All the chemicals and reagents were purchased from Sigma-Aldrich (St.Louis, MO, United States). MitoSOX Red mitochondrial superoxide indicator and JC-1 were obtained from Invitrogen by Thermo Fisher Scientific (Carlsbad, CA, United States). HO-1/HMOX-1 primary antibody was purchased from Proteintech (10701-1-AP, Proteintech, Illinois, USA).

Porcine ovaries were procured from a local abattoir and promptly transported to the laboratory within 3 h in a 0.9% (v/v) NaCl solution maintained at 37–39°C. Upon arrival, the ovaries were subjected to two washes with a 0.9% (v/v) NaCl solution. Medium-sized follicles (3–7 mm in diameter) were selected for the retrieval of cumulus-oocyte complexes (COCs) using an 18G needle and a 10 mL disposable syringe (32). Subsequently, the collected complexes were transferred to 15 mL conical tubes. COCs exhibiting evenly granulated cytoplasm and compact cumulus cell layers were selected, and 50–60 COCs were cultured in each well of a four-well dish (Nunc, Roskilde, Denmark) containing 500 μL of IVM medium and not covered with mineral oil. The IVM medium consisted of TCM199 (Invitrogen Corporation, Carlsbad, CA, United States) supplemented with 0.91 mM sodium pyruvate, 0.6 mM cysteine, 75 ug/mL kanamycin, 10 ng/mL epidermal growth factor, 10% (v/v) porcine follicular fluid, and 1 μg/mL insulin. During the initial 22 h of IVM, the COCs were incubated with 10 IU/mL equine chorionic gonadotropin (eCG) and 10 IU/mL human chorionic gonadotropin (hCG), followed by a subsequent 20 h incubation without these hormones. All IVM procedures were conducted in a humidified incubator (Astec, Fukuoka, Japan) maintained at 39°C with a 5% CO₂ atmosphere.

PA was conducted in accordance with a previously published protocol (33). Initially, metaphase II stage (MII) oocytes were collected following IVM and treated with 0.1% hyaluronidase for 1 min to facilitate the removal of cumulus cells through gentle mechanical pipetting. Subsequently, the MII oocytes were rinsed twice with an activation solution comprising 280 mM mannitol, 0.01 mM CaCl₂ and 0.05 mM MgCl₂ before undergoing activation in 2 mL of the same solution. Activation was achieved through the administration of two direct electrical pulses at 120 V/mm for 60 μs, delivered via a cell fusion generator (LF101; NepaGene, Chiba, Japan). Subsequently, the activated oocytes were cultured in PZM3 containing cytochalasin B within a humidified environment of 5% CO₂, 5% O₂, and 95% N₂ for 4 h (34). All embryos were subjected to electrical activation and underwent three washes with IVC medium droplets (containing 10 oocytes per 30 μL) fully covered with mineral oil. PA was initiated on day 0, with embryo transferring to fresh droplets on days 2 (48 h) and 4 (96 h) of development. The cleavage rates of the embryos were analyzed on the second day and categorized into five groups based on the number of cells: 1, 2–3, 4–5, 6–8 cells, and fragmented embryos. On the seventh day, embryonic development was quantitatively assessed by determining blastocyst formation rates across three groups categorized by blastocyst morphology: early, expanded, and hatched. To quantify cell numbers in blastocysts, all blastocysts displaying a blastocoel were stained with 10 μg/mL Hoescht-33342 for 10 min. Following staining, each blastocyst was mounted on a glass slide in a drop of 100% and examined under an epifluorescence microscope (TE 300; Nikon, Tokyo, Japan) at 200 x magnification. The cell numbers were manually counted, and blastocysts with a blastocoel and at least 20 cells were classified as blastocysts (34–36). Myo-Ins supplementation was introduced into the IVC media at concentrations of 0 (control), 5, 10, and 20 mM, without any prior research on its use. The optimal concentration for Myo-Ins supplementation was determined through analysis, as depicted in Supplementary Figure S1 and Supplementary Table S1.

