Dimethylacetamide can be used as an alternative to glycerol for the successful cryopreservation of koala (Phascolarctos cinereus) spermatozoa
Yeng Peng Zee A G , William V. Holt B , Jaime Gosalvez C , Camryn D. Allen A , Vere Nicolson D , Michael Pyne E , Michelle Burridge D , Frank N. Carrick F and Stephen D. Johnston AA The University of Queensland, Gatton, Qld 4343, Australia.
B Institute of Zoology, The Zoological Society of London, Regent’s Park, London, NW1 4RY, UK.
C Universidad Autónoma de Madrid, 20849 Madrid, Spain.
D Dreamworld, Coomera, Qld 4209, Australia.
E Currumbin Wildlife Sanctuary, Currumbin, Qld 4223, Australia.
F Koala Study Program, CMLR, The University of Queensland, St Lucia, Qld 4072, Australia.
G Corresponding author. Email: y.zee@uq.edu.au
Reproduction, Fertility and Development 20(6) 724-733 https://doi.org/10.1071/RD08036
Submitted: 2 March 2008 Accepted: 28 May 2008 Published: 9 July 2008
Abstract
Swelling of koala sperm chromatin following cryopreservation has largely been attributed to the absence of intermolecular disulfide cross-linkages in the marsupial sperm nucleus. Fish spermatozoa also lack disulfide bonds within their chromatin, but have been successfully cryopreserved. The present study examined the hypothesis that the cryoprotectants used for fish sperm cryopreservation would confer a similar degree of protection on koala spermatozoa. Three concentrations each of five cryoprotectants (dimethyl sulfoxide, methanol, propylene glycol, ethylene glycol and dimethylacetamide (DMA)) were evaluated. Each treatment was compared against an established koala sperm cryopreservation protocol that uses 14% glycerol. Post-thaw assessment of progressive motility, plasma membrane integrity and mitochondrial membrane potential (MMP) revealed that protocols using 15% DMA achieved 62.2 ± 3.6% (P < 0.05) sperm survival, of which 79% (P < 0.05) had high MMP, an improvement of 32% and 40%, respectively, over sperm frozen in 14% glycerol. The percentage of spermatozoa with swollen nuclei was also lowest when frozen in 15% DMA, both immediately after thawing (18.0 ± 3.5%; P < 0.05) and after 2 h incubation at 35°C (35.8 ± 4.4%; P < 0.05). A second study was conducted to determine the optimal concentration of DMA for use in the cryopreservation of koala spermatozoa. High DMA concentrations (17.5% and 20%) resulted in significantly lower proportions of live spermatozoa showing high MMP immediately after thawing compared with spermatozoa frozen in the lower concentrations. The percentage of koala spermatozoa with swollen chromatin following cryopreservation was not affected by DMA concentration.
Additional keywords: amides, JC-1, mitochondrial function.
Acknowledgement
The present study was financially supported by the Australian Research Council Linkage Grant Scheme (LP0455785).
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