Storage of cryopreserved in liquid nitrogen is very demanding in terms of maintenance, storage space, equipment, and costs. Such demands are predicted to increase as more women are resorting to oocyte preservation to preserve their future fertility options. This study therefore sought an alternative method for gamete-preservation: lyophilization and dry storage.
Three experiments in parallel compared various cooling methods on the recovery and survival after freeze/dry of in vitro-matured MII bovine oocytes (n=68). Ten oocytes were cryopreserved with slow freezing (using MTG 1314 device) at a cooling rate of 4⁰ C/minute (group A); 24 oocytes with rapid freezing (using MTG 516 device), at a cooling rate of 150⁰ C/minute (group B); and 34 with vitrification using minimum drop size in IMT-4 solution (mix of cryoprotectants and trehalose) at a cooling rate > 20,000⁰C/minute. The lyophilization process was carried out with the VirTis wizard for 24 hours with shelf temperature of -55⁰ C and vacuum 10 mTorr. The rehydration process took place at room temperature using equilibrated TCM199 supplemented with 0.5M trehalose. Oocyte survival was assessed with the live/dead staining (SYTO/PI).
For group A, 70% of the oocytes were recovered after rehydration but only 1/7 stained as viable (14%); for group B, 71% were recovered and 10/17 stained as viable (59%). For group C, 88% were recovered and 23/30 stained as viable (77%) (p < 0.05).
Lyophilization of oocytes is a groundbreaking innovation for gamete bio-banking; 2) Vitrification is confirmed as an essential method not only for preservation in liquid nitrogen but also for a dry state.
Cryopreservation of Oocytes and Embryos
Theriogenology, vol. 81, 96-102 (2014)
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Figure 1. Freeze-dried oocytes
Figure 2. Removal of MII nucleus from freeze-dried oocytes
Figure 3. First two cell embryo obtained from injection of a metaphase plate from a freeze dried oocyte into fresh enucleated oocyte
Figure 4. Vitrified lyophilized oocytes