsa Alexandra (CODIRECTOR)
• Tribunal: 
  • Dinis, Maria Teresa (PRESIDENTE)
  • Robles Rodríguez, Vanesa (SECRETARIO)
  • Asturiano Nemesio, Juan Francisco (VOCAL)
  • Sarasquete Reiriz, Maria del Carmen (VOCAL)
  • Chereguini Fernández-Maquieira, Olvido (VOCAL)
• Descriptores: 
  • PISCICULTURA
  • REPRODUCCION ANIMAL
  • FISIOLOGIA ANIMAL
• Localización:  BIBLIOTECA UNIVERSITARIA SAN ISIDORO
• 230124_621442.pdf
• Resumen:
  In aquaculture like in any other animal production industry, optimal broodstock husbandry management is a key issue for reproduction control. Even so, most approaches to this subject are still very empiric, the majority of cultivated species show some reproductive dysfunctions and in several species fish are captured from the wild for broodstock repopulation. Correct reproduction control implies control of gametogenesis and the development of appropriate techniques for its evaluation. In this context male reproductive status and the evaluation of spermatogenesis and sperm quality of cultivated species are of utmost importance. Sperm quality is the ability of a sperm sample to fertilize the egg and produce normal offspring. Nevertheless for aquaculture and research purposes, easiest and more rapid parameters/methods are needed, whereas researchers require complementary methodological approaches to understand the basic factors affecting sperm quality. There is not a single parameter predictive of sperm quality but the assessment of some parameters together could give a consistent idea of the sample quality. Thus, in the present work we have tested the applicability of some new tools for sperm quality analysis and at the same time we have improved some of the more commonly applied ones. Sperm motility, besides being easy to monitor, also reflects several attributes of sperm functionality and for this reason is often used as a quality measurement. In recent years computer analysis (CASA) has been applied for an objective evaluation of this parameter. CASA software analyzes several parameters of each spermatozoon individually and thus a huge amount of data is produced from each sample. Complete use of the data rendered by the CASA software implies the application of an elaborated statistical analysis such as cluster analysis, grouping spermatozoa with similar characteristics. While motility is the decisive factor on which spermatozoa achieve fertilization, DNA integrity is a decisive factor for normal embryonic development and in this context, the comet assay technique measures chromatin integrity in each single cell. DNA damage is usually correlated with the apoptosis process, and although this issue is not as extensively studied in fish sperm as in mammals, it could help explain the low observed sperm quality in some captive species and/or low sperm quality after some storage procedures. One of the easiest ways to measure cell apoptosis is the detection of the plasma membrane destabilization process that occurs in early steps of apoptotic process, through specific markers. In addition to the apoptosis process, membrane integrity is often assessed for sperm quality measurement, by the application of hydrophilic dyes such as propidium iodide (PI). A cell with an injured membrane will be unable to perform its functions, such as the reception of stimulus that triggers motility activation or gamete fusion. Furthermore, sperm plasma membrane composition, because of its unique function, is different from the somatic cells, presenting a high content in polyunsaturated fatty acids which increases membrane fluidity and allows the normal sperm function and mechanisms such as gamete fusion. All sperm plasma membrane components and their interaction are essential for the correct cell function and thus alterations on them can seriously affect the spermatozoa functionality. In this sense, it is also important to evaluate sperm membrane composition. In the present work we were interested in developing better tools for sperm evaluation of cultured marine teleost by means of: giving some clues about potential changes on broodstock husbandry conditions, mainly through dietary supplementation for improving sperm quality; and studying the effect that the cryopreservation technique (the easiest way to store sperm for long periods) has on sperm quality, mainly the effects on sperm motility and membrane composition. To achieve these goals we focus our attention on Senegalese sole, Solea senegalensis, sperm quality in captivity and tried to improve male reproductive performance. In this species, sperm quality seems to be one of the factors preventing good quality spawn from being obtained, which impairs S. senegalensis mass production. Additionally, we also focus our attention on gilthead seabream, Sparus aurata, using this species as a model for sperm cryopreservation in marine teleosts, due to high availability of sperm, knowledge of reproductive biology and its importance for the Mediterranean aquaculture industry. In a