Cryopreservation and Flow Cytometric Analysis of Ovarian Tissue in Murray River Rainbowfish, <i>Melanotaenia fluviatilis</i>
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...
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| Format: | Article |
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MDPI AG
2022-03-01
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| Series: | Animals |
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| Online Access: | https://www.mdpi.com/2076-2615/12/6/794 |
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| author | Nicola Rivers Jonathan Daly Robert Jones Peter D. Currie Peter Temple-Smith |
| author_facet | Nicola Rivers Jonathan Daly Robert Jones Peter D. Currie Peter Temple-Smith |
| author_sort | Nicola Rivers |
| collection | DOAJ |
| description | 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, <i>Melanotaenia fluviatilis</i> (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 × 10<sup>8</sup> ± 4.4 × 10<sup>7</sup> 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 (<i>p</i> = 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%; <i>p</i> = 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. |
| first_indexed | 2024-03-09T20:12:48Z |
| format | Article |
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| issn | 2076-2615 |
| language | English |
| last_indexed | 2024-03-09T20:12:48Z |
| publishDate | 2022-03-01 |
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| record_format | Article |
| series | Animals |
| spelling | doaj.art-9f175fe032534a6887ab9945df7ee3bb2023-11-24T00:10:11ZengMDPI AGAnimals2076-26152022-03-0112679410.3390/ani12060794Cryopreservation and Flow Cytometric Analysis of Ovarian Tissue in Murray River Rainbowfish, <i>Melanotaenia fluviatilis</i>Nicola Rivers0Jonathan Daly1Robert Jones2Peter D. Currie3Peter Temple-Smith4Department of Obstetrics and Gynaecology, School of Clinical Sciences, Monash University, Clayton, VIC 3168, AustraliaThe Australian Frozen Zoo, Clayton, VIC 3168, AustraliaThe Aquarium Vet, Moorabbin, VIC 3189, AustraliaAustralian Regenerative Medicine Institute, Clayton, VIC 3800, AustraliaDepartment of Obstetrics and Gynaecology, School of Clinical Sciences, Monash University, Clayton, VIC 3168, AustraliaFreshwater 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, <i>Melanotaenia fluviatilis</i> (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 × 10<sup>8</sup> ± 4.4 × 10<sup>7</sup> 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 (<i>p</i> = 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%; <i>p</i> = 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.https://www.mdpi.com/2076-2615/12/6/794fishbiobankingcryopreservationovaryconservation |
| spellingShingle | Nicola Rivers Jonathan Daly Robert Jones Peter D. Currie Peter Temple-Smith Cryopreservation and Flow Cytometric Analysis of Ovarian Tissue in Murray River Rainbowfish, <i>Melanotaenia fluviatilis</i> Animals fish biobanking cryopreservation ovary conservation |
| title | Cryopreservation and Flow Cytometric Analysis of Ovarian Tissue in Murray River Rainbowfish, <i>Melanotaenia fluviatilis</i> |
| title_full | Cryopreservation and Flow Cytometric Analysis of Ovarian Tissue in Murray River Rainbowfish, <i>Melanotaenia fluviatilis</i> |
| title_fullStr | Cryopreservation and Flow Cytometric Analysis of Ovarian Tissue in Murray River Rainbowfish, <i>Melanotaenia fluviatilis</i> |
| title_full_unstemmed | Cryopreservation and Flow Cytometric Analysis of Ovarian Tissue in Murray River Rainbowfish, <i>Melanotaenia fluviatilis</i> |
| title_short | Cryopreservation and Flow Cytometric Analysis of Ovarian Tissue in Murray River Rainbowfish, <i>Melanotaenia fluviatilis</i> |
| title_sort | cryopreservation and flow cytometric analysis of ovarian tissue in murray river rainbowfish i melanotaenia fluviatilis i |
| topic | fish biobanking cryopreservation ovary conservation |
| url | https://www.mdpi.com/2076-2615/12/6/794 |
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