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Dimethylacetamide can be used as an alternative to glycerol for the successful cryopreservation of koala (Phascolarctos cinereus) spermatozoa

Yeng Peng Zee

G, William V. Holt

Jaime Gosalvez

Camryn D. Allen

Vere Nicolson

Michael Pyne

Michelle Burridge

Frank N. Carrick

Stephen D. Johnston

The University of Queensland, Gatton, Qld 4343, Australia. Institute of Zoology,The Zoological Society of London, Regent’s Park, London, NW1 4RY, UK. Universidad Autónoma de Madrid, 20849 Madrid, Spain. Dreamworld, Coomera, Qld 4209, Australia. Currumbin Wildlife Sanctuary, Currumbin, Qld 4223, Australia. Koala Study Program, CMLR,The University of Queensland, St Lucia, Qld 4072, Australia. Corresponding author. Email:

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 andmitochondrialmembranepotential(MMP)revealedthatprotocolsusing15%DMAachieved62.2±3.6%(P <0.05) spermsurvival,ofwhich79%(P <0.05)hadhighMMP,animprovementof32%and40%,respectively,overspermfrozen 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 2h incubation at 35◦C (35.8±4.4%; P <0.05). A second studywasconductedtodeterminetheoptimalconcentrationofDMAforuseinthecryopreservationofkoalaspermatozoa. HighDMAconcentrations(17.5%and20%)resultedinsignificantlylowerproportionsoflivespermatozoashowinghigh MMPimmediatelyafterthawingcomparedwithspermatozoafrozeninthelowerconcentrations.Thepercentageofkoala spermatozoa with swollen chromatin following cryopreservation was not affected by DMA concentration.