Identification of the most stable reference gene for RT-qPCR analysis of vitrified-warmed bovine blastocysts:...

Accurate normalization using stable reference genes (RGs) is essential for RT-qPCR, particularly in single bovine blastocysts with limited RNA. To date, RG validation under vitrification and warming conditions remains limited. This study compared fresh control and vitrified-warmed in vitro produced bovine blastocysts to identify appropriate RGs for normalizing target gene expression in vitrification-related studies of bovine embryos. In the treatment group, embryos were vitrified at the morula stage, warmed, and cultured to the blastocyst before analysis, and blastocysts from both groups were analyzed at the single-embryo level. Cycle threshold values of candidate RGs (ACTB, GAPDH, H2A, HMBS, PPIA, SDHA, YWHAZ, and 18S) were analyzed for stability ranking using the comparative ΔCt method, geNorm, BestKeeper, and NormFinder, followed by comprehensive analysis with RefFinder. ACTB emerged as the most stable RG across both groups, whereas H2A exhibited the lowest stability. To validate the identified RGs, apoptosis-related genes (FOXO3a, BAX, and CASP3) were normalized using ACTB, H2A, and the traditionally used RG (GAPDH). When normalized with ACTB, CASP3 expression, a marker of the downstream apoptotic pathway, was significantly higher in the vitrified-warmed group than the control group, consistent with the increased proportion of TUNEL-positive apoptotic bodies observed in vitrified-warmed blastocysts. However, normalization with GAPDH or H2A abolished these differences or produced opposite expression patterns between the two groups. These findings reveal that appropriate RG selection is essential for reliable RT-qPCR analysis of vitrified bovine embryos and provide a framework for future studies to improve embryo cryopreservation efficiency.