Fresh fecal samples were collected from each bird as soon as excreta was discharged through the cloaca at 77 days of age with the average body weight 2.32 kg. Next, all the birds were humanely euthanized by cervical dislocation and subsequently dissected. The contents and mucosal surfaces of the duodenum, jejunum, ileum, and cecum were collected immediately after dissection. To ensure the consistency of samples among individuals, a 10-cm-long fixed section of the duodenum and jejunum, the whole ileum, and a pair of ceca were selected for sampling from each bird. The two ends of the selected section of duodenum, jejunum, and ileum were hold and locked by forceps. The selected intestinal section was cut off and the forceps at the end closer to cloaca were removed to let the content get into the storage tube with appropriate squeezing. Next, the intestinal section was opened and the mucosa was scraped from the end closer to proventriculus to the end closer cloaca. The contents and mucosa were mixed uniformly before storage. Since ceca are blind-ended and the content in ceca is stickier than that in the small intestine, we cut off ceca and collected samples as above described without using forceps. All samples were immediately placed in liquid nitrogen and then stored at −80°C. Both the intestinal contents and mucosa were sampled based on the consideration that the microbes from both sources may contribute to host interactions with respect to nutrient metabolism and immunity (Smith et al., 2015 (link)).
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