34 The KVS direct transfer system for vitrified-warmed bovine embryos provides an effective alternative to the traditional slow-freeze method

Currently, the transfer of slow-freeze blastocysts is widely used in the bovine commercial assisted reproduction industry. However, the transfer of vitrified blastocysts is not as common, even though they have superior viability compared with slow-freeze blastocysts. We recently developed a vitrification carrier called the Kitasato vitrification system (KVS), which has a highly absorbent surface that effectively removes excess free vitrification solution from specimens before the cooling step (Momozawa et al. 2017 Reprod. Biol. Endocrinol. 15, 29). The KVS has shown excellent results for bovine blastocyst vitrification (Momozawa et al. 2023 Cryobiology 113, 104568). In this study, we investigated the efficacy of the KVS direct system, which combines the vitrification of bovine blastocysts using KVS with a novel warming device that does not require the use of a stereomicroscope. Bovine blastocysts on Days 6 and 7 after IVF were used. First, blastocysts were transferred to an equilibration solution containing 7.5% (vol/vol) ethylene glycol, 7.5% (vol/vol) dimethyl sulfoxide (DMSO), and 0.25 M sucrose in a basic medium (BM) for 10 min. The BM consisted of modified TCM-199 supplemented with 20% fetal bovine serum. After equilibration, the blastocysts were transferred to a vitrification solution (VS) containing 15% (vol/vol) ethylene glycol, 15% (vol/vol) DMSO, and 0.5 M sucrose in BM for 30 s. They were then placed on the KVS with ~0.6 µL of VS. The excess solution was absorbed by the KVS absorber within 5–10 s, and the device with the embryo was plunged and stored into liquid N₂. For warming, the KVS with the vitrified embryo was transferred to 0.35 mL of warming solution (0.3 or 0.15 M sucrose in serum-free BM) in the novel warming device at 39°C. After 1 min, the warming device containing the embryos was kept for 10 min at room temperature (20–25°C). In the first experiment, we assessed the viability of vitrified blastocysts warmed with the novel warming device using 0.3 or 0.15 M concentration of sucrose. Vitrified-warmed blastocysts were cultured for 72 h at 39°C under 5% CO₂, 5% O₂, and 90% N₂, with high humidity. Embryos that developed to the re-expanded stage after 24 h of culture were considered viable. Development of blastocysts to the hatching stage was observed at 72 h postculture. The survival and hatching rates were analyzed using the Fisher’s exact text. There were no significant differences in the survival and hatching rates between the two sucrose concentrations in the warming solution (survival: 95.2% vs. 97.3%; hatching at 72 h postwarming: 79.0% vs. 83.8%). In the second experiment, we assessed the pregnancy rate after transfer of vitrified-warmed embryos (embryo transfer) to synchronized recipients. A single blastocyst, vitrified-warmed by the KVS and the novel warming device, was transferred nonsurgically into the uterine horn ipsilateral to the corpus luteum of each recipient (12 heifers and 15 cows). Pregnancy was confirmed by transrectal ultrasonography between Days 30 and 35 after embryo transfer. Pregnancy rates were 58.3% for the heifers and 53.3% for the cows. In summary, the results indicate that the KVS direct system is a useful procedure for the direct transfer of vitrified-warmed bovine embryos.