Fish Physiology and Biochemistry

, Volume 44, Issue 6, pp 1499–1507 | Cite as

Development of sperm vitrification protocols for two endangered salmonid species: the Adriatic grayling, Thymallus thymallus, and the marble trout, Salmo marmoratus

  • Eszter KásaEmail author
  • Jelena Lujić
  • Zoran Marinović
  • Tímea Kollár
  • Gergely Bernáth
  • Zoltán Bokor
  • Béla Urbányi
  • Kinga Katalin Lefler
  • Dušan Jesenšek
  • Ákos Horváth


Vitrification was applied to the sperm of two endangered fish species of Soča River basin in Slovenia, the Adriatic grayling (Thymallus thymallus) and marble trout (Salmo marmoratus) following testing different cooling devices and vitrifying media. Sperm was collected, diluted in species-specific non-activating media containing cryoprotectants, and vitrified by plunging directly into liquid nitrogen without pre-cooling. Progressive motility, curvilinear velocity, and straightness of fresh and vitrified-warmed sperm were evaluated with computer-assisted sperm analysis (CASA). Fertilization trials were carried out to test the effectiveness of vitrification in the case of grayling. A protocol utilizing a glucose-based extender, 30% cryoprotectants (15% methanol + 15% propylene glycol), 1:1 dilution ratio, and droplets of 2 μl on a Cryotop as cooling device yielded the highest post-thaw motility values for both Adriatic grayling (7.5 ± 6.5%) and marble trout (26.6 ± 15.8%). Viable embryos were produced by fertilizing eggs with vitrified grayling sperm (hatching 13.1 ± 11.7%, control hatching 73.9 ± 10.4%). The vitrification protocol developed in this study can be utilized in the conservation efforts for the two species as an alternative to slow-rate freezing when working in field conditions or when specific equipment necessary for slow-rate freezing is not available.


Spermatozoa vitrification Ultra-rapid cooling Fish sperm cryopreservation Adriatic grayling Marble trout Gene preservation 



Assistance by the staff of the Tolminka Fish Farm of the Angling club of Tolmin in Slovenia is acknowledged.

Funding information

The work was funded by the National Research, Development and Innovation Office of Hungary (grant number K-109847), the New Hungarian National Excellence Predoctoral Fellowship (grant to EK), the Stipendium Hungaricum Scholarship Programme (grant 106360 to ZM), and the EFOP-3.6.3-VEKOP-16-2017-00008 project. The project is co-financed by the European Union and the European Social Fund.


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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Eszter Kása
    • 1
    Email author
  • Jelena Lujić
    • 1
  • Zoran Marinović
    • 1
  • Tímea Kollár
    • 1
  • Gergely Bernáth
    • 1
  • Zoltán Bokor
    • 1
  • Béla Urbányi
    • 1
  • Kinga Katalin Lefler
    • 1
  • Dušan Jesenšek
    • 2
  • Ákos Horváth
    • 1
  1. 1.Department of AquacultureSzent István UniversityGödöllőHungary
  2. 2.Ribiška družina TolminTolminSlovenia

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