Abstract
Epigenetic modifications, particularly DNA methylation, are strongly associated with chronological age across mammalian species. This study developed cheetah-specific epigenetic clocks from methylation profiles generated from cheetah blood and liver samples tested on the HorvathMammalMethylChip40 Illumina Array. The resulting age clock used 52 CpG sites and predicted age across blood and liver samples (r = 0.97 and MAE = 0.86). When applied to a test set of blood collected from live cheetahs, the clock provided accurate predictions for adult individuals (age > 3 years) but was less precise at and around age of sexual maturity. A second clock, incorporating cheetah, lion, and tiger profiles, used 46 CpG sites and predicted age across these feline species (r = 0.94 and MAE = 1.16). Additionally, a sex clock using 67 CpG sites accurately predicted sex in all test samples. To explore the potential of methylation as a biomarker beyond age and sex, we conducted a differential methylation analysis to investigate disease-related methylation patterns in cheetahs diagnosed with hepatic sinusoidal obstruction syndrome (SOS). This analysis identified 4,377 CpG sites with significant differences between SOS-positive and SOS-negative cheetahs (adjusted p-value < 0.05). These findings advance the development of epigenetic clocks for precise age and sex prediction in cheetahs and related species and establish a foundation for leveraging methylation biomarkers to investigate diseases in wildlife conservation efforts.