Isabella Manenti ^1* Ugo Ala ¹ Elisabetta Macchi ¹ Irene Viola ¹ Paola Toschi ¹ Paolo Accornero ¹ Mario Baratta ² Silvia Miretti ¹ Eugenio Martignani ¹

• ¹ Department of Veterinary Sciences, University of Turin, Turin, Italy
• ² Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parma, Emilia-Romagna, Italy

The preservation of locally endangered breeds is essential for ecosystem services affecting both society and the environment. Reproductive fitness becomes a crucial consideration in this regard. MicroRNAs (miRNAs) are small noncoding RNA molecules that play a key role in post-transcriptional regulation. They usually act in tissues, where they are produced, but when released in extracellular fluid, they are termed circulating miRNAs (c-miRNAs). C-miRNAs may serve as potential biomarkers, whose profile changes under different physiological states. The purpose of this work is to establish a connection between distinctive variations in c-miRNAs’ expression and specific estrus cycle phases in Frabosana-Roaschina sheep, an endangered Piedmontese breed. Two trials, each involving twenty ewes, with different reproductive efficiency (nulliparous in the first trial, 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. 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 luteal phase (day 11). C-miRNAs of six representative animals from second trial were sent to be sequenced. Analyses on the sequencing data have highlighted twelve c-miRNAs differentially expressed (DE), when comparing day 11 with day 2: five miRNAs resulted upregulated, whereas seven miRNAs are 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 used to validatethe DE miRNAs in all ewes was performed and differences in gene expression between the two sampling points and the two trials emerged, in line with literature. The investigation of these miRNAs’ involvement in the regulation of the estrus could improve reproductive performance and welfare of Frabosana-Roaschina.