Date Published: April 18, 2019
Publisher: Public Library of Science
Author(s): Roman Franěk, Zoran Marinović, Jelena Lujić, Béla Urbányi, Michaela Fučíková, Vojtěch Kašpar, Martin Pšenička, Ákos Horváth, Marc Muller.
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.
Common carp (Cyprinus carpio) is one of the oldest domesticated fish species in the world and is mainly cultured in Europe and Asia. Nowadays, the common carp expanded to all continents with exception of Antarctica. Overall carp production from aquaculture in 2016 was more than 4 million tons—9.6% of the total freshwater finfish aquaculture production in that year . Fruitful history and lasting popularity of this species gave to rise many different strains and lines which became important for breed management and production of hybrids in Central Europe [2–4]. Due to this fact, significant efforts have been committed to preservation of carp genetic resources. Long-term cultivation of pure-breed livestock , methods for genetic diversity identification [6–9] as well as methods for creating and keeping gene banks through sperm cryopreservation [10–14] have been developed. However, ex situ preservation of valuable genetic material is still based only on sperm cryopreservation.
In the present study, we have developed for the first time a cryopreservation methodology for common carp spermatogonia through freezing and vitrification of testicular tissue. Transplanted spermatogonia were physiologically active since they were able to colonize the genital ridge and renew testicular fate as well switch and develop as female germ cells in recipient’s ovary. This finding suggest that reproduction is from a single germ cell donor is self-sustaining for future reproduction of germline chimeras. Results of this study can serve as an alternative way for long-term preservation of common carp germplasm which can be recovered through inter-specific transplantation into dnd-MO sterilized goldfish recipients.
This study developed an optimal protocol for cryopreservation of carp male germ cells by freezing with subsequent restoration in goldfish as a surrogate host. Post-thaw viability of cryopreserved spermatogonia was improved over 40% through optimizing factors such as cryoprotectants, their concentrations, cooling rate, tissue size, incubation time and lastly sugar supplementation and their concentration. Importantly, our study showed that cryopreservation can be successfully performed without advanced cooling equipment when a commercially available cooling box placed in a -80°C deep freezer can be used. Incorporation rates of fresh and cryopreserved spermatogonia were similar after inter-specific transplantation into surrogate goldfish and transplanted spermatogonia developed within both ovaries and testes. The donor-derived origin was further confirmed by vasa and dnd1 gene expression in germline chimera gonads. The results could serve as an alternative strategy in breeding programs for male germplasm cryopreservation with subsequent recovery in goldfish hosts. Additionally, cryopreservation gives a possibility to synchronize and carry out transplantation according to the availability of hosts. The results of the present study can be used in combination with the hypothermic storage described by Lujić et al.  where hypothermic storage is optimal for short-term storage of up to two weeks, while the freezing methodology developed in this study is optimal for long-term storage. Further steps will be taken to develop a protocol for female germ cell cryopreservation as well to improve transplantation success using younger recipients or developing germ-less carp hosts for allogenic germ cell transplantation.