29 Time-lapse analysis of bovine embryos derived after invitro fertilization from vitrified and fresh oocytes

Abstract

Oocyte vitrification enables the long-term conservation of female genetic resources. However, obtaining viable bovine embryos from vitrified oocytes has proven to be difficult. Embryo development is a dynamic process, and critical stages can go unnoticed with the use of traditional morphologic assessments. Therefore, the aim was to evaluate morphokinetic parameters in embryos derived from fresh versus vitrified oocytes. After 22h of IVM, oocytes were divided into (1) oocytes surrounded by multiple layers of cumulus cells (COCs; n=275) and (2) oocytes partially denuded, leaving only corona radiata (CR; n=178). Then, two-thirds of the CR oocytes were subjected to one of two vitrification protocols as follows: high concentration of cryoprotectants (CR-H; n=171; Ortiz-Escribano et al. 2016 Theriogenology 86, 635-641) and low concentration of cryoprotectants (CR-L; n=163; Ishii et al. 2018 J. Reprod. Develop. 6). After warming, vitrified oocytes were incubated ~2h in maturation medium. Invitro fertilization and culture were performed simultaneously for all groups. Four to eight zygotes from each group were assigned randomly to time lapse (Primo Vision; Vitrolife), and group culture was performed in leftovers as a control. Differences between groups in survival (intact zygotes after IVF), cleavage, and blastocyst rates were evaluated by logistic regression. Morphokinetic data (time to reach first (1-2 cells), second (3-4 cells), third (5-8), fourth (9-16 cells), fifth (>16 cells), cleavage, and blastocyst stage, and time in lag phase) were investigated. Survival rates in COCs (98%) and CR (96%) groups were not different; CR-H (87%) showed lower survival than COCs but similar survival to that of CR. Group CR-L (82%) had a lower survival rate than the rest of the groups (P<0.05). Higher cleavage rate (83%) was found in COCs compared with the rest of the groups. The CR and CR-H groups showed similar cleavage rate (65 and 55%, respectively), whereas CR-L had a lower cleavage rate (42%) than other groups (P<0.05). As expected, both vitrified groups showed lower blastocyst rates (4% for CR-H and 10% for CR-L) than fresh COCs (44%; P<0.05). Morphokinetics measures were affected by the treatments: time to reach first cleavage was similar for COCs (35.3h), CR (38.4h), and CR-L (34.8h), whereas CR-H was slower (42.4h). However, the fourth division was reached earlier by CR-H (51.7h), which was significantly faster than for CR-L (88.8 h; P<0.05) but similar for COCs (71.3h) and CR (60.7h). In conclusion, more morphokinetic data are needed to compare different vitrification methods and confirm whether time-lapse analysis can be used to predict blastocyst outcome after oocyte vitrification.