Effects of dietary protein-to-sea mud ratio on growth, immune response, intestinal digestion, and health of sea cucumber (<i>Apostichopus japonicus</i>)

Yi Xiong; Chunxiao Zhang; Zhixiang Wang; Ling Wang; Kai Song; Ruijuan Ma; Xueshan Li; Kangle Lu; Samad Rahimnejad; Yi Xiong; Chunxiao Zhang; Zhixiang Wang; Ling Wang; Kai Song; Ruijuan Ma; Xueshan Li; Kangle Lu; Samad Rahimnejad

doi:10.48130/animadv-0026-0004

Figures (6) Tables (7)

Figure 1.
Effects of dietary protein-to-sea mud ratio on intestinal morphology in A. japonicus (× 40). MT, muscularis thickness. FH, fold height. Histological observations (H&E staining) revealed a gradual thickening of the intestinal wall and an increase in fold height with rising dietary protein and sea mud levels.
Figure 2.
Rarefaction curves of species richness and evenness in the gut samples of A. japonicus.
Figure 3.
Nonmetric multidimensional scaling (NMDS) analysis of the intestinal bacterial community of A. japonicus. Notes: stress < 0.2 indicates that NMDS can accurately reflect the degree of difference between samples.
Figure 4.
Hierarchical clustering tree of the intestinal bacterial community of A. japonicus based on the Bray_Curtis distance algorithm.
Figure 5.
Composition of intestinal microbial flora of A. japonicus at the phylum level.
Figure 6.
Composition of intestinal microbial flora of A. japonicus at the genus level.

Ingredients (%)	PS no. (protein/sea mud)
Ingredients (%)	PS1(10/30)	PS2(10/45)	PS3(10/60)	PS4(13/30)	PS5(13/45)	PS6(13/60)
Sea mud	30.00	45.00	60.00	30.00	45.00	60.00
Sargassum meal	40.00	30.00	20.00	40.00	30.00	20.00
Fish meal¹	2.00	2.00	2.00	3.00	3.00	3.00
Soybean meal	3.00	3.00	3.00	4.00	4.00	4.00
Wheat flour	23.00	14.2	6.00	18.00	9.20	1.00
Casein	0.00	3.00	5.50	3.00	6.00	8.50
Microcrystalline cellulose	0.00	0.80	1.50	0.00	0.80	1.50
Vitamin premix²	1.00	1.00	1.00	1.00	1.00	1.00
Mineral premix³	1.00	1.00	1.00	1.00	1.00	1.00
Proximate composition (%)
Crude protein	10.50	10.70	10.22	13.20	13.60	13.20
Crude lipid	1.46	1.85	2.76	2.09	2.06	2.55
Protein/sea mud	0.34	0.23	0.17	0.44	0.30	0.22
¹ Fish meal, Xiamen Jiakang feeds group Corp., Ltd, imported from Peru. ^{2, 3} Mineral premix and vitamin premix were prepared following our recent study^[15].

Table 1.

Formulation and proximate composition of the experimental diets (% dry matter).

