Dimethyl tyrosine conjugated cell-penetrating peptides improved bovine blastocyst formation in vitro

In vitro embryo production and cryopreservation are assisted reproductive technologies to propagate genetically superior cattle. The objective of this study was to improve blastocyst formation in fresh and vitrified D5 (IVF day = D0) bovine morulae exposed to dimethyl tyrosine (DMT) conjugated cell-penetrating peptides (CPPs; DMT-SS31 and DMT-mTP4, antioxidants). In Experiment 1, D3 and D5 embryos were exposed to either 0.5 µM Alexa 635 labeled-SS31 or -mTP4 in CR1aa medium, at 37 °C for 30 min. Half of the morulae were examined immediately, whereas the remainder were cultured to D8 (blastocyst stage). Localization of SS31 and mTP4 were observed in D3, D5 and D8 embryos. In Experiment 2, ROS generation in the presence of SS31 or mTP4 was determined in D5 morulae using H₂FFDA marker; both CPPs reduced ROS generation in morulae (P < 0.03). In Experiment 3, fresh and vitrified morulae were treated with SS31 or mTP4 (0.5 µM each) or nothing (control) in CR1aa medium, at 37 °C for 30 min. Formation of D8 blastocysts was reduced (P < 0.001) in vitrified versus fresh embryos and both SS31 and mTP4 produced more blastocysts than control (P < 0.05). In Experiment 4, concentration-dependent effects of SS31 (0, 0.5, 1 or 2 µM) on the blastocyst formation were assessed. In fresh and vitrified morulae, there were linear relationships between SS31 concentration and blastocyst formation, with 2 µM SS31 maximizing blastocyst formation (P < 0.05). In conclusion, cell-penetrating peptides in culture medium improved blastocyst formation of fresh or vitrified bovine morulae.