Slow-freezing is more damaging than vitrification to post-thaw quality of large equine embryos collapsed by blastocoele aspiration

Abstract

Embryo flushing in mares is best performed on day 7 or 8 after ovulation because the likelihood of recovering an embryo(s) is higher. However, embryos larger than 300 µm are less tolerant of cryopreservation unless the blastocoele is first collapsed. While vitrification of collapsed blastocysts has yielded good results, there are no reports of the relative success of slow-freezing punctured blastocysts. The aim of this study was to compare the effects on post-thaw embryo quality of vitrifying and slow-freezing day 7-8 equine blastocysts after blastocoele puncture. Embryos were collected from donor mares on day 7 or 8 after ovulation, graded (1 to 4), measured (µm), and divided into two size categories (330-550 µm, n= 11 and >550 µm, n= 16). Blastocoele fluid was aspirated using a micromanipulator, and the embryos then either slow-frozen (n= 14; 2 steps, 10% glycerol) or vitrified (n= 13; 2 steps,16.5% ethylene glycol/16.5% DMSO/0.5M sucrose). After thawing or warming, embryos were cultured for 24h before being graded and measured to determine degree of (re)expansion (%). Next, the embryos were stained with DAPI to identify dead cells, before being fixed and further labeled with Phalloidin (cytoskeleton), WGA (capsule), and TOPRO-3 (DNA) to assess live/dead cell proportions, cytoskeleton quality, and capsule integrity. Control embryos (>300-550 µm, n=3 or >550 µm, n=3)were collapsed and cultured for 24h without cryopreservation. Data were analyzed using one-way ANOVA, Kruskal-Wallis, or Fisher's exact tests. For 300-550 µm embryos, quality grade was negatively affected by cryopreservation but did not differ between slow-frozen (3±0) and vitrified (1.7±1) embryos. On the other hand, re-expansion was significantly reduced in slow-frozen (73±34%) compared to vitrified (148±26%) and control (179±10%) embryos. Live/dead cell proportion, cytoskeleton quality and capsule integrity were not influenced significantly by cryopreservation technique. For larger (>550 µm) embryos, re-expansion was also significantly reduced in slow-frozen (52±24%) compared to vitrified (115±7%) and control (129±12%) embryos. In addition, embryo quality was compromised more after slow-freezing (3±0.5) than vitrification (1.9±0.4) or 24h culture alone (control: 2±0). Furthermore, the proportion of dead cells and degree of cytoskeleton disruption was higher after slow-freezing than vitrification, although the incidence of capsule loss was not influenced by freezing method. In conclusion, slow-freezing of micromanipulator-collapsed equine blastocysts compromises embryo quality more than vitrification; therefore, after aspiration of blastocoel fluid, it is preferable to vitrify rather than slow-freeze equine embryos >300 µm.