Declining fish populations highlight the importance of techniques that safeguard fish species against extinction. Biobanks or “frozen zoos” store cryopreserved cells from endangered species in the hope of generating new individuals in the future. However, cryopreservation of fish gametes and embryos has been challenging leaving endangered fish species without a backup plan in the event of extinction. Current research in fish now turns to germline progenitor cells: the spermatogonia and oogonia located in the gonadal tissue. We present a cryopreservation protocol for testes from the rare Murray River Rainbowfish (Melanotaenia fluviatilis).
Testes were cryopreserved in one of the following permeating cryoprotectants- DMSO, EG, methanol or glycerol at 1.3M with 0.1M trehalose in 1.5% BSA and mixed salt solution (296mOsm, pH=7.4). Viability of cells in post-thaw testis suspensions was determined using SYBR14/Propidium Iodide (PI) staining and flow cytometry. To isolate and assess spermatogonia from cell suspensions, size-specific beads were used to gate spermatogonia-sized cells, as determined from histological assessment of the testes. Analysis using one-way ANOVA and Tukey’s post hoc test showed 1.3M DMSO was the most effective permeating cryoprotectant, yielding the highest post-thaw viability at 72.6% ± 10.48%, significantly higher (p<0.05) than the next best-performing cryoprotectant, 1.3M EG (35.53% ± 2.37%), and cryoprotectant-free negative control (10.68% ± 7.09%). Changes to DMSO concentration (1.0M, 1.6M, and 2.0M) showed no significant difference in viability between DMSO cryopreserved tissue and fresh samples (78.76% ± 7.65).
Recovery of viable cells from cryopreserved gonadal tissue is the first step to developing new management and conservation options for vulnerable fish species. Future studies aim to generate offspring from cryopreserved tissue via a surrogate using germ cell transplantation techniques.