Transcriptome Sequencing and Differential Analysis of Testes of 1-Year-Old and 3-Year-Old Kazakh Horses

The Kazakh horse is an outstanding dual-purpose dairy and meat breed in China, characterized by early maturity, tolerance to coarse feed, and strong stress resistance. Previous studies have examined gene expression patterns in the testicular tissues of Kazakh horses at different age stages, but the molecular mechanisms regulating testicular sexual maturation remain unclear. To address this gap, this study conducted HE staining and in-depth transcriptome sequencing analysis of Kazakh horse testicular tissue before and after sexual maturity. HE staining showed that the G3 group had well-formed seminiferous tubule lumens, dense interstitial cells, and visible early spermatocytes and spermatozoa, indicating structural maturation. (G1 group: pre-sexual maturity; G3 group: post-sexual maturity), with four biological replicates per group (n = 4). Differentially expressed genes (DEGs) were called using the criteria of |log₂(fold change)| ≥ 1.5 and adjusted p-value ≤ 0.05. A total of 3054 differentially expressed genes (DEGs), including CABS1, RPL10, PGAM2, TMSB4X, and CYP17A1, were identified in the G1 and G3 groups. Among these, 402 genes showed upregulation and 2652 genes showed downregulation. GO annotation and KEGG enrichment analysis of DEGs revealed their predominant enrichment in the following categories: signaling pathways such as Focal adhesion, Pathways in cancer, and the PI3K-Akt signaling pathway. RT-qPCR validation confirmed the accuracy of the transcriptomic sequencing data. This study further elucidates the differentially expressed genes and associated signaling pathways in Kazakh stallion testes tissue before and after sexual maturity, providing a theoretical foundation and data reference for enhancing reproductive efficiency in equids and promoting biological processes such as testes development and spermatogenesis.