A novel approach to cryopreservation of porcine embryos by vitrification after blastocoelic micromanipulation.

In this study, the effects of the reduction of blastocoelic fluid prior to vitrification were examined on porcine embryos (n=44; 160-540 mu m in size; graded excellent (1) according to IETS guidelines). The embryos were either assigned to a control group (n=24) or to the experimental group (n=20) which differed in that blastocoelic fluid was aspirated before microinfusion of vitrification solution 1 (VS1; 1.4 M glycerol in PBS). During the vitrification procedure, the embryos were exposed to 2 cryopreservation solutions, VS1 and VS2 (1.4 M glycerol plus 3.6 M ethylene glycol in PBS), for 5 minutes each, and then to a third cryopreservative, VS3 (3.4 M glycerol and 6.6 M ethylene glycol in PBS), for 1 minute. The straws with embryos were placed in a cooled plastic goblet surrounded by liquid nitrogen vapors for 1 minute and then immersed into the liquid nitrogen for rapid freezing through the phase transition point. The embryos were thawed at 30 degrees C for 15 s in a water bath and cultured for a period of 24 hrs in NCSU media. Morphological evaluation of the embryos using a stereomicroscope revealed, that initial re-expansion was complete in 16 of the treated and 7 of the control embryos. This demonstrated a significant difference between treatment and control groups at thaw in the recovery of apparently functional embryos (p < 0.001). All embryos from both treatments were degenerated after 24 hrs of culture.