Sleep deprivation and Gut Microbiome--Peer J.

The study was conducted to investigate the impact of sleep deprivation on altering the puberty onset in juvenile Sprague-Dawley (SD) rats. Four pregnant SD rats were purchased from BioLASCO Taiwan Co. Ltd, and they were housed at the Laboratory Animal Center at Taipei Medical University in a controlled environment (12-hour light-dark cycle, 22–24°C, 40%–60% humidity). The juvenile SD rats were weaned and grouped at postnatal day 21 (PND 21). Then, they were randomly assigned to Control Female (CF) (n=6), Sleep-Deprivation Female (SDF) (n=6), Control Male (CM) (n=6), and Sleep-Deprivation Male (SDM) (n=6) groups, totally 24 rats. We ensured their body weight and sex were balanced across groups. They were subjected to 15 hours of sleep deprivation per day for 4 weeks after weaning. Sleep deprivation is a highly stressful condition; therefore, the body weights of rats were monitored every other day, and all rats survived until euthanization. The rats were euthanized by using cardiac puncture after 4 weeks of sleep deprivation, and blood samples and tissues were collected and stored in the -80 ℃ refrigerator until used.Gut microbiome compositionFecal samples were collected at vaginal opening days (PND 30~PND 40) from female rats and were collected at preputial separation days (PND 39~PND 49) from male rats during the experiment. In brief, collected fecal samples were transferred immediately to cold storage and remained stored at 80°C until processing near days of vaginal opening. Fecal genomic DNA was extracted using the QIAamp DNA Stool Mini Kit (cat. no. 51504, QIAGEN, Denmark) according to the manufacturer’s instructions, stored at -80°C, and underwent processing including polymerase chain reaction (PCR) assays and 16S rRNA sequencing. The Pacbio sequencing for full-length 16S genes (V1-V9 regions) was performed. The full-length 16S genes was amplified using barcoded 16S gene-specific primers. Subsequently, the PCR reaction was carried out by KAPA HiFi HotStart ReadyMix (Roche), and its products were purified using the AMPure PB Beads for SMRTbell library construction and sequencing processes. Consequently, multiple sequence alignment was performed by QIIME2 alignment MAFFT against the NCBI database to analyze the sequence similarities among the amplicon sequence variants (ASVs).