Exploratory Genome-Wide Computational Analysis of Candidate MiRNA Regulatory Variants in Thermotolerant Dairy Cows

Heat stress poses a critical threat to dairy cattle productivity, fertility and welfare, yet the genetic mechanisms underlying individual variation in thermotolerance remain poorly understood. While previous studies have focused predominantly on miRNA expression profiles or coding gene variants, genome-wide identification of variants within miRNA genes has remained largely unexplored. In this exploratory study, pooled whole-genome sequencing was applied to previously characterized thermotolerant and thermosensitive Holstein cows, classified based on reproductive performance under summer heat stress and supported by physiological and endocrine characterization, to identify candidate genetic variants within miRNA genes. Fifteen variants were detected in the thermotolerant sequencing pools but not in the thermosensitive pools under the applied variant-calling and filtering criteria, across 15 distinct miRNA loci, including one potentially novel SNP. Five variants were located within mature miRNA sequences, and several were associated with predicted changes in minimum free energy (ΔG), suggesting potential differences in pre-miRNA secondary structure and thermodynamic stability. Two seed-region variants in bta-miR-2883 and bta-miR-2388-3p were associated with large computationally predicted differences in miRNA target repertoires, with gains of approximately 3000 predicted target genes and losses of approximately 600 per miRNA. Functional enrichment analysis of the computationally predicted target sets identified over-represented biological processes and pathways related to stress-responsive signaling pathways and metabolism among gained targets, and intracellular trafficking, vesicle-mediated transport, and membrane dynamics among lost targets. Collectively, these computational findings identify candidate variants within miRNA genes and generate hypotheses regarding their potential regulatory consequences, without establishing their functional involvement in thermotolerance. Individual-level genotyping, genotype–phenotype association analyses, and functional validation are required to determine their biological relevance.