Elsevier

Cryobiology

Volume 81, April 2018, Pages 201-205
Cryobiology

Culture, cryobanking and passaging of karyotypically validated native Australian amphibian cells

https://doi.org/10.1016/j.cryobiol.2018.03.004Get rights and content

Abstract

This study describes the culture, cryobanking, thawing and passaging of karyotypically validated cells from two life history stages of the “least concern” Australian native amphibian species Litoria infrafrenata. Adult frog toe and tadpole macerates were generated from animals euthanized due to ill health following injury. Cultured cells proliferated and formed colonies after one to two weeks of culture. Cultures were cryopreserved in liquid nitrogen for a minimum of one month, thawed, passaged for expansion and karyotyped. Post-thaw karyotypes revealed the expected 2N = 24 diploid chromosome number in approximately 90% of all metaphase spreads. Further, metacentric, submetacentric and subtelocentric configurations were the same as previously described karyotype configurations obtained from living frogs of this same species. Using cryobanked and prospectively validated cell lines, conservation programs including assisted reproduction technologies and genomic, mitochondrial and proteomic mining initiatives may therefore be complemented with minimal or no disturbance to living and healthy animals.

Section snippets

Brief communication

Amphibians are experiencing a critical existence juncture, with approximately 32% of the currently recognised species under immediate threat [1]. The main driver of this crisis is human activity and the spread of chydridiomycosis [1]. Conservation programs involving habitat conservation and assisted reproduction techniques (ART) have demonstrated success in safeguarding many threatened species from imminent extinction. A greater number of complementary techniques, including cryobanking

Funding

Richard Mollard provided all funds for the materials and the execution of experimental procedures described in this study.

Conflicts of interest

The author is the owner of Amphicell Pty Ltd, an Australian native frog conservation advocacy aiming to safeguard the future of amphibian biodiversity within Australia.

Acknowledgments

The author thanks Deborah Pergolotti from Frog Safe Inc. (funded through public donations) for provision of tissues from deceased animals. The author also thanks Associate Professor Jean-Paul Scheerlinck for access to tissue culture facilities, and Dr Charlie Pagel for the use of microscopic equipment for karyotyping; both at the University of Melbourne's Department of Veterinary and Agricultural Sciences.

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  • Cryopreservation of embryos and larvae of the edible sea urchin loxechinus albus (Molina, 1782)

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    Cryopreserving gametes and larvae is a highly beneficial biotechnological tool for aquaculture for both conserving species and for increasing stock in marine environments [2,24–26,42,59] by culturing them in controlled settings. Cryopreserving larvae can provide individuals for cultivating throughout the year without the need to condition reproducers [3], as well as for genetic selection programs [4,58] for monitoring environmental quality [12,30,43], and for contributing to cryobanks [27,32,34,45,60]. There have been successful cryopreservation studies with the gametes and larvae of diverse aquatic species, mainly fish and mollusks of ecological and commercial importance [E.g Refs. [14,18,24–26,29,31,38,40,42,55]].

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