Routray Padmanav, Suzuki Toru, Strüssmann Carlos Augusto, Takai Rikuo
Department of Food Science and Technology, Tokyo University of Fisheries, Japan.
Theriogenology. 2002 Nov;58(8):1483-96. doi: 10.1016/s0093-691x(02)01076-2.
High performance liquid chromatography (HPLC) was used to assess the uptake dynamics of the cryoprotectant DMSO by intact unfertilized eggs (stage 0), 8-cell (stage 5) and eyed embryos (stage 30) of medaka, Oryzias latipes, the relation of the internal concentration (Cin) of DMSO with fertilization and survival rates, and the effects of several factors on these processes. The factors examined were: cryoprotectant concentration (0.6, 1.2, 1.9 and 2.5 M), impregnation time (1, 3, 5, 10, 15 and 20 min), temperature (0, 5 and 20 degrees C), hydrostatic pressure (0 and 50 atm), and the osmotic conditions of the materials (normal or partially dehydrated). Cryoprotectant permeation, estimated from the initial rates of DMSO uptake, was higher in embryos than in eggs and increased with embryonic development; however, the DMSO Cin in eyed embryos reached a plateau at 1-5 min and could not be increased by prolonging impregnation. The highest fertilization and survival rates for any given DMSO Cin were obtained with high concentrations and short times of impregnation rather than low concentrations and long impregnation times. Application of hydrostatic pressure (50 atm) and exposure for 3 min to a 1 M trehalose solution prior to impregnation induced a substantial increase in the DMSO Cin of 8-cell embryos in comparison to untreated controls with no significant effect on survival. Hydrostatic pressure also promoted DMSO uptake in unfertilized eggs, but with rapid loss of viability, and was ineffective in eyed embryos. The uptake of DMSO and its toxicity to 8-cell embryos were directly proportional to the temperature of impregnation. The results of this study reveal important interactions between cryoprotectant concentration, impregnation time and the developmental stage (or type) of the materials and provide evidence that hydrostatic pressure, temperature of impregnation and the osmotic conditions of the materials can be manipulated to increase the uptake of cryoprotectant by fish eggs and embryos.
采用高效液相色谱法（HPLC）评估了青鳉（Oryzias latipes）完整未受精卵（0期）、8细胞期胚胎（5期）和眼点期胚胎（30期）对冷冻保护剂二甲基亚砜（DMSO）的摄取动力学、DMSO内部浓度（Cin）与受精率和存活率的关系，以及几个因素对这些过程的影响。所研究的因素包括：冷冻保护剂浓度（0.6、1.2、1.9和2.5 M）、浸渍时间（1、3、5、10、15和20分钟）、温度（0、5和20摄氏度）、静水压力（0和50个大气压）以及材料的渗透条件（正常或部分脱水）。根据DMSO摄取的初始速率估算，胚胎中冷冻保护剂的渗透高于卵，且随胚胎发育而增加；然而，眼点期胚胎中的DMSO Cin在1 - 5分钟时达到平台期，延长浸渍时间无法使其增加。对于任何给定的DMSO Cin，高浓度和短时间浸渍比低浓度和长时间浸渍能获得更高的受精率和存活率。与未处理的对照组相比，施加静水压力（50个大气压）并在浸渍前将8细胞期胚胎暴露于1 M海藻糖溶液3分钟可使DMSO Cin大幅增加，且对存活率无显著影响。静水压力也促进了未受精卵对DMSO的摄取，但会导致活力迅速丧失，对眼点期胚胎则无效。DMSO的摄取及其对8细胞期胚胎的毒性与浸渍温度成正比。本研究结果揭示了冷冻保护剂浓度、浸渍时间与材料发育阶段（或类型）之间的重要相互作用，并提供了证据表明可以通过控制静水压力、浸渍温度和材料的渗透条件来增加鱼卵和胚胎对冷冻保护剂的摄取。
