School of Animal Biology, Faculty of Natural and Agricultural Sciences, the University of Western Australia, Crawley, WA, Australia.
Theriogenology. 2012 Oct 15;78(7):1557-69. doi: 10.1016/j.theriogenology.2012.06.025.
Three experiments were conducted to optimize the protocol for cryopreservation of emu sperm. Ejaculates were collected from trained male emus then diluted 1:1 and pooled before allocation to treatments and measured for sperm viability, motility, egg membrane penetration ability, membrane stability, and morphology. In Experiment 1, semen was either cooled to 5 °C after dilution or diluted with a precooled to 5 °C diluent before cooling to 5 °C and then frozen at liquid nitrogen vapor temperatures of -140 °C and -35 °C, with 6% or 9% dimethylacetmide (DMA; a permeating cryoprotectant) and compared for sperm functions. The percentages of viable (42.8 ± 1.1%), normal (39.0 ± 1.3%), and motile (29.8 ± 1.3%) sperm were higher (P < 0.001) for semen frozen at -14 °C with 9% DMA (path 2) than for all other combinations. In Experiment 2, we assessed the value of combining DMA and trehalose in the diluent. Combining trehalose (3% to 9%) with DMA (3% to 9%) prior to freezing reduced (P < 0.001) the percentages of postthaw viable (by 4 to 9 ± 1.2%), normal (by 5 to 11 ± 1.3%), and motile sperm (by 13 to 17 ± 2.5%) and the number of holes on the perivitelline layer (by 27 to 29 holes/mm(2)). Postthaw function was best preserved with 9% DMA alone. In experiment 3, we investigated the possibility of increasing DMA concentrations from 6% to 24%. Postthaw sperm viability (52 to 55 ± 2.3%) and morphology (48 to 51 ± 1.7%) were higher (P < 0.05) with 18% and 24% than with 6% to 12% DMA and did not differ between 18% and 24% DMA. However, sperm motility (36 to 43 ± 2.9%) and the number of perivitelline holes were similar (P > 0.05) for 9% to 18% DMA (36 to 55 ± 12%). We concluded that adding 6% to 9% trehalose to the diluent offered no advantage, and that the current best practice for preserving postthaw function in emu sperm is to dilute semen with a precooled to 5 °C diluent and use 18% DMA.
进行了三项实验以优化鸸鹋精子的冷冻保存方案。从经过训练的雄性鸸鹋中采集精液，然后稀释 1:1 并混合，然后分配到处理组中，测量精子的活力、运动能力、卵膜穿透能力、膜稳定性和形态。在实验 1 中，精液在稀释后要么冷却至 5°C，要么用预冷却至 5°C 的稀释剂稀释，然后冷却至 5°C，然后在液氮蒸气温度为-140°C 和-35°C 下冷冻，使用 6%或 9%二甲基乙酰胺（DMA；一种渗透保护剂）进行比较，以评估精子功能。与所有其他组合相比，用 9% DMA（路径 2）冷冻至-14°C 的精子活力（42.8±1.1%）、正常（39.0±1.3%）和运动（29.8±1.3%）更高（P<0.001）。在实验 2 中，我们评估了在稀释剂中组合使用 DMA 和海藻糖的价值。在冷冻前将海藻糖（3%至 9%）与 DMA（3%至 9%）组合使用，可降低（P<0.001）解冻后精子活力（降低 4%至 9±1.2%）、正常（降低 5%至 11±1.3%）和运动（降低 13%至 17±2.5%）以及卵周层上的孔数（降低 27%至 29 个孔/mm²）。单独使用 9% DMA 可更好地保存解冻后的精子功能。在实验 3 中，我们研究了将 DMA 浓度从 6%增加到 24%的可能性。解冻后精子活力（52 至 55±2.3%）和形态（48 至 51±1.7%）分别高于 18%和 24%（P<0.05），而 6%至 12% DMA 之间的差异不显著，18%和 24% DMA 之间也没有差异。然而，精子运动能力（36 至 43±2.9%）和卵周层孔数（P>0.05）在 9%至 18% DMA 之间相似（36 至 55±12%）。我们得出的结论是，在稀释剂中添加 6%至 9%的海藻糖没有优势，目前保存鸸鹋精子解冻后功能的最佳实践是用预冷却至 5°C 的稀释剂稀释精液，并使用 18% DMA。
