| Objective This study tends to improve the conditions of vitrification in large pieces of ovarian cortex, explore the most simple, economic and effective ovarian tissue cryopreservation through the adding of FSH before sheep and fetal ovary cryopreserved and transplanted to preserve ovarian stroma cells and strorage of follicles.To choose the best grafting position through autoplastic and xenogenic transplantation of different positions providing technical methods to enhance the practicality and feasibility of clinical.Methods 1.The study of the stroma cell morphology of vitrification on different size of ovarian cortex of sheep: The ovarian cortex from 3 to 6 month old sheep was divided into three different groups, 40mm~3: 5mm×4mm×2mm,80mm~3: 10mm×4mm×2mm and 160 mm~3: 10mm×8mm×2mm.A two-step osmotic equilibrium was used to explore the equilibrium time in different size ovarian tissue of sheep,the effects of cryopreservation were assessed by common microscopy and isolating stroma cell examination.2.The study of fresh and vitrificated ovarian tissue xenotransplantation in sheep adding FSH: after adding FSH in culturing fluid and cryoprotectants, the sheep ovarian tissue was transplanted into the nude mice neck subcutaneous tissue, evaluate the effects of FSH on large ovarian cortex tissue after transplanted through the determination of serum E2, the number of survival follicles in transplanted ovarian tissue.3.The study of vitrificated ovarian tissue autotransplantation in sheep: after vitrificated and thawed 40mm~3 of ovarian cortex, the sheep ovarian cortex was transplanted into the orthotopic and heterotopic tissue to evaluate the better position of ovarian cortex transplantation and the conditions of ovarian cortex after transplantation through the the number of survival follicles in transplanted ovarian tissue.4.The study of vitrificated ovarian tissue xenogenic transplantation in sheep: after vitrificated and thawed 40mm~3 of ovarian cortex, the sheep ovarian cortex was transplanted into the orthotopic and heterotopic xenogenic tissue to evaluate the position of ovarian cortex transplantation and the conditions of ovarian cortex after transplantation through the determination of serum E2 and FSH, the the number of survival follicles in transplanted ovarian tissue.5.By using the best equilibrium time of vitrification on different size of ovarian cortex of sheep aadding FSH, the fresh tissue was cryopreserved and cultured. Evaluate the vitrification on different size of ovarian cortex and the effects of FSH through the follicles'development and morphologic structure .Results 1.The three size of ovarian cortex were vitrificated after appropriate equlilibrated.Histology:A 40mm~3:The number of stroma cells at 7min osmotic equilibrium group (16.00±2.16) were similar to fresh control group (16.00±2.83) the number of stroma cells at 5min,6min,8min osmotic equilibrium group were significantly more than that of in fresh group, P<0.05,the percentage of morphologically normal stroma cells at 7min osmotic equilibrium group (79.50±6.95) and 8min osmotic equilibrium group (80.25±4.03) were similar to fresh control group (88.00±3.56) the percentage of morphologically normal stroma cells at 5min,6min osmotic equilibrium group were significantly less than that of in fresh group, P<0.05。80mm~3: The number of stroma cells at 11min osmotic equilibrium group (20.050±2.38) and 12min osmotic equilibrium group (19.25±0.50) were similar to fresh control group (18.00±2.45) the number of stroma cells at 10min,13min osmotic equilibrium group were significantly more than that of in fresh group, P<0.05;the percentage of morphologically normal stroma cells at 11min osmotic equilibrium group (84.00±0.81) were similar to fresh control group (86.25±1.50) the percentage of morphologically normal stroma cells at 10min,12min,13min osmotic equilibrium group were significantly less than that of in fresh group, P<0.05;160mm~3: The number of stroma cells at 19min osmotic equilibrium group(19.50±2.38)were similar to fresh control group (18.50±1.73) the number of stroma cells at 17min,18min,20min osmotic equilibrium group were significantly more than that of in fresh group, P<0.05;the percentage of morphologically normal stroma cells at 19min osmotic equilibrium group (81.50±4.40) were similar to fresh control group (85.25±4.99) the percentage of morphologically normal stroma cells at 17min,18min,20min osmotic equilibrium group were significantly less than that of in fresh group, P<0.05. B The survival rate of stroma cells in sheep ovary after vitrification: The number and survival rate of stroma cells in every group was similar, P>0.05. 2. The results of fresh and vitrificated sheep ovarian tissue xenotransplanted into nude mice neck subcutaneous tissue showed that : the ratio of oestrum recovery and the number of follicles in vitrificated transplanted adding FSH group was s similar to fresh transplanted group,higher than that of frozen-thawing transplanted group, P<0.05. The serum E2 in vitrificated transplanted adding FSH group was s similar to fresh transplanted group,higher than that of frozen-thawing transplanted group, P<0.05. 3. The result of vitrificated ovarian tissue autotransplantation in sheep: comparing with the same size of fresh ovarian cortex,only 6.38% of follicles survived after fresh autoallergic orthotopic transplantation for 6 monthes, only 5.74% of follicles survived after vitrification autoallergic orthotopic transplantation for 3 monthes.There were secondary follicles in graft of both above. Both of two heterotopic transplantation had no follicle survived.4. The result of vitrificated ovarian tissue xenogenic transplantation in sheep: Except of fresh xenogenic orthotopic transplantation group, the serum FSH in other transplantation groups were significantly higher than antithetical group, P<0.05. the serum FSH in heterotopic transplantation groups were higher than that in orthotopic transplantation groups, P<0.05. Except of fresh xenogenic orthotopic transplantation group, the serum E2 in other transplantation groups were significantly lower than antithetical group, P<0.05. The serum E2 in fresh xenogenic orthotopic transplantation group was higher than the other three transplantation groups, P<0.05. The serum E2 in vitrification xenogenic orthotopic transplantation group was higher than heterotopic transplantation groups, P<0.05. The follicle survival rate in grafts: after synchronization of estrus at 3 monthes, 6.79% to 2.42% of follicles survived in orthotopic transplantation group. Both of two heterotopic transplantation had no follicle survived.5. The fetal ovarian tissue after cryopreserved and cultured: A.constituent ratio of normal follicles: Both of two vitrification groups were similar to fresh control group, P<0.05.Two vitrification groups were similar, P<0.05.The vitrification groups and cultivation for 4.5 hours after thawing groups were different, P<0.05. B follicular diameter: Both of two vitrification groups were similar to fresh control group, P<0.05.Two vitrification groups were similar, P<0.05.The follicular diameter of cultivation after thawing groups were longer than the vitrification groups, P<0.05.Conclusion 1.Altra-rapid vitrification can effectively preserve stroma cells in sheep ovary tissue of 40mm~3,80mm~3 and 160mm~3, providing cells for vasculogenesis after transplantation.2. The number of survival follicles in the frozen-thawn graft and the development of culturing ovarian tissue were increased by adding 10μg/mL FSH.3.The number of normal follicles in vitrificated group and fresh group were similar.The suitable position of sheep ovarian tissue was orthotopic transplantation. The rate of survival follicles in fresh xenogenic orthotopic transplantation group and vitrificated xenogenic orthotopic transplantation group were similar, but massive follicles were lost during transplantation.4. The suitable equilibrium time of vitrification can effectively preserve fetal ovary tissue of larger pieces.Adding FSH during vitrifiation and thawing process did not influence constituent ratio of follicles, but some primordial follicles could develop to primary follicle after culturing for 4.5 hours.
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