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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 µm ± 1.75 µm 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 × 10⁸ ± 4.4 × 10⁷ 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. Full article

Rainbowfishes (Melanotaeniidae) are the largest monophyletic group of freshwater fishes occurring in Australia and New Guinea, with 112 species currently recognised. Despite their high taxonomic diversity, rainbowfishes remain poorly studied from a cytogenetic perspective. Using conventional (Giemsa staining, C banding, chromomycin A₃ staining) and molecular (fluorescence in situ hybridisation with ribosomal DNA (rDNA) and telomeric probes) cytogenetic protocols, karyotypes and associated chromosomal characteristics of five species were examined. We covered all major lineages of this group, namely, Running River rainbowfish Melanotaenia sp., red rainbowfish Glossolepis incisus, threadfin rainbowfish Iriatherina werneri, ornate rainbowfish Rhadinocentrus ornatus, and Cairns rainbowfish Cairnsichthys rhombosomoides. All species had conserved diploid chromosome numbers 2n = 48, but karyotypes differed among species; while Melanotaenia sp., G. incisus, and I. werneri possessed karyotypes composed of exclusively subtelo/acrocentric chromosomes, the karyotype of R. ornatus displayed six pairs of submetacentric and 18 pairs of subtelo/acrocentric chromosomes, while C. rhombosomoides possessed a karyotype composed of four pairs of submetacentric and 20 pairs of subtelo/acrocentric chromosomes. No heteromorphic sex chromosomes were detected using conventional cytogenetic techniques. Our data indicate a conserved 2n in Melanotaeniidae, but morphologically variable karyotypes, rDNA sites, and heterochromatin distributions. Differences were observed especially in taxonomically divergent species, suggesting interspecies chromosome rearrangements. Full article