Gut Microbiome Profiling of Shennongjia Snub-nosed Monkeys

Approximately, a total of 85 R. roxellanae were available for research in Shennongjia Nature Reserve. The monkeys were distributed across three regions: Region I: there were about 60 free-ranging monkeys lived in the Dalongtan area, which were composed of four one-male units (OMU) and one all male unit (AMU); Region II: Shennongjia snub-nosed monkey breeding base where 15 monkeys were kept and randomly divided into three OMUs by animal keepers; each unit was independently caged in one steel house; and Region III: Xiaolongtan area is the place where the injured monkeys rescued from different areas of Shennongjia Reserve were cared; 10 monkeys in a cage were taken as one unit. In total, we collected fresh fecal samples from 38 monkeys including 30 wild monkeys from Regions I and eight captive monkeys from II (six, three healthy and three diarrheal) and III (two, one healthy and one diarrheal monkeys) respectively in July 2012. Monkey age, raising patterns, sex, and health status were recorded (additional file 1). The monkey age was determined by their coat color, body length and wart-like growth at the corners of the mouth as described previously [27 ]. Meanwhile, the health status of the monkeys was clinically determined by observing their bright coats, movement (lively, powerful), good appetite, fecal shape, and color.
All the wild and captive monkeys were fed three times per day (10:00–11:00, 14:00–15:00, and 18:00–19:00). The wild monkeys from region I usually came quickly from the wild to the trees in the feeding place at the fixed time after hearing the call of the staff. Then they jumped down from the trees to get the food and ate it. After feeding, they immediately went back to the mountain for their free-living in the wild and could get more food in the forest. Meanwhile, the monkeys in regions II and III were kept and fed in their cages at similar timepoints.
Fecal samples were collected from the monkeys by using the protocol described previously [2 (link)]. Briefly, fecal samples were collected during one week by two researchers before feeding time in the morning (10:00–11:00) and afternoon (14:00–15:00), when the monkeys had already gone down the mountain, gathered in the trees at the feeding place and waited for their food. Each researcher was responsible for one monkey unit per time. Fresh fecal samples were immediately collected into sterile tubes. For the diarrheal fecal samples, sterile cotton swabs were used and dipped the feces in the center carefully for several times to collect feces as many as possible, and stored into sterile tubes as well. Samples were stored at -80 °C at Shennongjia Reserve and transported to our laboratory on dry ice and stored at -80 °C until DNA extraction. Microbial DNA from fecal samples was extracted using QIAamp DNA stool mini kits (Qiagen, CA, USA) following standard protocols.