84 Improvement in blastocyst formation in vitro following exposure to cell-penetrating antioxidants at the morula stage

In vitro embryo production (IVP) is a useful technology for exploiting and conserving genetically superior animals. The present study aimed to improve blastocyst formation of fresh and vitrified-warmed bovine morulae (Day [D]6 post-IVF) by exposure to cell-penetrating antioxidants (DMT-SS31 and -mTP4). Bovine embryos were produced in vitro from slaughterhouse-derived oocytes after maturation and fertilization with frozen-thawed semen. Morulae were either vitrified or remained as such (fresh). Both fresh and vitrified-warmed morulae were cultured in the presence of SS31 and mTP4 up to the blastocyst stage (D8 post-IVF). The specific objectives were to determine (1) the internalization of SS31 and mTP4 in embryonic cells, (2) the beneficial effect of SS31 and mTP4 in embryo culture media on the blastocyst formation from fresh and vitrified morulae, and (3) the concentration-dependent effect of SS31 on the post-warmed blastocyst formation. In Experiment 1, bovine zygotes (D3 post-IVF; n = 5) and fresh morulae (D5 post-IVF; n = 10) were each exposed to 0.5 µM Alexa 635-conjugated SS31 or mTP4 in Charles Rosenkrans 1+amino acids (CR1aa) culture medium, for 30 min. A batch of morulae were allowed to culture in 100-µL droplets of CR1aa up to the blastocyst stage. Both Alexa 635-SS31 and -mTP4 penetrated the zygote and morulae. The fluorescence observed in morulae remained up to 32- to 64-cell blastocysts. In Experiment 2, The fresh and vitrified-warmed morulae were cultured with 0 (control), 0.5 µM SS31, or 0.5 µM mTP4 in 100-µL droplets of CR1aa culture medium. Chi-squared test revealed that the fresh morulae exposed to SS31 yielded more blastocysts than morulae without SS31 (70% vs. 57%, respectively; P < 0.005); however, the blastocyst formation in the vitrified-warmed morulae was nonsignificant (42% vs. 34%, respectively). In contrast, mTP4 generated more blastocysts in the vitrified-warmed morulae than morulae without mTP4 (52% vs. 38% respectively; P < 0.05); however, the blastocyst formation in the fresh morulae was nonsignificant (69% vs. 62% respectively). In Experiment 3, the concentration-dependent effect of SS31 on the blastocyst formation was tested. Both fresh and vitrified-thawed morulae were treated with 0, 0.5, 1, or 2 µM SS31 in 100-µL droplets of CR1aa culture medium. The blastocyst formation was observed in both fresh and vitrified-warmed morulae at D8 post-IVF. Regression analysis showed significant relationships between SS31 concentration and blastocyst formation in fresh (R² = 0.91, P < 0.05) and vitrified-warmed (R² = 0.96, P < 0.05) morulae. Tukey’s test demonstrated that SS31 (2 µM) in CR1aa culture medium yielded the maximum blastocyst formation in the fresh (42/56; 75%) and vitrified-thawed (51/93; 55%) morulae, compared with other concentrations (P < 0.05). In conclusion, the addition of cell-penetrating antioxidants (SS31 and mTP4) to embryo culture media may be useful for improving blastocyst formation in the fresh and vitrified morulae.

This research was supported by A-base funding from Agriculture and Agri-Food Canada, The Government of Canada.