51 Bovine embryos with distinct early morphokinetic pathways present different post-embryonic genome activation transcriptomic patterns and different cryotolerance

We recently developed and validated a methodology based on early morphokinetics to predict 6 categories of bovine embryos, including 4 categories of blastocysts: early hatching blastocysts (EHB), hatching blastocysts (HB), low hatching blastocysts (LHB), and arrhythmic blastocysts (AB) (Reis et al. 2018 Anim. Reprod. 15, 601). We hypothesised that different early morphokinetic pathways could (1) be accompanied by different biological patterns (EGA, etc.) and (2) influence interaction with environmental constraints (cryopreservation, etc.). The objective of this study was (1) to investigate whether transcriptomic differences between the predicted EHB, HB, LHB, or AB could be observed immediately after the fourth embryonic cycle (just after EGA); (2) to assess cryotolerance of these morphokinetic categories. Time lapses were produced during 4 days post invitro insemination (dpi) (Reis et al. 2018). Study 1: 128 embryos having finished the fourth embryonic cycle were individually dry frozen at 4.7 dpi, classified as EHB, HB, LHB, or AB, and pooled into four batches of eight embryos/category (total 16 samples). Total RNA was extracted, amplified using the SMARTseq V4 ultralow input kit (Clontech), libraries were prepared using the Nextera XT Illumina library preparation kit, and sequenced (paired-end 50-34bp) on an Illumina NextSEqn 500 instrument. Identification of differentially expressed genes was achieved using Limma package (R). The P-values were adjusted using the Benjamini and Hochberg false discovery rate (Saenz-de-Juano et al. 2014). Fold change >2 or