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: VS 1 (20% ethylene glycol ( EG ) + 20% propanediol ( PROH ) + 0.25  m trehalose (Tr)), VS 2 (20% EG  + 1M Tr) or VS 3 (30% EG  + 0.75  m Tr). Re‐expansion and hatching rates were higher (p < 0.05) for embryos vitrified in VS 3 (72.2 ± 1.9 and 58.2 ± 0.8) than VS 1 (64.4 ± 0.9 and 37.2 ± 2.5) or VS 2 (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 VS 1 or VS 3 solutions. No significant differences were detected between the two methods; but re‐expansion and hatching rates were higher (p < 0.05) with VS 3 (73.5 ± 3.1 and 47.1 ± 2.1) than VS 1 (63.3 ± 3.3 and 39.7 ± 2.8). In experiment 3, embryos were vitrified by solid surface in VS 1 or VS 3 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 VS 3 did not differ between those exposed to 0.25  m 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 VS 1 were higher (p < 0.05) in those exposed to 0.25  m 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.