Expression profiles of circulating miRNAs in an endangered Piedmontese sheep breed during the estrus cycle.

Introduction: The preservation of locally endangered breeds is essential for maintaining ecosystem services that benefit both society and the environment. Reproductive fitness becomes a crucial consideration in this context. MicroRNAs (miRNAs) are small non-coding RNA molecules that play a key role in post-transcriptional regulation. Typically, they function within the tissues where they are produced. However, when they are released into extracellular fluid, they are referred to as circulating miRNAs (c-miRNAs). C-miRNAs may serve as potential biomarkers, whose profile changes under different physiological states. The purpose of this study is to establish a connection between distinctive variations in the expression of c-miRNAs and specific estrus cycle phases in Frabosana-Roaschina sheep, an endangered Piedmontese breed. Methods: Two trials, each involving 20 ewes with different reproductive efficiencies (nulliparous in the first trial and pluriparous in the second trial), were sampled on alternate days after synchronization for blood, saliva, and feces. Ultrasound scans were performed during the induced estrus cycle. The animals' behaviors were assessed through video recordings. Results: In the first trial, play behaviors were detected without sexual behaviors, whereas in the second trial, sexual behaviors were observed without play behaviors. Based on plasma trends of 17β-estradiol and progesterone and ultrasound images, two moments were identified for miRNAs analyses: the beginning of the follicular phase (day 2) and the beginning of the luteal phase (day 11). C-miRNAs of six representative animals from the second trial were sequenced. Analyses of the sequencing data have identified 12 c-miRNAs that were differentially expressed (DE) when comparing day 11 with day 2: five miRNAs were found to be upregulated, whereas seven miRNAs were downregulated. An enrichment analysis, based on predicted targets, using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases was performed. Many of these genes regulate reproductive pathways with the possible involvement of miRNAs. Finally, qRT-PCR was conducted to validate the DE miRNAs in all ewes. Differences in gene expression between the two sampling points and the two trials were observed, in line with existing literature. Discussion: Investigating the role of these miRNAs in regulating estrus could improve the reproductive performance and welfare of Frabosana-Roaschina ewes.