In vitro assessment of a direct transfer vitrification procedure for bovine embryos

Abstract

We developed a simple vitrification technique for bovine embryos that could permit direct transfer. Embryos were produced in-vitro by standard procedures. The base medium for cryopreservation was a chemically defined medium similar to SOF + 25 mM Hepes and 0.25% fatty acid free bovine serum albumin (FAF-BSA) (HCDM2). In experiment 1, embryos were first exposed to 3.5 M ethylene glycol (V1) for 1, 2 or 3 min at room temperature (20–24 °C), and then moved to 7 M ethylene glycol (V2) at 4 or 20–24 °C and loaded in 0.25-mL straws. After 45 s in 7 M ethylene glycol, straws were placed in liquid nitrogen. Embryos that were loaded at 20–24 °C had higher survival rates than those loaded at 4 °C ( P < 0.05). Exposure for 1 min was best for morulae, while 3 min was best for blastocysts. In experiment 2, blastocysts were handled at 24 °C and exposed to two concentrations of ethylene glycol in V1 (3.5 or 5 M) followed by V2 as in experiment 1, two warming temperatures (20 or 37 °C) and two post-warming holding times until culture (5 or 15 min). Exposure to 5 M ethylene glycol and warming at 37 °C was the optimal combination of procedures, and embryos survived well after 15 min in straws if warmed at 37 °C. In experiment 3, ethylene glycol concentration (3, 4 or 5 M) and exposure time (0.5 or 1 min) during two-step addition of cryoprotectant were studied for bovine morulae. In experiment 4, morulae were exposed to V2 for 30 or 45 s in HCDM2 or Vigro holding medium and then held in 22–24 °C air or 37 °C water post-warming. Experiment 5 was like experiment 4 except blastocysts were used. Overall survival rates of blastocysts in experiment 5 averaged 80% of non-vitrified controls after 48 h culture. The survival rates with in vitro-produced morulae in experiments 1, 3 and 4 were unacceptable. Vitrification solutions based on Vigro tended to result in higher survival than HCDM2 for blastocysts, but not morulae. In experiment 6, the survival rate in vitro of in vivo-produced morulae and blastocysts after two-step vitrification was nearly 100%. Our vitrification technique was very effective for in vitro produced blastocysts, but not for in vitro-produced morulae.