miRNA Sequencing and Differential Analysis of Testes from 1-Year-Old and 3-Year-Old Kazakh Horses.

This study aims to elucidate the miRNA regulatory mechanisms during the developmental process of Kazakh horse testes at 1 and 3 years of age. Through miRNA sequencing and bioinformatics analysis of testicular tissues from 1-year-old and 3-year-old horses, a developmentally stage-specific miRNA expression profile was constructed. A total of 1640 miRNAs were identified, among which 437 (380 up-regulated and 57 down-regulated) exhibited significant differential expression between the two age groups, including eca-miR-16, eca-miR-17, eca-miR-103, and eca-miR-199a-5p. Functional enrichment analysis revealed that the target genes of these differentially expressed miRNAs were primarily involved in key processes such as oxidative stress response, hormone receptor signaling regulation, and cytoskeletal remodeling, suggesting that testicular maturation depends on a complex post-transcriptional regulatory network. Further KEGG analysis revealed significant enrichment of classic reproductive signaling pathways, including PI3K/AKT, Wnt/β-catenin, Hippo, and TGF-β, indicating their synergistic roles in spermatocyte proliferation/differentiation and testicular homeostasis establishment. Although limited by a small sample size, this study elucidates the molecular mechanisms underlying male reproductive maturation in Kazakh horses at the post-transcriptional regulatory network level, providing preliminary theoretical support and potential markers for evaluating stallion reproductive performance and molecular breeding.