A practical technique for enzymatic dissociation and isolation of pikeperch (Sander lucioperca) (Percidae, Teleostei) early stage germ cells (eGC), including spermatogonia and spermatocytes, is reported in this study. Their potential to differentiate into functional gametes, and transmit genetic information to the next generations makes them suitable for cryopreservation and surrogate reproduction studies by germline chimera. Two different age groups (14 month old and 18 month old) of pikeperch were used to adjust the correct stage of eGC isolation. Finally the 18 month old samples were selected due to their high average gonadal volume (0.513 g). 10 ml PBS + 0.3% trypsin (304 mOsm/kg, pH 8) were used for enzymatic dissociation of testicular cells and they were sorted by centrifugation in Percoll density gradient. eGCs were identified on the basis of their ploidy level by CYSTAIN DNA 1 steps kit (PARTEC) and morphological characteristics trough by light microscopy. Cell counting was performed on histological sections and Percoll gradient layers whit the method of random square counting. The method of isolation enriched the number of eGC from 41.3% to 84.7%, obtained from the 33% of Percoll concentration.
| Identifer | oai:union.ndltd.org:nusl.cz/oai:invenio.nusl.cz:188876 |
| Date | January 2015 |
| Creators | GÜNGÖR, Ege |
| Source Sets | Czech ETDs |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/masterThesis |
| Rights | info:eu-repo/semantics/restrictedAccess |
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