Fecal ARGs characterized progressive feralization of Przewalski's horses via re-introduction

Re-introduction of captive endangering or extinct animals to their native habitats holds profound ecological significance. But evaluating the feralization progress of re-introduced “wild” animals requires concise and quantitative indicator(s) to integrate traditional assessments on self-sustainability and reproduction. A fecal ARGs’ profiling was conducted for Przewalski’s horses, an extinct flagship herbivore, in Xinjiang, China from the Breeding Center (captive), Tianshan Wildlife Zoo (semi-captive), and Kalamaili Nature Reserve (re-introduction) with Mongolian wild asses serving as a reference in the shared Kalamaili range. Over 60 ARGs were quantified across 6 samples at each site for Przewalski’s horses while 46 ARGs for Mongolian wild asses. The re-introduced Przewalski’s horses had significant losses of fecal ARGs in sharing number and relative abundance in comparison to the captive ones. The re-introduced Przewalski’s horses had complex internal connections among the quantified fecal ARGs in expression potential (average relative abundance per quantified ARGs by antimicrobial category) and even more significant external connections with Mongolian wild asses than with captive horse. Overall, the re-introduction of Przewalski’s horses achieved notable and progressive success in feralization. Tetracyclines resistance genes (Tetra-RGs) alone, with full quantification frequency, could quantitatively characterized this achievement. Ratio of Tetra-RGs to aminoglycosides resistant genes (Amino-RGs) would differentiate the feralization progress with orders of magnitude, for the re-introduced Przewalski’s horses at 279 and the captive one at 11.7, against Mongolian wild asses at 0.86 in this study. The two ARGs-based indicators might be applicable to quantify feralization progress in re-introduction of other captive endangering or extinct animals with validations. • Significant losses of fecal ARGs were observed in re-introduced Przewalski’s horses than the captive ones. • Tetra-RGs were significant declined in expression intensity after re-introduction of Przewalski’s horses. • Tetra-RGs and a ratio of Tetra-RGs to Amino-RGs were recommended to delineate the feralization progress.