Methodologies to Identify Metabolic Pathway Differences Between Emaciated and Moderately Conditioned Horses: A Review of Multiple Gene Expression Techniques

Starvation in horses presents critical welfare, economic, and management challenges with underlying molecular mechanisms of metabolic modification and recovery left poorly defined. Prolonged caloric deprivation induces significant systemic shifts in carbohydrate, protein, and lipid metabolism, reflected in coordinated changes in tissue-specific gene expression. This review synthesizes current knowledge on equine metabolic responses to starvation, emphasizing pathways found through RNA sequencing (RNA-seq) and real-time quantitative polymerase chain reaction (RT-qPCR) studies. Molecular investigations using RNA-seq and RT-qPCR have provided insight into transcriptional reprogramming during starvation and subsequent refeeding. Shifts in gene expression reflect the metabolic transition from carbohydrate dependence to lipid use, suppression of anabolic signaling, and activation of proteolytic pathways. However, interpretation of these data requires caution, as factors such as post-mortem interval, tissue handling, and euthanasia methods particularly the use of sodium barbiturates can influence transcript stability and abundance, potentially confounding results. The literature shows that starvation-induced molecular changes are not uniform across tissues, with skeletal muscle, liver, and adipose tissue showing distinct transcriptional signatures and variable recovery patterns during refeeding. Cross-species comparisons with hibernation, caloric restriction, and cachexia models provide context for understanding these changes, though equine-specific studies remain limited. Identified gaps include the scarcity of longitudinal data, inconsistent tissue sampling protocols, and lack of standardized reference genes for transcriptomic analyses in horses. Addressing these limitations will improve the accuracy of molecular evaluations and enhance our ability to predict recovery trajectories. A more comprehensive understanding of systemic and tissue-specific responses to starvation will inform evidence-based rehabilitation strategies, reduce the risk of refeeding syndrome, and improve survival and welfare outcomes for affected horses.