52 Metabolic profile of in vitro-produced bovine embryos is affected by cryopreservation

Metabolic profiles can be used to quantify the viability of early in vitro-produced (IVP) embryos. In cattle, differences between vitrification and slow freezing of embryos have been measured in terms of cryosurvival, fertility, DNA fragmentation, and gene expression, but not yet at the metabolic level. This study has compared the metabolic profile of surviving embryos cryopreserved by vitrification or slow freezing. IVP expanded blastocysts were randomly allocated into vitrification (n = 33) or slow freezing (n = 33) groups. For vitrification, embryos were placed in 7.5% ethylene glycol (EG) + 7.5% dimethyl sulfoxide (DMSO) in holding medium (HM: TCM-199 HEPES + 20% FBS) for 3 min, then transferred to HM with 15% EG + 15% DMSO + 0.5 M sucrose for 30–40 s, loaded onto cryotops, and plunged into LN. For slow freezing, embryos were exposed to 1.5% EG in Hepes-SOF for 10 min and loaded into 0.25-mL straws in a central column of Hepes-SOF + 1.5 M EG surrounded by four columns of 0.75 M EG in HM separated by air columns. Straws were placed in an embryo freezer and, after seeding at −6°C, the freezing curve lowered 0.5°C min⁻¹ to −32°C. Fresh blastocysts (n = 33) were used as a control group. For warming, cryotops were submerged into 1 M sucrose in HM for 1 min and washed in 0.5 M sucrose in HM for 3 min. For thawing, straws were exposed to air for 10 s and immersed in water at 35°C for 30 s. After warming/thawing, blastocysts were transferred to SOF medium and cultured singly in 5-µL drops for 24 h at 38.5°C in a 5% CO₂, 5% O₂ humidified atmosphere. Survival and hatching yields were recorded at 24 h after warming. Metabolic profiles were determined by measuring in spent culture 5-µL drop the depletion and appearance (pmol/embryo/hour) of glucose, pyruvate, and lactate using ultramicrofluorometric assays. Data were analysed by ANOVA (P ≤ 0.05) and are presented as mean ± standard deviation. Survival of blastocysts cryopreserved by vitrification after 24 h postwarming was higher (81.8 ± 9.1%) than those cryopreserved by slow freezing (66.6 ± 5.2%). Moreover, vitrified blastocysts showed similar hatchability (27.3 ± 9.1%) to fresh nonvitrified blastocysts (30.3 ± 13.9%) and higher than slow frozen (18.2 ± 7.3%). Slow-frozen blastocysts depleted more glucose from the culture media than vitrified (75.2 ± 36.3 vs 43.7 ± 18.4 pmol/e/h) or fresh (53.0 ± 30.5 pmol/e/h). By contrast, fresh blastocysts depleted more pyruvate (107.3 ± 24.8 pmol/e/h) than for those cryopreserved (vitrified: 61.7 ± 19.6 pmol/e/h; slow frozen: 62.3 ± 14.0 pmol/e/h). Fresh blastocysts released the greatest quantity of lactate (135.1 ± 60.2 pmol/e/h) compared with vitrified (94.8 ± 61.4 pmol/e/h) and slow-frozen blastocysts (42.6 ± 16.9 pmol/e/h). These data show that the cryopreservation process affects embryo metabolism. Given links between metabolic patterns and viability, our observations may relate to ongoing developmental capacity. Future research on the relationship between metabolic profile of cryopreserved embryos and their ability to produce a pregnancy is needed.

This research was supported by Spanish Ministry of Science and Innovation (Project PID2020-116531RB-I00).