Omic technology to monitoring resilience and adaptation to exercise and heat stress in endurance horses

Samanta Mecocci^1,2Elisabetta Porzio³Elisabetta Chiaradia³Marco Pepe^3,4Angelo Paris⁵Stefania Bergagna⁶Daniele Pietrucci²Giovanni Chillemi^7,8Francesca Beccati^3,4*Katia Cappelli^3,4

• ¹Department of Veterinary Medicine, University of Perugia, Perugia, Italy
• ²Department for Innovation in Biological, Agro-Food and Forest Systems (DIBAF), University of Tuscia, Viterbo, Italy
• ³Universita degli Studi di Perugia Dipartimento di Medicina Veterinaria, Perugia, Italy
• ⁴Sports Horse Research Center, Department of Veterinary Medicine, University of Perugia, Perugia, Italy
• ⁵Private Practitioner, Via Flaminia 12, 06028, Sigillo, Perugia, Italy
• ⁶Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d'Aosta (IZSPLV), Torino, Italy
• ⁷Department of Experimental Medicine, University of Rome “Tor Vergata”, Roma, Italy
• ⁸Bioinformatics Research Unit in Infectious Diseases, National Institute for Infectious Diseases Lazzaro Spallanzani - IRCCS, Via Portuense, Roma, Italy

In horses, heat exposure modulates the hypothalamic–pituitary–adrenal axis, autonomic nervous system, and hypothalamic–pituitary–thyroid axis to maintain body temperature and prevent excessive heat accumulation. However, during strenuous exercise under hot and humid conditions, heat production may exceed dissipation, leading to heat stress, anhidrosis, heat stroke, or brain damage. Incremental field standardized exercise tests (fSETs) provide a reliable approach to assess training and fitness levels. This study aimed to investigate the molecular features underlying resilience and adaptation to combined heat-and exercise-induced stress in horses. Six Arabian horses from Italia Endurance Stable and Academy were monitored during fSETs performed under heat stress (HS) and thermoneutral (TN) conditions, with blood samples collected before and after each test. Hematocrit, lactate, and biochemical parameters were measured, and total serum RNA was sequenced. Lactatemia and hematocrit were significantly higher in HS vs TN, while alanine aminotransferase, creatinine, and creatine kinase increased in HS POST vs PRE fSET. Differentially expressed small RNAs included eca-myomir-206, eca-mir-301, eca-mir-3613-3p, eca-mir-142, and eca-mir-144, which were modulated by temperature and exercise. A protein–protein interaction (PPI) network of miRNA targets was constructed and analyzed for Gene Ontology (GO) enrichment. In POST vs PRE fSET, enriched terms involved transcriptional regulation, glucose and LDL response, intracellular trafficking, cytoskeleton organization, cardiac conduction, ion channels, and immune regulation. In HS POST vs PRE fSET, enrichment was observed for positive regulation of dendritic cell cytokine production, negative regulation of inflammation, and attenuation of oxidative stress-induced apoptotic signaling. Overall, these findings indicate that heat amplifies the physiological burden of endurance exercise and alters the molecular mechanisms supporting performance and recovery. Circulating small RNAs may act as early signals for homeostatic restoration and could help elucidate adaptive responses to stress, guiding personalized training strategies.