microRNAs for qPCR Normalization Under Morphofunctional Conditions in Bovine Sperm (Bos taurus)

Cattle represents one of the most common and widely distributed categories of large ruminants, with well‐established production practices. Fertility is a key factor that significantly influences the success of this production. Studies have shown that microRNAs (miRNAs) present in sperm cells play a crucial role as regulators of processes related to sperm functionality. miRNAs quantification by qPCR is one of the most accurate and straightforward methods, but this technique requires data normalization, and there is no universal consensus on which miRNAs should be used. The present study aimed to identify suitable miRNAs normalizers for qPCR analysis of Bos taurus semen. To achieve this, normalization candidates were assessed under different semen quality conditions, considering sperm morphology and motility. A small nuclear RNA (U6) and six miRNA candidates (Let‐7c‐5p, miR‐100‐5p, miR‐25‐3p, miR‐26a‐5p, miR‐204‐5p, miR‐92a‐3p) were selected. The expression stability of each candidate was analyzed using four independent methods (delta Ct, geNorm, NormFinder, and BestKeeper), under the semen quality conditions. Additionally, a comprehensive stability analysis was conducted using RefFinder, for each condition individually and for the combined conditions. The results indicated that miR‐92a‐3p was the most stable reference miRNA for motility‐related analyses, while Let‐7c‐5p emerged as the best candidate for morphology‐focused analyses. As a normalizer to analyze samples concomitantly, Let‐7c‐5p was identified as the optimal normalizer, while miR‐26a‐5p was the least stable candidate. This study provides the first identification of miRNA normalizers for qPCR analysis of Bos taurus semen, enabling more accurate miRNA quantification in this biological matrix and species.