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.

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.

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1. Introduction

Advances in assisted reproductive technologies (ART) have allowed novel solutions for germplasm preservation and infertility management [1,2]. Nonetheless, several constraints persist, with physicochemical or biological origins, impairing a broad application of these ART techniques. Currently, oxidative stress is considered a major constraint affecting the quality of embryos in in vitro production systems, as it has been proven to cause damage by oxidizing lipids, nucleic acids, and macromolecules, resulting in embryonic arrest or death [3,4,5,6]. This is a major problem for ART success [7,8,9,10,11,12].

In physiological conditions, Reactive Oxygen Species (ROS), namely hydrogen peroxide, play a critical role as signaling molecules [13,14,15], as well as by activating mitophagy, regulating the glutathione (GSH) pool, and leading to cellular differentiation [16,17,18]. Several ROS, including hydrogen peroxide (H_2_O_2_) and superoxide, are also involved in the immune and inflammatory cellular response [19,20,21] as well as in the reproductive function, interfering with the quality of the gametes and fertilization process. Specifically, H_2_O_2_ plays a role in sperm capacitation and acrosome reaction in a dose–response manner [22,23]. Due to their cytotoxic effects, the levels of ROS are highly controlled by endogenous antioxidants, namely SOD, which is responsible for transforming O_2_^−^ into H_2_O_2_ that in turn is converted by CAT or GPX into H_2_O and stable O_2_. However, the high developmental rate, coupled with the embryos’ high oxygen consumption, makes them a significant source of increased ROS production [10,24,25]. When antioxidant defenses are unable to compensate for an increased generation of ROS, biological effects can include the deactivation of different enzymes or even damage to macromolecular components, including DNA and proteins, or leading to carbohydrate oxidation and lipid peroxidation [14,21,26]. When this damage occurs in mitochondria, it can generate a detrimental cycle in the production of ROS, often ending in cell death [6,14,21].

Especially under pathological conditions, mitochondria are an important source of ROS [6,27,28]. This organelle generates ATP through oxidative phosphorylation (OXPHOS), essential for the main cellular activities [14,18,29]. In the embryo, OXPHOS is needed to produce energy for the essential developmental events, such as the first cleavages, genome activation, morula compaction, and blastocyst formation, emphasizing the need to control oxidative stress levels at this stage of development [11,30,31]. The oxidation of NADH and succinate by the electron transport chain (ETC) generates the electrochemical gradient necessary for ATP synthesis, but it can also result in incomplete oxygen reduction and superoxide anion formation, primarily at complexes I and III [14,32].

Several antioxidants displaying different chemical structures and targets have been used to control oxidative stress in different cells. For instance, the natural antioxidant melatonin is targeted to the embryo’s inner cell mass [33,34], achieving high results in the reduction in H_2_O_2_ and hydroxyl radical (^−^OH), as well as in the improvement of mitochondrial function and oocyte in vitro maturation in several species [33,34,35,36]. In addition, there are other interventions to reduce oxidative stress in mitochondria based on vitamins and metabolic cofactors, such as ascorbate, L-carnitine, folic acid, and CoQ10 [37,38,39,40]. However, these molecules have some associated problems due to the high levels of toxicity, lack of target specificity, or off-target toxicity to mitochondria [41]. On the other hand, MitoQ, a mitochondria-targeted antioxidant synthesized by coupling the ubiquinone core with the delocalized lipophilic cation and triphenylphosphonium (TPP^+^), was also tested during IVF procedures. MitoQ can permeate the negative lipid bilayer of the mitochondria, being oxidized in complex II [42,43], exhibiting beneficial effects, such as promotion of oocyte maturation by reducing ATP and ROS levels, inducing autophagy, decreasing mitochondrial membrane potential (MMP), and increasing thermogenesis [34]. When supplemented to embryo culture medium, MitoQ did not affect the rate of development to the blastocyst stage [3]. However, MitoQ causes toxicity on the mitochondrial bioenergetic apparatus, even at low concentrations [44,45], and cannot contribute to the inhibition of iron toxicity [44,46]. Therefore, novel therapeutics are needed to minimize oxidative stress, especially when artificially produced during in vitro fertilization and embryo production techniques [3,47,48,49].

