Department of Agricultural, Environmental and Food Sciences, University of Molise, Campobasso, Italy.
Theriogenology. 2012 Oct 1;78(6):1381-9. doi: 10.1016/j.theriogenology.2012.06.009. Epub 2012 Aug 13.
This study was designed to identify a suitable freezing protocol for rabbit semen by comparing the effects of different concentrations and equilibration times of dimethylacetamide (DMA) and dimethylsulfoxide (DMSO) on the postthaw quality of the semen. After establishing the best protocols for each cryoprotectant, their efficacy was compared by examining the in vivo fertilizing capacity of the semen samples. Pooled semen samples diluted in freezing medium containing 4%, 6%, or 8% DMA or DMSO (all combined with 1% sucrose as a nonpermeating cryoprotectant) were loaded in straws and equilibrated for 5, 15, or 45 min before freezing in liquid nitrogen vapor. The variables assessed after thawing were sperm motility, viability, osmotic resistance, and acrosome and DNA integrity. Marked effects on these variables were shown by the cryoprotectant concentration and equilibration time, with best results obtained using DMA 6% or DMSO 8% and equilibration times of 45 min. These freezing protocols were selected to compare the two cryoprotectants in an insemination trial. Three groups of 114 rabbit does (28 nulliparous and 86 multiparous in each group) were inseminated with fresh semen or with semen frozen using the optimized DMA or DMSO protocols. Fertility rates and numbers of kids born were similar, respectively for the DMSO-frozen (79.8% and 7.7 ± 0.3 young per kindling) and fresh semen (81.6% and 8.6 ± 0.3) yet higher (P ≤ 0.05) than the rates returned using the DMA-frozen semen (47.4% and 6.7 ± 0.4). Moreover, the numbers of rabbits born alive when DMSO was used in the freezing protocol, despite being lower than those recorded using fresh semen, were higher than when DMA was used as the cryoprotectant (P < 0.05). The physiological status of the does (nulliparous or multiparous) had no influence on the fertility and prolificacy results. Our findings indicate that the cryosurvival of rabbit sperm frozen using DMSO or DMA as the cryoprotectant is highly influenced by the concentration of cryoprotectant used and the time the semen is exposed to the agent before freezing. According to our in vivo fertility and prolificacy data, DMSO emerged as more effective than DMA for the cryopreservation of rabbit sperm.
本研究旨在通过比较不同浓度和平衡时间的二甲基乙酰胺（DMA）和二甲基亚砜（DMSO）对精液解冻后质量的影响，来确定一种适合兔精液的冷冻方案。在为每种冷冻保护剂建立最佳方案后，通过检查精液样本的体内受精能力来比较它们的效果。将含有 4%、6%或 8% DMA 或 DMSO（均与 1%蔗糖作为非渗透冷冻保护剂结合使用）的冷冻液稀释的混合精液样本装入 straws 中，在冷冻前在液氮蒸气中平衡 5、15 或 45 分钟。解冻后评估的变量包括精子活力、存活率、渗透压、顶体和 DNA 完整性。冷冻保护剂浓度和平衡时间对这些变量有明显影响，DMA6%或 DMSO8%和平衡时间 45 分钟的效果最佳。选择这些冷冻方案是为了在授精试验中比较两种冷冻保护剂。将 3 组 114 只母兔（每组 28 只初产和 86 只经产）分别用新鲜精液或用优化的 DMA 或 DMSO 方案冷冻的精液授精。DMSO 冷冻组（79.8%和 7.7±0.3 只每窝）和新鲜精液组（81.6%和 8.6±0.3）的受孕率和产仔数相似，但均高于 DMA 冷冻组（47.4%和 6.7±0.4）（P≤0.05）。此外，尽管使用冷冻方案时 DMSO 组产活仔兔的数量低于新鲜精液组，但高于使用 DMA 作为冷冻保护剂时的数量（P<0.05）。母兔的生理状态（初产或经产）对受孕率和繁殖力结果没有影响。我们的研究结果表明，使用 DMSO 或 DMA 作为冷冻保护剂冷冻兔精子的冷冻存活率受使用的冷冻保护剂浓度和冷冻前精子暴露于冷冻保护剂的时间的影响。根据我们的体内生育力和繁殖力数据，DMSO 比 DMA 更适合兔精子的冷冻保存。
