An improved vitrification protocol for equine immature oocytes, resulting in a first live foal.

BackgroundThe success rate for vitrification of immature equine oocytes is low. Although vitrified-warmed oocytes are able to mature, further embryonic development appears to be compromised. ObjectivesThe aim of this study was to compare two vitrification protocols, and to examine the effect of the number of layers of cumulus cells surrounding the oocyte during vitrification of immature equine oocytes. Study designExperimental invitro and invivo trials. MethodsImmature equine oocytes were vitrified after a short exposure to high concentrations of cryoprotective agents (CPAs), or a long exposure to lower concentrations of CPAs. In Experiment 1, the maturation of oocytes surrounded by multiple layers of cumulus cells (CC oocytes) and oocytes surrounded by only corona radiata (CR oocytes) was investigated. In Experiment 2, spindle configuration was determined for CR oocytes vitrified using the two vitrification protocols. In Experiment 3, further embryonic development was studied after fertilisation and culture. Embryo transfer was performed in a standard manner. ResultsSimilar nuclear maturation rates were observed for CR oocytes vitrified using the long exposure and nonvitrified controls. Furthermore, a lower maturation rate was obtained for CC oocytes vitrified with the short exposure compared to control CR oocytes (P=0.001). Both vitrification protocols resulted in significantly higher rates of aberrant spindle configuration than the control groups (P<0.05). Blastocyst development only occurred in CR oocytes vitrified using the short vitrification protocol, and even though blastocyst rates were significantly lower than in the control group (P<0.001), transfer of five embryos resulted in one healthy foal. Main limitationsThe relatively low number of equine oocytes and embryo transfer procedures performed. ConclusionsFor vitrification of immature equine oocytes, the use of 1) CR oocytes, 2) a high concentration of CPAs, and 3) a short exposure time may be key factors for maintaining developmental competence.