Reference microRNA selection for qPCR normalization in cryopreserved Bos taurus semen

The accurate quantification of microRNA (miRNA) expression in cryopreserved bovine semen is essential for studies aiming to identify fertility biomarkers. However, reliable normalization of qPCR data remains a challenge due to the lack of validated endogenous reference miRNAs for frozen–thawed semen samples. Sperm quality parameters, including motility, membrane integrity, acrosomal status, and DNA fragmentation, were assessed using CASA and flow cytometry. The stability of five candidate reference miRNAs was evaluated in both fresh and frozen–thawed semen samples obtained from ten Angus bulls. Although stability was assessed across fresh and frozen–thawed conditions, the main objective was to identify a reliable reference miRNA for normalization in cryopreserved semen. Expression stability was analyzed using four independent algorithms (ΔCt, geNorm, NormFinder, and BestKeeper), and a comprehensive ranking was generated by RefFinder. Cryopreservation induced significant disruption of the plasma membrane and acrosomal damage, while DNA integrity remained unaffected. Among the candidates, miR-25-3p consistently ranked as the most stable across multiple algorithms and was identified as the optimal reference miRNA by RefFinder. In contrast, U6 and miR-204-5p showed high variability and poor stability, suggesting their unsuitability for use as normalizers in this context. miR-25-3p is a reliable endogenous reference miRNA for qPCR normalization in cryopreserved bovine semen. These findings support the implementation of experimentally validated normalizers to improve the accuracy and reproducibility of miRNA expression studies in reproductive biotechnology.