We previously developed several scaffolds inspired by dietary antioxidants as promising strategies to target excessive ROS production in different cell lines, such as the two antioxidants AntiOxCIN_4_ and AntiOxBEN_2_, synthesized from caffeic and gallic acids, respectively [44,45]. Moreover, promising results were obtained through the supplementation of AntiOxBEN_2_ during the maturation of bovine oocytes [50] and also during the capacitation and fertilization processes [23]. In the present work, our objective was to test, for the first time, AntiOxCIN_4_ and AntiOxBEN_2_ supplementation in the embryo culture media to study their effects on embryo developmental competence, mitochondrial function, and cryosurvival. The cleavage and blastocyst rates, as well as the quality of the embryos and the number of viable embryos available for transfer (both fresh and post-vitrification), were evaluated. The results suggest that these two antioxidants, which were developed by our team to reduce mitochondrial oxidative stress, could be a promising new therapeutic strategy for ART.

2. Materials and Methods

2.1. Chemicals and Reagents

Cell culture medium, water for embryo culture (W1503), media components, chemicals, and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.

2.2. Synthesis of AntiOxCIN4 and AntiOxBEN2

The synthetic strategy and procedures used for the synthesis of the mitochondriotropic antioxidants AntiOxCIN_4_ [44] and AntiOxBEN_2_ [45] have been previously described. Briefly, the synthesis of the mitochondria-targeting antioxidants AntiOxCIN_4_ (derived from caffeic acid) and AntiOxBEN_2_ (derived from gallic acid) has been performed by binding the natural molecule to a TPP cation. This binding ensures the antioxidants’ specific targeting and accumulation in mitochondria.

2.3. Oocyte Collection and In Vitro Maturation

Bovine ovaries were collected from a local slaughterhouse at Santarém and kept at 35–37 °C, in a phosphate-buffered saline (PBS) supplemented with 0.15% of bovine serum albumin (w/v, BSA) and 0.05 mg mL^−1^ of kanamycin. From each ovary, follicles of 2–8 mm in diameter were aspirated with a 19-gauge needle. Oocytes with at least three layers of compact cumulus cells and an evenly granulated cytoplasm were washed and selected for maturation [51]. Maturation was accomplished for 22–24 h, in an incubator at 38.8 °C with humidified air and 5% CO_2_. The maturation medium was composed of tissue culture medium (TCM) 199 (M4530) with 10% of fetal bovine serum, 0.2 mM sodium pyruvate, 10 ng mL^−1^ of epidermal growth factor, and 10 µL mL^−1^ of gentamicin [50].

2.4. Oocyte Fertilization and Embryo Culture

Oocyte fertilization was performed with frozen–thawed semen from the same Holstein bull following the Percoll procedure. In vitro fertilization medium consisted of modified Tyrode’s medium supplemented with 5.4 USP mL^−1^ heparin, 10 mM penicillamine, 20 mM hypotaurine, and 0.25 mM epinephrine. Sperm concentration was adjusted to 2 × 10^6^ spermatozoa mL^−1^ [52]. Sperm and oocytes were co-incubated for 22 h (IVF = day 0). Then, presumptive zygotes were placed into droplets (25 µL, 25 each) of SOF supplemented with BME and MEM amino acids, glutamine, glutathione, BSA, and antioxidants, according to the experimental design, layered with mineral oil, and cultured at 38.8 °C in a humidified atmosphere with 5% O_2_, 5% CO_2_, and 90% N_2_. After assessing cleavage at 48 h (evaluated by examining the embryos; those with two or more cells were considered cleaved), cleaved embryos were transferred to SOF plus 10% FCS, supplemented or not (control) with antioxidants to proceed with development during 8 to 10 days. Cleavage (2–4 cell embryo) and day 7/8 embryo (morula and blastocysts, with most embryos at the blastocyst stage) development rates were calculated as a proportion of inseminated oocytes and cleaved embryos, respectively. Embryo quality was evaluated based on their morphology and structural integrity and categorized into one of four quality grades (adapted from Lindner and Wright, 1983 [53]). Grade 1 embryos were considered excellent. They had a uniform, symmetrical, and compact structure, homogeneous cytoplasm, and no visible defects. Grade 2 embryos were considered good. They exhibited minor irregularities, such as slight variations in blastomere size or shape, but remained viable. Grade 3 embryos had fair quality, showing some fragmentation or uneven blastomeres, which could reduce their developmental capacity. Grade 4 embryos were classified as dead or degenerated and presented lysed or collapsed blastomeres and clear signs of degeneration, rendering them nonviable [53].

