World Aquaculture 2023

May 29 - June 1, 2023

Darwin, Northern Territory, Australia


V. Kašpar*,M.Pšenicka, R. Franek


University of South Bohemia in CeskéBudejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, 389 25, Vodnany, Czech Republic, e-mail:


Surrogacy using germ stem cell transplantation into sterilized host is well suited for isogenic line production in fish or conservation of genetic resources because cryopreservation of milt has still limited use for restoration. We attempted to overcome disadvantages connected with the conventional approach for conservation of genetic resources orisogenic line production using germ cell manipulation involving cryopreservation and surrogate reproduction technology.Moreover, novel methods of uniparental inheritance induction or cultivation of germ cells in vitro were developed.

Series of experiments were performed to develop optimal procedure for cryopreservation of common carp male and female early-stage germ cells. A cryopreservation protocol for tissue of ovary using slow-rate freezing (1?°C/min) was developed using a Me2SO-based cryomedia with addition of 0.3 M trehalose. Testing of available cryoprotectants and protocols for testicular tissue resulted in the highest survival achieved using 2 M Me2SO and cooling rate of -1 °C/min. Recovery and physiological activity of cryopreserved germ cells was confirmed by transplantation into sterile goldfish when cryopreserved germ cells retained colonization rate comparable to non-cryopreserved control.Goldfish surrogates were stimulated for spawning and collected gametes were used for fertilization and genotyping using carp and goldfish specific primers.Goldfish surrogates produced viable common carp progeny as confirmed by genotyping. Both male and female gametes were obtained, even from a single double haploid donor confirming feasibility of isogenic line production.

Next aim of this study was to develop a cold shock androgenesis protocol for common by testing combination of different temperature treatments and different cold-shock durations applied shortly after gamete activation.Results of cold-shock treatment testing showed that optimal condition for egg nucleus elimination was a cold-shock at 2 °C for 60 min duration and double haploid induction performed in larger scale using 2 °C cold-shock for 60 min subsequently treated by a heat shock arresting first mitotic cleavage resulted in reduced fertilization and hatching rates for all replicates and low yield of double haploid progeny (1.09–1.28% in experimental incubation, <1% in hatchery incubation) confirmed by RADseq.

Optimization of in vitro germ cell culture condition for short term cell culture involved testing of basal mediaand different types of feeder cells withcontinual monitoring of mitotic proliferation.We found that germ cells cultured with hESC media and RTG2 cell line as feeder possessed significantly higher proliferation and survival.

We developed a widely applicable strategy for managing isogenic lines in common carp which is including production of homozygous donors, cryopreservation and in vitro culture of germ stem cells and their propagation by surrogate host.

The work was supported by National Agriculture Agency project number QK1910428 and Czech Science Foundation 22-01781O