Hydroxycinnamic and Hydroxybenzoic-Based Mitochondriotropic Antioxidants Improve Bovine Embryo Quality and Cryo-Survival

Assisted reproductive technologies (ART) use has increased over the past decades. However, reports concerning ART's low efficiency continue to emerge, citing causes related to lower embryo quality and pregnancy rates compared to their in vivo counterparts. One of the setbacks of ART is oxidative stress, which can impair embryo developmental rates. Mitochondrial redox and energetic homeostasis determine both cell survival and death, so mitochondria are a key target for therapeutic intervention strategies. In the present work, our objective was to improve the quality of viable embryos by adding new mitochondria-targeted antioxidants in the embryo culture media to reduce oxidative stress. Two naturally derived antioxidants synthesized by our team, AntiOxBEN₂ and AntiOxCIN₄, based on hydroxybenzoic and hydroxycinnamic scaffolds, respectively, were studied in two different experimental protocols (here called experiments). The first experiment investigated the effects of the antioxidants on embryo development to determine their optimal concentrations. The first assay of the first experiment focused on the effects of AntiOxCIN₄ at concentrations of 1, 2.5, and 10 μM, while the second assay focused on the effects of AntiOxBEN₂ at the same concentrations. A control group without supplementation was run simultaneously. The second experiment aimed to compare the best concentrations of these antioxidant molecules in the embryo culture media and their effect on embryos' resistance to vitrification/warming. In each experiment, the embryos were morphologically evaluated, and the total and viable cell numbers were examined. Reactive oxygen species (ROS) and mitochondrial polarization were also evaluated using specific fluorescent dyes. In experiment 1, an increased embryo quality was identified by using 2.5 μM AntiOxCIN₄ (<i>p</i> = 0.03) and 2.5 μM AntiOxBEN₂ (<i>p</i> = 0.001). Moreover, blastocysts supplemented with 2.5 μM AntiOxCIN₄ had higher viability (<i>p</i> = 0.008), while those supplemented with 2.5 μM AntiOxBEN₂ presented a greater total cell number (<i>p</i> = 0.01). An improvement in embryo cryosurvival following the supplementation during the culture process with either antioxidant was identified in experiment 2, with superior expansion scores after vitrification/warming and culture (2.5 μM AntiOxCIN₄, <i>p</i> = 0.056 and 2.5 μM AntiOxBEN₂, <i>p</i> = 0.059). In conclusion, both AntiOxCIN₄ and AntiOxBEN₂ had a beneficial effect on embryo development and cryosurvival, suggesting a potential intervention to reduce oxidative stress in assisted reproductive technologies.