Studies On Vitrification Of Mouse Ovarian Tissue

Ovary is one of the most important reproductive organ of female mammals. It is of great practical significance to cryopreserve ovary or ovarian tissue of mammal for species protection, medicine and domestic animal breeding and reproduction. The cryopreservation of ovary or ovarian tissue of mammal could make full use of the ovary resources from slaughtered female mammals, which could provide oocyte materials for biological basic researches and practicle applications, such as in vitro fertilization, nuclear transfer and transgenic technique. To optimize ovarian tissue vitrification procedures in which the kind of cryoprotectants, the equilibrium time before cryopreservation, carrier and the size of the ovarian tissue were investigated, the ovaries from five-week-old Kunming mice were used in this experiment.In order to decide the optimal cryoprotectant and equilibrium time before cryopreservation, mouse ovarian tissue were treated with 7.5% EG (ethylene glycol) +7.5% DMSO (dimethyl sulfoxide) and 7.5% PD (7.5% PROH +7.5% DMSO) pretreatment of liquid handling oocytes were 10 min, 15 min and 20 min, then 15% EG +15% DMSO and 15% PD (15% PROH +15% DMSO) vitrification of the liquid handling 3min, cryopreserved in liquid nitrogen, thawed and then removed cryoprotectants from the ovarian tissue, punctured follicles in ovarian tissue, isolated oocytes from ovarian tissue. And the isolated oocytes were cultured and fertilized in vitro after maturity. The isolated oocytes from non-frozen ovarian tissue were taken as controls. The results showed that there were significant differences in maturity rate and the cleavage rate between the oocytes isolated from ovarian tissue treated with15% ED and 15 min of equilibrium time before cryopreservation (43.2% and 18.8% respectively) and the oocytes isolated from ovarian tissue treated with 15% PD and 15 min of equilibrium time before cryopreservation (37.4% and 10.5% respectively) (P <0.05); and there were significant differences in maturation rate and the cleavage rate between the oocytes isolated from ovarian tissue treated with 15% ED or 15% PD and both with 20 min of equilibrium time before cryopreservation (36.9% and 11.4% respectively) and the oocytes isolated from control ovaries (81.04% and 65.62% respectively) (P <0.05): were significantly lower than the control group ( P <0.05); of all the maturity groups 20 min and cleavage rate were significantly lower than the control group (P <0.05).). It was considered that ED be better cryoprotectant for cryopreservation of mouse ovarian tissue than PD. To investigate the effects ovarian tissue carrier on cryopreservation of mouse ovarian tissue, three kinds of carriers, e.g. hemi-straw, centrifuge tube and cryovial, were used in this experiment. The result showed that there were significant differences in cultured maturation rates among oocytes isolated from ovarian tissue cryopreserved in hemi-straw, centrifuge tube and cryovial (41.8%, 40.8% and138.3% respectively)(P<0.05); and there were significant differences in cleavage rates after in vitro ferrtilization among oocytes isolated from ovarian tissue cryopreserved in hemi-straw, centrifuge tube and cryovial (15.3%1, 6.9% and 11.3% respectively)(P<0.05);and there was most significant differences in cleavage rates after in vitro ferrtilization between oocytes isolated from ovarian tissue cryopreserved in hemi-straw and cryovial)(P<0.01). The result indicated that hemi-straw be more suitable to be use as carrier of mouse ovarian tissue cryopreservation.There was significant difference in maturation rate and cleavage rate after in vitro ferrtilization between oocytes isolated from cut frozen ovarian tissue(44.4% and 19.0% respectively) and the whole frozen ovarian tissue(36.2% and 10.4%)(P<0.05). It indicated that the cut ovary be more suitable to be cryopreserved.Base on our experiment results the optimum proceduree of ovarian tissue vitrification was suggested as follows: cutting ovary into four, pretreating with 7.5%EG for 10 min, treating with 15% EG for 5 min, and using hemi-straw as carrier.