James E R, Peacock R
J Helminthol. 1986 Mar;60(1):65-73. doi: 10.1017/s0022149x00008269.
Various cooling (0.1-5,100 degrees C min-1) and warming (20-6,800 degrees C min-1) rates, stepped cooling schedules and four cryoprotective additives (dimethyl sulphoxide, methanol, ethanediol and glycerol) were investigated in cryopreservation studies with Dictyocaulus viviparus third-stage larvae. Exsheathment with sodium hypochlorite was essential to achieve significant survival. With uninterrupted cooling, highest survival (30% normally motile) was achieved with rates of 10-70 degrees C min-1. Survival was higher (50-75%) using 1 degree C min-1 to -10 degrees C followed by plunging into liquid nitrogen. The optimum warming rate was 6,800 degrees C min-1. The use of cryoprotectants led to marginally lower survival while varying the suspending media had no significant effect on survival. X-irradiated, exsheathed third-stage larvae cryopreserved by the optimum protocol yielded 38.3 +/- 4.2% survival. Two calves each infected with 45,000 (15,000 viable) exsheathed, unirradiated, cryopreserved third-stage larvae harboured 494 worms (1.1% infectivity) and 355 worms (0.8%) at necropsy. Numbers of first-stage larvae in the faeces reached 420/g and 105/g respectively 27 days after infection.
在对胎生网尾线虫第三期幼虫进行的冷冻保存研究中，研究了各种冷却（0.1 - 5,100℃/分钟）和升温（20 - 6,800℃/分钟）速率、分步冷却方案以及四种冷冻保护添加剂（二甲基亚砜、甲醇、乙二醇和甘油）。用次氯酸钠脱鞘对于实现显著的存活率至关重要。在连续冷却的情况下，以10 - 70℃/分钟的速率可实现最高存活率（30%正常活动）。先以1℃/分钟降至 - 10℃，然后投入液氮中，存活率更高（50 - 75%）。最佳升温速率为6,800℃/分钟。使用冷冻保护剂导致存活率略有降低，而改变悬浮介质对存活率没有显著影响。按照最佳方案冷冻保存的经X射线照射、脱鞘的第三期幼虫存活率为38.3±4.2%。两头小牛分别感染45,000条（15,000条存活）脱鞘、未照射、冷冻保存的第三期幼虫，尸检时分别发现494条虫（感染率1.1%）和355条虫（感染率0.8%）。感染后27天，粪便中第一期幼虫数量分别达到420条/克和105条/克。
