Many small, Australian, freshwater fish species are at risk of extinction within the next 20 years, meaning immediate and targeted efforts for conservation are required. An emerging tool in conservation is the cryopreservation and storage of reproductive cells and tissues in facilities known as “frozen zoos”. This approach creates a cellular “back-up” and could play a critical role in future conservation efforts if a species were to go extinct. Unfortunately, the cryopreservation of fish oocytes and embryos has faced significant challenges, limiting the use of gamete cryopreservation in threatened fish species. One alternative is the cryopreservation of gonadal tissue which contains early germ line cells, the spermatogonia and oogonia.
We describe the successful cryopreservation of testicular and ovarian tissue in Melanotaenia fluviatilis, from the family Melanotaeniidae (order: Atheriniformes) which contains several species listed as endangered or critically endangered on the IUCN Red List. Post-thaw viability of cells from cryopreserved gonadal tissue was comparative to fresh controls in both testis and ovarian tissue, with viabilities of 72.6%±10.5% and 63.5%±18.2%, respectively. Additional comparative experiments were conducted in M. australis which indicated our cryopreservation protocol could be applied across the Melanotaenia genus. Using flow cytometry, we also optimised a method to produce enriched samples of spermatogonia and oogonia from gonadal tissue. The isolation of gonial cells is critical for future downstream methods used to produce gametes from these cells such as cell surrogacy or in vitro differentiation.
We present the first protocol for the biobanking of reproductive tissue from members of Melanotaeniidae, and the order Atheriniformes, and in doing so, provide a framework for further investigation into the use of biobanking and assisted reproduction in the conservation of rainbowfish, and other Australian fish species.