Paradigm shift in frog sperm cryopreservation: reduced role for non-penetrating cryoprotectants

in Reproduction
Authors:
Rose UptonConservation Biology Research Group, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW, Australia
FAUNA Research Alliance, PO Box 5092, Kahibah, NSW, Australia

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https://orcid.org/0000-0002-1324-6873
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Simon ClulowFAUNA Research Alliance, PO Box 5092, Kahibah, NSW, Australia
Centre for Conservation Ecology and Genomics, Institute for Applied Ecology, University of Canberra, Bruce, ACT, Australia

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Kim ColyvasCollege of Engineering, Science and Environment, The University of Newcastle, Callaghan, NSW, Australia

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Michael MahonyConservation Biology Research Group, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW, Australia
FAUNA Research Alliance, PO Box 5092, Kahibah, NSW, Australia

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, and
John ClulowConservation Biology Research Group, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW, Australia
FAUNA Research Alliance, PO Box 5092, Kahibah, NSW, Australia

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Correspondence should be addressed to R Upton; Email: rose.upton@newcastle.edu.au
DOI:
https://doi.org/10.1530/REP-22-0486
Volume/Issue:
Volume 165: Issue 6
Page Range:
583–592
Article Type:
Research Article
Online Publication Date:
26 Apr 2023
Copyright:
© Society for Reproduction and Fertility 2023
Restricted access

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In brief

Sperm cryopreservation has been recognised as a tool for preventing loss of genetic diversity in amphibians; however, the combined effect of penetrative and non-penetrative cryoprotectants in cryodiluents is poorly understood. We demonstrate a clear benefit of using low concentrations of non-penetrative cryoprotectants when cryopreserving sperm of Australian tree frogs.

Abstract

Sperm cryopreservation protocols have been developed for an increasing number of amphibian species since the recognition of a global amphibian decline. Yet, the development of these protocols has neglected to elucidate the combined effect of the penetrative(PCP) and non-penetrative cryoprotectant (NPCP) on the recovery of live, motile sperm. The two-factor hypothesis of cryoinjury recognises a trade-off between cooling cells slowly enough to allow osmotic dehydration and therefore avoid intracellular ice formation, but fast enough to minimise effects from increasing extracellular osmolality as the frozen fraction of the media increases during freezing. We tested the effect of two concentrations of a PCP (10 and 15% v/v dimethyl sulfoxide (Me2SO)) and two concentrations of an NPCP (1 and 10% w/v sucrose) in various combinations on the sperm of six pelodryadid frogs. In all species, 15% v/v Me2SO with 1% w/v sucrose provided superior post-thaw recovery with high proportions of forward progressive motility, live cells and intact acrosomes (typically >50% for each). Theoretically, it has been suggested that increased NPCP concentration should improve cell survival by increasing the rate and extent of cell dehydration. We suggest, however, that the elevated osmolality in the unfrozen water fraction when 10% sucrose is used may be causing damage to cells via excessive cell shrinkage and solute effects as proposed in the two-factor hypothesis of cryoinjury. We showed this response in sperm across a range of frog species, providing compelling evidence for this hypothesis. We suggest protocol development using the PCP/NPCP ratios demonstrated in our study will be broadly applicable to many amphibian species.

Supplementary Materials

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Full Text Views 37 37 28
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Print ISSN:
1470-1626
Online ISSN:
1741-7899

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