Item	PS no. (protein/sea mud)						ANOVA (p value)
Item	PS1(10/30)	PS2(10/45)	PS3(10/60)	PS4(13/30)	PS5(13/45)	PS6(13/60)	Protein	Sea mud	Protein x sea mud
IBW (g)¹	5.37 ± 0.01	5.40 ± 0.02	5.42 ± 0.02	5.40 ± 0.01	5.40 ± 0.03	5.39 ± 0.03
FBW (g)²	4.74 ± 0.72^b	5.36 ± 0.14^b	5.76 ± 0.84^b	5.29 ± 0.34^b	7.55 ± 0.93^a	7.78 ± 0.45^a	0.005	0.022	0.321
WGR (%)³	−11.67 ± 13.59^b	−0.71 ± 2.30^b	6.31 ± 15.67^b	−2.22 ± 6.10^b	39.57 ± 16.68^a	44.28 ± 7.80^a	0.005	0.021	0.297
SGR (%)⁴	−0.27 ± 0.30^c	−0.01 ± 0.04^bc	0.07 ± 0.28^abc	−0.05 ± 0.11^c	0.58 ± 0.24^ab	0.65 ± 0.10^a	0.011	0.038	0.553
IBR (%)⁵	2.69 ± 1.22^c	4.13 ± 0.36^bc	3.94 ± 0.45^bc	3.46 ± 0.44^bc	4.82 ± 0.16^ab	6.48 ± 0.20^a	0.017	0.011	0.245
SR (%)⁶	100.00 ± 0.00^a	100.00 ± 0.00^a	93.33 ± 1.67^b	96.67 ± 1.67^ab	96.67 ± 3.33^ab	98.33 ± 1.67^ab	0.712	0.312	0.061
Values in the same row (multiple comparisons or main effects analysis) with different superscript letters are significantly different (p < 0.05), while the values for two-factor analysis of variance are less than 0.05, indicating significant differences at p < 0.05. Data are presented as mean ± S.E.M. (n = 3).¹ IBW (g) = initial body weight. ² FBW (g) = final body weight. ³ Weight gain rate (WGR, %) = (final body weight–initial body weight)/initial body weight × 100. ⁴ Specific growth rate (SGR, % day⁻¹) = (ln final body weight–ln initial body weight)/feeding days × 100. ⁵ Intestinal to body wall ratio (IBR, %) = intestinal weight/body weight (wet weight). ⁶ Survival rate (SR, %) = 100 × (final number/initial number of sea cucumbers).wall

Table 2.

Effects of dietary protein-to-sea mud ratio on growth performance of A. japonicus.

Item	PS no. (protein/sea mud)						ANOVA (p value)
Item	PS1(10/30)	PS2(10/45)	PS3(10/60)	PS4(13/30)	PS5(13/45)	PS6(13/60)	Protein	Sea mud	Protein × sea mud
Moisture	89.50 ± 0.17^ab	89.39 ± 0.10^ab	89.80 ± 0.09^a	88.95 ± 0.49^b	90.03 ± 0.06^a	90.19 ± 0.27^a	0.441	0.027	0.074
Crude protein	54.83 ± 0.75^b	57.17 ± 1.41^ab	61.67 ± 1.58^a	58.53 ± 2.03^ab	57.30 ± 1.67^ab	54.57 ± 0.97^b	0.381	0.626	0.009
Crude lipid	2.30 ± 0.34	3.12 ± 0.21	2.84 ± 0.83	2.52 ± 0.23	2.60 ± 0.23	2.43 ± 0.09	0.481	0.544	0.625
Values in the same row (multiple comparisons or main effects analysis) with different superscript letters are significantly different at p < 0.05, while the values for two-factor analysis of variance are less than 0.05, indicating significant differences at p < 0.05. Data are presented as mean ± SEM (n = 3).

Table 3.

Effects of dietary protein-to-sea mud ratio on body-wall composition of A. japonicus (%).

Item	PS no. (protein/sea mud)						ANOVA (p value)
Item	PS1(10/30)	PS2(10/45)	PS3(10/60)	PS4(13/30)	PS5(13/45)	PS6(13/60)	Protein	Sea mud	Protein × sea mud
TPS	196.35 ± 60.27^c	324.31 ± 30.08^bc	362.3 ± 103.23^bc	385.85 ± 29.88^bc	570.93 ± 47.55^ab	736.57 ± 194.26^a	0.005	0.059	0.631
LPS	1.52 ± 0.12^b	1.34 ± 0.01^b	2.07 ± 0.10^ab	1.97 ± 0.29^ab	2.51 ± 0.40^a	2.56 ± 0.19^a	0.002	0.071	0.245
AMS	0.12 ± 0.01	0.13 ± 0.07	0.12 ± 0.01	0.13 ± 0.01	0.14 ± 0.01	0.16 ± 0.01	0.506	0.841	0.807
Values in the same row (multiple comparisons or main effects analysis) with different superscript letters are significantly different at p < 0.05, while the values for two-factor analysis of variance are less than 0.05, indicating significant differences at p < 0.05. Data are presented as mean ± SEM (n = 3). TPS, Trypsin; LPS, Lipase; AMS, Amylase. The unit 'U/(mg prot)' indicates the amount of enzyme activity units per mg of soluble protein in the sample, reflecting the specific activity of the enzyme.

