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- Improvement of the cryopreservation protocols for the dusky grouper, Epinephelus marginatusPublication . F Riesco, Marta; Raposo de Magalhães, Cláudia; Engrola, Sofia; Martinez-Paramo, Sonia; Mira, Sara; Cunha, Maria Emilia; Cabrita, ElsaThe dusky grouper, Epinephelus marginatus, is a potential species for aquaculture production although the limited number of males kept in captivity has been the cause of some constraints in its production. Sperm cryopreservation emerged as a solution for this problem. However, spermatozoa can be severely affected by freezing/thawing processes and poor sperm quality is a limiting factor in reproduction success. The present study aimed at evaluating two main aspects in the design of a cryopreservation protocol-extender additives (taurine, glucose, cholesterol, BSA) and sperm containers (0.5 mL straws, 2 mL cryovials and 5 mL macrotubes). Sperm quality was assessed through the evaluation of the percentage of motile cells, viable cells, DNA fragmentation, lipid peroxidation and apoptosis. Some specific techniques, such as Caspase 3/7 detection, which provides information on membrane integrity and cell death, detecting early and late apoptotic and necrotic events, were required to establish an optimized cryopreservation protocol for this species. Taurine was the most suitable cryopreservation additive in terms of viable cells and cholesterol presented the highest percentage of necrotic cells in this study. Caspase 3/7 assay enabled us to detect necrotic damage induced by cryopreservation. Statement of relevance: The development of reproductive tools in dusky grouper, a potential species for aquaculture production, emerges as important tool to decrease the number of wild males maintained in captivity. A cryopreservation protocol was previously described for this species although several constraints in terms of sperm quality were detected. Our work provided new evidences that cryopreservation protocols can be improved through the addition of certain additives and use of appropriate sperm containers. Specific sperm analysis was crucial to identify and establish the most suitable conditions for breeders management and species conservation purposes. (C) 2016 Elsevier B.V. All rights reserved.
- 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.
- Biology of teleost primordial germ cells (PGCs) and spermatogonia: Biotechnological applicationsPublication . Robles, Vanesa; F RIESCO, MARTA; Psenicka, Martin; Saito, Taiju; Valcarce, David G.; Cabrita, Elsa; Herraez, PazThis review provides a general view on teleost germline development using primordial germ cells (PGCs) or spermatogonia (SG), highlighting recent progress in research on these two cell types in teleost fishes. Due to the interest of these cells for gene banking purposes, this chapter also reviews the available protocols for cryopreservation and the techniques that have been employed to assess cellular and molecular status after the process. Protocols for in vitro culture and the possibility of generating PGCs in vitro from non-committed embryonic cells are also discussed. Furthermore, the potential of these cells in surrogate production is presented, analyzing the transplantation and xenotransplantation experiments performed in fish. (C) 2016 Elsevier B.V. All rights reserved.
- First study in cryopreserved Crassostrea angulata spermPublication . F RIESCO, MARTA; Félix, Francisca; Matias, Domitilia; Joaquim, Sandra; Suquet, Marc; Cabrita, ElsaSperm cryopreservation is a widely employed technique that promotes alternative techniques to contribute to broodstock management or restoration programs for species of commercial interest, endangered species or species with an interesting genotype. The preservation of genetic material from improved stocks or from the original population is extremely important for the oyster aquaculture industry to prevent the potential impacts of epidemic diseases and natural disasters. The Portuguese oyster, Crassostrea angulata, was the most important species commercialized by the shellfish industry. However, inadequate management of this industry and pathology occurrences resulted in a significant decrease in natural populations. For this reason, in this work a successful sperm cryopreservation protocol for this important species has been developed for the first time. Different internal cryoprotectants (DMSO, ethylene glycol, polyethylene glycol and methanol) at several concentrations (5, 10, 20%), containers (straws vs cryovials) and freezing rates (slow and fast rates) were tested. Cryoprotectant toxicity tests corroborated that this assay did not take into account the following steps of cryopreservation protocol as sperm agglutination. A fast freezing rate of cells diluted in 10% DMSO and the use of straws as containers were the best cryopreservation conditions for Portuguese oyster sperm. Finally, fertilization assays confirmed the efficiency of the cryopreservation protocol in oyster sperm. These results demonstrated that different susceptibilities have been detected concerning sperm cryopreservation depending on oyster species or genetic material composition. (C) 2016 Elsevier Inc. All rights reserved.
- Molecular basis of spermatogenesis and sperm qualityPublication . Robles, Vanesa; Herraez, Paz; Labbe, Catherine; Cabrita, Elsa; Psenicka, Martin; Valcarce, David G.; F RIESCO, MARTASpermatozoan quality can be evaluated in different ways, here we focus on the analysis of DNA, RNA and epigenetic status of germ cells. These characterizations also can be the bases for explaining sperm quality at other levels, so we will see how some of these molecules could affect other sperm quality markers. Moreover, we consider the possibility of using some of these molecules as predictors of sperm quality in terms of the ability to produce healthy offspring. The relevant effect of different types of RNA molecules in germ line specification and spermatogenesis and the importance of germ cell DNA integrity and a proper epigenetic pattern will be also discussed. Although most studies at this level have been performed in mammals, some information is available for fish; these recent discoveries in fish models are included. We provide a general overview on how these molecules could have a deep influence in the final sperm quality.(C) 2016 Elsevier Inc. All rights reserved.
- Comparative study on cellular and molecular responses in oyster sperm revealed different susceptibilities to cryopreservationPublication . F RIESCO, MARTA; Félix, Francisca; Matias, Domitilia; Joaquim, Sandra; Suquet, Marc; Cabrita, ElsaThe Portuguese (Crassostrea angulata) and Pacific oyster (Crassostrea gigas), both from the genus Crassostrea, are two important species for production and conservation. Although they have common characteristics, different susceptibilities to rearing conditions have been described in these species. Overall, in the case of C. angulata, only a few remaining populations are present in the south of Portugal and Spain. The preservation of genetic material from improved stocks or from the original population is crucial in oysters to prevent the potential impacts of epidemic diseases and natural disasters. Sperm cryopreservation in oysters has progressed in recent years. However many issues, such as protocol standardization, are still unsolved for the application of research results. In the present study a sperm cryopreservation protocol, previously published in C. angulata by our group, were analyzed in terms of cellular and molecular damage, in an effort to determine the most sensible parameters to standardize the cryopreservation protocols for both species. Different approaches in the analysis of sperm quality were performed for the first time in this genus to detect different susceptibilities between the two valuable species. Our results revealed that our previously published protocol containing 10% DMSO as cryoprotectant is more suitable comparing to 10% EG in both species. In addition, an integrative analysis was performed in both oyster species comparing all cellular parameters and C. gigas showed a higher susceptibility to cryopreservation using this optimized protocol. Moreover, higher susceptibility to transcript degradation was detected in C. gigas using this optimized cryopreservation protocol. This study highlights the importance of using different techniques and exhaustive analyses for selecting the most suitable cryopreservation protocol and its standardization, thus ensuring the total safety of the technique.