Browsing by Author "Almeida, Maria Mafalda Amaral Fonseca de"
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- Methodological approaches for spermatogonia population enrichment and effect of cryopreservation on Senegalese sole spermatogonia qualityPublication . Almeida, Maria Mafalda Amaral Fonseca de; Cabrita, Elsa; Fatsini, ElviraSenegalese sole (Solea senegalensis) is an emerging species for aquaculture that shows reproductive issues in aquaculture conditions. The transplantation of cryopreserved spermatogonia into closely related species allows the production of surrogated broodstocks. However, the cryopreservation of spermatogonia might cause damage on the quality of spermatogonia, meaning quality assays must be performed. For these assays, it is important to perform cell enrichment protocols to achieve only pure spermatogonia population. Therefore, the aim of this project was to develop an efficient protocol for Senegalese sole spermatogonia enrichment and observe how the cryopreservation protocol affects these germ cells. For this purpose, two experiments were performed using fresh and cryopreserved using dimethyl sulphoxide (DMSO) as cryoprotectant solution. In experiment 1, cells were cultivated in several extracellular matrixes (uncoated, collagen, gelatine and laminin) to perform differential plating for cell enrichment. The results showed that gelatine and collagen plates showed higher spermatogonia presence, however due to the low percentage of spermatogonia recovery obtained this technique was considered unfit for the target species. Afterwards, serial strainers were used to observe that the appropriate size was the 5 μm strainer, which contained higher percentage of spermatogonia recovery. Then, two 5 μm strainers were used and showed slightly better and promising results. This method was validated by RT- PCR where three relevant spermatogonia markers (gfra1, pou5f1/oct4 and nanos2) were up regulated in the samples from the strainers, yet no significance difference was observed. In experiment 2, effect of cryopreservation on the quality of spermatogonia was evaluated where the results showed that viability, DNA integrity and epigenetic were affected by cryopreservation. No difference in lipid peroxidation was observed. Future studies should focus on the limitations of the enrichment techniques to improve this method and improving the cryopreservation protocol to decrease the damaged suffered by cells during this technique.