Cryopreservation and Flow Cytometric Analysis of Ovarian Tissue in Murray River Rainbowfish, Melanotaenia fluviatilis

Simple Summary Freshwater fish populations are in global decline, with many Australian freshwater species expected to become extinct in the next twenty years. The storage of reproductive cells and tissues at extremely cold temperatures in bio-banks known as "Frozen Zoos", allows for the indefinite storage of genetic material, meaning that in the event of an extinction, we have a genetic blueprint available to produce new individuals and reintroduce a species into the wild. Here we have developed a cryopreservation protocol for the storage of ovarian tissue from the threatened Murray River Rainbowfish. Many Australian freshwater fish species are threatened with extinction, our methodology provides a framework for the conservation of other fish species in Australia and globally. Freshwater fish populations are declining with many small, Australian fish species at risk of extinction within the next twenty-years. Cryopreservation of reproductive cells and tissues makes it possible to reproduce individuals from a species even after they are extinct in the wild. We describe the successful cryopreservation of ovarian tissue in the Murray River Rainbowfish, Melanotaenia fluviatilis (Order: Atheriniformes). Histology showed that oogonia are 13.70 mu m +/- 1.75 mu m in size, stain positive for germ-line marker Vasa, and represent approximately 2.29 +/- 0.81% of cells in the ovary. Flow cytometry was used to analyse ovarian cell suspensions, requiring an optimised tissue digestion protocol. We found that 0.25% trypsin with 1.13 mM EDTA produced cell suspensions with the highest viability (76.28 +/- 4.64%) and the highest number of cells recovered per gram of tissue (1.2 x 10(8) +/- 4.4 x 10(7) cells/g). Subsequent sorting of ovarian cell suspensions by flow cytometry increased oogonial cells in suspension from 2.53 +/- 1.31% in an unsorted sample to 5.85 +/- 4.01% in a sorted sample (p = 0.0346). Cryopreservation of ovarian tissue showed DMSO-treated samples had higher cell viability post-thaw (63.5 +/- 18.2%) which was comparable to fresh samples (82.5 +/- 7.1%; p = 0.36). Tissue cryopreserved in 2.0 M DMSO had the highest cell viability overall (76.07 +/- 3.89%). This protocol could be applied to bio-banking programs for other species in the Melanotaeniidae, and perhaps species in other families and orders of Australian fish.