Transcriptome Sequencing and Differential Expression Analysis of Ovaries from Kazakh Mares During Seasonal Quiescence and Estrus Activation.

Kazakh horses are typical seasonal breeders, and ovarian activity is markedly reduced during the non-breeding period. Hormonal stimulation can induce ovarian functional responses during seasonal anestrus; however, the early ovarian transcriptional changes associated with this response remain incompletely understood. In this study, ovarian transcriptome sequencing was performed in 12 Kazakh mares examined during the seasonal anestrous period. The mares were classified into a non-hormonally stimulated seasonal quiescent group (DB, n = 6) and a hormone-induced ovarian activation group (DY, n = 6) following cloprostenol and eCG treatment. RNA sequencing and bioinformatics analyses were conducted to identify differentially expressed genes and enriched biological pathways. Using false-discovery-rate-corrected criteria (FDR < 0.05 and |log2FC| > 1), a total of 2119 differentially expressed mRNAs and 530 differentially expressed lncRNAs were identified between the two groups. FDR-corrected GO and KEGG enrichment analyses revealed significant changes associated with immune regulation, cellular communication, metabolic processes, and reproductive functions. KEGG pathway analysis demonstrated significant enrichment of complement and coagulation cascades, cytokine-cytokine receptor interaction, chemokine signaling pathway, PI3K-Akt signaling pathway, and ovarian steroidogenesis-related pathways (q ≤ 0.05). C1QB, CREB5, and IRS1 were prioritized as transcriptomic candidate genes based on their differential expression and pathway associations, but their functional roles were not experimentally tested. RT-qPCR analysis of selected DEGs showed expression trends concordant with the RNA-seq results and was used as a technical consistency assessment. Because the DY group was pharmacologically induced during seasonal anestrus and tissues were collected shortly after ultrasound confirmation of ovarian activation, the observed transcriptional profile should be interpreted as an early hormone-responsive state rather than a stable estrous state. Furthermore, because untreated naturally cycling mares during the reproductive season were not included, endogenous seasonal effects cannot be distinguished from pharmacological effects of hormonal stimulation.