Resumen
- The aim of the present experiments was to determine whether treatment with the antioxidants ascorbic acid and dithiothreitol, or an inhibitor of caspase-3, Z-DEVD-FMK, during cryopreservation could improve the cryotolerance of bovine embryos produced in vitro. For all experiments, bovine embryos were produced in vitro using abattoir-derived ovaries. At Day 7 after insemination, blastocyst and expanded blastocyst stage embryos were harvested and subjected to controlled-rate freezing following equilibration for 8 to 10 min in freezing medium [Hepes-TALP (Parrish et al. 1986) plus 1.5 M ethylene glycol and 0.5 M sucrose] with treatments as described below. Embryos were thawed and then cultured for 72 h in SOF-BE1 (Fields et al. 2011) supplemented with 10% (vol/vol) fetal bovine serum at 38.5°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2. Re-expansion and hatching rates were recorded at 24, 48, and 72 h. For Experiment 1, embryos (n = 578) were equilibrated in freezing medium containing 0, 0.1, 0.3, or 0.5 mM ascorbic acid. For Experiment 2, embryos (n = 243) were equilibrated in freezing medium containing 0, 50, 100, or 200 μM dithiothreitol. For Experiment 3, embryos (n = 227) were equilibrated in freezing medium containing 0, 50, 100, or 200 μM Z-DEVD-FMK. Embryos frozen in freezing medium containing ascorbic acid had increased (P < 0.05) re-expansion and hatching rates at 24, 48, and 72 h compared with embryos not treated with ascorbic acid (Table 1). The optimal concentration of ascorbic acid for post-thaw cryosurvival was 0.1 mM. In particular, embryos treated with 0.1 mM ascorbic acid during cryopreservation had increased (P < 0.05) re-expansion rates at 24, 48, and 72 h, as well as hatching rates at 48 and 72 h, compared with control-treated embryos (Table 1). There was no effect of treatment with dithiothreitol or Z-DEVD-FMK on re-expansion or hatching rates at 24, 48, or 72 h after thaw. In conclusion, addition of ascorbic acid to freezing medium improves the cryosurvival of bovine embryos produced in vitro. Further research is necessary to determine whether treatment with ascorbic acid can increase pregnancy rates.Table 1.Effect of ascorbic acid during cryopreservation of bovine embryos (%, means ± standard error of the mean)