Development of cryopreservation method of the farm animal ovary

7日龄大鼠卵巢用2M甘油缓慢冷冻法或改良VS1玻璃化冷冻法冷冻，再用分步冷冻法或0.5M蔗糖-PBS冷冻法加热稀释。在每一次冷冻保存过程中，对它们进行组织学评估。通过器官培养证实玻璃化冷冻卵巢的存活率。将玻璃化冷冻和蔗糖稀释的卵巢移植到去势的成年大鼠体内，检测卵巢的存活率。取0.7~1 mm的山羊卵巢组织块，用2M甘油或1.5M DMSO缓慢降温，或用玻璃化法快速降温，加温稀释后进行组织学和细胞学评价。在大鼠卵巢的冷冻保存中，玻璃化冷冻的卵巢表现出较好的组织学保存，有轻微的细胞收缩和固缩，由冷冻保护剂的渗透作用和化学作用引起，也有轻微的细胞因渗透作用和结冰而破裂。卵巢冷却缓慢，颗粒细胞扩张破裂，卵母细胞严重破坏和收缩。培养4d后，玻璃化冷冻卵巢内可见2层或2层以上颗粒细胞生长的卵泡(24.7×28.8个/卵巢)。17只大鼠中有12只(70.6%)可观察到卵巢的内分泌活动和/或单侧或双侧卵巢的生长，但大多数表现为形态和功能异常。它们中的一部分交配，大多数成为假孕，但没有产生后代。这些结果表明，玻璃化冷冻保存的大鼠卵巢仍能保持部分存活率。在冷冻保存的山羊卵巢组织切片中，组织学观察到的损伤与大鼠卵巢相似。在1.5M DMSO中缓慢冷却的样品具有较好的保存效果。在2M甘油中缓慢冷却或玻璃化分步稀释的切片损伤严重。在玻璃化冷冻和蔗糖稀释的切片中，部分滤泡的细胞学结构保存良好，但大多数滤泡表现出轻微的损伤。

Seven day-old rat ovaries were cooled by slow freezing in 2 M glycerol or by the vitrification method using modified VS1, warmed and diluted by stepwise method or in 0.5 M sucrose-PBS. In each cryopreservation process, they were evaluated histologically. Viability of vitrified ovaries were confirmed by organ culture. Viability of vitrified and sucrose-diluted ovaries were also assayed by orthotropic transplantation into spayed adult rats. Pieces of goat ovarian tissue (0.7-1 mm cubes) were cooled slowly in 2 M glycerol or 1.5 M DMSO, or cooled rapidly by vitrification method, warmed, diluted and evaluated histologically and cytologically. In cryopreservation of rat ovaries,vitrified ovaries showed better histological preservation, with mild cell shrinkage and pyknosis caused by osmotic and chemical effects of cryoprotectants and mild cell rupture by osmotic effect and ice formation. In ovaries cooled slowly, expansion and rupture of granulosa cells, and severe destruction and shrinkage of oocytes were noted. After 4 day-culture, growing follicles with 2 or more layers of granulosa cells were observed in vitrified ovaries (24.7*28.8 follicles/ovary). Endocrine activity of ovaries and/or growth of unilateral or bilateral ovaries were observed in 12/17 (70.6%) rats with grafts, but most showed abnormality in morphology and function. A part of them were mated and most became pseudopregnant, but none yielded progenies. These results indicate that rat ovaries cryopreserved by vitrification method have kept partial viability. In cryopreserved pieces of goat ovarian tissue, damage observed histologically were similar to those in rat ovaries. Pieces cooled slowly in 1.5 M DMSO showed better preservation. Pieces cooled slowly in 2 M glycerol or vitrified and diluted by stepwise method were severely damaged. In vitrified and sucrose-diluted pieces, cytological structure was well preserved in some of follicles, however, most showed mild damage.