Viability Of Bovine Blastocysts Obtained After 7, 8 Or 9 Days Of Culture Following Vitrification And One-Step Rehydration

Abstract

This study examined the morphological appearance, hatching rates and live:dead cell ratios following vitrification of in vitro produced (IVP) bovine blastocysts. Expanded blastocysts obtained after 7, 8 or 9 d of culture were vitrified following 2-step equilibration in 10% ethylene glycol (EG) plus 0.3M trehalose in Dulbecco's PBS (DPBS) supplemented with 10% calf serum and 0.6% BSA(m-PBS) for 5min at 22°C and then a vitrification solution consisting of 40% EG plus 0.3M trehalose and 20% PVP in DPBS supplemented with 0.3% BSA for 1 min. Embryos were aspirated into 0.25 ml insemination straws and cooled rapidly by plunging into liquid nitrogen. Straws were thawed in 30°C water for 20 sec and the contents were expelled into 2 ml of m-PBS. Embryos were washed 2 to 3 times in fresh m-PBS and their immediate post-thaw survival was assessed morphologically. Then, embryos were cultured in TCM199 and viability of frozen-thawed embryos recorded visually by reexpansion/ hatching of embryos at 24, 48 and 72 h. The live:dead cell ratios of hatched blastocysts was determined by differential fluorochrome staining. Briefly, hatched blastocysts were rinsed in fresh culture medium, incubated in PBS containing propidium iodine (PI, 10 ug/ml) and bisbenzimide (10 ug/ml) for 30 min in an incubator at 38.5°C and 5% C02 in air. Embryos were washed in PBS supplemented with 3% BSA and squashed on a microscope slide and examined with a fluorescence microscope. The total cell number and live:dead cell numbers of 10 embryos were determined for each day of culture (Day 7, 8 or 9).