Piglets born after vitrification of embryos using the open pulled straw method

Morulae and unhatched blastocysts from Large White hyperprolific (LWh) and Meishan (MS) gifts were selected to test an ultrarapid open pulled straw (OPS) vitrification method with two media, The viability of vitrified/warmed embryos was estimated by the percentage of embryos that developed to the hatched blastocyst stage in vitro or by birth after transfer. In Experiment 1, two cryoprotectant dilution media were compared for cryopreservation of MS and LWh blastocysts: TCM was a standard Hepes-buffered TCM199 + 20% NBCS medium and PBS was a PBS + 20% NBCS medium. After a two-step equilibration in ethylene glycol, dimethyl sulfoxide, and sucrose, 2-5 blastocysts were loaded into OPS and plunged into liquid nitrogen. Embryos were warmed: a four-step dilution with decreasing concentrations of sucrose was applied. In PBS, LWh blastocysts (27%) had a lower viability in vitro than MS blastocysts (67%; P = 0.001). In TCM, no significant difference was observed between genotypes (41% for LWh and 43% for MS blastocysts) and both viability rates were lower than that of the control groups. In Experiment 2, morula-stage LWh and MS embryos were vitrified and warmed using PBS. The viability rate was low and did not differ between LWh (11%) and MS (14%). In Experiment 3, 200 MS and 200 LWh blastocysts were vitrified/warmed as described in Experiment 1 (PBS). In each of 20 MS recipients, 20 embryos were transferred. The farrowing rate was 55% and recipients farrowed four and five piglets (median) for MS and LWh blastocysts, respectively. The OPS method is therefore appropriate for cryopreservation of unhatched porcine blastocysts. (C) 2000 Academic Press.