Kontes pellet pestle

Thirty-six Asian seabass at the age of three months (body weight: ~50 g) originally maintained in a 1,000 L tank containing 500 L of freshwater were divided equally into two tanks containing 3,00 L of freshwater. Eighteen fishes, used as a control, were fed twice daily with pelleted feed (Biomar, Nersac, FR, France), and the other eighteen fishes in the test tank were not given access to feed before sampling. Six fishes from each group were sacrificed at three, six and twelve days post fasting, respectively. Before experiments, the pestles and dissecting tools were first soaked in 5% concentration of Clorox bleach (The Clorox Company, Oakland, USA) for ~15 minutes, and autoclaved for 20 minutes after washing and rinsing in ddH2O. The working surface area and the deceased fish were decontaminated with 70% ethanol. Small sections (~1 cm of length) from the middle part of the intestine samples were taken and homogenized in 1 ml of Trizol reagent (Invitrogen, Carlsbad, USA) with sterile KONTES® pellet pestle driven by a cordless motor (Fisher Scientific, New Hampshire, USA). RNA isolation was conducted using the Trizol reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. The remaining intestine samples from each fish was taken for DNA isolation using the QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions.

We extracted metabolites from the colon tissues based on previous methods [28 (link),29 (link),30 (link),31 (link)]. Briefly, colon tissues were accurately weighed by difference using an analytical balance (Ohaus; VWR, Mississauga, ON, Canada) into a 1.5 mL microcentrifuge tube (Eppendorf, Mississauga, ON, Canada). Tissues were homogenized in 70% ethanol (200 µL 70% ethanol/100 mg tissue) with a disposable tissue grinder (Kontes Pellet Pestle; Fisher Scientific, Ottawa, ON, Canada). The resulting suspension was centrifuged at 3000× g for 3 min (Galaxy 16DH centrifuge, VWR, Mississauga, Ontario, Canada), and the supernatant was decanted and centrifuge-filtered using a 0.2 µm Ultrafree-MC centrifugal filter (Millipore-Sigma, Oakville, ON, Canada) at 3000× g for 3 min (Galaxy 16DH centrifuge). One hundred µL of the filtrate was transferred to an autosampler vial (300 µL polypropylene with pre-slit Teflon-coated caps; Waters Corp., Mississauga, ON, Canada) fitted with a conical bottom spring insert (250 µL glass; Canadian Life Science, Peterborough, ON, Canada) for ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) analysis.