80 EFFECT OF PRE-EQUILIBRATION TIME ON THE SURVIVAL RATE OF IN VITRO-FERTILIZED BOVINE EMBRYOS AFTER VITRIFICATION

Abstract

Successful cryopreservation of embryos depends on the pre-equilibration time. This study was designed to compare 2 pre-equilibration times – short (1 min) and long (5 min) – and to evaluate the re-expansion and hatching rates of different stages of embryos using the short pre-equilibration method. Cumulus–oocyte complexes (COCs) from ovaries obtained from a slaughterhouse were matured, fertilized, and cultured in vitro. In Experiment 1, expanded blastocysts between 7 and 9 days of culture were pre-equilibrated for the short time (1 min) in 100 μL of vitrification solution 1 (VS1: containing 7.5% ethylene glycol (EG), 7.5% dimethyl sulfoxide (DMSO), and 20% calf serum (CS) in TCM-199), followed by incubation in 100 μL of vitrification solution 2 (VS2: containing 15% EG, 15% DMSO, 20% CS, and 1 m sucrose (Suc) in TCM-199) for 30 s. Another group of blastocysts was subjected to the long pre-equilibration (5 min) in 100 μL VS1, followed by incubation in 100 μL of VS2 for 30 s. The embryos were placed into Cryotops^® (Kitasato Supply Co., Tokyo, Japan) and immediately submerged in liquid nitrogen and kept there for 1 week. Blastocysts were warmed by plunging the Cryotops into 1 m Suc in TCM-199 for 1 min, placed in 0.5 m Suc in TCM-199 for 3 min, and finally placed in CR1aa alone for 5 min before being cultured. In Experiment 2, 8-cell embryos, morulae, and expanded blastocysts were vitrified by the previously described short equilibration method. The re-expansion and hatching rates of embryos were determined as the percentage of vitrified–warmed embryos undergoing further development in the in vitro culture. The data were analyzed by the chi-square test. Results are presented in Table 1. There was no difference between the short and long pre-equilibration times in terms of survival (94.0% v. 94.1%, respectively) and morphological appearance immediately after warming. However, re-expansion of blastocysts (ability to develop further) was slightly higher with the short pre-equilibration than with the long pre-equilibration (90.0% v. 85.9%, respectively). In Experiment 2, there were no differences in embryo re-expansion, but the hatching rates of 8-cell embryos were lower than those of morulae, blastocysts, and controls. In conclusion, the results of this study indicate that the length of pre-equilibration time prior to vitrification does not influence embryo re-expansion, and that bovine morulae and blastocysts can be vitrified with equal success. We also conclude that insufficient permeation of cryoprotectants may occur in 8-cell embryos.

Table 1. Survival and hatching rates of vitrified bovine embryos after warming