Jousan F D, Utt M D, Whitman S S, Hinshaw R H, Beal W E
Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0306, USA.
Theriogenology. 2004 Apr 15;61(6):1193-201. doi: 10.1016/j.theriogenology.2003.07.009.
The objective of this study was to evaluate the in vitro development of frozen-thawed bovine embryos held at room temperature or refrigerated for 2, 6 or 12 h prior to freezing. After recovery, embryos were randomly assigned to be placed in holding media for 2 h (n=131), 6 h (n=136) or 12h (n=133) prior to freezing. Approximately one-half of the embryos were refrigerated (5 degrees C; n=203) while the remaining half were held at room temperature (22 degrees C; n = 197) until freezing. Embryos were frozen in 10% ethylene glycol and stored in liquid nitrogen. After thawing, embryos were cultured for 72 h in Ham's F-10 media supplemented with 4% fetal bovine serum. Embryos were evaluated for quality and stage of development prior to freezing and after culture. At the end of culture, it was determined if each embryo had developed beyond the stage recorded prior to freezing and if the embryo had hatched from the zona pellucida. The percentage of embryos that developed during culture was greater (P < 0.001) for Grade 1 (81%) than for either Grade 2 (65%) or Grade 3 (48%) embryos. Likewise, a greater proportion (P < 0.001) of Grade 1 embryos developed to hatched blastocysts (60%) than either Grade 2 (40%) or Grade 3 (24%) embryos. The holding temperature from collection to freezing did not influence embryo development, regardless of the interval from embryo collection to freezing. The proportion of embryos that developed to expanded blastocysts and hatched was greater (P < 0.005) for embryos held 2 h prior to freezing (64%) than for embryos held for 12 h (33%). Hatching rate of embryos held 6 h prior to freezing (54%) tended (P < 0.08) to be lower than the hatching percentage for embryos held for 2 h. Thus, post-thaw embryonic development was impaired the longer embryos were held prior to freezing and temperature during the interval from collection to freezing did not affect post-thaw development.
本研究的目的是评估在冷冻前于室温下保存或冷藏2、6或12小时的冻融牛胚胎的体外发育情况。回收后，胚胎被随机分配在冷冻前置于保存培养基中2小时（n = 131）、6小时（n = 136）或12小时（n = 133）。大约一半的胚胎被冷藏（5摄氏度；n = 203），而其余一半在室温（22摄氏度；n = 197）下保存直至冷冻。胚胎在10%乙二醇中冷冻并储存在液氮中。解冻后，胚胎在补充有4%胎牛血清的Ham's F - 10培养基中培养72小时。在冷冻前和培养后对胚胎的质量和发育阶段进行评估。在培养结束时，确定每个胚胎是否发育到超过冷冻前记录的阶段以及胚胎是否从透明带孵化。培养期间发育的胚胎百分比对于1级胚胎（成功率81%）高于2级胚胎（成功率65%）或3级胚胎（成功率48%）（P < 0.001）。同样，1级胚胎发育到孵化囊胚的比例（60%）高于2级胚胎（40%）或3级胚胎（24%）（P < 0.001）。从采集到冷冻的保存温度不影响胚胎发育，无论从胚胎采集到冷冻的间隔时间如何。冷冻前保存2小时的胚胎发育到扩张囊胚并孵化的比例（64%）高于保存12小时的胚胎（33%）（P < 0.005）。冷冻前保存6小时的胚胎孵化率（54%）略低于保存2小时的胚胎（P < 0.08）。因此，解冻后胚胎发育会因冷冻前保存时间延长而受损，并且从采集到冷冻期间的温度不影响解冻后发育。