2.5. Assessment of Embryo Viability and Oxidative Stress

The protocol for the assessment of embryo viable cells was adapted from Romão et al. [54] using the fluorescent DNA staining dye (Hoechst 33342, B2261, Sigma-Aldrich, St. Louis, MO, USA) to stain cell nuclei in the fixed cells, allowing to count total cell number. Concomitantly, the embryo was stained with Propidium Iodide (PI, P4170 Sigma-Aldrich, St. Louis, MO, USA), a dye that penetrates the damaged membranes of dead cells (red fluorescent cells). The number of damaged and total cells was used to calculate embryo viability percentage.

The quantification of ROS level was performed with CellRox (C10444, Thermo Fisher Scientific, Waltham, MA, USA) through the quantification of the cell-permeant dye that exhibits fluorescence upon oxidation by ROS. Briefly, excellent, good, and fair embryos on day 9 of development or vitrified/warmed embryos after 24 h of culture were selected and transferred to SOF plus 20% FCS. Then, embryos were washed in PBS, moved to 100 µL of Pronase containing droplets for the digestion of the zona pellucida, for 3 min, and to a droplet of Tyrodes medium for another 1.5 min to finalize the removal of the zona pellucida. Finally, embryos were washed in PBS and incubated in SOF + 20% FCS plus 5 µM CellRox for 1 h in the dark, at 38.8 °C in a humidified atmosphere with 90% N_2_, 5% O_2,_ and 5% CO_2_. Afterward, embryos were fixed in a 4% paraformaldehyde solution for 1 h and then transferred to a solution of 50 µg mL^−1^ of Hoechst 33342 and 5 µg mL^−1^ of PI in SOF + 20% FCS for 30 min. Ultimately, 2 µL of Mowiol (Calbiochem 475904, Merck, Darmstadt, Germany) were placed on a glass slide with each stained embryo and covered with a coverslip. The mounted slide was kept in the dark and refrigerated for a couple of hours being observed by florescence microscopy (Olympus BX51 (Olympus, Tokyo, Japan)), with an excitation wave of 450 nm collected in the blue, 461 nm in the ultraviolet and 540 nm in the red fluorescence channels (BP 470–490, objective UPlanFI 20 × 0.50) to acquire the fluorescence of the CellRox, Hoechst and PI, respectively (Figure 1). Images were processed by ImageJ software (v. 1.54p (National Institutes of Health, Bethesda, MD, USA)) (adapted from Romão et al., 2013) [54,55].

2.6. Assessment of Mitochondrial Membrane Potential

The lipophilic cation fluorescent probe, the 5, 5′, 5′-tetrachloro-1, 1′, 3, 3′- tetraethylbenzimidazolcarbocyanine iodide (JC1, T4069 Invitrogen, Carlsbad, CA, USA), can penetrate selectively into the membrane, being potential-dependent. When the membrane presents a high MMP, JC-1 has a big accumulation forming J aggregates emitting red fluorescence, an emission with a maximum of 590 nm. Conversely, when the mitochondria have a low MMP, JC1 accumulates as a monomer emitting green fluorescence, an emission with a maximum of 529 nm [50,56].

On day 9 of development, the transferable embryos achieving at least the blastocyst stage were selected and incubated with 5 µg mL^−1^ of JC1 in Hank’s solution for 20 min at 38.8 °C and 5% CO_2_. Then each embryo was placed on a warmed slide and observed in a fluorescence microscope (Olympus BX51) with an excitation wave of 450 nm collected in the blue fluorescence channels (BP 470–490, objective UPlanFI 20 × 0.50) (adapted from Ferreira et al., 2026) [50]. Images were processed by ImageJ. The intensity of red and green fluorescence and the ratio between the two colors (r = red/green) were calculated.

