92 BLASTOCYST FORMATION FROM VITRIFIED BOVINE OOCYTES, ZYGOTES, AND TWO-CELL EMBRYOS

Abstract

The objective of this study was to devise a protocol to preserve bovine oocytes and early cleavage-stage embryos by vitrification and to compare their subsequent embryonic development after in vitro fertilization (IVF). Mature bovine oocytes from a commercial source (BoMed; Madison, WI, USA) were randomly allocated (in four replicates) to four treatment groups. Group I: control oocytes were subjected to IVF and cultured in CR1aa medium in a humidified atmosphere of 5% O₂/5% CO₂/90% N₂ at 38°C. Group II: MII-stage oocytes were subjected to vitrification and then fertilized by IVF. Group III: presumptive zygotes were vitrified after IVF. Group IV: two-cell embryos resulting from IVF that were cultured for ∼28 h before vitrification. The vitrification solution consisted of TCM199 medium supplemented with 10% fetal bovine serum (mTCM) and containing 20% ethylene glycol (EG)/20% dimethyl sulfoxide (DMSO)/0.65 M trehalose. The oocytes/embryos to be vitrified were rinsed in mTCM, then in 5% EG/5% DMSO, then in 10% EG/10% DMSO, and finally for 45 s in the vitrification solution. For vitrification, groups of 6 to 12 oocytes/embryos were pipetted in <1-μL volume of vitrification medium onto the tip of a CryoTop (Katayama et al. 2003 Fertil. Steril. 80, 223); plunged directly into liquid nitrogen (LN₂), and stored for ∼2 h. Vitrified samples were warmed and liquefied by rapidly transferring the Cryotops from LN₂ into 0.25 M trehalose in mTCM at 37°C and then sequentially at 1-min intervals into 0.188 M and 0.125 M trehalose. Cleavage was evaluated on Day 3 post-insemination, and blastocyst development was assessed on Days 7 and 9 post-insemination. Of the 251 oocytes in Group I, 71% cleaved by Day 3, 21% formed blastocysts by Day 7, and 29% did so by Day 9; 3% of the total hatched. Of the 116 oocytes in Group II, fewer cleaved (P > 0.05) by Day 3 (54%) and developed into blastocysts by Day 7 (4%) and by Day 9 (8%); none hatched. Group III zygotes (n = 131) responded like Group II oocytes, 53% cleaved, and 5% formed blastocysts on Day 7 and 7% on Day 9; none hatched. In contrast, 19% of the 122 two-cell embryos formed blastocysts by Day 7 and 28% by Day 9, and 3% hatched. Although significantly fewer oocytes/embryos in Groups II and III cleaved compared with Group I, more than 50% of them did so after vitrification. After fertilization and cleavage, the two-cell embryos were much more resistant to the deleterious effects of cryoprotectants and vitrification. Higher survival of two-cell embryos may result from their increased permeability to cryoprotectants, and to water due to their higher surface area to volume ratio.