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

Rose Upton, Simon Clulow, university newcastle, Michael Mahony, John Clulow

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)


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 cryoprotectant (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 (Me 2SO)) 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 Me 2SO 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.

Original languageEnglish
Pages (from-to)583-592
Number of pages10
JournalJournal of Reproduction and Fertility
Issue number6
Early online date26 Apr 2023
Publication statusPublished - 2023


Dive into the research topics of 'Paradigm shift in frog sperm cryopreservation - reduced role for non-penetrating cryoprotectants'. Together they form a unique fingerprint.

Cite this