Direct Transfer Of Vitrified Sheep Embryos

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...