【作者】 王丽娟；

【导师】 李武；

【作者基本信息】 东北农业大学 ， 动物遗传育种与繁殖， 2003， 硕士

【摘要】 本研究应用EFS40玻璃化液对6.5～7日龄的绵羊胚胎进行玻璃化冷冻及解冻后直接移植试验。所用胚胎都是经FSH超排处理绵羊获得的。胚胎经形态鉴定后，用胚胎存放液(含20％FCS的PBS液)清洗。冷冻前，胚胎在室温(18～25℃)下置于含10％乙二醇的PBS液中平衡10分钟，移入玻璃化液EFS40中装管并平衡30秒钟，液氮气中熏蒸2分钟后投入液氮中保存，解冻时在室温(18～25℃)空气浴10秒钟后，32℃水浴20秒钟解冻。 试验1：胚胎解冻后以0.5M蔗糖液一步(6分钟)除去保护剂或用胚胎存放液一步直接除去保护剂，然后胚胎移入胚胎存放液中洗3次，将形态正常的胚胎手术移植至同期发情的受体绵羊子宫内；试验2：玻璃化冷冻胚胎解冻后在0.25ml聚乙烯塑料细管内脱除防冻剂直接装管移植，以常规冷冻解冻为对照组。试验结果：试验1共冷冻道赛特与东北半细毛羊杂交青年羊胚胎32枚，解冻回收27枚，其中21枚形态正常，解冻后透明带完整率为100％(27／27)；形态完整率为77.78％(21／27)，移植后的妊娠率为47.37％(9／19)，胚胎存活率为42.86％(9／21)，其中桑椹胚、囊胚的冻后形态完整率分别为81.82％(9／11)、75.00％(12／16)(P＞0.05)，差异不显著；桑椹胚和囊胚移植后的妊娠率分别为37.50％(3／8)、54.55％(6／11)，胚胎存活率分别为33.33％(3／9)、50.00％(6／12)，差异均不显著(P＞0.05)。13枚冷冻胚胎解冻后用0.5M蔗糖脱防冻剂，其中10枚形态恢复正常(76.92％)，与直接用胚胎存放液脱除防冻剂的结果(78.57％，11／14)无差异(P＞0.05)。将两种方法解冻的形态正常的胚胎移植，移植后的妊娠率分别为44.44％(4／9)、50.00％(5／10)、胚胎存活率分别为40.00％(4／10)、45.45％(5／11)(P＞0.05)差异不显著；试验2将10枚解冻后的胚胎细管内脱防冻剂后，直接装管移植给8只受体(其中2只受体移植双胚)，妊娠率为50.00％(4／8)，胚胎成活率为40.00％(4／10)，(其中受体1只在妊娠后第2个月流产，最后有3只受体产3只羔羊)与同期常规冷冻解冻组相比无显著差异(P＞0.05)。 本研究结果表明：绵羊胚胎应用EFS40进行玻璃化冷冻，解冻后用胚胎存放液细管内脱除防冻剂后直接移植获得较高的的妊娠率和胚胎存活率。该方法与常规方法比较简便、省时、可普及，特别是能降低绵羊胚胎移植的成本，加速绵羊品种改良。更多还原

【Abstract】 Direct transfer of vitrified sheep embryos by the method of vitrification (EFS40) was studied. Embryos were obtained surgically from superovulated donors on 6.5~7 days after the onset of estrus (onset of estrus=0 day). After being graded according to morphology, embryos were washed in embryo holding solution (PBS supplemented with 20%FCS) , only those classified as excellent and good were processed. Before vitrification, embryos were kept at room temperature (18-25 C) for 10 min in PBS-FCS containing 10% ethylene glycol (EG), and finally plunged for 30sec into vitrification solution EFS40 (PBS-FCS with EG 40% and ficoll 18% and sucrose 0.3M) , then loaded into 0.25ml straws. The straws were fumigated with liquid nitrogen vapor for 2min before they were plunged into liquid nitrogen (LN2) . Straws were thawed 10 sec in air (18-25 C) followed 20 sec in a 32 C water bath. In experiment 1, after being thawed, embryos were washed for cryoprotectant removal by one step of 0.5M sucrose or embryo holding solution for 6 min, then embryos were washed three times in embryo holding solution, and morphologically intact embryos (MIEs) were transferred surgically to the uterus of recipient 6.5-7days after the onset of synchronization of estrus; In experiment 2, embryos were directly transferred without cryoprotectant removal nor embryo evaluation after thawing. Data of this study were analyzed using Chi-square test.In experiment 1, thirty-two embryos of excellent and good quality were vitrified. Afterthawing, 27 embryos were recovered, all of 27 embryos’ zona pellucid were found intact(100%),21 ofwhich (77.78%) wereMIEs,81.82%(9/ll)and75.00%( 12/16)formorulaeand blastocysts respectively (P>0.05) .There was no significant different of developmentalstage of the embryos on the pregnant rate, with 37.50% (3/8) for morulae and 54.55% (6/11)for blastocysts, similar to the different of the embryo survival rate (33.33% vs 50.00%). Afterthawing, cryoprotectants were diluted by 0.5M sucrose from 13 embryos, 10 of which(76.92) were MIEs, similar to the result of by embryo holding solution (11/14,78.57%)(PX).05). The methods of cryoprotectant removal didn’t affect on the pregnant rate (44.44%vs50.00%) and embryo survival rate (40.00% vs 45.45%) . In experiment 2, after thawing,10 vitrified sheep embryos following in straw-dilution were directly transferred to 8 recipientsand three recipients gave birth to three living lids (1 female, 2 male) , is not different tonon-vitrified embryos.The results of this paper indicated that sheep embryos can be successfully cryopreserved by a simple, rapid procedure, after being thawed, embryos were washed for cryoprotectant removal by embryo holding solution in straw-dilution, then embryos were directly transferred to recipients. This method of sheep embryo cryopreservation and thawing provided high pregnancy and embryo survival, this method is easy and rapid and may be popularized easily, especially it may help to reduce the costs of embryo transfer in sheep and increase the use of this technique genetic improvement of this species compare to traditional methods.Postgraduate: Wang LijuanMajor: Animal genetics breeding and reproductionSupervisor: Prof. Li Wu更多还原