As previously described (37) intracellular GSH and ROS levels were assessed following established procedures. Embryos at the 4–5 cell stage were selected from each experimental group on day 2 for analysis. Intracellular GSH (indicated by blue fluorescence) and ROS (indicated by green fluorescence) levels within the embryo cytoplasm were quantified utilizing CellTracker Blue CMF₂HC (Invitrogen) and H₂DCFDA (Invitrogen), respectively. The embryos were stained by incubating them in a solution of TLH-PVA medium containing either 10 μM CMF₂HC or H₂DCFDA for 30 and 10 min, respectively. Following this, the samples were washed three times with TLH-PVA before being transferred to 8 μL droplets of TLH-PVA. The levels of glutathione (GSH) and ROS were quantified using a fluorescence microscope (TE300; Nikon, Tokyo, Japan) equipped with ultraviolet filters (370 nm for GSH and 460 nm for ROS). The fluorescence intensity of each embryo was then assessed using Image J software and normalized against that of the control group.

The number of apoptotic cells in the blastocysts stained with TUNEL was determined using an in-situ cell death detection kit as previously described (38). On the seventh day, Myo-Ins-treated and control group blastocysts were washed three times in 0.1% PBS-PVA (PVS). The blastocysts were then fixed in 4% paraformaldehyde (PFA) in PBS at 25°C (room temperature (RT)) for 30 min. In the subsequent stage, the blastocysts underwent two washes in a solution containing 0.1% PVS, in conjunction with 0.1% Tween 20 and 0.01% Triton X-100 (v/v). Subsequently, the blastocysts were treated with 0.3% Triton X-100 in PBS for 1 h at 37°C in order to facilitate permeabilization. Subsequently, the TUNEL assay was conducted utilizing fluorescein-conjugated deoxy uridine triphosphate (dUTP) and terminal deoxynucleotidyl transferase (obtained from Roche, Mannheim, Germany) for 90 min at 37°C. Subsequently, the blastocysts were subjected to two further washes in 0.1% PVS, after which they were counterstained with 5 μg/mL Hoechst-33342 for 10 min at RT in order to visualize the nuclei.

Blastocysts on day 7 were cultured in PZM 3 supplemented with 2.5 μM 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) (Cat # T3168, Invitrogen, Eugene, OR, USA) for 30 min at 38.5°C in 5% CO₂. Membrane potential was determined by calculating the ratio of red fluorescence, which corresponds to activated mitochondria (J-aggregates), to green fluorescence, which corresponds to less activated mitochondria (J-monomers) (39). Images were captured using an epifluorescence microscope (TE300; Nikon, Tokyo, Japan), and Image J software was used to quantify the relative fluorescence intensities.

To examine the colocalization of the mitochondria and mitochondrial ROS, blastocysts were incubated with 500 nM MitoTracker Red CMXRos (Cat #M7512, Invitrogen, Eugene, OR, USA) and 0.4 μM MitoSOX red (Cat # 2201584, Invitrogen, Oregon, USA) at 38.5°C for 30 min (39, 40). The fluorescence signals were visualized using an epifluorescence microscopy (TE300; Nikon, Tokyo, Japan) and the relative fluorescence intensities were quantified using the Image J software.

Blastocysts (n = 20/group) were collected from the control and Myo-Ins treated groups and washed with DPBS before being stored in 1.5 mL microcentrifuge tubes (SPL Life Sciences, Co., Ltd., Pocheon, Gyeonggi-do, Republic of Korea) and placed at −80°C until analysis. For the extraction of total RNA, TRIzol reagent (TaKaRa Bio, Inc., Otsu, Shiga, Japan) was used. Following the manufacturer’s protocol, cDNA was synthesized using a 5× reverse transcription master mix (Elpis Bio, Inc., Chungcheongnam-do, Daejeon, Republic of Korea). For qRT-PCR, the synthesized cDNA (0.5 μg/μL) was mixed with 2× SYBR Premix Ex Taq (TaKaRa Bio Inc.) and 10 pmol of specific primers (Macrogen, Inc., Seoul, Republic of Korea). The primers used in this study are listed in Supplementary Table S2. A CFX96 Touch real-time PCR detection system (Bio-Rad, Hercules, CA, United States) was used for the qRT-PCR analysis. The reactions were initiated by pre-denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 57°C for 15 s, and extension at 72°C for 30 s. Data were collected during the extension phase of each cycle, and the relative quantification (R) value was calculated using the following equation: R = 2-^{[ΔCtsample − ΔCtcontrol]} (41). The expression levels of genes were normalized to RN18S as a control.

