Cyanidin supplementation during in vitro maturation improves bovine embryo development and blastocyst quality and is associated with a more favorable oocyte redox status.

Reactive oxygen species (ROS) generated during in vitro maturation (IVM) are considered major contributors to impaired oocyte maturation and subsequent embryo development. Consequently, supplementation of IVM media with antioxidant compounds has emerged as a promising strategy for improving embryo quality. This study investigated the effects of cyanidin supplementation during IVM on bovine oocyte maturation, subsequent embryo development, and blastocyst quality. Cumulus-oocyte complexes (COCs) recovered from Holstein cattle ovaries were morphologically evaluated, and only Category I COCs were included. In Phase I, oocytes were randomly allocated to four groups according to the concentration of cyanidin supplemented during IVM: 0 μM (Control), 10 μM (Cyn10), 50 μM (Cyn50), and 100 μM (Cyn100). The Cyn10 group exhibited the highest meiotic resumption rate and the lowest proportion of oocytes with Grade I cumulus expansion. ROS-associated fluorescence decreased and GSH-associated fluorescence increased with increasing cyanidin concentration in all treated groups (P < 0.001), with the most pronounced changes observed in the Cyn100 group; these changes, however, were not accompanied by improved embryo development. The blastocyst formation rate differed significantly among groups (P < 0.05) and was highest in the Cyn10 group (55.00%). Based on the combined Phase I findings, 10 μM was identified as the best-performing concentration among those tested and was selected for subsequent detailed analyses. In Phase II, oocytes assigned to the Cyn10 and Control groups underwent IVM, and relative transcript abundance was assessed using reverse-transcription quantitative real-time PCR (RT-qPCR). Transcript levels of GPX4, GDF9, GREM1, PTGS2, and mTOR were significantly higher in the Cyn10 group than in the Control group (P < 0.001). In Phase III, additional in vitro embryo production (IVEP) cycles were performed using the Cyn10 and Control groups, and Day 7-8 blastocysts were evaluated for oxidative status, cell allocation, and apoptosis. Cleavage (89.66% vs. 83.39%; P < 0.05), blastocyst formation (48.28% vs. 31.53%; P < 0.001), and blastocyst reaching rates (45.75% vs. 27.68%; P < 0.001) were higher in the Cyn10 group than in the Control group. Blastocysts from the Cyn10 group had numerically higher inner cell mass (ICM) cell numbers (53.57 ± 14.44 vs. 47.20 ± 17.50; P = 0.068), whereas trophectoderm (TE) cell numbers (92.97 ± 16.66 vs. 71.88 ± 18.73; P < 0.001) and total cell count (TCC; 146.53 ± 27.62 vs. 119.08 ± 34.09; P = 0.002) were significantly higher than those in the Control group. Conversely, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) analysis showed that the apoptotic cell number (8.10 ± 3.14 vs. 13.80 ± 5.06) and apoptotic index (5.64 ± 2.75% vs. 12.10 ± 4.77%) were significantly lower in the Cyn10 group (P < 0.001). Blastocysts from the Cyn10 group also exhibited significantly lower ROS-associated fluorescence (14.59 ± 7.45 vs. 25.57 ± 6.92) and higher GSH-associated fluorescence (32.89 ± 5.30 vs. 15.87 ± 4.50) than those from the Control group (P < 0.001). Collectively, among the concentrations tested, supplementation with 10 μM cyanidin during IVM was associated with the most favorable overall developmental profile, characterized by improved oocyte maturation and subsequent embryo development, favorable redox-associated changes, higher total blastocyst cellularity, and reduced apoptosis. These findings suggest that cyanidin supplementation during IVM may represent a promising approach for improving bovine IVEP outcomes.