The hollow fiber vitrificaiton (HFV) method, we developed (Matsunari et al., JRD 58, 2012) has been shown to be very effective in the cryopreservation of highly cryosensitive embryos, such as in vitro matured-fertilized pig morulae. The objective of this study was to verify the effectiveness of the HFV method to the in vitro matured-fertilized bovine embryos in early developmental stage. Crossbred (Holstein×Japanese Black) in vitro matured-fertilized bovine embryos were cultured in IVD101 medium (IFP, Japan) for 8 days after insemination (day-0). Vitrification was performed on the embryos at the 2-4 cell (day-1), 8-16 cell (day-3), and morula (day-5) stages, respectively. Survival of the vitrified embryos were evaluated by in vitro development to blastocysts. Vitrificaiton of the embryos was performed according to our previous report. Seven to 19 embryos were loaded in cellulose triacetate hollow fibers (internal/external diameter 185/200 μm, length 25 mm), and vitrified with 15% DMSO, 15% ethylene glycol, and 0.5M trehalose in liquid nitrogen. After thawing at 38.5 °C in 1M sucrose solution, the cryoprotectants were removed by a stepwise manner. The blastocyst formation rates of the vitrified embryos at each developmental stage were similar to those of the control embryos (2-4 cell: 71.4% (10/14) vs 85.7% (12/14), 8-16 cell: 70.0% (7/10) vs 77.8% (7/9), morula: 89.5% (17/19) vs 84.2% (16/19)). These data show that the HFV method is effective for cryopreserving early stage in vitro matured-fertilized bovine embryos.
This study was supported by JST, ERATO, Nakauchi Stem Cell and Organ Regeneration Project.
02 Sep 2014 - 04 Sep 2014