The immunofluorescence technique was employed in accordance with the methodology described by Yoon et al. (42) with certain modifications. In brief, 4% PFA in PBS was employed to fix the blastocysts for 30 min at RT. Subsequently, the blastocysts were permeabilized in 0.5% Triton X-100 at RT for 1 h, after which they were washed twice with 0.1% PVS. The Image-iT™ FX Signal Enhancer (Invitrogen, Carlsbad, CA, United States) was employed to treat the blastocysts for a period of 30 min at RT. Subsequently, they were incubated in PBS containing 3% BSA and 0.05% Tween 20 for 1 h and 30 min at RT. The blastocysts were incubated overnight at 4°C with a rabbit anti-HO-1/HMOX-1 primary antibody (10701-1-AP, 1:100 dilution in blocking buffer, Proteintech, Illinois, USA). On the following day, the blastocysts were washed three times with 0.1% Tween 20 and 0.01% Triton X-100 in 0.1% PVS (TTVS) at RT for 5 min. They were then incubated with the appropriate secondary antibody, goat anti-rabbit IgG (H + L) Alexa Fluor 488 (A11029, 1:200; Invitrogen Corporation, Carlsbad, CA, USA), for 2 h at RT. Following three washes in TTVS, the blastocysts were counterstained with Hoechst-33342 for 10 min and mounted on glass slides in an anti-fade mounting medium (Molecular Probes, Inc., Eugene, OR, USA). The stained blastocysts were analyzed using an epifluorescence microscope (TE 300; Nikon) equipped with UV filters. The ImageJ software was employed to quantify the fluorescence intensity of the stained blastocysts.

In experiment 1, a total of 954 embryos were used across three replicates to assess the effects of different concentrations of Myo-Ins (0, 5, 10, 20, 40, and 80 mM) on the in vitro development of porcine PA embryos. The embryos were cultured in IVC medium supplemented with the specified concentrations of Myo-Ins throughout the entire IVC period. Developmental competence was evaluated by measuring cleavage rate, blastocyst formation rate, and total cell number in blastocysts. Based on the results, high concentrations of Myo-Ins (40 and 80 mM) decreased the blastocyst formation rate, suggesting that these levels may negatively impact embryo development. Therefore, lower concentrations (0, 5, 10, and 20 mM) were selected for further experiments to optimize the in vitro development of porcine PA embryos. In experiment 2, 352 total embryos were used in three biological replicates to assess various concentrations of Myo-Ins (0, 5, 10, and 20 mM). We aimed to investigate whether Myo-Ins can reduce oxidative stress in early 4-cell staged PA embryos by monitoring intracellular GSH and ROS levels. In experiment 3, a total of 168 blastocysts were used across three independent replicates to evaluate the effect of different concentrations of Myo-Ins (0, 5, 10, and 20 mM) on apoptosis. The number of TUNEL-positive cells and cell number were measured to assess apoptosis levels in the blastocysts. In experiment 4, a total of 280 blastocysts were used in at least three replicates to investigate mRNA expression levels of antioxidant and mitochondrial related genes in porcine PA blastocysts. In experiment 5, a total of 281 blastocysts were used across three biological replicates to evaluate the (∆Ψm) in porcine blastocysts after PA with different concentrations of Myo-Ins (0, 5, 10, and 20 mM). In experiment 6, a total of 144 blastocysts were used across three biological replicates to assess mitochondrial distribution in porcine blastocysts after PA with different concentrations of Myo-Ins (0, 5, 10, and 20 mM). In experiment 7, a total of 167 blastocysts were used across three replicates to assess mitochondrial reactive oxygen species (ROS) levels in porcine blastocysts after PA with different concentrations of Myo-Ins (0, 5, 10, and 20 mM). In experiment 8, a total of 140 blastocysts were used across three replicates to investigate the effect of various concentrations of Myo-Ins (0, 5, 10, and 20 mM) on HO-1 protein levels after immunofluorescence assay.

Statistical analysis was performed using SPSS (version 12.0; SPSS, IBM, Armonk, NY, United States) and GraphPad Prism (GraphPad Software, San Diego, CA, United States). All the experiments were performed at least three times unless stated otherwise. Data were presented as the mean ± SEM. Percentage data (cleavage and blastocysts formation rates) and average data (intracellular GSH and ROS in 4–5 cell stage embryos, TUNEL assay in blastocysts, mitochondrial membrane potential, Mito tracker, MitoSoX, and relative gene expression levels) were analyzed using one-way analysis of variance. Statistical significance was set at p < 0.05.

