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

## TL;DR

Two new antioxidants improved embryo quality and survival after freezing in lab-based reproductive techniques.

## Contribution

Novel mitochondria-targeted antioxidants, AntiOxCIN4 and AntiOxBEN2, were developed and shown to enhance bovine embryo development and cryosurvival.

## Key findings

- 2.5 μM AntiOxCIN4 and AntiOxBEN2 improved embryo quality and viability.
- Both antioxidants enhanced embryo cryosurvival after vitrification/warming.
- Optimal concentrations were identified for each antioxidant in embryo culture.

## Abstract

The efficiency of assisted reproductive technologies (ART) remains suboptimal, partly due to oxidative stress, which has a negative impact on embryo development. As mitochondrial redox and energy homeostasis are essential for cellular function, the mitochondria represent a promising therapeutic target. This study evaluated two novel mitochondria-targeted antioxidants, AntiOxBEN2 and AntiOxCIN4, during embryo culture. Results showed that AntiOxBEN2 and AntiOxCIN4 improved embryo development and cryosurvival, highlighting their potential as therapeutic agents to mitigate oxidative stress in assisted reproductive technology.

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 (p = 0.03) and 2.5 μM AntiOxBEN2 (p = 0.001). Moreover, blastocysts supplemented with 2.5 μM AntiOxCIN4 had higher viability (p = 0.008), while those supplemented with 2.5 μM AntiOxBEN2 presented a greater total cell number (p = 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, p = 0.056 and 2.5 μM AntiOxBEN2, p = 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.