Analysis of Differential Gene Expression and Alternative Splicing in Ovaries of High- and Low-Prolificacy Sheep Using Nanopore Full-Length Transcriptome Sequencing

Abstract

Reproductive efficiency determines the economic benefits of the sheep industry, yet the molecular mechanisms underlying prolificacy remain incompletely understood. To investigate transcriptomic differences associated with sheep prolificacy, we performed Oxford Nanopore Technologies (ONT) full-length transcriptome sequencing on ovarian tissues collected during the estrous phase from high-prolificacy Small-tailed Han sheep and comparatively lower-prolificacy Wadi sheep (n = 3 biological replicates per group), with an average sequencing depth of approximately 6.3 Gb per sample. With screening thresholds of |log2FoldChange| > 1 and p < 0.05, transcriptomic analysis identified 457 differentially expressed genes (DEGs; 207 upregulated, 250 downregulated) and 1033 differentially expressed transcripts (DETs). In total, 55% of DETs exhibited expression changes independent of overall gene abundance, highlighting the potential role of alternative splicing (AS)-mediated post-transcriptional regulation. We detected 55,478 AS events and screened 96 significant differential alternative splicing (DAS) events (|∆PSI| > 0.1, p < 0.05) across 78 differentially spliced genes (DSGs). Functional enrichment showed DEGs were primarily associated with reproduction pathways (e.g., TGF-β, MAPK, and ovarian steroidogenesis), whereas DSGs were enriched in p53 signaling and ribosome pathways. Protein–protein interaction network analysis highlighted highly connected candidate genes, including INHBA, CYP19, TNFAIP6, TK1, RRM2, BIRC5, BCL2, ISG15, PCLAF, and MX1, potentially involved in follicular development and reproductive signaling. The results of this study enrich the full-length transcriptomic resources for Small-tailed Han sheep and Wadi sheep, and provide candidate genes and transcriptomic resources for further functional investigation of sheep prolificacy.