Departamento Reproducción Animal, INIA, 28040 Madrid, Spain.
Poult Sci. 2011 Sep;90(9):2047-53. doi: 10.3382/ps.2011-01355.
A sperm cryopreservation protocol requiring dimethylacetamide (DMA, 6%) as a cryoprotectant was optimized via assays involving different prefreezing equilibration times (1, 10, 30, 60, and 120 min at 5°C) and different freezing rates achieved by the following: 1) using nitrogen vapor to reduce the temperature from 5°C to -85°C at 10°C/min (slow freezing rate); 2) using a biological freezer unit in a 2-step method to reduce the temperature from 5°C to -35°C at 7°C/min and then from -35°C to -140°C at 60°C/min (medium freezing rate); or 3) using a biological freezer unit in a 1-step freezing method to reduce the temperature from 5°C to -180°C at 60°C/min (rapid freezing rate). Heterospermic semen samples from chicken breeds raised as part of a Spanish genetic resource conservation program were used in all assays. The 1-min equilibration treatment was associated with a lower percentage of viable thawed spermatozoa than the 30-min treatment (P < 0.05). The remaining sperm variables studied were not affected by equilibration time. The medium-rate 2-step freezing method was associated with a higher percentage of motile spermatozoa after thawing and with greater acrosome integrity (P < 0.05) than the slow nitrogen vapor or rapid 1-step methods. Thawed sperm movement quality and plasma membrane integrity (as assessed by the hypoosmotic swelling test) were better (P < 0.05) in samples frozen by the medium-rate 2-step freezing method than in those subjected to the slow nitrogen vapor method. Fertility was not influenced by freezing method, although that achieved with the medium rate 2-step freezing method showed a trend toward being greater than that achieved with the rapid 1-step method (P = 0.07). Together, the present results suggest that slow cooling rates are not recommendable when using dimethylacetamide. The 2-step freezing method may be useful in the establishment of a germplasm bank for Spanish chicken breeds.
一种精子冷冻保存方案需要使用二甲基乙酰胺（DMA，6%）作为冷冻保护剂，通过不同的预冷冻平衡时间（5°C 下的 1、10、30、60 和 120 分钟）和不同的冷冻速率进行优化：1）使用氮气蒸气将温度从 5°C 降低至-85°C，降温速度为 10°C/min（缓慢冷冻速率）；2）使用两步法的生物冷冻器将温度从 5°C 降低至-35°C，降温速度为 7°C/min，然后从-35°C 降低至-140°C，降温速度为 60°C/min（中速冷冻速率）；或 3）使用一步法的生物冷冻器将温度从 5°C 降低至-180°C，降温速度为 60°C/min（快速冷冻速率）。所有实验均使用西班牙遗传资源保护计划中饲养的鸡种的异精精液样本。与 30 分钟处理相比，1 分钟平衡处理与活解冻精子的百分比较低相关（P <0.05）。研究的其余精子变量不受平衡时间的影响。与缓慢的氮气蒸气或快速的 1 步方法相比，中速 2 步冷冻法解冻后具有更高比例的运动精子和更高的顶体完整性（P <0.05）。解冻精子运动质量和质膜完整性（通过低渗肿胀试验评估）在中速 2 步冷冻法冷冻的样品中比在缓慢的氮气蒸气法中更好（P <0.05）。冷冻方法不影响受精能力，尽管中速 2 步冷冻法的受精能力有高于快速 1 步法的趋势（P = 0.07）。总的来说，这些结果表明，当使用二甲基乙酰胺时，不建议使用缓慢的冷却速率。两步冷冻法可能对建立西班牙鸡种的种质库有用。
