Zee Yeng Peng, Holt William V, Gosalvez Jaime, Allen Camryn D, Nicolson Vere, Pyne Michael, Burridge Michelle, Carrick Frank N, Johnston Stephen D
The University of Queensland, Gatton, QLD, Australia.
Reprod Fertil Dev. 2008;20(6):724-33. doi: 10.1071/rd08036.
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 degrees 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.
考拉精子染色质在冷冻保存后发生肿胀，这在很大程度上归因于有袋类动物精子细胞核中缺乏分子间二硫键交联。鱼类精子染色质内也缺乏二硫键，但已成功实现冷冻保存。本研究检验了这样一个假设，即用于鱼类精子冷冻保存的冷冻保护剂对考拉精子也能提供相似程度的保护。评估了五种冷冻保护剂（二甲基亚砜、甲醇、丙二醇、乙二醇和二甲基乙酰胺（DMA））各自的三种浓度。将每种处理方法与使用14%甘油的既定考拉精子冷冻保存方案进行比较。解冻后对精子的渐进性运动能力、质膜完整性和线粒体膜电位（MMP）进行评估，结果显示使用15% DMA的方案实现了62.2±3.6%（P<0.05）的精子存活率，其中79%（P<0.05）具有高MMP，与用14%甘油冷冻的精子相比，分别提高了32%和40%。当在15% DMA中冷冻时，解冻后立即（18.0±3.5%；P<0.05）以及在35℃孵育2小时后（35.8±4.4%；P<0.05），细胞核肿胀的精子百分比也是最低的。进行了第二项研究以确定用于考拉精子冷冻保存的DMA的最佳浓度。与在较低浓度下冷冻的精子相比，高浓度的DMA（17.5%和20%）导致解冻后立即显示高MMP的活精子比例显著降低。冷冻保存后考拉精子染色质肿胀的百分比不受DMA浓度的影响。
