Experimental Study On Vitrification Of Rabbit Ovarian Tissue

Objective:To compare the effect of direct cover vitrification with hemi straw vitrification on the morphology and ultra microstructure of different stages of follicles of rabbit ovarian tissue.Methods:10healthy female rabbits were bilateral ovariectomized and each ovary were randomly divided into three groups which were the fresh group,the direct cover vitrification group and the hemi straw vitrification group. DMSO+EG was used as the cryoprotectant in vitrification. The ovarian tissues were frozen with direct cover vitrification and hemi straw vitrification. The last two groups were preserved in the liquid nitrogen for7days.Then we thawed the tissues of by rapid rewarming. Histological and ultrastructural observation and comparison of influences on the different stages of follicles in two methods frozen-thawed ovarian tissues.Results:1.The distribution of primordial and primary follicles in the fresh ovarian tissues was not different from that in the frozen tissues(P>0.05).2.After thawing rabbit ovarian tissue by using of direct cover vitrification and hemi straw vitrification, the rates of both the primordial follicles and the primary follicles with normal morphology were68.32%and68.01%respectively, compared with fresh group (84.51%) there were significant difference(P<0.05), but there was no significant difference between two freezing methods (P>0.05).3. After thawing rabbit ovarian tissue by using of direct cover vitrification and hemi straw vitrification, the rates of the primordial follicles, the primary follicles with normal morphology were72.66%and71.73%,55.08%and56.18%respectively, compared with fresh group (88.11%and72.86%) there were significant difference(P<0.05), but there was no significant difference between two freezing methods (P>0.05). After thawing, the rate of the normal primary follicles (55.68%) was lower than the rate of the normal primordial follicles(72.20%), there were significant difference(P<0.05).4.Under the obversation with transmitting electronic microscope,the majoy ultrastructure of follicles in ovarian tissue was preserved well after cryopreservation, but the ultrastructure of follicles were caused a certain degree of damagement by application of direct cover vitrification and hemi straw vitrification, there was the most damagement in mitochondria. The characteristic ultrastructure of cryoinjury in cells represented cytoplasmic vacuolization,swelling and vacuolization in mitochondria, pyknosis and so on.Conclusion:1.Cryopreserved rabbits ovarian tissues by direct cover vitrification and hemi straw vitrification can also maintain partial primordial and primary follicles. There was a certain degree of damagement in follicles at every stage, but cryoperserving methods have more damagement to the primary follicles than to the primordial follicles. There was no signifinant differences of primordial follicles on morphology between direct cover vitrification group and hemi straw vitrification group.2.Follicles of rabbits ovarian tissue were caused a certain degree of damagement on ultrastructure by application of direct cover vitrification group and hemi straw vitrification, there was the most damagement in mitochondria.