74 Dimethyl sulfoxide supplementation increases twin rates and SOX2, OCT4, and CDX2 protein presence in bovine embryos and demi-embryos

During the early embryo development, key genes such as SOX2 and OCT4 play an important role on pluripotency maintenance of inner cell mass and indirectly by CDX2 regulation on trophectoderm. Previous reports have shown that lower doses of dimethyl sulfoxide (DMSO) during in vitro maturation (IVM) significantly increase SOX2 and OCT4 gene expression (Ynsaurralde-Rivolta et al. 2020 Theriogenology 148,140–148). This effect could be used not only for in vitro embryo production but also in demi-embryo production as a strategy to improve its quality. The aim of this work was to study the effect of DMSO supplementation during the IVM on embryos and demi-embryos in the production rates and quality through regulatory pluripotency proteins presence. To this, cumulus-oocytes complexes (COCs) collected from slaughterhouse ovaries were in vitro matured in TCM 199 containing 10% FBS, 10 µg/mL FSH, 0.3 mM sodium pyruvate, 100 mM cysteamine, and 2% antibiotic-antimycotic, supplemented with 0.75% v/v DMSO for 22 h, at 6.5% CO₂ in humidified air and 38.5°C. IVF was performed with 16 × 10⁶ spermatozoa/mL for 5 h. Presumptive zygotes were cultured in SOF medium for 7 days at 38.5°C. After 24 h of culture, blastomeres of two-cell stage embryos were separated and each one was cultured individually in a microwell (n = 20) to day 7. Whole embryos were cultured in groups (n = 20). At Day, 7 embryos (control n = 20, DMSO N = 23) and demi-embryos (control n = 39, DMSO N = 43) were fixed in paraformaldehyde 4% and stored at 4°C. Immunofluorescence was performed to label nuclear presence of proteins SOX2, OCT4, and CDX2; cell nuclei were identified using DAPI. Cell numbers were analysed by ANOVA and percentages were arcsine transformed. Production rates were similar for embryos and demi-embryos (embryos: control 39% vs DMSO 41%; demi-embryos: control 37% vs DMSO 47%); however, the DMSO group obtained higher rates of twin embryos (74%^a vs 61%^b in control group) over embryos separated (P < 0.05). Embryos showed higher cell numbers than demi-embryos (embryos: control 94 and DMSO 101; demi-embryos: control 39 and DMSO 41) (P < 0.05). SOX2 positive cells were increased in embryos and demi-embryos from the DMSO group (31 and 24 in embryos and demi-embryos, and 15 and 18 in control embryos and demi-embryos) (P < 0.05). OCT4 positive cells were higher in the DMSO embryo group (73), followed by control (53), and then DMSO and control demi-embryos (37, 32) (P < 0.05). CDX2 positive cells were also higher in the DMSO embryo group (54), than in control embryos (31) and DMSO and control demi-embryos (24, 22) (P < 0.05). Interestingly, SOX2 % cells were increased in demi-embryos with respect to embryos in both groups (DMSO 58, control 52 demi-embryos vs DMSO 32, control 18 embryos). OCT4 and CDX2 % were lower in control embryos (59 and 35) than the other groups (DMSO embryos 75 and 54, control demi-embryos 85 and 61, and DMSO demi-embryos 85 and 62) (P < 0.05). The use of lower doses of DMSO during in vitro maturation result in a viable strategy to increase twin rates and the expression of regulatory proteins SOX2, OCT4, and CDX2.