Ex situ reared black-footed ferrets exhibit altered sperm DNA methylation.

Many endangered species rely on ex situ management for survival when external threats exist on the landscape. Yet ex situ settings pose challenges through space limitation, altered environment, and diet. This can lead to environmentally-determined inbreeding depression, where ex situ animals exhibit reduced reproductive fitness compared to their in situ counterparts, despite originating from the same genetic stock. We investigated epigenetic differences as a potential mechanism underlying environmentally-determined inbreeding depression in black-footed ferrets (Mustela nigripes), an North American endemic species reliant on ex situ conservation. More specifically, we explored how environmental context may influence sperm DNA methylation in samples collected from 12 ex situ and 5 in situ males. Average sperm DNA methylation was significantly higher in ex situ individuals. We additionally identified >500 differentially methylated regions between ex situ and in situ sperm samples that were enriched for gene ontology terms pertaining to reproduction and development. Putative genes of interest included NPR2, WEE2, SLC15A1, PDE10A, PIP5K1B, CACNA1E, and CACNA1A, all of which have previously been linked to spermatogenesis, sperm motility, or fertilization in mammals. Results suggest that environmental conditions may alter sperm DNA methylation in black-footed ferrets, with possible links to decreased reproductive success in ex situ settings. These findings provide valuable insights to the molecular mechanisms underlying environmentally-determined inbreeding depression in black-footed ferrets and other conservation-reliant species, and can serve as a foundation for future research on improving reproductive health in endangered wildlife.