The vitrification of in vitro fertilized cow blastocysts by the open pulled straw method

Abstract

In 6 replicates, a total of 450 immature oocytes recovered from 144 slaughterhouse-derived bovine ovaries were matured and fertilized in vitro, then cultured for 7 to 9 d on a granulosa cell monolayer in tissue culture medium 199 (TCM-199) supplemented with fetal calf serum. Of 126 blastocysts (28% of oocytes cultured), 117 (26% of oocytes cultured) were vitrified in Hepes/bicarbonate-buffered TCM-199 medium and 20% fetal calf serum, with ethylene glycol and dimethylsulfoxid as the cryoprotectants. After thawing in 1.2 mL holding medium with 0.25- M sucrose and after l min in holding medium with 0.15-M sucrose, blastocysts were cultivated in vitro for 24 h. The re-expansion rate of blastocysts was 69.2% (81 blastocysts), and 39.5% (32 blastocysts) were hatched. Re-expansion and hatching rates differed between the blastocysts vitrified on 7 and 8+9 days (74.6% and 46% vs. 62% and 29%, respectively). After transfer to recipient cows, 3 out of 6 were diagnosed by ultrasonography as pregnant. Three calves were born from 18 transfered embryos (16.7%). The open pulled straw (OPS) method seems to be a convenient, simple and effective method for cryopreservation of 7 to 9 d bovine embryos.