Small noncoding RNAs play critical regulatory roles in development across organisms. This study profiled microRNAs (miRNAs) and tRNA-derived fragments (tRFs) during bovine conceptus elongation. Elongating conceptuses were obtained via superovulation of eight Angus heifers. Twenty samples from ovoid (OV, n = 6; 0.5–3 mm), tubular (TUB, n = 7; 5–15 mm), and filamentous (FIL, n = 7; 20–34 mm) stages underwent small RNA sequencing. Differential expression of miRNAs and tRFs was analyzed using DESeq2, accounting for donor-sire effects. No tRFs showed differential abundance across any pairwise comparisons. For miRNAs, the expressions of six miRNAs were upregulated in OV versus TUB conceptuses (padj < 0.05), including four let-7 family members (bta-let-7g, bta-let-7f, bta-let-7a-5p, and bta-let-7c) and two additional miRNAs (bta-miR-224 and bta-miR-449a). Furthermore, there were 3 miRNAs differently abundant between the ovoid and filamentous transition (padj < 0.04), including two members of the let7 family (bta-let-7g and bta-let-7f) and bta-miR-449a. Predicted targets of these differentially abundant miRNAs were identified using miRanda. Enrichment analyses of the targeted genes included pathways regulating cellular proliferation, pathways in cancer, and immune-related pathways. The let-7 family, along with miR-449a and miR-224, are candidate regulators of the balance between cellular proliferation and differentiation during elongation, based on their differential abundance and in silico target predictions.

Background/Objectives: Long non-coding RNAs (lncRNAs) regulate gene expression and developmental processes, yet their expression and potential functions during bovine conceptus elongation are poorly understood. This study characterized stage-associated lncRNA expression, genomic context, associations with neighboring protein-coding genes, and sex-associated expression during this developmental window. Methods: RNA-sequencing data were generated from bovine conceptuses collected at ovoid (n = 6), tubular (n = 7), and filamentous (n = 7) developmental stages and analyzed to identify differentially expressed lncRNAs. Stage-associated lncRNAs were classified by genomic relationships with nearby protein-coding genes, and correlations with neighboring genes were assessed to identify candidate local regulatory relationships. Coding potential and spliced-read evidence were assessed for each lncRNA. In individually sequenced tubular and filamentous conceptuses, sex was assigned using XIST and Y-linked gene expression, followed by assessment of sex-associated transcription. Results: Forty-two lncRNAs were differentially expressed during conceptus elongation. Most changes occurred between the ovoid and tubular stages and were maintained through the filamentous stage. Genomic-context classification and expression-correlation analyses identified four candidate local relationships between lncRNAs and neighboring protein-coding genes, one of which (MKLN1) was robust to adjustment for donor, consistent with potential cis-regulatory activity. Coding-potential analysis supported the non-coding status of these transcripts, although one sex-associated locus showed a clear protein-coding signature and is likely misannotated. Sex-associated analyses identified female-biased XIST expression and widespread female-biased X-linked transcription. Conclusions: This study provides the first characterization of lncRNA expression during bovine conceptus elongation. The identified stage-associated lncRNAs and candidate local regulatory relationships provide targets for functional investigation of mechanisms supporting conceptus development. Sex-associated transcriptional patterns further support incomplete X-chromosome dosage compensation during this developmental interval.