Bio-loggers and miRNAs are innovative tools for measuring physiological changes in lambs during transport.

For livestock, transport can involve several potential stressors including human handling, stocking density, air temperature, noise, vibration, and loading/unloading procedures. The animal's perception of and its ability to adapt to those stimuli are not fully understood, which makes it difficult to carry out welfare assessments. This study aimed to detect stressful moments in lambs during transport through changes in physiological and molecular markers. Data obtained from bio-loggers that record physiological variables and an evaluation of molecular biomarkers such as cortisol and circulating microRNAs (c-miRNAs) identified the most stressful moments of transport, which can be a valuable tool for evaluating and improving transport conditions for livestock. Rasa Aragonesa lambs were implanted with bio-loggers that record subcutaneous body temperature (BT) and heart rate (HR). Plasma and saliva were sampled for molecular analyses; specifically, saliva for cortisol concentrations, plasma for creatine kinase (CK), and lactate dehydrogenase (LDH), and plasma and saliva for c-miRNAs expression profiles. Immediately after the lambs were unloaded, the bio-loggers registered a significant (P < 0.05) spike-in HR and a drop in BT, and salivary cortisol concentrations increased significantly (P < 0.05), which indicated unloading as one of the main stressful points for the lambs. Out of the 17 miRNAs tested, 4 showed a significant difference in expression (P < 0.05). MiR-23a and -27a were both expressed in plasma and saliva, while miR-17 and -24 were most expressed in saliva after unloading. Finally, the expression of plasmatic miR-23a, -24, and 27a were significantly (P < 0.05) positively correlated with the LDH whose concentrations together with those of CK are significantly increased (P < 0.01) after unloading. The study identified the timing of a lamb's adaptation response during and after transport, which reflected the dynamic nature of ovine plasma and salivary miRNAs during transport-induced stress, giving them the potential to be biomarkers that can be useful in animal welfare assessments.