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 The coelomic fluid was withdrawn from the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.