2.7. Embryo Cryopreservation

For cryopreservation, the vitrification protocol was adapted from Pereira et al. [57] using glycerol and ethylene glycol (EG) as permeant cryoprotectants. This protocol allows the direct transfer of the embryos to the recipient cows. Briefly, grade 1, 2, and 3 blastocysts were selected and washed in TCM supplemented with 20% newborn calf serum (NBCS) previously equilibrated at 38.8 °C, and 5% CO_2_. Then the protocol proceeded at room temperature, in three steps as follows: 10% glycerol for 5 min, 10% glycerol and 20% EG for 5 min, and finally 25% glycerol and 25% EG for 30 s, in TCM–NBCS. During the last step, embryos were quickly aspirated into the center of a 0.25 mL plastic straw (IVM, L’Aigle, France) within 20–30 µL of vitrification solution. Embryos were separated by two air bubbles from two surrounding segments of TCM–NBCS containing 0.8 M galactose. The straws were sealed and immediately plunged directly into liquid nitrogen. For warming, the straws were removed from the liquid nitrogen and warmed up in air (5 s), and then in water at 22 °C (15 s), followed by the cutting of the tip of the straw to empty the embryo into a Petri dish still immersed in the vitrification medium and galactose, at room temperature. The embryo was transferred to the TCM–NBCS at room temperature and then to the same medium at 38.8 °C [57]. Embryos were evaluated for their integrity and expansion (expanded, semi-expanded, or shrunken) rate. After the warming and evaluation protocol, embryos were placed in culture medium (SOF + 10% FCS) in an incubator at 38.8 °C, 90% N_2_, 5% O_2_, and 5% CO_2_, for 24 h. Then, embryos were morphologically evaluated (expanded, semi-expanded, or shrunken) [58] and total and viable cells were examined, as well as ROS levels and the mitochondrial state, through the membrane potential evaluation, using specific fluorescent dyes, as described above.

2.8. Experimental Design

To study the effects of the antioxidants AntiOxCIN_4_ and AntiOxBEN_2_ on bovine embryonic development, two experiments were performed. The first experiment studied the effects of the antioxidants (assay 1: AntiOxCIN_4_ and assay 2: AntiOxBEN_2_) on embryo development and quality to determine their optimal concentrations. These molecules were added to the embryo culture media at the concentrations of 0 (control), 1, 2.5, and 10 µM (based on previous doses successfully applied during oocyte maturation [59] (Figure 2)).

A total of 3925 oocytes were collected over sixteen sessions for the first experiment (8 sessions per assay). In each session, bovine oocytes were kept in the maturation medium for 22 h, in an incubator at 38.8 °C with humidified air and 5% CO_2_ and then inseminated with frozen-thawed semen (Day 0 = D0). After 20 h of co-incubation, the presumptive zygotes were randomly distributed into the groups. For the first assay, four groups were constituted: control (n = 295), 1CIN (n = 256), 2.5CIN (n = 317), and 10CIN (n = 298), corresponding to 0, 1, 2.5, and 10 µM of AntiOxCIN_4_ supplemented to the embryo culture media, respectively. The second assay used the same concentrations but with the AntiOxBEN_2_: control group (n = 442) and 1BEN (n = 372), 2.5BEN (n = 377), and 10BEN (n = 422) groups, corresponding to 0, 1, 2.5, and 10 µM of AntiOxBEN_2_, respectively, added to the embryo culture media. In each assay, embryo development was evaluated morphologically (cleavage and blastocyst production rates and quality). The transferable blastocysts (grades 1, 2, and 3) were analyzed further to determine total cell number (Hoechst dye), viability (PI dye), MMP (JC-1 dye), and ROS production (CellRox dye). The percentage of viable cells and the total number of cells were calculated using 31 blastocysts (control, n = 6; 1CIN, n = 8; 2.5CIN, n = 7; and 10CIN, n = 10) and 25 blastocysts (control, n = 5; 1BEN, n = 6; 2.5BEN, n = 8; and 10BEN, n = 6), which were stained with Hoechst and PI. JC-1 dye was used to analyze mitochondrial membrane potential in 32 blastocysts (Experiment 1, first assay: control, n = 8; 1 CIN, n = 10; 2.5 CIN, n = 8; and 10 CIN, n = 6) and 15 embryos (Experiment 1, second assay: control, n = 4; 1 BEN, n = 4; 2.5 BEN, n = 4; and 10 BEN, n = 3). Finally, to estimate oxidative stress in the embryos’ cells, the CellROX dye was applied to 18 embryos in the first assay (control, n = 6; 1CIN, n = 4; 2.5CIN, n = 3; and 10CIN, n = 5) and 17 embryos in the second assay (control, n = 5; 1BEN, n = 3; 2.5BEN, n = 5; and 10BEN, n = 4) of experiment 1.

