Reducing antibiotic usage in livestock animals has become an urgent issue worldwide to prevent antimicrobial resistance. Here, abuse of chlortetracycline (CTC), a versatile antibacterial agent, on the performance, blood components, fecal microbiota, and organic acid concentration in calves was investigated. Japanese Black calves were fed milk replacer containing CTC at 10 g/kg (CON) or 0 g/kg (EXP). Growth performance was not affected by CTC administration. However, CTC administration altered the correlation between fecal organic acids and bacterial genera. Machine learning methods such as association analysis, linear discriminant analysis, and energy landscape analysis revealed that CTC administration affected according to certain rules the population of various types of fecal bacteria. It is particularly interesting that the population of several methane-producing bacteria was high in the CON, and that of Lachnospiraceae, a butyrate-producing bacteria, was high in the EXP at 60 d of age. Furthermore, statistical causal inference based on machine learning data estimated that CTC treatment affects the entire intestinal environment, inhibiting butyrate production for growth and biological defense, which may be attributed to methanogens in feces. Thus, these observations highlight the multiple harmful impacts of antibiotics on intestinal health and the potential production of greenhouse gas in the calves.
