136 BLASTOCOELE COLLAPSE IMPROVES POST-THAW SURVIVAL OF SLOW FROZEN AND VITRIFIED IN VITRO-PRODUCED BOVINE EMBRYOS

Abstract

Slow-freeze cryopreservation of in vitro-produced bovine embryos often results in poor post-thaw embryo viability. Results with vitrification tend to be better but do not always produce consistent results for a variety of reasons. Experiments with human and equine embryos have demonstrated improved post-thaw survival when blastocoele fluid is removed before vitrification. We hypothesised that removing blastocoele fluid before vitrification or slow-freeze cryopreservation would improve post-thaw survival of in vitro-produced bovine embryos. Three replicates of embryos were generated from abattoir-derived ovaries using standard techniques. Seven days post-fertilization, embryos were evaluated for quality, and grade 1 or 2 embryos were allocated to 1 of 4 treatments: slow-freeze control (n = 103); slow-freeze collapsed (n = 106); vitrified control (n = 117); vitrified collapsed (n = 99). Blastocoele fluid was removed by aspiration with a pipette advanced to the centre of the blastocoele while the embryos were stabilised with a holding pipette. After collapse, embryos were immediately slow frozen in commercial medium [1.5 M ethylene glycol (EG) + 0.1 M sucrose, Bioniche] in 0.25-mL straws or vitrified in 2 steps: 3 min in 1.5 M EG, 30 s in 7 M EG + 0.6 M galactose + 18% Ficoll in open-pulled straws. After thawing or warming, embryos were cultured in chemically defined medium-2 + 5% FCS in 5% CO₂, 5% O₂, 90% N₂ at 38.5°C. Embryos were evaluated for survival based on re-expansion of the blastocoele after 24 h in culture. Data were analysed by GLIMMIX (SAS Institute Inc., Cary, NC, USA). There were significant replicate effects but there were no significant differences in survival between embryos that were vitrified or slow frozen. Approximately twice as many collapsed embryos survived compared to intact control embryos regardless of whether the embryos were vitrified or slow frozen (collapsed: 50.5 ± 4.1%, control: 26.3 ± 3.5%; P < 0.001). Advanced blastocysts (fully expanded and hatched blastocysts) survived better than early blastocysts (pre-expansion blastocysts and expanding blastocysts, 45.9% ± 3.9 v. 30.0% ± 4.4, respectively; P < 0.01). The only significant interaction (P < 0.05) was that vitrification was superior to freezing for early blastocysts (39.5 ± 5.9% v. 22.1 ± 5.0% survival) but not advanced blastocysts (44.4 ± 5.3% v. 47.4 ± 5.8% survival). Removal of blastocoele fluid before cryopreservation of in vitro-produced bovine embryos may be a useful technique for improving post-thaw survival regardless of cryopreservation method or blastocyst stage.