A second experiment was conducted to compare the best results of the previous two assays and to further investigate the impact of the selected doses of the two antioxidants on embryo cryosurvival. In this experiment, during 12 sessions, 2033 presumptive zygotes were divided into three groups: control (n = 657), 2.5CIN (n = 751), and 2.5BEN (n = 625), where embryos were supplemented during culture as above. The cleavage rate at 48 h post-insemination and embryos on day 8 (number and quality) were evaluated, as above. Then only transferable embryos (grades 1, 2, and 3) were vitrified (control, n = 25; 2.5CIN, n = 25; and 2.5BEN, n = 30). After warming, the integrity and expansion of the embryos were evaluated. These embryos were maintained in culture for 24 h. Then, their expansion rates, total and viable cells (control, n = 7; 2.5CIN, n = 4; and 2.5BEN, n = 5), mitochondrial membrane potential (control, n = 7; 2.5CIN, n = 5; and 2.5BEN, n = 9), and levels of ROS (control, n = 7; 2.5CIN, n = 4; and 2.5BEN, n = 5) were measured, as in the previous experiments, to signal the best antioxidant performance in vitrified-warmed embryos.

2.9. Statistical Analysis

Data from embryo development and quality and post-warmed integrity and expansion were analyzed with the Proc GLIMIX from SAS 9.4 M9 (Statistical Analysis Systems, SAS Inst., Inc., Cary, NC, USA) using the binomial distribution and logit as a link function. The statistical test was the residual PL. Analysis of embryo total and viable cells, CellRox, and JC1 data was processed with the Proc MIXED model from SAS using restricted maximum likelihood (REML). Sessions were considered as a random effect and the treatment as a fixed effect. The Mann–Whitney U Test was used to compare embryo expansion analysis (expansion, semi-expansion, and shrunken) between groups.

The results were considered statistically significant when p ≤ 0.05.

3. Results

3.1. AntiOxCIN4 and AntiOxBEN2 Dose-Dependently Improved Embryo Quality

The supplementation of 1 and 2.5 µM of AntiOxCIN_4_ to the embryo culture media tended to improve the cleavage rate (p = 0.09, Figure 3). No statistical differences were found in the rate of day 7/8 embryos when the culture media were supplemented with AntiOxCIN_4_ or AntiOxBEN_2_ (Figure 3). However, significant effects of both antioxidants on the quality grades of the produced embryos were identified (p ≤ 0.03; Figure 4 and Figure 5).

In the first assay, the control group had a higher rate of poor-quality embryos (Grade 4) compared to the 2.5CIN and 10CIN groups (p = 0.03 and p = 0.01, respectively). In addition, the 10CIN group had fewer poor-quality embryos than the 1CIN group (p = 0.05) (Figure 4).

In the second assay, the 2.5BEN group had more good-quality embryos (p = 0.005) compared to the other groups. Moreover, the 1BEN and 2.5BEN groups tended to have fewer poor-quality embryos (p = 0.08) compared to the control and 10BEN groups (Figure 5).

