L-proline supplementation in the freezing medium enhances the viability and quality of bovine blastocysts after slow freezing and thawing.

L-proline (Pro) is a natural amino acid with known antioxidant and cryoprotectant activity. This study aimed to assess the impact of Pro supplementation in freezing medium on blastocyst survival and quality. In vitro fertilization (IVF) was conducted using oocytes collected from Korean cattle, and Day 7 blastocysts were cryopreserved through slow freezing. Optimal post-thaw blastocyst survival was determined by adding various Pro concentrations to the freezing medium. Additionally, the effect of Sucrose (Suc) alone or in conjunction with Pro was evaluated. To assess blastocyst quality, we analyzed reactive oxygen species (ROS) levels, apoptosis, and gene expression in blastocysts that survived 24 h after slow freezing-thawing. The hatching rate at 72 h was significantly higher in the 0.3 M Pro group than that in the 0 M group (p = 0.0466). The hatching rates at 48 and 72 h were significantly higher in the Pro group than in the Suc and Suc + Pro groups (48 h: Suc, p = 0.0037; Suc + Pro, p = 0.0052; 72 h: Suc, p = 0.0024; Suc + Pro, p = 0.0009). ROS levels and the apoptosis index were significantly lower in the Pro group than in the Suc group (p = 0.0099, and 0.0098, respectively). Furthermore, mRNA expression of HSPA1A was significantly lower in the Pro and Suc + Pro groups than in the Suc group (p = 0.0074, and p = 0.01174, respectively). Additionally, GCLC mRNA expression was significantly higher in the Pro group than in the Suc group (p = 0.0308). These findings indicate that Pro supplementation in a slow freezing medium enhances the viability and quality of embryos.