Abstract
Globally, fish populations are in decline from overfishing, habitat destruction and poor water quality. Recent mass fish deaths in Australia’s Murray–Darling Basin highlight the need for improved conservation methods for endangered fish species. Cryopreservation of testicular tissue allows storage of early sperm precursor cells for use in generating new individuals via surrogacy. We describe successful isolation and cryopreservation of spermatogonia in an Australian rainbowfish. Testis histology showed rainbowfish spermatogonia are large (> 10 μm) and stain positive for Vasa, an early germ line-specific protein. Using size-based flow cytometry, testis cell suspensions were sorted through “A” (> 9 μm) and “B” gates (2–5 μm); the A gate produced significantly more Vasa-positive cells (45.0% ± 15.2%) than the “B” gate (0.0% ± 0.0%) and an unsorted control (22.9% ± 9.5%, p < 0.0001). The most successful cryoprotectant for “large cell” (> 9 μm) viability (72.6% ± 10.5%) comprised 1.3 M DMSO, 0.1 M trehalose and 1.5% BSA; cell viability was similar to fresh controls (78.8% ± 10.5%) and significantly better than other cryoprotectants (p < 0.0006). We have developed a protocol to cryopreserve rainbowfish testicular tissue and recover an enriched population of viable spermatogonia. This is the first step in developing a biobank of reproductive tissues for this family, and other Australian fish species, in the Australian Frozen Zoo.
Highlights
Total biomass of global marine environments is reported to have declined by 49% between 1970 and 2012 mostly due to continued degradation of marine ecosystems and the impacts of overfishing[1]
Melanotaenia fluviatilis has paired, partially-fused testes attached to the dorsal body wall that converge to form a single opening at the urogenital pore
In other fish species, spermatogonia typically appear as large, lightly-stained cells, while the later spermatogenic stages appear as smaller cells with more tightly packed chromatin and darker nuclear staining[24]
Summary
Total biomass of global marine environments is reported to have declined by 49% between 1970 and 2012 mostly due to continued degradation of marine ecosystems and the impacts of overfishing[1]. Using the research of Lee et al.[14,15] and Hagedorn et al.[16] as a starting point for our experimental design, we have developed a cryopreservation protocol for testis tissue from M.fluviatilis that produces viable spermatogonial cells post-thaw To our knowledge, this is the first time cryopreservation of gonadal tissue has been attempted in an Australian fish species as well as within the order of Atheriniformes which comprises silver-sides and rainbowfishes distributed across the globe. We have validated cell sorting methods previously described by Hagedorn et al[18], to provide an optimised method for the cryopreservation and isolation of an enriched population of viable, target germ cells from the testis of M.fluviatilis for use in future applications, such as cell culture or germ cell transplantation We see this as a vital first step towards the expansion of biobanking programs for Australian fish species
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