Ten animals were assigned to each of three groups based on their mean body weights to obtain an even distribution. Caffeine (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in distilled water (10 mL/kg) at concentrations calculated to deliver 120 and 180 mg/kg body weight/day (these Caffeine groups are designated CF1 and CF2, respectively) and administered by gavage to ensure complete consumption of the established daily dose in the morning (9 to 11 a.m.). The control group (CT) received distilled water daily for 4 weeks. The choice of dose levels was based on the literature, coupled with range finding studies to avoid sub-lethal effects at the highest dose [10 (link),16 (link)].
Animals were examined for any treatment-related clinical signs and weighed daily. Body weight was measured to the nearest 0.1 g with an electronic scale (Dretec Corp., Seoul, South Korea) and recorded from the day before the start of feeding of Caffeine for the four weeks of treatment. All the animals were killed 24 h after their last treatment, using established protocols and ethical procedures. Terminal blood samples were collected by heart puncture, and sera were stored at −70 °C.
Animals were examined for any treatment-related clinical signs and weighed daily. Body weight was measured to the nearest 0.1 g with an electronic scale (Dretec Corp., Seoul, South Korea) and recorded from the day before the start of feeding of Caffeine for the four weeks of treatment. All the animals were killed 24 h after their last treatment, using established protocols and ethical procedures. Terminal blood samples were collected by heart puncture, and sera were stored at −70 °C.
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