first work we analyzed sperm quality of a S. senegalensis broodstock using different cellular approaches during the reproductive season. Sperm motility was measured with CASA and a clustering procedure was applied that allowed the detection of four distinct subpopulations. These subpopulations varied with time after activation and the fast and linear subpopulation (probably responsible for fertilization) just 60 s after motility activation was nearly absent. Also, we measured cell plasma membrane resistance to seawater hyperosmolarity and noticed that there was a fast decrease in percentage of cells with intact membrane after sperm motility activation (no more than 12% viable cells 45 s after motility activation). This fact seemed to be modulating sperm subpopulation composition of the different males. Sperm plasma membrane resistance to seawater hyperosmolarity was negatively correlated with mean percentage of DNA fragmentation presented by the sperm cells (measured with the comet assay). Nonetheless, the majority (64.1%) of the spermatozoa in the different males presented DNA fragmentation below 30%, which is considered relatively low when compared with other marine teleosts. We also measured the percentage of apoptotic cells, using the fluorescent probe Annexin-V FITC. The apoptotic population ranged from 6 to 20%, their presence in the ejaculate indicating some spermiation deregulation. Since in S. senegalensis males spermiate all year round, in a second work we applied a similar approach monthly throughout the year to better understand S. senegalensis sperm quality limitations. 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 water temperature, and recovering slightly in the second spawning season (September-October). In this sense, the percentage of progressive motile sperm and sperm velocity showed a decrease from March to July (when the highest temperatures were observed), followed by a slight increase in August and October. DNA fragmentation values showed highest values between the two spawning seasons (summer months) and decreased to the end of the year. The percentage of apoptotic cells was lowest in March and highest in November. The percentage of cells resistant to seawater exposure was constantly low throughout the year and in summer months presented values lower than 5% of cells with intact membrane just 15 s after motility activation. However, there were two peaks of cells with intact membrane 15 s after motility activation in May and October, related with the two spawning seasons. The low sperm plasma membrane resistance to seawater hyperosmolarity observed in the two works can be part of a semen selection process to positively choose cells with better performance, eliminating overripe cells, and preventing them from achieving fertilization. Additionally, S. senegalensis presents a reproductive behavior that allows the sperm to achieve oocytes in a short period after ejaculation. The different males present in the broodstocks reached their sperm quality peak at different times of the year and of the spawning seasons, which will result in an unequal contribution for the next generation since females only spawn in specific periods. In the third work we designed a strategy to improve Solea senegalensis sperm quality through dietary improvement, feeding three broodstocks on different diets supplemented with fatty acids (docosahexaenoic acid (DHA)) and antioxidants (selenium (Se) and vitamin E (vitE)). Our aim was to improve S. senegalensis sperm quality, especially its membrane resistance to seawater hyperosmotic shock, to which they are exposed after ejaculation. In the males fed on the supplemented diet (DHA plus antioxidants) there was an increase in sperm motility parameters (percentage of progressive spermatozoa and curvilinear velocity (VCL)). These motility parameters were positively correlated with both polyunsaturated fatty acids (PUFAs) and DHA proportion in the sperm. Fish fed on DHA only did not show any improvement because of the harmful effects of the generation of reactive oxygen species, causing the high lipid peroxidation values observed in the sperm of the males fed on this diet. This value was positively correlated with the amount of lysophosphatidyl-choline and sphingomyelin. Glutathione peroxidase activity, measured as an indirect contribution of Se, was significantly higher in blood plasma of males fed on DHA plus antioxidants. The addition of the antioxidants to the diets circumvented the higher lipid peroxidation. Opposed to what was initially proposed, cell resistance to seawater hyperosmotic shock was not improved. There was an increase in cholesterol and in several unsaturated fatty acids (DHA, oleic acid, linoleic acid, etc.) in sperm from males fed on DHA plus antioxidants, and at the same time, there was a decrease in the proportion of saturated fatty acids. The components added to the diets were clearly incorporated in the semen composition and positively affected