Development of vitrified porcine primordial follicles in xenografts

The objective was to cryopreserve porcine primordial follicles by vitrification and to assess the development of these follicles in xenografts. Ovarian tissues containing primordial follicles were collected from neonatal (15-d-old) piglets. They were vitrified in modified tissue culture medium (TCM)-199 containing 15% (v/v) ethylene glycol, 15% (v/v) dimethylsulfoxide, 20% (v/v) fetal calf serum, and 0, 0.25, or 0.5 M sucrose. After 1 wk of storage in liquid nitrogen (LN₂), the tissues were warmed, and the morphology of follicles and oocytes was examined histologically. After vitrification in sucrose-free medium, there were 50 ± 2 (mean ± SEM; n = 10) follicles per tissue, in contrast with 108 ± 10 (n = 10) in fresh tissues. Losses were attributed to puncturing oocytes during the vitrification-warming process, as oocytes were apparently normal after treatment of the sucrose-free vitrification solution without plunging into LN₂. When tissues were vitrified in sucrose-supplemented medium, loss of oocytes decreased (P < 0.05). However, the number of abnormal oocytes having nuclear shrinkage was increased (P < 0.05) by the addition of 0.5 M sucrose; this occurred in a small number of oocytes treated with sucrose-supplemented vitrification solutions without vitrification. After 2 mo of xenografting of vitrified-warmed tissues in SCID (severe combined immune deficiency) mice, primordial follicles developed to the secondary stage (accompanied by oocyte growth), whereas there was development to the antral stage in xenografts of fresh tissues. In conclusion, primordial follicles from neonatal pigs maintained their developmental ability after vitrification and warming, although their developmental rate was slower than that of the fresh control in xenografts.