3.2. AntiOxCIN4 and AntiOxBEN2 Did Not Disturb the Mitochondrial Membrane Potential of Viable Embryos

In the present work, the red to green JC-1 fluorescence, as well as the ratio between them, was measured. However, no statistical difference was found among concentrations of both antioxidants (p > 0.05, Figure 6).

3.3. AntiOxCIN4 and AntiOxBEN2 Did Not Alter the Oxidative Balance on Viable Embryos

No statistical differences (p > 0.05) were found in ROS levels when embryo culture media were supplemented with either AntiOxCIN_4_ or AntiOxBEN_2_, and the viable embryos were stained with CellRox dye.

3.4. AntiOxCIN4 Enhanced Embryo Viability, and AntiOxBEN2 Increased Embryo Total Cell Number in a Dose-Dependent Manner

After staining the embryos with Hoechst 33342 and PI, the number of total and damaged cells was counted, and embryo viability was calculated. Embryo total cell number and viability have been used as an indicative measure of embryo quality and developmental potential after cryopreservation and transplantation by several authors [38,58,60]. In the present study, the supplementation of AntiOxBEN_2_ increased the total number of embryo cells at the concentration of 2.5 µM (Figure 7). In fact, the 2.5 BEN group had more total cells than the control group (p = 0.01).

On the other hand, embryo viability was higher in the 2.5CIN and 1CIN groups compared to the control group (p ≤ 0.01). Embryo viability was also higher in the 2.5CIN group than in the 10CIN (p = 0.03) (Figure 7).

The above-mentioned results obtained in the first experiment have indicated that 2.5 µM was the best concentration for both antioxidants, which should be used in the second experiment. In summary, in the first assay, the 2.5CIN group showed better embryo quality and viability. In the second assay, the 2.5 BEN group also had higher embryo quality and total cell number.

3.5. AntiOxCIN4 and AntiOxBEN2 Increased Embryo Cryosurvival

In experiment 2, no significant differences were achieved in cleavage and D8 embryo rates between groups (p > 0.05). However, more grade 2 embryos were produced by supplementing embryo culture media with a concentration of 2.5 µM of AntiOxCIN_4_ compared to the control (p = 0.01, Figure 8). No differences were found between groups for the other grades (G1, p = 0.51; G3, p = 0.44; G4, p = 0.36) (Figure 8).

After cryopreservation and storage of transferable embryos, they were warmed and evaluated. No differences were found for their immediate integrity (p = 0.58) and expansion (p = 0.37) rates. However, after 24 h of culture, better expansion scores were identified after adding 2.5 µM of AntiOxCIN_4_ (p = 0.056) or 2.5 µM of AntiOxBEN_2_ (p = 0.059) compared to control (Figure 9).

3.6. AntiOxCIN4 or AntiOxBEN2 Supplementation During Embryo Culture Did Not Disturb the Viability and Mitochondrial Function of Vitrified-Warmed Embryos

After culture, no statistical differences were found concerning embryo total and viable cells examined after staining with Hoechst and PI dyes, as well as in embryo ROS levels evaluated with CellRox dye (p = 0.73, p = 0.39, and p = 0.41, respectively). Also, no differences in the embryo mitochondrial state (JC1 dye) for either red or green emitted fluorescence, as well as for the ratio between them (p = 0.53), were identified.

4. Discussion

Our team has developed mitochondria-targeted derivatives of gallic and caffeic acid by conjugating them to TPP+ via an alkyl chain [44,45]. These cationic antioxidants are expected to accumulate in the mitochondria due to their negative inner membrane potential (Δψ), thereby enhancing protection against oxidative damage [28,45]. In this study, AntiOxBEN_2_ and AntiOxCIN_4_ were added to bovine embryo culture media to evaluate their ability to reduce oxidative stress and improve cryosurvival. For the first time, the results have demonstrated that supplementing in vitro produced (IVP) bovine embryos with these antioxidants improved cryotolerance, with both compounds at 2.5 μM enhancing embryo morphology and post-warming expansion. Since the birth of the first IVF baby, the use of assisted reproductive technologies (ART) has grown substantially to support animal and human reproduction, species and breeds preservation, and the generation of genetically superior animals [1,61,62,63]. However, IVP embryos still exhibit lower quality than their in vivo counterparts, largely due to mitochondrial dysfunction and oxidative stress [1,3,61,64,65,66]. Therefore, the oxidative stress therapy demonstrated in the present study is highly relevant and could help to close the quality gap between in vivo and in vitro embryos.

