2.2 | Experimental design and sample collection
At least 40 individuals per regeneration stage [the beginning (10 d), middle (14 d) and end (21 d) of intestinal regeneration] were used for analyses. Ten individuals with the longest or shortest intestines were classified as the faster (F) and slower (S) regenerating individuals, respectively. The samples were labeled F10, S10, F14, S14, F21 and S21, respectively. After 7 d of intestine regeneration, lumen formation of the new intestine began; the intestine gradually developed to form a complete structure in which the digestive and absorptive functions were restored during 14-21 d of intestine regeneration (L. Sun et al., 2011; Lina Sun et al., 2013). The 10th d was therefore an appropriate time point for sampling the new intestine.
The GF and conventionally reared (CV) groups were subjected to two different treatments: 80 sea cucumbers were cultured normally in seawater in the laboratory as the CV group. 160 sea cucumbers were soaked for 3 h in sterile filtered seawater containing 100 U/ml penicillin and 100 μg/ml streptomycin, and were then cleaned twice in 0.003% sodium hypochloride (Bates et al., 2006). No developmental defects and similar rates of sterility were observed with the antibiotic mixtures used. Then, 80 sea cucumbers were transferred to sterile filtered seawater tanks at a density of 10 sea cucumbers per tank in a sterile room as the GF group; the tanks remained sterile for the duration of the experiment. And the other 80 sea cucumbers were cultured normally in seawater in the laboratory as the XGF group. At least 10 individuals of similar size were randomly selected per regeneration stage (10 and 14 d) were used for analyses.
In the experiments, individuals were rinsed with sterile seawater and moved into sterile plates. The coelomic fluid was withdrawn from the coelom of sea cucumbers using sterile syringes. The intestines were aseptically dissected and measured the length, and immediately transferred into sterile tubes and measured the weight, and then preserved at -80°C until DNA extraction.