Toward surrogate-based conservation: Preservation of European eel (Anguilla anguilla) spermatogonial stem cells.

In this study, we optimized cryopreservation and hypothermic storage protocols for the European eel (Anguilla anguilla) spermatogonial stem cells (SSCs). As the European eel is listed as a critically endangered fish species on the IUCN red list, there is a strong need to develop and advance ex situ conservation strategies beyond the current conservation measures. Cryopreservation of SSCs was done through freezing and vitrification of testicular tissue pieces. Freezing was optimized through five sequential experiments and has yielded SSC post-thaw viability above 50%. The physiological competence of frozen/thawed cells was tested by transplanting donor-derived testicular cells into sterilized common carp larvae. After two months, gonads of 32% of the recipients displayed a positive fluorescent signal indicating that frozen/thawed cells retained physiological competence. Needle-immersed vitrification was optimized by testing different equilibration and vitrification solutions. SSCs displayed viability rates above 70% after protocol optimization. Hypothermic storage experiments displayed that storage of isolated testicular cells is more favorable than storing testicular tissue pieces, and that there was no significant reduction of SSC viability for 6 days when storing them in testicular cell suspension. Results obtained in this study are the first validated protocols for SSC preservation in the European eel and present the foundation for biobanking and future development of surrogate-based ex situ conservation programs for this and phylogenetically-related eel species.