Antrodia cinnamomea Extraction Waste Supplementation Promotes Thermal Stress Tolerance and Tissue Regeneration Ability of Zebrafish
Abstract
Antrodia cinnamomea (AC) has been shown to have anti-inflammatory, anti-tumor, and immunomodulation activities. It is estimated that hundreds of metric tons of AC extraction waste (ACEW) are produced per year in Taiwan. This study aims to assess the feasibility of applying ACEW as feed supplement in the aquaculture industry. ACEW significantly inhibited the growth of microorganisms in the water tank, by around 39.4% reduction on the fifth day with feed supplemented of 10% ACEW. The feed conversion efficiency of zebrafish with 10% ACEW supplementation for 30 days was 1.22-fold compared to that of the control. ACEW dramatically improved the tolerances of zebrafish under the heat and cold stresses. When at water temperature extremes of 38 °C or 11 °C, compared to the 100% mortality rate in the control group, the 10% ACEW diet group still had 91.7% and 83.3% survival rates, respectively. In a caudal fin amputation test, the fin recovery of zebrafish was increased from 68.4% to 93% with 10% ACEW diet after 3-week regeneration. ACEW effectively down-regulated the gene expression of TNF-α, IL-1β, IL-6, and IL-10, and up-regulated the gene expression of IL-4/13A. Additionally, the supplement of ACEW in the feed can maintain and prevent the fish's body weight from dropping too much under enteritis. Taken together, ACEW has beneficial potential in aquaculture.
Figures 
Figure 1
Total bacterial plate count of…
Figure 1
Total bacterial plate count of tank water during 4-day cultivation. Thirty fish were…
Figure 1 Total bacterial plate count of tank water during 4-day cultivation. Thirty fish were used in each group and placed in the same aquarium with one fifth of tank water changed every day during the feeding experiment period. Significant difference compared with the vehicle group was denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001.
Figure 2
Effect of Antrodia cinnamomea extraction…
Figure 2
Effect of Antrodia cinnamomea extraction waste (ACEW) supplementation on ( A ) weight…
Figure 2 Effect of Antrodia cinnamomea extraction waste (ACEW) supplementation on (A) weight gain, (B) fertilization rate, and (C) hatching rate of zebrafish during 30-day cultivation. For the body weight gain study, thirty adult fish were used in each group and put in the same aquarium. For the reproduction study, three males were paired with three female fish for each group in the mating tank one night before spawning event. Significant difference compared with the vehicle group was denoted as *** p < 0.001.
Figure 3
Effect of ACEW supplementation on…
Figure 3
Effect of ACEW supplementation on survival rate of zebrafish under rapid temperature changes.…
Figure 3 Effect of ACEW supplementation on survival rate of zebrafish under rapid temperature changes. Each group of 12 adult zebrafish was fed with a specified diet for 30 days before the experiment, and then underwent the rapid temperature changes as: (A) the heating challenge experiment; (B) the cooling challenge experiment. The time course of temperature change is described in Section 4.6.
Figure 4
Effect of ACEW supplementation on…
Figure 4
Effect of ACEW supplementation on caudal fin regeneration ability. ( A ) Length…
Figure 4 Effect of ACEW supplementation on caudal fin regeneration ability. (A) Length changes of caudal fins; (B) Typical photographs of the original, amputated and regenerated fins. Each group of 20 adult zebrafish was fed with specified diet for 30 days before the experiment, and then half-length of the caudal fin was amputated. After 21-day recovery, the length of caudal fin was measured. Significant difference compared with the vehicle (group 0) was denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001.
Figure 5
Effect of ACEW supplementation on…
Figure 5
Effect of ACEW supplementation on gene expression of inflammation-associated cytokines in zebrafish under…
Figure 5 Effect of ACEW supplementation on gene expression of inflammation-associated cytokines in zebrafish under the intestinal stimulation by oxazolone in prevention mode experiment. Each group of 10 adult zebrafish was fed with specified diet for 30 days before the experiment, and then 0.2% oxazolone were intrarectally instilled to induce acute enteritis. Significant difference of gene expression between the ACEW supplemented diet group and the control diet group (group OZ without ACEW addition) was denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001.
Figure 6
Effect of ACEW supplementation on…
Figure 6
Effect of ACEW supplementation on gene expression of inflammation-associated cytokines in zebrafish under…
Figure 6 Effect of ACEW supplementation on gene expression of inflammation-associated cytokines in zebrafish under the intestinal stimulation by oxazolone in therapeutic mode experiment. To each group of 10 adult zebrafish, 0.2% oxazolone were intrarectally instilled to induce acute enteritis. After 5-h induction, fish were fed with specified diet twice a day. On day-1, -3, and -5, gene expression of (A) TNF-α, (B) IL-1β, (C) IL-6, (D) IL-10, and (E) IL-4/13A was analyzed by qRT-PCR. Significant difference of gene expression between the ACEW supplemented diet and the control diet group (group OZ without ACEW addition) was denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001.
Figure 6
Effect of ACEW supplementation on…
Figure 6
Effect of ACEW supplementation on gene expression of inflammation-associated cytokines in zebrafish under…
Figure 6 Effect of ACEW supplementation on gene expression of inflammation-associated cytokines in zebrafish under the intestinal stimulation by oxazolone in therapeutic mode experiment. To each group of 10 adult zebrafish, 0.2% oxazolone were intrarectally instilled to induce acute enteritis. After 5-h induction, fish were fed with specified diet twice a day. On day-1, -3, and -5, gene expression of (A) TNF-α, (B) IL-1β, (C) IL-6, (D) IL-10, and (E) IL-4/13A was analyzed by qRT-PCR. Significant difference of gene expression between the ACEW supplemented diet and the control diet group (group OZ without ACEW addition) was denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001.
Figure 7
Effect of ACEW supplement on…
Figure 7
Effect of ACEW supplement on weight gain of zebrafish in therapeutic mode experiment…
Figure 7 Effect of ACEW supplement on weight gain of zebrafish in therapeutic mode experiment of oxazolone-induced acute enteritis. Zebrafish were fed with different experimental diets after induction, and were weighted at day 1, 3, and 5. Significant difference of gene expression between the ACEW supplemented diet and the control diet group (group OZ without ACEW addition) was denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001. All figures (8)