Laboratorio de Producción in vitro de Embriones, Biotecnología de la Reproducción, Departamento de Producción Animal, INTA, CC 276 (7620) Balcarce, Buenos Aires, Argentina.
Anim Reprod Sci. 2010 Mar;118(1):19-24. doi: 10.1016/j.anireprosci.2009.06.015.
The aim of the present research was to develop a low cost and easy to perform vitrification method for in vitro-produced cattle embryos. Effect of container material was evaluated (plastic straw compared to glass capillary, experiment 1), two volume sample (1 compared to 0.5 microL, experiment 2) and warming solution composition medium (Tissue Culture Medium 199 (TCM-199) compared to phosphate buffered saline (PBS), experiment 3) as modifications of the open pulled straw (OPS) system in order to reduce embryo damage caused by exposure to cold. In all experiments, day 7 and expanded blastocysts of cattle were exposed to the vitrification solution 1 for 3 min and 30s in solution 2. After this, embryos were placed in a droplet and loaded in a narrow end container, and immediately submerged into liquid nitrogen. For warming, vitrified embryos were plunged into warming solution 1 for 3 min, and transferred into warming solution 2 for 1 min. Fresh embryos kept in culture were used as control group. Hatching rates were recorded in all cases at day 13. In experiment 1 there was no significant effect of container material on hatching rates. Postwarming survival rate of vitrified embryos was lower than control (27.5% plastic straws, 18.9% glass capillary and 80.5% control, P<0.05). In experiment 2, there was no significant effect of volume in hatching rates (58.3% 1 microL, 61.3% 0.5 microL and 80.5% control, P<0.05). In experiment 3, the composition of the holding medium of warming solution influenced hatching rates (84.1% TCM-199, 74.8% PBS and 91.1% control P<0.05). These data suggest that neither glass capillaries nor reduced sample volume could improve hatching rates after vitrification-warming with open pulled straw (OPS) procedure, and that PBS can replace TCM-199 in warming solutions, but lesser hatching rates should be expected.
本研究旨在开发一种低成本、易于操作的牛体外胚胎玻璃化方法。评估了容器材料（塑料 straw 与玻璃 capillary 的对比，实验 1）、样本量（1μL 与 0.5μL 的对比，实验 2）和解冻液成分（Tissue Culture Medium 199（TCM-199）与磷酸盐缓冲盐水（PBS）的对比，实验 3）对开放式拉 straw（OPS）系统的影响，以期降低胚胎在冷暴露过程中受到的损伤。在所有实验中，第 7 天的牛囊胚和扩张囊胚均在 vitrification solution 1 中暴露 3 分钟和 30 秒，然后放入液滴中，装入窄端容器中，立即浸入液氮中。解冻时，将 vitrified 胚胎浸入解冻液 1 中 3 分钟，然后转移至解冻液 2 中 1 分钟。新鲜胚胎在培养中作为对照组。所有情况下均在第 13 天记录孵化率。在实验 1 中，容器材料对孵化率没有显著影响。玻璃化胚胎解冻后的存活率低于对照组（27.5%塑料 straws，18.9%玻璃 capillary 和 80.5%对照组，P<0.05）。在实验 2 中，样本量对孵化率没有显著影响（58.3% 1μL，61.3% 0.5μL 和 80.5%对照组，P<0.05）。在实验 3 中，解冻液中保持液成分影响孵化率（84.1% TCM-199，74.8% PBS 和 91.1%对照组，P<0.05）。这些数据表明，玻璃毛细管和减少样本量都不能改善开放式拉 straw（OPS）程序玻璃化-解冻后的孵化率，并且 PBS 可以替代解冻液中的 TCM-199，但孵化率可能会降低。