S. senegalensis sperm quality traits. Improvement of motility could reduce the time required for fertilization, thus minimizing the effect of the high sensitivity of cells to seawater. In the fourth work we studied the effect of different cryopreservation protocols on fish sperm motile subpopulations using S. aurata sperm and applying a Two-Step Cluster analysis to the data rendered by CASA. We identified 3 different sperm subpopulations. The fast and linear subpopulation (SP3) was the better correlated with the hatching rate. This same subpopulation was the best represented 15 s after activation and was also the one showing a greater decrease in time, being the least represented after 60 s. There was a significant variation in the subpopulation composition between the different cryopreservation protocols (three cryoprotectants (dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG)) each one at two different concentrations; two packaging volumes (0.5 ml straws, and 1.8 ml cryovials); and different freezing rates (1, 2, 3, 4 and 8 cm above the liquid nitrogen surface for the straws and 1, 2 and 4 cm for the cryovials)). The different composition in subpopulations obtained between the protocols pointed out which were the less efficient freezing/thawing procedures. According to the analysis of the subpopulations, and their variation through time after motility activation, samples frozen in straws with 5% DMSO and in cryovials with 10% DMSO at 2 and 1 cm from the liquid nitrogen, respectively, produced the best results (closer to the control). Samples cryopreserved with cryovials presented a significant decrease in SP3 compared with the same treatment in straws. Clustering analysis allowed the detection of fish sperm subpopulations according to their motility pattern and showed that sperm composition in terms of subpopulations was differentially affected by the cryopreservation protocol depending on the cryoprotectant used, freezing rates and packaging systems. In the fifth work we proposed to test a new tool for fish sperm, YO-PRO 1 nucleic acid stain, to detect early plasma membrane destabilization for the evaluation of spermatozoa viability after the freezing/thawing procedures. Our results showed that instead of the two sperm subpopulations detected with the traditional evaluation with the PI and SYBR 14 assay, the application of YO-PRO 1 together with PI allowed the detection of a third subpopulation of cells permeable to YO-PRO 1 but not to PI. This subpopulation was composed by cells with a disturbed membrane which were non-permeable to PI, but whose permeability was already compromised, thus YO-PRO 1 stain was more sensitive than PI. There was an increase in the YO-PRO 1 subpopulation after sperm cryopreservation and, as observed by flow cytometry, these changes in membrane permeability and integrity were also accompanied by spermatozoa morphometric modifications. After detecting this membrane destabilization in the sixth and last work we decided to study the effects that the cryopreservation process has on the head plasma membrane (HM) and flagella lipidic composition. Simultaneously we attempted to stabilize membrane composition by the addition of antifreeze proteins- AFPs (AFPI and AFPIII). The addition of AFPIII (1µg/mL) significantly increased straight line velocity (VSL) and the percentage of viable cells compared with samples only cryopreserved with 5% DMSO. The same concentration of AFPI did not produce any significant improvement in sperm quality. Also, we observed different effects depending on the cryoprotectant solution used (whether AFPI and AFPIII were used or not) on the phospholipid and fatty acids composition of the spermatozoa. Freezing with 5% DMSO alone increased the phosphatidyl-serine (PS) content, increased the saturated fatty acids and decreased the unsaturated fatty acids (mainly PUFA) in both HM and flagella. Nevertheless flagella lipidic composition was not so affected as the HM by the cryopreservation procedure. On the other hand, AFPs, especially AFPIII, seemed to have interacted with unsaturated fatty acids avoiding changes in lipidic profiles during cryopreservation and stabilizing the plasma membrane organization both in head and flagella. Also it was observed that flagella had a higher amounts of unsaturated fatty acids and higher amounts of phosphatidyl-serine and phosphatidyl-ethanolamine than the HM. This will probably confer higher fluidity to the flagella and explain its lower degree of lipid modifications after cryopreservation. Finally the HM lipid composition but not the flagella composition was correlated with sperm quality parameters such as viability, motility and velocity, suggesting that mainly the HM lipid composition influenced sperm performance. These sperm quality parameters were positively affected by the higher amount of unsaturated fatty acids. Our data support the hypothesis of a direct interaction of AFPs with specific hydrophobic moieties of the membrane lipids.