2.2.3. Experiment 4: digestive physiology
Sea cucumbers were maintained under the same experimental conditions as Section 2.3.3 Experiment 3: feeding and ingestion for two weeks. Subsequently, six individuals of each size group (small, medium and large) were sampled to obtain intestines every 2 h during a 24 h cycle (12 sampling points). The intestines were cut longitudinally and washed thoroughly in ice-cold 0.84% normal saline. After rinsing, all samples were frozen quickly in liquid nitrogen and then stored at −80 °C until analysis. One intestine sample of sea cucumbers was insufficient to determine three digestive enzyme activities. So we mixed two replicate samples into one sample. In order to avoid the effect of different feeding times, all animals were fed excessively at 08:00 on the sampling day. Sampling during the dark phase was performed under a dim light.