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Rose Upton, Simon Clulow, Kim Colyvas, Michael Mahony, and John Clulow

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.


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.

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Brett Nixon, Katherine A Ewen, Klara M Krivanek, John Clulow, Grahame Kidd, Heath Ecroyd, and Russell C Jones

The role of the avian epididymis in post-testicular development and capacitation was examined to assess whether avian spermatozoa undergo any processes similar to those characteristic of mammalian sperm development. We found no evidence of a need for quail sperm to undergo capacitation and 90% of testicular sperm could bind to a perivitelline membrane and acrosome react. However, computer-assisted sperm analysis showed that 20% of testicular sperm from the quail were capable of movement and only about 12% of the motile sperm would have a curvilinear velocity greater than the mean for sperm from the distal epididymis. Nevertheless, epididymal transit was associated with increases in mean sperm velocity and the proportion of motile sperm. Together, these findings explain why earlier workers have achieved some fertilizations following inseminations of testicular spermatozoa and also demonstrate the need for some epididymal maturation of avian spermatozoa. Analysis of the electrophoretic profile of quail epididymal luminal proteins revealed that only one major protein (∼16 kDa) is secreted by the epididymis and it was virtually the only protein secreted by the ipsilateral epididymis following unilateral orchidectomy. Mass spectrometry showed that this protein is hemoglobin; this finding was confirmed using anti-hemoglobin antibodies. It is suggested that hemoglobin may support sperm metabolism in the quail epididymis, aid in motility, and/or serve as an antioxidant.