42 Differential effects of vitrification and slow freezing on mitochondrial respiratory properties after thawing of expanded bovine blastocysts

In recent years, demand for frozen-thawed in vitro-derived bovine embryos steadily increased. However, in vitro-derived embryos still display reduced freezeability, which reflects impaired qualities going along with reduced pregnancy rates after transfer compared with ex vivo-derived counterparts. Since it is generally accepted that freezing technique implicates cryosurvival, the aim of the present study was to analyse the mitochondrial metabolism of bovine embryos cryopreserved by two techniques compared with fresh ones. Consequently, in vitro-derived expanded blastocyts (D7) generated by routine procedures (SOFaa + 5% ÖCS, 5% CO₂ & 5% O₂, 38.8°C) were cryopreserved either by vitrification or slow freezing, and fresh blastocysts served as control. Comparisons were conducted in terms of nonmitochondrial and mitochondrial oxygen consumption and ATP production, as well as relative mitochondrial reserve capacity. Metabolic measurements of fresh, vitrified-thawed (vitrification), and freeze-thawed (slow frozen) embryos (6–12 replicates, pools of 10 expanded blastocysts) were conducted using re-expanded embryos 4 h after thawing by an extracellular FLUX-Analyser under application of a Cell-Mito Stress Test Kit (Seahorse SFp, Agilent). As a main result, basal embryo respiration as indicated by oxygen consumption rate (pmol/min) was significantly lower (0.25-fold) for cryopreserved embryos compared with fresh counterparts, irrespective of freezing technique (ANOVA, P < 0.05). In agreement, cryopreserved embryos displayed nearly 0.3-fold reduction in terms of mitochondrial oxygen consumption compared with fresh ones and illustrated 0.2-fold reduction with respect to ATP production (P < 0.05). Moreover, mitochondrial reserve capacity was significantly reduced (P < 0.05) in both vitrified and slow-frozen embryos compared with fresh ones (235.4% vs 244.3% vs 281.8%, respectively). It is noteworthy that there was no obvious effect of freezing technique on mitochondrial respiratory properties except the nonmitochondrial respiration, with slow-frozen embryos demonstrating nearly 2-fold higher values. In summary, the present study clearly illustrates a negative impact of cryopreservation on mitochondrial respiratory properties. In detail, we identified an effect on mitochondrial capacity, which could be a leading cause for impaired embryo qualities after cryopreservation. In that context, higher nonmitochondrial respiration of slow-frozen embryos might explain lower embryo qualities than the bovine following slow-freezing compared with vitrification. Collectively, the findings of this work identified several points related to mitochondrial fitness. These could be useful for further improvement of cryopreservation techniques for in vitro-derived bovine embryos.