The optimal concentration of Myo-Ins was investigated by adding different concentrations (0, 5, 10, 20, 40, and 80 mM) during IVC for porcine embryonic development following PA, as shown in Supplementary Figure S1 and Supplementary Table S1. Considering these findings, we selected the 5, 10 and 20 mM Myo-Ins treatment groups for this experiment. On day 2, the embryonic cleavage rate was evaluated in all the Myo-Ins treatment groups (82.7% ± 4.7, 82.0% ± 3.6, 85.0% ± 4.8%) compared with the control group (77.5% ± 3.1%) (Table 1) (Figures 1A,B). Myo-Ins treatment did not show any differences in the cleavage rate when compared with the control group. The blastocyst formation rates were found to be significantly increased (p < 0.05) in the 10 (50.9% ± 2.5%) and 20 mM (61.9% ± 6.7%) Myo-Ins treated groups compared to the control group (36.7% ± 1.7%) (Figures 1A,C). The supplementation of Myo-Ins at concentrations of 5-(37.0% ± 0.7%), 10-(38.7% ± 2.6%) and 20-(40.2% ± 3.2%) did not result in a significant increase in the average total cell number of blastocysts in comparison to the control group (40.5% ± 2.2%) (Table 1).

To investigate the antioxidative effect of Myo-Ins, we analyzed the intracellular GSH and ROS levels in 4–5 cell stage embryos cultured with Myo-Ins-supplemented medium on day 2 after PA (Figure 2). The 20 mM Myo-Ins group showed significantly higher (p < 0.05) intracellular GSH levels than those in the control group. Furthermore, embryos in the 10 and 20 mM treated groups displayed significantly lower (p < 0.05) intracellular ROS levels compared to than those in the control group.

To evaluate the quality of porcine PA blastocyst, the incidence of apoptosis and total nuclei were counted. Myo-Ins supplementation during IVC did not affect the total number of nuclei compared to the control group (Figures 3A,B). Nonetheless, the number of apoptotic nuclei and the apoptotic index significantly decreased (p < 0.05) in the Myo-Ins-treated groups compared to those in the control group (Figures 3A,C,D).

To investigate whether Myo-Ins supplementation during IVC after PA affects the expression levels of mitochondrial function-related genes and antioxidant pathway genes such as NRF2/HO-1, blastocysts from each group were analyzed (Figure 4). Myo-Ins significantly increased (p < 0.05) the expression of mitochondrial function-related genes, such as solute carrier family 2 member 1 (SLC2A1) and ATP synthase (ATP5F1A), in the 10 and 20 mM groups compared to the control group (Figure 4). Additionally, the mRNA transcript levels of NRF2, HO-1, and GCLC were significantly higher (p < 0.05) in the 20 mM Myo-Ins group than in the control group. SOD1 transcript levels were significantly higher in the 10 mM Myo-Ins-treated group than in the control group. However, no significant differences were observed in the other mitochondrial-related genes or antioxidant genes when compared to the control group (Figure 4).

Mitochondria are the main organelles responsible for ROS production, and any malfunction regarding them may compromise embryonic development (43, 44). Therefore, Myo-Ins supplementation in the IVC medium was assessed in three ways: mitochondrial membrane potential (MMP), mitochondrial distribution (Mitotracker), and mitochondrial ROS (MitoSox). Myo-Ins at 20 mM significantly improved (p < 0.05) mitochondrial dysfunction by enhancing MMP (Figures 5A,B). Moreover, the mitochondrial quantity also significantly increased (p < 0.05) in the 20 mM Myo-Ins-treated group compared to that in the control group (Figures 6A,B). In addition, mitochondrial oxidative stress was reduced (p < 0.05) in the Myo-Ins-treated groups compared to that in the control group (Figures 7A,B).

Previous study reported the antioxidant role of Myo-Ins against oxidative stress in boar sperm in which NRF2 gene expression was significantly increased compared to that in the control group. However, downstream genes of the NRF2 pathway have not been discussed before in porcine IVC (30). Therefore, to investigate whether Myo-Ins supplementation during IVC stimulates the NRF2/HO-1 pathway at the porcine blastocyst stage, we analyzed the expression of the HO-1 protein using immunofluorescence. The blastocysts treated with 20 mM Myo-Ins showed a significant increase (p < 0.05) in the level of HO-1 protein compared to that in the control group (Figures 8A,B).