Several studies have shown that adding antioxidants to culture media improves embryo development and viability, such as Gardner and Sakkas (2023) [67] and Guseva et al. (2024) [68], which are consistent with the results presented here after AntiOxBEN_2_ and AntiOxCIN_4_ supplementation. On the other hand, Hosseini et al. (2009) [69] and de Mattos et al. (2022) [70] have reported that continuous supplementation with β-mercaptoethanol (βME), even after thawing, offers greater protection against oxidative stress. In the present study, antioxidant supplementation was only performed during embryo culture, and only transferable embryos were analyzed. Although higher expansion rates were observed after embryo vitrification, warming, and culture, no differences were measured in oxidative stress when measured using CellROX dye. Several authors have demonstrated that high ROS production led to embryonic cell death, rendering embryos nonviable [71,72]. These findings are consistent with the results presented here, which showed that the supplementation of AntiOxBEN2 (1 and 2.5 µM, p = 0.08) and AntiOxCIN4 (2.5 and 10 µM, p = 0.03 and p = 0.01, respectively) reduced the number of poor-quality blastocysts compared to control.

Dietary polyphenol antioxidants and derivatives, such as AntiOxBEN_2_ and AntiOxCIN_4_, can prevent and minimize oxidative stress, acting primarily as ROS scavengers, as well as by minimizing the formation of hydroxyl radicals dependent on metals, mainly through a chelation mechanism. They can also modulate enzymes that remove ROS and/or inhibit enzymes that produce ROS [44,45]. Previously, Ferreira et al. [50] and Santos et al. [23] have supplemented the maturation, the capacitation, and the fertilization media with AntiOxBEN_2,_ showing the positive effects of this mitochondriotropic antioxidant in gamete functionality. When added to the semen capacitation and fertilization media, the concentration of 1 µM AntiOxBEN_2_ has reduced ROS, increased MMP in spermatozoa, and improved embryonic development [23]. The supplementation of the maturation medium demonstrated that a concentration of 10 µM AntiOxBEN_2_ optimized nuclear maturation and increased the cleavage rate (79.4% versus 67.7% in the control group) [50]. There was also an increase in the number of copies of mt-ND5, which was indicative of mitochondrial biogenesis, without any significant alteration to ATP, ROS, or oxygen consumption rate levels [50]. In the present study, when added to the embryo culture media, this antioxidant improved embryo total cell number and morphological quality, as well as cryopreservation survival (higher expansion scores) at a concentration of 2.5 µM. This improvement in the quality of embryos with 2.5 µM of AntiOxBEN_2_ demonstrates that this antioxidant may be a good approach for IVP and embryo cryopreservation, since some authors have shown the correlation between the diameter of embryos and their cryopreservation survival based on the capacity for re-expansion and cell allocation [73,74,75]. Additionally, after transferring embryos with a higher total cell number, pregnancy rates have improved [38,60,76].

Concerning AntiOxCIN_4_, Ferreira et al. (2022) have supplemented semen capacitation and fertilization media with this mitochondriotropic antioxidant at concentrations of 0.1 and 1 μM, reporting that the supplementation with 1 μM AntiOxCIN_4_ during the bull sperm capacitation process improved some of the cinematic parameters of sperm, such as total and progressive motility, number of rapid spermatozoa, beat cross frequency and lower number of static spermatozoa, and that both concentrations (0.1 and 1 μM) increased the number of good quality embryos [77] as in the present study. AntiOxCIN_4_ also increased cell stress resistance in human hepatoma-derived cells (HepG2), induced through cell damage, by activating the Nrf2-p62-Keap1 axis, leading to up-regulation of antioxidant defenses, triggering macroautophagy and/or mitochondrial autophagy (mitophagy), and mitochondrial biogenesis [44,78]. Additionally, Teixeira et al. (2020) have supplemented the bovine oocyte maturation medium with concentrations of 10, 20, 50, and 100 μM of AntiOxCIN_4_, which was shown to dose-dependently improve oocyte maturation [59]. Recently, Teixeira et al. (2025) demonstrated that AntiOxBEN_2_ and AntiOxCIN_4_ counterbalance oxidative stress in primary human skin fibroblasts by activating ROS-protective mechanisms and concluded that, despite their similar chemical structure and antioxidant capacity, AntiOxBEN_2_ and AntiOxCIN_4_ display both common (redox-adaptive) and specific (bioenergetic-adaptive) effects [79].

