74 Gene expression analysis of developmental key genes in in vitro bovine twin embryos produced by blastomeres separation and embryo bisection

Bovine twin production is a technique to multiply embryos of high genetic value and an ideal model for research. Our previous work has shown differences in the cellular compositions between bisection twins compared with embryos produced by blastomere separations (Ynsaurralde et al. Reprod. Fertil. Dev. 31, 164 abst). However, it is not known whether both methods produce embryos with differences in the expression of key genes for embryo development and survival. In this work, we compared the gene expression pattern of SOX2, NANOG, OCT4, CDX2, IFNt, BAX, and BCL2 in monozygotic twin embryos produced by blastomere separation and embryo bisection fromIVF embryos and in vitro whole embryos with and without zona pellucida as control group. To this aim, cumulus-oocyte complexes (COCs) collected from slaughterhouse ovaries were in vitro matured in TCM 199 containing 10% fetal bovine serum, 10 µg mL⁻¹ FSH, 0.3 mM sodium pyruvate, 100 mM cysteamine, and 2% antibiotic-antimycotic for 24 h, at 6.5% CO₂ in humidified air and 38.5°C. IVF was performed with 16 × 10⁶ spermatozoa mL⁻¹ for 5 h. Afterwards, presumptive zygotes were cultured in synthetic oviductal fluid (SOF) medium for 7 days at 38.5°C and 5% O₂. As the microwell control, a group of presumptive zygotes without zona pellucida were individually cultured in microwells. After 24 h of culture, blastomeres of 2-cell stage embryos were separated and each was cultured individually in a microwell for 7 days. Embryo bisection was performed manually under stereoscope, on Day 7 blastocysts in which the zona pellucida was previously removed. Three pools of hemi embryos (N = 20) and embryos (N = 13) were stored in RNAlater at −20°C until RNA extraction. Two technical replicates were analysed. Total mRNA was extracted using the PicoPure RNA kit (ThermoFisher Scientific) and the data were analysed by ΔΔCT method using ACTB and GAPDH as endogenous control genes. Results were analysed using ANOVA followed by Tukey’s test (P < 0.05). Twin embryos produced by blastomere separation and control embryos showed higher expression levels of IFNt than twins obtained after embryo bisection and controls cultured in microwell. Control embryos cultured in microwells resulted in the lowest expression of the proapoptotic gene BAX, and hemi embryos showed the highest levels (P < 0.05). No statistical differences were registered between treatments (both controls and twins) with respect to SOX2, NANOG, OCT4, CDX2, and BCL2 (P > 0.05). Our results indicate that there is no difference in expression of key developmental genes among twins produced by blastomere separation and embryo bisection. This may indicate that both strategies are useful to produce healthy embryos. However, the statistical difference observed in the expression levels of IFNt could imply a difference in expression levels of genes related to establishment of pregnancy in advanced embryonic stages.