MicroRNA (miRNA) in Plasma Small Extracellular Vesicles (sEV) as Potential Early Indicators of Dairy Cow Subfertility

Pregnancy and live birth rates are commonly used metrics to define fertility in humans and animals. The impact of aberrant microRNA (miRNA) expression on fertility-related genes represents a significant knowledge gap in understanding post-transcriptional regulatory mechanisms associated with reproductive dysfunction. Identifying subfertility markers is therefore critical to the success of fertility intervention strategies, particularly in agriculture, where sustainable farming practices are linked to overall economic performance. Here, we explore the expression patterns and association of blood plasma small extracellular vesicles (sEV)-derived miRNA with Holstein-Friesian dairy cow (Bos taurus) subfertility. Small RNA-seq identified 14 differentially expressed plasma sEV-derived miRNAs (FDR < 0.05 and -logFC > 2) between divergent low fertile (LF) and high fertile (HF) primiparous dairy cows (n = 10/group) with known reproductive outcomes. qRT-PCR miRNA assay validation of these plasma sEV miRNA candidates isolated from a different sample population of young heifers (10 month old) (LF and HF; n = 8/group) confirmed that the abundance of miR-181b-2-3p was significantly higher in LF sEVs compared to HF sEVs [p value = 0.0093, relative expression ratio = 2.665 (2^-ΔΔCT; LF = 19.6095, HF = 7.35636)]. Our results suggest that circulating sEV miRNA may contribute, in part, to fertility traits in dairy cows. The association of miR-181b-2-3p with the subfertility phenotype suggests that this miRNA may serve as a putative early indicator of LF status.