In the present study, AntiOxCIN_4_ and AntiOxBEN_2_ have improved blastocyst quality in a dose-dependent manner. The reduction in poor-quality embryo production with 2.5CIN, allied to the improvement of embryo viability (68% when compared to 15% of the control group), with an increase in good-quality embryos with 2.5BEN, suggests that our antioxidants may mitigate oxidative damage and improve the intracellular environment during early development. Our results reinforce the importance of assessing embryo quality and not just developmental rates. Studies involving the mitochondria-targeted antioxidant MitoQ supplementation into the embryo culture medium showed that it also did not interfere with the development to the blastocyst stage [3]. Other studies have reported that MitoQ caused toxicity to the mitochondrial bioenergetic apparatus even at low concentrations [44,45] and could not inhibit iron toxicity [44,46]. Presented results indicate that our antioxidants had more promising effects in improving blastocyst quality, without cytotoxicity or alterations in ROS levels, while maintaining the functionality of mitochondria. Moreover, an increase in cryopreserved embryos’ survival, showing a better expansion after warming and culture without harming the performance of mitochondria or the viability of embryos, was shown in the last experiment. These results support the importance and effectiveness of these mitochondria-directed antioxidants in the embryo culture media, which is consistent with the results obtained by the above-mentioned authors.

More studies should be performed by simultaneously supplementing these antioxidants to the maturation and embryo culture media to further improve the cleavage and D8 embryo rates and viability, as performed by Tonekam et al. (2025) [80]. However, the effectiveness of each antioxidant on embryo production rates and quality depends on various factors, such as species, breed, age, and management of the gamete donor, the stage of development, and the concentration (high concentrations can be harmful) [1,52,68,81] that should be taken into consideration. Additionally, the supplementation of mitochondriotropic antioxidants prior to and during vitrification should also be tested, since Sprícigo et al. (2017) described a decrease in spindle damage, modulated apoptosis, and positively affected gene expression when the embryos were supplemented with resveratrol and/or L-carnitine during these processes [82]. Since no significant differences were found in the reduction in ROS in the present study, this issue could be further approached through molecular biology analyses, as shown in various types of cells where AntiOxBEN_2_ and AntiOxCIN_4_ have reduced oxidative stress; this effect was reinforced in the most recent study by Teixeira et al. (2025) [79]. Other beneficial effects of these antioxidants, inducing mitochondrial biogenesis and improving bioenergetic efficiency without cytotoxicity [50,59] maybe responsible for the identified higher embryo developmental competence and cryosurvival. Further field studies should be performed to transfer these produced embryos to recipient cows in order to evaluate AntiOxBEN_2_ and AntiOxCIN_4_ effects on pregnancy and calving rates, the ultimate goal of ART.

5. Conclusions

This study aims to investigate the effect of the two novel mitochondria-directed antioxidants, AntiOxCIN_4_ and AntiOxBEN_2_, during embryo culture as a solution to mitigate oxidative stress and improve embryo quality and cryosurvival. Our results showed for the first time that embryo supplementation with these antioxidants at a concentration of 2.5 µM increased the quality and cryosurvival of the produced blastocyst. Moreover, the blastocysts supplemented with 2.5 μM AntiOxCIN4 during culture exhibited higher viability, while those supplemented with 2.5 μM AntiOxBEN2 presented a greater total cell number. Therefore, the two antioxidants developed by our team are a promising novel therapeutic strategy to be used in ART.