Skidmore J A, Billah M, Loskutoff N M
Camel Reproduction Centre, Dubai, United Arab Emirates.
Reprod Fertil Dev. 2004;16(6):605-9. doi: 10.1071/rd03094.
The present paper describes experiments designed to investigate methods for cryopreserving embryos from dromedary camels. Because preliminary studies had shown ethanediol to be the best cryoprotectant to use for camel embryos, the current experiments were performed to determine the minimum exposure time to 1.5 m ethanediol required to achieve cryoprotection. The uteri of 30 donor camels were flushed non-surgically 8 days after mating. Embryos were recovered and 158 were assigned to one of three groups, which were exposed to 1.5 m ethanediol for either 10 min (n = 67), 5 min (n = 51) or 1 min (n = 40). Embryos were subsequently thawed and rehydrated by expelling either directly into holding medium (HM; HEPES-buffered Tyrode's medium containing sodium lactate and 3 mg mL(-1) bovine serum albumin, 10% fetal calf serum, 100 IU mL(-1) penicillin G, 100 microg mL(-1) streptomycin and 25 microg mL(-1) amphotercin B) or initially into HM containing 0.2 m sucrose for 5 or 10 min. The survival rate of all embryos immediately post-thawing, as judged by the morphological appearance of the embryos, was high (91%), but was greatly reduced after 2 h culture (59%). Ninety-two embryos were transferred to recipient camels resulting in 18 viable fetuses (1 min ethanediol exposure, n = 1/15; 5 min ethanediol exposure, n = 3/34; 10 min ethanediol exposure, n = 14/43). Of the embryos rehydrated directly in HM, six of 65 resulted in viable fetuses and those rehydrated initially in 0.2 m sucrose for 5 or 10 min resulted in nine of 47 and three of 46 fetuses respectively. From these experiments, we conclude that camel embryos can be cryopreserved using ethanediol as a cryoprotectant when the embryos are cooled slowly (to 33 degrees C) before being plunged into liquid nitrogen for storage.
本文描述了旨在研究单峰骆驼胚胎冷冻保存方法的实验。由于初步研究表明乙二醇是用于骆驼胚胎的最佳冷冻保护剂,因此进行了当前实验以确定实现冷冻保护所需的1.5 m乙二醇的最短暴露时间。30头供体骆驼在交配后8天进行非手术子宫冲洗。回收胚胎,将158个胚胎分配到三组中的一组,分别暴露于1.5 m乙二醇中10分钟(n = 67)、5分钟(n = 51)或1分钟(n = 40)。随后将胚胎解冻并通过直接排入保存培养基(HM;含有乳酸钠和3 mg mL(-1)牛血清白蛋白、10%胎牛血清、100 IU mL(-1)青霉素G、100 μg mL(-1)链霉素和25 μg mL(-1)两性霉素B的HEPES缓冲的台氏液)或最初排入含有0.2 m蔗糖的HM中5或10分钟进行复水。根据胚胎的形态外观判断,所有胚胎解冻后立即的存活率很高(91%),但在培养2小时后大幅降低(59%)。92个胚胎被移植到受体骆驼体内,产生了18个存活胎儿(1分钟乙二醇暴露组,n = 1/15;5分钟乙二醇暴露组,n = 3/34;10分钟乙二醇暴露组,n = 14/43)。在直接在HM中复水的胚胎中,65个中有6个产生了存活胎儿,而最初在0.2 m蔗糖中复水5或10分钟的胚胎分别产生了47个中的9个和46个中的3个胎儿。从这些实验中,我们得出结论,当骆驼胚胎在投入液氮储存之前缓慢冷却(至33摄氏度)时,可以使用乙二醇作为冷冻保护剂进行冷冻保存。