Improvement in post-thaw viability of in vitro-produced bovine blastocysts vitrified by glass micropipette (GMP)

Abstract

The purpose of this study was to investigate the use of a glass micropipette (GMP) as a vessel for vitrification of in vitro-produced (IVP) bovine blastocysts and to compare the results with post-thaw survival rate of bovine blastocysts frozen in GMP with those frozen in open pulled straw (OPS) that have been previously investigated. The GMP vessel permitted higher freezing and warming rates than the OPS due to the higher heat conductivity of the glass and the lower mass of the solution that contained the embryos. Groups of three bovine IVP blastocysts were sequentially placed into vitrification solution before being loaded into either the OPS or GMP vessels and they immersed into LN(2) within 20-25 s. Post-thaw blastocysts were serially washed in 0.25 and 0.15 M sucrose in a holding medium (HM: D-PBS supplemented with 5% FCS) and then in TCM-199 for 5 min in both cases. They was then cultured in TCM 199 supplemented with 10% FCS for 24 or 48 h. The rate of blastocyst re-expansion was significantly different between OPS (79.6%) and GMP (90.4%) methods. Neither was the hatching rate significantly different among OPS (51.8%), GMP (57.1%) methods and non-vitrified group (67.3%). Only the rate of post-thaw re-expanding of blastocysts loaded in narrow column was significantly higher than that of the wide column (83.3% versus 56.7%) (P < 0.05), although the GMP straw was loaded with three blastocysts per vessel. These results indicated that the GMP vessels provided high survival rates of bovine IVP blastocysts. The location of the embryos loaded into a narrow or wide portion was considered to be a limiting factor to the viability of bovine IVP embryos.