Faculty of Veterinary Science, University of Sydney, New South Wales, Australia.
Theriogenology. 2012 Jul 15;78(2):315-25. doi: 10.1016/j.theriogenology.2012.02.005. Epub 2012 Mar 22.
Effective sperm cryopreservation protocols are limited to a small number of marsupial species. In this study, postmortem gamete rescue (PMGR) epididymal sperm samples from Tasmanian devils (N=34) euthanized due to the fatal Devil Facial Tumor Disease were used to develop long-term sperm storage techniques for the species. Cryoprotectant toxicity associated with equilibration of sperm samples in a TEST yolk diluent (TEST; 189 mM N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, 85 mM Trizma base [Tris], 11 mM glucose, 20% vol/vol egg yolk; pH 7.1, and 315.0±5.0 mOsm/kg) with a final concentration of 0.06 M trehalose, or 4%, 10%, and 18% vol/vol of either glycerol or dimethyl sulfoxide (DMSO), was examined over 12 h at 15 °C. Trehalose supplementation resulted in an immediate decline (P<0.05) of total motility. After 12 h, total motility was reduced (P<0.05) in treatments containing 18% glycerol, and 10% and 18% dimethyl sulfoxide. The effects of final glycerol concentration (4% and 10%), glycerol equilibration duration (10 min 1 h, or 3 h) prefreeze, freezing rate and the addition of 0.10 M lactose or a combination of 0.10 M lactose and 0.11 M raffinose were assessed during three experiments on the cryopreservation of postmortem gamete rescue samples in TEST. In all experiments, motility and viability were reduced (P<0.01 postthaw). Samples cryopreserved in TEST supplemented with lactose or lactose with raffinose using a fast freezing rate (-8 °C/min from 4 to -40 °C, then -65 °C/min until -165 °C) produced the highest (P<0.05) postthaw motility (18.6±5.5% and 16.9±8.5%, respectively), which represented 35% to 48% retention of prefreeze motility. These results apparently were the best postthaw results of dasyurid sperm reported to date and will help lay the foundations for developing assisted reproductive technologies for marsupial species.
有效的精子冷冻保存方案仅限于少数有袋目物种。在这项研究中，使用了因致命的袋獾面部肿瘤病而安乐死的袋獾（N=34）死后配子抢救（PMGR）附睾精子样本，为该物种开发长期精子储存技术。在 15°C 下，用 0.06M 海藻糖或 4%、10%和 18%的甘油或二甲基亚砜（DMSO）平衡精子样品 12 小时，研究了与 TEST 卵黄稀释液（TEST；189mM N-三（羟甲基）甲基-2-氨基乙磺酸、85mM 三羟甲基氨基甲烷[Tris]、11mM 葡萄糖、20%体积/体积卵黄；pH7.1 和 315.0±5.0mOsm/kg）中的平衡相关的精子保护剂毒性。海藻糖补充剂导致总活力立即下降（P<0.05）。12 小时后，含 18%甘油、10%和 18%二甲基亚砜的处理组总活力降低（P<0.05）。在三个实验中评估了最终甘油浓度（4%和 10%）、甘油平衡时间（10 分钟、1 小时或 3 小时）、预冷冻时的冷冻速度以及添加 0.10M 乳糖或 0.10M 乳糖和 0.11M 棉子糖的组合对 TEST 中死后配子抢救样品冷冻保存的影响。在所有实验中，解冻后活力和活力均降低（P<0.01）。使用快速冷冻速度（4 至-40°C 时为-8°C/min，然后为-65°C/min 直至-165°C），在补充有乳糖或乳糖和棉子糖的 TEST 中冷冻保存的样品产生了最高的解冻后活力（分别为 18.6±5.5%和 16.9±8.5%），这代表冷冻前活力的 35%至 48%的保留。这些结果显然是迄今为止报道的袋鼬精子解冻后效果最好的结果，这将有助于为有袋目物种开发辅助生殖技术奠定基础。
