36 Morphodynamics of isosmotic warming of vitrified in vitro-produced bovine embryos

Cryopreservation of in vitro-produced (IVP) embryos in animals and humans by vitrification is a validated technology. Even though vitrification of IVP bovine embryos results in a high re-expansion rate in vitro (>80%) and pregnancy rates similar to fresh transfers, the technology has not been widely adopted. Some of the disadvantages of vitrification is the time-consuming stepwise (SW) warming procedures. Recently, isosmotic (ISO) warming of vitrified IVP embryos was described in equids with pregnancy rates similar to SW warming (Canesin et al. 2020 Theriogenology 151, 151–158). The objective of this study was to evaluate the morphodynamics of ISO and SW warming of bovine IVP embryos. We hypothesized that re-expansion dynamics, hatching time, embryo size, and zona pellucida damage are similar between ISO and SW warming. Embryos (n = 32) from three replicates and two different bulls were obtained in vitro as previously described (Owen et al., 2022 J. Anim. Sci. 100, skac043). Day 7, stage 7 grade 1 blastocysts were vitrified in open pulled straws (OPS). Briefly, embryos were exposed to 1.5 M ethylene glycol for 5 min and then to 7 M ethylene glycol and 0.6 M galactose in embryo holding medium (HM) for 30 s. Embryos were then loaded (by capillary action) into OPS with ~1 μL of medium, and the OPS containing the embryo was immediately plunged into liquid nitrogen (Campos-Chillon et al. 2009 Theriogenology 71, 349–354). Step-wise warming (n = 14) took place by placing the tip of the OPS into 200 μL of 0.3 M sucrose in HM at 37°C; after 1 min, the embryos were moved to 0.15 M sucrose for 5 min and then washed in HM. Isosmotic warming (n = 18) took place by placing the tip of the OPS directly in 200 μL of HM at 37°C. Embryos were then cultured in a Time-Lapse incubator, and morphodynamics were annotated at 0, 2, 4, 6, and 12 h until embryo hatching using PhotoTune Imaging Software and analyzed with ANOVA. Furthermore, re-expansion and hatching rates and the integrity of the ZP were evaluated with Chi-squared test. Results indicate no differences (P = 0.97) between ISO and SW for re-expansion size (μm) after warming at 2 h (173 ± 5.0 and 177 ± 5.3), 4 h (174 ± 5.0 and 176 ± 5.3), 6 h (177 ± 5.0 and 178 ± 5.3), 12 h (186 ± 5.0 and 185 ± 5.3), and 18 h (200 ± 5.0 and 195 ± 5.3). Re-expansion (89% and 100%) and hatching (75% and 78%) rates, hatching time (20.9 ± 3.9 h and 20.1 ± 4.2 h), and zona pellucida damage (28% and 21%) were similar (P > 0.49) for ISO and SW, respectively. In conclusion, ISO warming of vitrified IVP bovine embryos could potentially be a viable and practical option for field implementation when warming in one step; additional replicates and clinical trials are on the way.