猪冷冻(-20℃)胚胎在我国移植成功

PIGLETS DELIVERED FROM FROZEN(-20℃)EMBRYOS

实验Ⅰ将129枚猪胚胎,用于冷冻-解冻-体外培养研究。以含有1.5M甘油的PBS液为冷冻液,以1℃/分的速率从35℃降至-7℃,在-7℃自动诱发给冰,然后以0.3℃/分的速率降至-35℃,再以0.1℃/分的速率降至-36℃,立即投入液氮(-196℃);胚胎解冻在37℃水浴中进行、分步脱出甘油后以改进的mBMOC-Ⅱ为培养液进行体外培养。结果,囊胚、扩张囊胚、刚孵出囊胚和孵化出后期囊胚、解冻后体外培养存活率分别为0％(0 /2)、64％(16/25)、0％(0/9)。在液氮中(-196℃)保存80～103天,扩张囊胚、刚孵出囊胚和孵出后期囊胚解冻后,体外培养存活率分别为41.6％(5/12)、63.7％(7/11)和0％(0/70)。结果表明,在液氮温度(-196℃)保存下,刚孵出囊胚有较高的耐冻性和存活能力。实验Ⅱ进行猪冷冻胚胎解冻后体内发育能力研究。96枚扩张囊胚和刚孵出囊胚,分批冷冻和保存在-20℃、-35℃和-196℃。解冻后移植3头受体,其中2头返情,1头妊娠并于1990年7月31日产仔2头,公母各1头,初生重均为1.35kg,妊娠期为116夭。该结果为我国首例猪冷冻(-20℃)胚胎移植成功,并为今后开展猪胚胎超低温(-196℃)冷冻保存技术研究奠定了基础。

Experiment 1: the survivat of pig embryos after freezing at -35℃ and - 196℃ and subsequent thawing has been investigated. Blastocyst (BL) to later hatched blastoc-yst (LHBL) were recovered at 5~8 days after mating. Total of 129 embryos were frozen in 1.5M glycerol and 0.25 ml straws, according to Hayashi's method (1989) slightly modified: from 35℃ to -7℃ at cooling rate l℃/min, kept at -7℃ for 7~10 min, while seeding was performed and then from -7℃ to -35℃ at 0.3℃/min, from -35℃ to -36℃ at 0.1℃/min and immediately plunged into liquid nitrogen (-196℃). Frozen embryos were thawed in 37℃ water bath for 20 sec. Glycerol was removed step-wise, with sucrose free buffer (A) or buffer with sucrose (B). After washing, embryos were cultured with 2ml modified KRB for 4~llh at 38℃ under condition of 5% CO2 in air. The survival rate of EXBL frozen at -35℃ was the highest 61.5% (16/25), when they were frozen at -196℃, only 41.6% (5/12) of them survived. HBL frozen at -196℃ had the highest survival rate, LHBL could not survive both at - 35℃ and -196℃. Experiment 2: A total of 231 embryos were recovered (25.7/donor) from 9 supe-rovulated donor sows. Ninety six EXBL and HBL were frozen at -20℃ (42), -35℃ (17), -196℃ (37), and tranferred to 3 recipients. One of them (-20℃) was pregnant and gave a birth of 2 piglets. The result showed that pig embryos could be frozen, embryos at different stages has changed freezing tolerance; frozen and thawed EXBL and HBL may be used for transfer to recipients.