Solid-surface vitrification and in-straw dilution after warming of in vitro-produced bovine embryos.

摘要

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)+0.25 m trehalose (Tr)), VS2 (20% EG+1M Tr) or VS3 (30% EG+0.75 m Tr). Re-expansion and hatching rates were higher (p<0.05) for embryos vitrified in VS3 (72.2+or-1.9 and 58.2+or-0.8) than VS1 (64.4+or-0.9 and 37.2+or-2.5) or VS2 (68.5+or-1.5 and 49.6+or-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+or-3.1 and 47.1+or-2.1) than VS1 (63.3+or-3.3 and 39.7+or-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, 0.25 m sucrose solution or holding media. Survival rates of embryos vitrified in VS3 did not differ between those exposed to 0.25 m sucrose (74.7+or-1.3 and 57.2+or-2.2) or holding (77.3+or-1.4 and 58.0+or-2.5) medium after warming; however, survival rates of embryos vitrified in VS1 were higher (p<0.05) in those exposed to 0.25 m sucrose (67.7+or-2.3 and 47.0+or-1.7) than holding medium (54.5+or-1.0 and 27.7+or-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.