Molecular biomarkers and subcutaneous bio-loggers to assess the lambs' adaptive physiological response during transport

Transportation is one of the most stressful conditions for farm animals [1]. Handling and management of lambs during transport are challenges that perturb homeostasis. The consequent adaptive response with the hypothalamic-pituitary-adrenal axis (HPA) activation attempts to restore balance and welfare conditions. Thus far, the digital revolution has enabled the quantification of animal welfare through the use of parameters analysis gathered by sensors, and this has led to innovative approaches to define the welfare condition of food producing animals. On the other hand, from molecular biology comes identifying new molecules as biomarkers. Stressors that alter behaviour, physiological parameters, and molecular expression—such as those of microRNAs—quickly activate the HPA axis. These molecules play a role in the post-transcriptional gene regulation of several cellular processes. Expressed by tissues, miRNAs are released in body fluids and therefore called circulating miRNAs (c-miRNAs) [2]. They can be used as minimally invasive biomarkers by changing their profile under physiological, pathological, and psychological conditions [3]. This study aims to evaluate the effects of transport on the Aragonesa breed lambs' adaptive response and welfare status, evaluated in terms of physiological (Heart rate and Body temperature) and molecular (saliva cortisol and c-miRNAs) parameters. Plasma and saliva samples of fourteen lambs, implanted with subcutaneous temperature (BT) and heart rate (HR) bio-loggers (DST micro-HRT, Star Oddi, Iceland), were collected five times/animal: 2 PRE (T0-24h; T1-4h before loading) and 3 POST (T2-immediately after unloading; T3-4h; T4-24h) a transport of 75 min. Salivary cortisol concentration was determined with an enzyme immunoassay. Based on their involvement in the adaptive response, 17 c-miRNAs were selected from literature and extracted from plasma and saliva. C-miRNAsʼ expression analysis was performed in real time q-PCR. BT and HR data were analysed with the Star-Oddi HRT Analyzer software. Cortisol analysis showed a significant concentration immediately after the unloading procedure (T2) (ANOVA one-way test; p<0.05) when compared with T0, T1, T3 and T4 identified as basal undisturbed points (rest time). At T2, lambs also presented a significant drop of BT (38.72±0.01 °C) (p<0.05) and a peak of HR (155.14±5.44 bpm) (p<0.05). Out of the 17 c-miRNAs analyzed, 5 were found expressed in plasma and saliva matrices of all sampling points. MiR-17, -23a, and -27a were differentially expressed in T2 only in saliva samples, while miR-24 in both matrices. Lambs presented the same BT at basal undisturbed points (mean 39.02±0.19), reaching a significant drop at T2 (38.72±0.3) (p<0.05), while they reached the maximum HR (bpm) during loading (150.09±4.89) and unloading procedures (T2: 155.14±5.44). In conclusion, detecting the most stressful times during routine farm procedures can be accomplished by combining molecular and classical biomarkers with physiological data collected by innovative devices like as bio-loggers. [1] De la Fuente et al. Physiological response and carcass and meat quality of suckling lambs in relation to transport time and stocking density during transport by road, Animal, 4(2):250-8, 2010. [2] O'Brien et al. Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation, Front Endocrinol, 9:402, 2018. [3] Do et al. miRNA Regulatory Functions in Farm Animal Diseases, and Biomarker Potentials for Effective Therapies, Int J Mol Sci, 22(6):3080, 2021.