New paper accepted in PlosOne

A new paper in collaboration with our colleagues from University of South Bohemia in Česke Budejovice, Faculty of Fisheries and Protection of Waters (Czech Republic) has been accepted for publication in PlosOne.

Franěk R., Marinović Z., Lujić J., Urbányi B., Fučíková M., Kašpar V., Pšenička M., Horváth Á. 2019. Cryopreservation and transplantation of common carp spermatogonia. PLoS One. 14(4):e0205481. DOI: 10.1371/journal.pone.0205481

Abstract

Common carp (Cyprinus carpio) is one of the most cultured fish species over the world with many different breeds and plenty of published protocols for sperm cryopreservation. However, data regarding preservation of gonadal tissue and surrogate production is still missing. A protocol for freezing common carp spermatogonia was developed through varying different factors along a set of serial subsequent experiments. Among the six cryoprotectants tested, the best survival was achieved with dimethyl sulfoxide (Me2SO). In the next experiment, a wide range of cooling rates (0.5–10°C/min) and different concentrations of Me2SO were tested resulting in the highest survival achieved using 2 M Me2SO and cooling rate of -1°C/min. When testing different tissue sizes and incubation times in the cryomedia, the highest viability was observed when incubating 100 mg tissue fragments for 30 min. Finally, sugar supplementation did not yield significant differences. When testing different equilibration (ES) and vitrification solutions (VS) used for needle-immersed vitrification, no significant differences were observed between the tested groups. Additionally, varied exposure time to VS did not improve the vitrification outcome where the viability was 4-fold lower than that of freezing. The functionality of cryopreserved cells was tested by interspecific transplantation into sterilized goldfish recipients. The exogenous origin of the germ cells in gonads of goldfish recipient was confirmed by molecular markers and incorporation rate was over 40% at 3 months post-transplantation. Results of this study can serve for long-term preservation of germplasm in carp which can be recovered in a surrogate recipient.

New paper accepted in Cyobiology

A new paper in collaboration with our colleagues from University of South Bohemia in Česke Budejovice, Faculty of Fisheries and Protection of Waters (Czech Republic) has been accepted for publication in Cryobiology.

 

Roman Franěk, Tomáš Tichopád, Christoph Steinbach, Xuan Xie, Jelena Lujić, Zoran Marinović, Ákos Horváth, Vojtěch Kašpar, Martin Pšenička. Preservation of female genetic resources of common carp through oogonial stem cell manipulation. Cryobiology. DOI:10.1016/j.cryobiol.2019.01.016

Abstract

Several experiments were conducted in order to develop an optimal protocol for slow-rate freezing (-1°C/min) and short-term storage (-80 or 4 °C) of common carp oogonial stem cells (OSCs). Dimethyl sulfoxide with concentration of 1.5 M was identified as the best cryoprotectant in comparison to propylene glycol and methanol. When comparing supplementation of sugars (glucose, trehalose, sucrose) in different concentrations (0.1, 0.3, 0.5M), glucose and trehalose in 0.3 M were identified as optimal. Finally, different equilibration times revealed a positive impact of prolonged equilibration on post-thaw viability. Short-term storage options for ovarian tissue pieces at -80 °C and 4 °C were tested as alternatives to cryopreservation and storage in liquid nitrogen. The presence of oogonia was confirmed by immunocytochemistry and viability after storage was determined by the trypan blue exclusion test. This study identified the best protocol for OSC cryopreservation using slow rate freezing resulting in ~65% viability. The frozen/thawed OSCs were labelled by PKH-26 and transplanted into goldfish recipients. The success of the transplantation was confirmed by presence of fluorescent cells in the recipient gonad and later on by RT-PCR with carp vasa specific primers.