83 DEVELOPMENT OF BOVINE IN VITRO-PRODUCED BLASTOCYSTS AFTER CRYOPRESERVATION IN CHEMICALLY DEFINED SOLUTIONS AND ONE-STEP DILUTION

Abstract

Efficient embryo cryopreservartion using a protein-free defined solution would be beneficial for hygienic commercial embryo transport. In addition, one-step rehydration of frozen–thawed embryos could allow direct transfer of cryopreserved embryos after thawing. The objective of this study was to assess the viability in vitro of bovine in vitro-produced (IVP) embryos after freezing and thawing in a base cryoprotective solution [modified synthetic oviduct fluid (mSOF) medium containing 1.8 m ethylene glycol and 0.05 m trehalose] supplemented with various concentrations of polyvinyl alcohol (PVA). The methods used for in vitro embryo production and embryo cryopreservation were modified from those described previously (Murakami et al. 1998 Cryobiology 36, 206–212). Briefly, oocytes collected from cow ovaries were matured, fertilized in vitro for 5 h, and cultured in mSOF containing 0.4% bovine serum albumin (BSA) at 38.5°C in a humidified atmosphere of 5% CO₂, 5% O₂, and 90% N₂ (reduced-O₂ atmosphere; Day 0). On Day 3, only cleaved embryos were co-cultured with bovine cumulus cells in mSOF containing 5% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO₂ in air. On Day 7, embryos that reached the blastocyst stage were selected and immersed directly into the base cryoprotective solution supplemented with 0.4% BSA (0.4BSA group, the protein-containing control group) or 1.5, 3.0, or 6.0% PVA (1.5PVA, 3PVA, and 6PVA groups, respectively) at room temperature. After exposure to solution, embryo–cryoprotectant solutions were loaded into 0.25-mL plastic straws and cryopreserved by the standard slow freezing method (about 60 to 100 embryos in each group). The frozen straws were placed in air for 10 s, and plunged into a 38.5°C water bath for 10 s; the contents were expelled into a plastic dish. The embryos were then transferred directly to pre-incubated minimum essential medium alpha medium containing 5% FBS and MEM nonessential amino acids solution (mαMEM) for one-step rehydration. These frozen.thawed embryos were washed well and cultured in fresh mαMEM in a reduced-O₂ atmosphere for 3 days to examine the developmental potential in vitro. Data were analyzed by ANOVA. The percentage of re-expanded embryos after freezing and thawing was significantly higher (P < 0.05) in 0.4BSA and 1.5PVA groups than in the 3PVA and 6PVA groups (95.4% in 0.4BSA and 90.9% in 1.5PVA v. 73.4% in 3PVA and 57.7% in 6PVA). In addition, there was no significant difference between 0.4BSA and 1.5PVA groups in the percentage of embryos developed into hatched blastocysts (82.0 v. 80.2%), but the percentage decreased with increasing PVA concentration (51.1% in 3PVA and 29.3% in 6PVA, respectively). These results suggest that the biological product BSA added in our standard cryoprotective solution can be replaced with 1.5% PVA to support similar viability of frozen.thawed bovine IVP embryos after one-step dilution. Further studies, including direct transfer of these frozen embryos after thawing, are needed to substantiate these results.