Mitochondrial bioenergetic profiles of warmed bovine blastocysts are typically altered after cryopreservation by slow freezing and vitrification

The widespread use of cryopreserved in vitro produced (IVP) bovine embryos is limited due to their low post-warming viability compared to their ex vivo derived counterparts. Therefore, the present study aimed to analyse in detail the consequences of cryopreservation (vitrification and slow freezing) on the bioenergetic profile of the embryo and its mitochondria. To accomplish that, day 7 IVP embryos were separated in a non-cryopreserved control group (fresh, n = 120, 12 replicates) or were either slow frozen (slow frozen, n = 60, 6 replicates) or vitrified (vitrified, n = 60, 6 replicates). An in-depth analysis of the bioenergetic profiles was then performed on these 3 groups, analysing pools of 10 embryos revealing that embryo cryopreservation both via vitrification and slow freezing causes profound changes in the bioenergetic profile of bovine embryos. Noteworthy, fresh embryos demonstrate a significantly (P < 0.05) higher oxygen consumption rate (OCR) compared to vitrified and slow frozen counterparts (0.858 ± 0.039 vs. 0.635 ± 0.048 vs. 0.775 ± 0.046 pmol/min/embryo). This was found to be largely due to significantly reduced mitochondrial oxygen consumption in both vitrified and deep-frozen embryos compared to fresh counterparts (0.541 ± 0.057 vs. 0.689 ± 0.044 vs. 0.808 ± 0.025 pmol/min/embryo). Conversely, slow-frozen thawed blastocysts showed 1.8-fold (P < 0.05) higher non-mitochondrial OCR rates compared to fresh embryos. Maximum mitochondrial respiration of vitrified and slow-frozen embryos was significantly reduced by almost 1.6-fold compared to fresh embryos and the proportion of ATP-linked respiration showed significantly lower values in vitrified thawed embryos compared to fresh embryos (1.1-fold, P < 0.05). Likewise, vitrification-warming and freeze-thawing reduced reactive glycolytic capacity (1.4 fold, 1.2-fold)as well as compensatory glycolytic capacity to provide energy in response to mitochondrial deficiency (1.3-fold and 1.2-fold, P < 0.05). In conclusion, the present study has, to the best of our knowledge, identified for the first time a comprehensive overview of typical altered metabolic features of the bioenergetic profile of bovine embryos after cryopreservation, which have great potential to explain the detrimental effects of cryopreservation on embryo viability. Avoidance of these detrimental effects through technical improvements is therefore suggested to be mandatory to improve the viability of bovine embryos after cryopreservation-warming.