Application of circulating-miRNAs expression profiles in an endangered Piedmontese sheep breed during the oestrous cycle

The conservation of local and endangered livestock breeds is crucial to maintaining biodiversity and ecosystem services, while ensuring their resilience and adaptability to harsh environments. Within this framework, understanding the molecular mechanisms underlying reproduction and stress adaptation becomes essential for improving welfare and management strategies. MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression at the post-transcriptional level. When released into extracellular fluids, they are referred to as circulating miRNAs (c-miRNAs) and can serve as potential biomarkers of physiological states. This PhD project investigated the role of c-miRNAs in reproduction and stress response in sheep, combining studies on two breeds: the endangered Italian Frabosana-Roaschina and the Spanish Rasa Aragonesa. In the first part, the focus was on the reproductive performance of Frabosana-Roaschina ewes. Two trials involving twenty animals each—nulliparous in the first and pluriparous in the second—were conducted after oestrus synchronisation. Blood, saliva, and faecal samples were collected on alternate days throughout the induced oestrus cycle, alongside behavioural observations and ultrasound scans. Hormonal analyses (17β-oestradiol, progesterone, cortisol) were used to identify two key moments: the beginning of the follicular phase (day 2) and the beginning of the luteal phase (day 11). Differential expression analyses of plasma c-miRNAs revealed twelve significantly modulated miRNAs between these two phases, several of which are involved in reproductive pathways according to Gene Ontology and KEGG enrichment analyses. These results suggest a role for specific miRNAs in regulating the oestrus cycle and, consequently, in maintaining reproductive efficiency in this endangered breed. The second part of the research focused on stress-related responses. Farm management practices can activate the hypothalamic–pituitary–adrenal axis, leading to physiological and molecular changes that affect animal welfare. A study on Frabosana-Roaschina ewes investigated salivary miRNAs associated with adaptation responses to farm stressors. Based on clustering analysis of cortisol trends and concentration, animals were grouped into two clusters showing differential expression of miR-16b and miR-21, indicating their potential cooperation in the integrated stress response. These findings highlight, for the first time, the presence of several miRNAs in sheep saliva and their variation in relation to stress profiles. Finally, a transport trial was conducted on Rasa Aragonesa lambs to assess the dynamic nature of c-miRNAs during acute stress. Animals were equipped with subcutaneous bio-loggers to continuously monitor heart rate and body temperature. Blood and saliva samples were collected before, during, and after transport to evaluate cortisol and c-miRNA profiles. The unloading phase was identified as the most stressful moment, marked by increased heart rate, elevated salivary cortisol, and significant modulation of miR-17, miR-23a, miR-24, and miR-27a. Notably, plasma miR-23a, -24, and -27a belong to the same cluster and are involved in muscle physiology and health, supporting their role as indicators of muscle activation and stress adaptation. Overall, this research identified c-miRNAs involved in both reproductive regulation and stress response in sheep. The integrated approach combining behavioural, physiological, and molecular analyses provides novel insights into the adaptive mechanisms of local breeds. These findings contribute to the conservation and sustainable management of Frabosana-Roaschina and Rasa Aragonesa sheep and underline the potential of circulating miRNAs as biomarkers for assessing reproductive status, welfare, and resilience in livestock species.