Solid-Surface Vitrification and In-Straw Dilution After Warming of In Vitro-Produced Bovine Embryos

Contents Three experiments were designed to test a solid-surface vitrification system for bovine in vitro-produced embryos and to develop a simple method of in-straw dilution after warming, which can be potentially used for direct transfer in the field. Experiment 1 evaluated embryo survival rates (i.e. re-expansion and hatching) after vitrification and warming in three different solutions: VS1 (20% ethylene glycol (EG)+20% propanediol (PROH)+0.25m trehalose (Tr)), VS2 (20% EG+1M Tr) or VS3 (30% EG+0.75m Tr). Re-expansion and hatching rates were higher (p<0.05) for embryos vitrified in VS3 (72.2 +/- 1.9 and 58.2 +/- 0.8) than VS1 (64.4 +/- 0.9 and 37.2 +/- 2.5) or VS2 (68.5 +/- 1.5 and 49.6 +/- 1.0; p<0.05). Experiment 2 was designed to compare two methods of vitrification: glass micropipettes or solid surface, using the VS1 or VS3 solutions. No significant differences were detected between the two methods; but re-expansion and hatching rates were higher (p<0.05) with VS3 (73.5 +/- 3.1 and 47.1 +/- 2.1) than VS1 (63.3 +/- 3.3 and 39.7 +/- 2.8). In experiment 3, embryos were vitrified by solid surface in VS1 or VS3 solutions and cryoprotectants were diluted in-straw after warming in a TCM 199, 0.25m sucrose solution or holding media. Survival rates of embryos vitrified in VS3 did not differ between those exposed to 0.25m sucrose (74.7 +/- 1.3 and 57.2 +/- 2.2) or holding (77.3 +/- 1.4 and 58.0 +/- 2.5) medium after warming; however, survival rates of embryos vitrified in VS1 were higher (p<0.05) in those exposed to 0.25m sucrose (67.7 +/- 2.3 and 47.0 +/- 1.7) than holding medium (54.5 +/- 1.0 and 27.7 +/- 3.1). In conclusion, solid-surface vitrification using simplified EG-based solutions and in-straw dilution with holding media may be a practical alternative for cryopreservation and direct transfer of in vitro-produced bovine embryos.