117 VITRIFICATION OF CHAROLAIS AND HOLSTEIN BLASTOCYSTS USING LN SLUSH

Abstract

Cryopreservation of embryos for transfer in outdoor conditions is a challenge; therefore a rapid and simple technique should be applied. Two different experiments were performed to develop a functional and efficient vitrification technique. Charolais (for Exp. 1) and Holstein (for Exp. 2) female cows were superovulated and artificially inseminated (AI). Seven days after AI, embryos were flushed from the uterus and vitrified. For vitrification, blastocyst were exposed to 10% vitrification solution (VS) for 3 min, transferred into 50% VS, and immediately thereafter into 87.5% VS (100% VS containing 38% (v/v) ethylene glycol (EG), 0.5 m trehalose, and 6% BSA in PBS). The embryos were then loaded into super open pulled straws (SOPS) (Minitub, Tiefenbach, Germany) in a minimum-volume droplet (0.5 μL). The SOPSs containing the embryos were sealed by applying a soldering device to the narrow end and by inserting an identification rod into the wide end. Samples were vitrified at a rapid cooling rate in LN Slush (VitMaster apparatus, IMT Ltd, Ness-Ziona, Israel). On the day of transfer, blastocysts were warmed by plunging the SOPS into the warming chamber of the device, which contained 70% ethanol at 37°C, for 5 s. The straws were then withdrawn from the warming chamber and the sealed end was cut off carefully. The embryos were immersed in a 200-μL drop of 0.6 m trehalose in PBS solution for 4 min and then transferred through a series of solutions containing decreasing concentrations (0.5, 0.4, 0.3, 0.2, and 0.1 m) of trehalose for 2 min each. Viability was evaluated according to the ability of the embryos to re-expand. Blastocysts with the highest morphology rank were selected for transfer and loaded into the transfer gun (Minitub). Holstein recipients were in estrus 6–8 days prior to transfer. In Exp. 1, all blastocysts (n = 15) re-expanded after the vitrification and warming processes and were transferred into the uteruses of 15 Holstein recipient cows. One recipient cow became pregnant and gave birth to a healthy calf. In Exp. 2, all blastocysts (n = 6) re-expanded after warming and were transferred, one each into the uterus of a recipient Holstein heifer. Three pregnancies are still ongoing. In summary, the results obtained demonstrate that plunging embryos in SOPSs into LN Slush in outdoor conditions offers a potential technique for embryo cryopreservation and transfers. Further field trails are required to examine the effect of recipient age (cow vs. heifer) and embryo breed (Charolais vs. Holstein) on the suggested procedure.