Fischer 344 rats

Male six-week-old Fischer 344 rats (CLEA Japan, Inc., Osaka, Japan) were housed in a room under controlled temperature and a 12/12 h light-dark cycle. The animals were divided into the following three experimental groups and fed for 8 weeks: i) Choline-deficient amino acid diet (CDAA; n=10); ii) choline-deficient amino acid diet plus antibiotics (CDAA+AB; n=10) and iii) choline-supplemented amino acid diet (CSAA; n=5). All rats were sacrificed at the end of week 8. For selective intestinal decontamination, poorly absorbable antibiotics (1 g/l polymyxin B sulfate salt (Fluka, Buchs, Switzerland) and 3 g/l neomycin trisulfate salt hydrate (Sigma-Aldrich, St. Louis, MO, USA) were administered to the rats in the CDAA+AB group by adding them to drinking water during the experimental period, excluding the first and fifth week. All animal procedures were performed according to standard protocols and in accordance with the standard recommendations for the proper care and use of laboratory animals. This study was approved by the ethics committee of Nara Medical University, Kashihara, Japan.