Brief Communication Cryopreservation of kangaroo spermatozoa using alternative approaches that reduce cytotoxic exposure to glycerol q Rhett McClean a, * , Yeng Peng Zee a , William V. Holt b , Stephen D. Johnston a a School of Animal Studies, The University of Queensland, Gatton Q 4343, Australia b Institute of Zoology, Zoological Society of London, Regent’s Park London NW1 4RY, UK article info Article history: Received 24 April 2008 Accepted 28 August 2008 Available online 11 September 2008 Keywords: Kangaroo Cauda epididymidal spermatozoa Glycerol Dimethylsulphoxide (DMSO) Dimethylacetamide (DMA) Immediate dilution post-thaw Vitrification abstract Alternative techniques for the cryopreservation of kangaroo spermatozoa that reduced or eliminated the need for glycerol were investigated including; (1) freezing spermatozoa with 20% glycerol in pre-pack- aged 0.25 mL Cassou straws to enable rapid dilution of the glycerol post-thaw, (2) investigating the effi- cacy of 20% (v/v) dimethyl sulphoxide (DMSO) and dimethylacetamide (DMA—10%, 15% and 20% v/v) as cryoprotectants and (3) vitrification of spermatozoa with or without cryoprotectant (20% v/v glycerol, 20% v/v DMSO and 20% v/v DMA). Immediate in-straw post-thaw dilution of 20% glycerol and cryopres- ervation of spermatozoa in 20% DMSO produced no significant improvement in post-thaw viability of kangaroo spermatozoa. Spermatozoa frozen in 20% DMA showed post-thaw motility and plasma mem- brane integrity of 12.7 ± 1.9% and 22.7 ± 5.4%, respectively, while kangaroo spermatozoa frozen by ultra-rapid freezing techniques showed no evidence of post-thaw viability. The use of 10–20% DMA rep- resents a modest but significant improvement in the development of a sperm cryopreservation procedure for kangaroos. Ó 2008 Published by Elsevier Inc. Despite significant recent advances in understanding those fac- tors that contribute to the cryopathology of kangaroo spermatozoa [7,8] there has been little progress in the development of even a moderately reliable cryopreservation protocol. Probably the most significant finding to date, has been the discovery that glycerol, the chemical used to protect the kangaroo sperm cell during cryo- preservation, is paradoxically also extremely toxic to the same cell at body temperature, both in terms of its effect on sperm metabo- lism (motility and mitochondrial function) [6] and impact on ultra- structural integrity [8]. It is now clear that if cryopreservation of kangaroo spermatozoa is going to be successful then new alternative strategies will need to be employed to counter the detrimental cytotoxic effects of glycerol. The first alternative cryopreservation procedure to be used in this study involved the rapid dilution of glycerol immedi- ately on thawing the semen sample to reduce the sperm cell’s exposure to a high glycerol concentration. This was achieved through the specialised packaging of chilled diluted spermatozoa into plastic Cassou straws surrounded by washing media. Immedi- ately upon thawing, the straw was vigorously flicked so that media columns holding the spermatozoa and washing media were thor- oughly mixed and the glycerated sample diluted. Despite the pos- sibility of osmotic injury, it was anticipated that removal of glycerol immediately upon thawing, might help to improve post- thaw motility and plasma membrane integrity. The current study also explored the cryopreservation ability of dimethylsulphoxide (DMSO) and dimethylamide (DMA) across a range of cryoprotectant concentrations and compared these to a standard 20% glycerol based protocol. McClean et al. [6] have re- cently shown that 20% DMSO was substantially less damaging to the physiology and ultrastructure of kangaroo spermatozoa than that of 20% glycerol or a combination of 10% glycerol + 10% DMSO. Recent cryopreservation studies on koala spermatozoa have re- vealed that DMA is at least equal to glycerol with respect to its ability to maintain post-thaw viability [11] and therefore an obvi- ous alternative cryoprotectant choice for kangaroo spermatozoa. We hypothesised that the use of alternative cryoprotectants such as DMSO and DMA would improve kangaroo sperm post-thaw motility and plasma membrane integrity. The final cryopreservation technique to be tested in this study involved the ultra-rapid freezing of small volumes of semen in what can be defined as a modified vitrification procedure. Isa- chenoko et al. [4] have argued that as spermatozoa are small cells, they can be held in microlitre volumes which should be capable of being frozen very rapidly in a glass-like phase without the need for cryoprotectant. While this specific technique is obviously re- stricted to small volumes of semen and is therefore not of practical significance for the purposes of convention artificial insemination, 0011-2240/$ - see front matter Ó 2008 Published by Elsevier Inc. doi:10.1016/j.cryobiol.2008.08.007 * Corresponding author. E-mail address: rhettmcclean@gmail.com (R. McClean). Cryobiology 57 (2008) 304–307 Contents lists available at ScienceDirect Cryobiology journal homepage: www.elsevier.com/locate/ycryo