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- Cryopreservation of germ cells for the production of marine speciesPublication . Cabrita, Elsa; Martinez-Paramo, Sonia; Dinis, Maria TeresaGerm cell cryopreservation is a safe method to store and preserve genetic material. Cryobanks in aquatic species were developed with different aims, benefiting fish farming, from management of reproduction to genetic selection of sperm from males with high reproductive value. Research has been conducted on the development of protocols for new/problematic species, for commercial species to improve gamete quality during storage or for conservation purposes.
- 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.
- Cryobanking of aquatic speciesPublication . Martinez-Paramo, Sonia; Horvath, Akos; Labbe, Catherine; Zhang, Tiantian; Robles, Vanesa; Herraez, Paz; Suquet, Marc; Adams, Serean; Viveiros, Ana; Tiersch, Terrence R.; Cabrita, ElsaThis review is focused on the applications of genome cryobanking of aquatic species including freshwater and marine fish, as well as invertebrates. It also reviews the latest advances in cryobanking of model species, widely used by the scientific community worldwide, because of their applications in several fields. The state of the art of cryopreservation of different cellular types (sperm, oocytes, embryos, somatic cells and primordial germ cells or early spermatogonia) is discussed focusing on the advantages and disadvantages of each procedure according to different applications. A special review on the need of standardization of protocols has also been carried out. In summary, this comprehensive review provides information on the practical details of applications of genome cryobanking in a range of aquatic species worldwide, including the cryobanks established in Europe, USA, Brazil, Australia and New Zealand, the species and type of cells that constitute these banks and the utilization of the samples preserved. Statement of relevance: This review compiles the last advances on germplasm cryobanking of freshwater and marine fish species and invertebrates, with high value for commercial aquaculture or conservation. It is reviewed the most promising cryopreservation protocols for different cell types, embryos and larvae that could be applied in programs for genetic improvement, broodstock management or conservation of stocks to guarantee culture production. (C) 2016 Elsevier B.V. All rights reserved.
- Fertilization capacity with rainbow trout DNA-damaged sperm and embryo developmental successPublication . Perez-Cerezales, S.; Martinez-Paramo, S.; Beirao, J.; Herraez, M. P.Mammalian spermatozoa undergo a strong selection process along the female tract to guarantee fertilization by good quality cells, but risks of fertilization with DNA-damaged spermatozoa have been reported. In contrast, most external fertilizers such as fish seem to have weaker selection procedures. This fact, together with their high prolificacy and external embryo development, indicates that fish could be useful for the study of the effects of sperm DNA damage on embryo development. We cryopreserved sperm from rainbow trout using egg yolk and low-density lipoprotein as additives to promote different rates of DNA damage. DNA fragmentation and oxidization were analyzed using comet assay with and without digestion with restriction enzymes, and fertilization trials were performed. Some embryo batches were treated with 3-aminobenzamide (3AB) to inhibit DNA repair by the poly (ADP-ribose) polymerase, which is an enzyme of the base excision repair pathway. Results showed that all the spermatozoa cryopreserved with egg yolk carried more than 10% fragmented DNA, maintaining fertilization rates of 61.1+/-2.3 but a high rate of abortions, especially during gastrulation, and only 14.5+/-4.4 hatching success. Furthermore, after 3AB treatment, hatching dropped to 3.2+/-2.2, showing that at least 10% DNA fragmentation was repaired. We conclude that trout sperm maintains its ability to fertilize in spite of having DNA damage, but that embryo survival is affected. Damage is partially repaired by the oocyte during the first cleavage. Important advantages of using rainbow trout for the study of processes related to DNA damage and repair during development have been reported. Reproduction (2010) 139 989-997