51 In silico-designed vitrification protocols based on in vitro-produced bovine embryos’ permeability at different cryoprotectants, temperatures, and lengths of in vitro culture

Cryopreservation of in vitro-produced (IVP) embryos has become an important part of the bovine embryo-transfer industry. While vitrification seems more efficient for IVP embryos, there is not yet a standardised protocol that results in pregnancy rates comparable to those obtained when fresh embryos are transferred, especially when blastocysts are vitrified after eight days (D8) of culture instead of seven days (D7). Current vitrification protocols greatly differ in the time and temperature of exposure to the equilibration solution (ES). Moreover, they have rarely considered the osmotic properties of bovine embryos. Knowledge of the permeability parameters of bovine blastocysts to water and cryoprotectants (CPA) will allow using mathematical modelling to optimise vitrification procedures. This study aimed to determine the permeability to water (L_p) and solutes (P_s) of bovine IVP blastocysts in the presence of ethylene glycol (EG) and dimethyl sulfoxide (Me₂SO) at different temperatures (25°C; 38.5°C) and different culture lengths (D7; D8). D7 (n = 46) or D8 (n = 41) blastocysts at the expanded stage were placed in a 25-μL drop of holding medium (HM: TCM199-Hepes with 20% (v/v) FBS) in a dish covered with mineral oil and held with a holding pipette on an inverted microscope. After blastocysts collapse with an intracytoplasmic sperm injection pipette, they were covered with a larger inner diameter pipette and introduced in a 25-µL drop of HM with 1.55 M EG or 1.55 M Me₂SO at 25°C or 38.5°C (5 replicates). The volumetric response of the blastocysts was recorded every 5 s during 10 min with a time-lapse recorder. The hydraulic conductivity (L_p) and CPA permeability (P_s) of blastocyst cell membranes were determined and then fitted to a two-parameter transport formalism to obtain four in silico predictions of the blastocyst osmotic behaviour when exposed to the ES (7.5% Me₂SO and 7.5% EG in HM) at each temperature and in vitro culture length. After checking normality, data were compared by ANOVA. No differences were found when comparing L_p values among groups (P > 0.05). However, P_s values were different when comparing CPAs, being higher (P = 0.03) for EG than for Me₂SO; temperatures, being higher (P = 0.01) at 38.5°C than at 25°C; and in vitro culture lengths, being higher (P = 0.04) for D8 than for D7 blastocysts. In silico predictions indicated that D7 blastocysts would need 8 min in ES at 25°C and 3 min 50 s at 38.5°C to recover their initial volume. D8 blastocysts would swell back to their initial volume after 4 min exposure to ES at 25°C, again, slower than at 38.5°C, when they would only need 3 min. These results are promising not only to improve current D7 vitrification protocols but also for D8 embryos to achieve similar results to when D7 embryos are cryopreserved. Future experiments are guaranteed to validate in silico-designed CPA loading steps for the optimisation of vitrification/warming at each temperature and length of culture.

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