PREGNANCIES FOLLOWING TRANSFER OF EQUINE EMBRYOS CRYOPRESERVED BY VITRIFICATION
PREGNANCIES FOLLOWING TRANSFER OF EQUINE EMBRYOS CRYOPRESERVED BY VITRIFICATION
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DOI：
10.1016/0093-691x(94)90686-d
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发表时间：
1994-08-01
期刊：
影响因子：
2.8
通讯作者：
OGURI, N
中科院分区：
文献类型：
--
作者：
HOCHI, S;FUJIMOTO, T;OGURI, N
本研究的目的是探讨玻璃化冷冻保存的马胚胎在体外和体内的发育能力。在第5 - 7天，通过非手术子宫冲洗（排卵检测=第0天）从本地小马和纯种马中回收28个胚胎。玻璃化溶液含有40%乙二醇、18%Ficoll和0.3M蔗糖的PBS溶液。将胚胎在玻璃化溶液中放置1至2分钟（第1组）或暴露于20%乙二醇的PBS溶液中10至20分钟（第2组和第3组）。将单个胚胎装载在0.25-ml吸管中，在液氮蒸气中冷却1分钟并浸入液氮中。将吸管在水中加热（20 ℃，20秒），并将内容物用PBS中的0.5M蔗糖排出。然后将蔗糖以1步（第1组和第2组）或4步（第3组）稀释。将胚胎（n=21）在补充有10%胎牛血清的TCM 199中在37 ℃、5%CO2的空气中培养120小时，并进行形态学评价。第1、2和3组中分别有0/7、4/7和4/7个胚胎发育至孵化或孵化囊胚期。根据第2组（4个胚胎）和第3组（3个胚胎）的处理，对另外7个胚胎进行玻璃化加温。在体外培养4 h后选择5个胚胎，并将其非手术移植到第4天受体子宫角中。移植2个胚胎（均为第-6天囊胚：第2组处理）导致妊娠期第-60天妊娠，胎仔存活。
The objective of this study was to investigate the in vitro and in vivo developmental abilities of equine embryos cryopreserved by vitrification. Twenty-eight embryos were recovered from Native pony and Thoroughbred mares at Days 5 to 7 by nonsurgical uterine flushing (detection of ovulation=Day 0). The vitrification solution contained 40% ethylene glycol, 18% Ficoll, and 0.3 M sucrose in PBS. The embryos were placed for 1 to 2 min in vitrification solution (Group 1) or following exposure to 20% ethylene glycol in PBS for 10 to 20 min (Groups 2 and 3). Single embryos were loaded in 0.25-ml straws, cooled for 1 min in liquid nitrogen vapor and immersed in liquid nitrogen. Straws were warmed in water (20 degrees C, 20 sec), and the contents were expelled with 0.5 M sucrose in PBS. Then the sucrose was diluted in 1-step (Groups 1 and 2) or 4-steps (Group 3). Embryos (n=21) were cultured for 120 h in TCM199 supplemented with 10% fetal bovine serum at 37 degrees C in 5% CO2 in air and evaluated morphologically. Development to the hatching or hatched blastocyst stage was obtained in 0/7, 4/7 and 4/7 embryos in Groups 1, 2 and 3, respectively. An additional 7 embryos were vitrified-warmed according to the treatment of Group 2 (4 embryos) and Group 3 (3 embryos). Five embryos were selected after in vitro culture for 4 h and were transferred nonsurgically into the uterine horn of Day-4 recipient mares. Transfer of 2 embryos (both Day-6 blastocysts: Group-2 treatment) resulted in pregnancies with a viable fetus at Day-60 of the gestation period.