Adaptation Response in Sheep: Ewes in Different Cortisol Clusters Reveal Changes in the Expression of Salivary miRNAs

SIMPLE SUMMARY: Monitoring animals’ welfare is a tricky challenge. The responsiveness to stressors is subjective and its magnitude varies among species, breeds, and individuals. Small molecules named miRNAs obtained from body fluid samples are promising biomarkers to classify animal welfare. Under common farming conditions, we identified several miRNAs in the saliva of ewes. Based on the concentration and trend of individual salivary cortisol, one of the molecules historically recognized and used to define the stress level in vertebrates, we divided the animals into two clusters. The expression of two miRNAs involved in regulating genes related to the stress pathway resulted in significant differences between the two phenotypes, and their trends were correlated. These results have produced objective data to improve the knowledge about sheep physiology and the basis to develop new tools for welfare assessment. In the animal production field, the assessment of biomarkers that are useful in identifying individuals with different levels of vulnerability or resiliency to stress stimuli may help to introduce new strategies in animal farms and genetic research for the selection of animals. ABSTRACT: Farm procedures have an impact on animal welfare by activating the hypothalamic-pituitary-adrenal axis that induces a wide array of physiological responses. This adaptive system guarantees that the animal copes with environmental variations and it induces metabolic and molecular changes that can be quantified. MicroRNAs (miRNAs) play a key role in the regulation of homeostasis and emerging evidence has identified circulating miRNAs as promising biomarkers of stress-related disorders in animals. Based on a clustering analysis of salivary cortisol trends and levels, 20 ewes were classified into two different clusters. The introduction of a ram in the flock was identified as a common farm practice and reference time point to collect saliva samples. Sixteen miRNAs related to the adaptation response were selected. Among them, miR-16b, miR-21, miR-24, miR-26a, miR-27a, miR-99a, and miR-223 were amplified in saliva samples. Cluster 1 was characterized by a lower expression of miR-16b and miR-21 compared with Cluster 2 (p < 0.05). This study identified for the first time several miRNAs expressed in sheep saliva, pointing out significant differences in the expression patterns between the cortisol clusters. In addition, the trend analyses of these miRNAs resulted in clusters (p = 0.017), suggesting the possible cooperation of miR-16b and -21 in the integrated stress responses, as already demonstrated in other species as well. Other research to define the role of these miRNAs is needed, but the evaluation of the salivary miRNAs could support the selection of ewes for different profiles of response to sources of stressors common in the farm scenario.