MIT-001 mitigates vitrification-induced mitochondrial stress and regulates F-actin reorganization, enhancing the survival and development of vitrified-warmed bovine blastocysts

In this study, we investigated whether MIT-001, a small-molecule reactive oxygen species (ROS) scavenger, improves the re-expansion and viability of bovine blastocysts in response to vitrification-induced mitochondrial dysfunction and stress. Therefore, this study aimed to analyze the protective effects of MIT-001 on the mitochondrial function of bovine blastocysts following vitrification warming. In this experiment, MIT-001 (0.1 μM) was allocated to three culture conditions based on treatment period: (I) warming only (WARM); (II) vitrification only (VITR); and (III) both vitrification and warming (VITR-WARM), compared with the control (Non-treated). Survival analysis of cryopreserved bovine blastocysts revealed that MIT-001 supplementation during the warming period (WARM group) significantly improved (<italic>p</italic> &lt; 0.01; Non-treated: 57.3 ± 2.3% vs WARM: 74.2 ± 7.3%) post-warm survival rates. It is noteworthy that surviving blastocysts in the WARM groups demonstrated significantly (<italic>p</italic> &lt;<italic> </italic>0.05) lower TUNEL positive cells (%) and a higher ratio of expanded blastocyst development compared to the other groups. Intracellular ROS, as well as mitochondrial and nuclear superoxide levels, were significantly reduced (<italic>p</italic> &lt; 0.001) in the MIT-001-treated WARM group, accompanied by enhanced mitochondrial activation (MitoTracker Orange staining). Simultaneously, mitochondrial membrane potential (MMP), assessed using JC-1 staining, was elevated, whereas a reduction in mitochondrial fission marker dynamin-related protein 1 (DRP1) expression was observed in surviving blastocysts from the MIT-001-supplemented WARM group (<italic>p </italic>&lt; 0.01). In addition, MIT-001 improved cytoskeletal stability by decreasing the aggregation thickness of filamentous actin (F-actin, <italic>p</italic> &lt; 0.001; Non-treated: 10.73 μm vs. WARM: 6.03 μm) in bovine blastocyst of the WARM group. Finally, the enhanced developmental potential of vitrified-warmed blastocysts was linked to increased phospho-p38 mitogen-activated protein kinase (MAPK) expression exclusively in the WARM group compared to the other groups. Consequently, MIT-001 mitigates cryopreservation-induced cellular stress by improving mitochondrial function and regulates F-actin stabilization to enhance the viability and developmental potential of vitrified-warmed bovine blastocysts. These findings highlight the potential of MIT-001 to support cellular recovery and developmental capacity during cryopreservation, suggesting that it may play an effective protective role in bovine blastocyst cryopreservation.