37 Liquid preservation of bovine in vivo-derived embryos under field conditions

Commercial application of embryo transfer is dependent on the optimal storage of embryos. The most common and routinely used preservation technique is cryopreservation. Liquid preservation at temperatures around 4°C offers an alternative to cryopreservation for shorter storage periods. Damage to the embryos associated with freezing and thawing can be avoided. Other obvious advantages of liquid storage are that it is more simple, cheap, and does not require special equipment and liquid nitrogen, and therefore, it is applicable under field conditions. Furthermore, short-term storage also allows the use of recipients, which would be excluded when they are too early in their cycle compared to the age of the embryo to be transferred. In this study we examined the feasibility of a short-term nonfreezing method of preservation of bovine in vivo-derived embryos compared to cryopreserved and fresh embryos. Bovine embryos were collected from superovulated donor animals (n = 9, Simmental breed) using a standard protocol employing FSH. Artificial insemination was performed with different bulls. Transferable morulae and blastocysts (IETS code) were randomly distributed to the following groups: group 1, embryos for fresh transfer (fresh); group 2, embryos for cryopreservation and transfer after thawing (cryo); group 3, embryos for liquid preservation (2–3 days) and transfer after warming (liquid). Whenever possible, embryos collected from one flushing were allocated to all groups. Cryopreservation (1.5 M ethylene glycol, ViGRO^® Freeze Plus, Vetoquinol), recipient management (2 mL prostaglandin, Veyx^®forte, Germany at Day 3 or natural heat, both at Day 3 in relation to the superovulation schedule) and embryo transfer (Day 7 of the cycle) are routine procedures used at the AI centre. Liquid preservation was performed in straws with TCMair (HEPES buffered) medium supplemented with 25% fetal bovine serum (FBS) at 4°C for 2 to 3 days. At the day of transfer embryos were placed in phosphate-buffered saline plus 20% FBS and transported at 20°C. Statistical analyses were performed via an ANOVA followed by a Tuckey test. A P-value of 0.05 was considered as significant. In total, 117 transfers were performed resulting in the following pregnancy rates: fresh 69.9% (29/41), cryo 77.4% (37/45), liquid 43.5% (14/31). These data show the pregnancy rate after transfer of liquid preserved embryos was significantly reduced compared to those of the other groups. The pregnancy rates in relation to the developmental stage comparing morulae (IETS code 4) and blastocysts (IETS code 5 and 6, pooled as one group) differed significantly only at the morula stage. Liquid preserved morulae did show a significant reduction in the pregnancy rate after transfer (27.8%, 5/18) compared to those from the fresh (67.9%, 15/23) and cryo (68.4%, 24/30) group. However, at the blastocyst stage similar pregnancy rates were obtained (fresh: 75.6%, 14/18; cryo: 86.7%, 13/15; liquid: 80.0%, 9/13). These data indicate that at least at the blastocyst stage of in vivo-derived bovine embryos liquid preservation could be used for a short-term storage as an alternative to cryopreservation.

The financial support of Dr. Karl-Eibl-Stiftung is gratefully acknowledged.