Table 4.

Effects of dietary protein-to-sea mud ratio on digestive enzyme activity in the intestine of A. japonicus (U/[mg prot]).

Item	PS no. (protein/sea mud)						ANOVA (p value)
Item	PS1(10/30)	PS2(10/45)	PS3(10/60)	PS4(13/30)	PS5(13/45)	PS6(13/60)	Protein	Sea mud	Protein × sea mud
MT (μm)	5.08 ± 0.33^c	6.65 ± 0.52^bc	5.68 ± 0.25^c	5.72 ± 0.49^c	9.34 ± 0.61^a	7.44 ± 0.79^b	0.002	0.001	0.194
FH (μm)	192.3 ± 45.58	215.64 ± 52.19	163.72 ± 24.81	187 ± 12.49	247.88 ± 36.67	224.01 ± 18.91	0.327	0.437	0.650
Values in the same row (multiple comparisons or main effects analysis) with different superscript letters are significantly different at p < 0.05, while the values for two-factor analysis of variance are less than 0.05, indicating significant differences at p < 0.05. Data are presented as mean ± SEM (n = 3). MT, muscularis thickness, FH, fold height.

Table 5.

Effects of dietary protein-to-sea mud ratio on intestinal structure of A. japonicus.

Item	PS no. (protein/sea mud)						ANOVA (p value)
Item	PS1(10/30)	PS2(10/45)	PS3(10/60)	PS4(13/30)	PS5(13/45)	PS6(13/60)	Protein	Sea mud	Protein × sea mud
ACP (King unit/[L])	2.20 ± 0.18^c	2.36 ± 0.42^c	2.72 ± 0.35^c	4.98 ± 0.28^b	5.02 ± 0.25^b	6.15 ± 0.47^a	0.000	0.057	0.494
AKP (King unit /[L])	0.23 ± 0.02^bc	0.16 ± 0.01^c	0.16 ± 0.03^c	0.22 ± 0.02^c	0.32 ± 0.01^ab	0.36 ± 0.05^a	0.000	0.420	0.003
SOD (U/[ml])	10.72 ± 0.24	10.72 ± 0.77	10.62 ± 0.70	11.64 ± 0.24	12.32 ± 0.3	11.20 ± 0.78	0.045	0.571	0.667
CAT (U/[ml])	1.64 ± 0.08^c	2.20 ± 0.02^b	2.11 ± 0.07^b	2.14 ± 0.03^b	3.10 ± 0.29^a	2.17 ± 0.16^b	0.001	0.000	0.034
LZM (U/[ml])	120.83 ± 7.13^bc	89.92 ± 9.78^c	137.5 ± 18.29^b	107.93 ± 4.22^bc	169.74 ± 4.66^a	89.38 ± 8.98^c	0.458	0.226	0.000
Values in the same row (multiple comparisons or main effects analysis) with different superscript letters are significantly different at p < 0.05, while the values for two-factor analysis of variance are less than 0.05, indicating significant differences at p < 0.05. Data are presented as mean ± SEM (n = 3). ACP, Acid phosphatase; AKP, Alkaline phosphatase; SOD, Superoxide dismutase, CAT, Catalase, LZM, Lysozyme. The unit 'U/mL' indicates the number of enzyme activity units per mL of sample, reflecting the absolute enzyme activity concentration. The unit 'King unit/L' indicates the number of King units of enzyme activity per L of sample, based on the classic colorimetric assay method.

Table 6.

Effects of dietary protein-to-sea mud ratio on antioxidant capacity in coelomocytes lysate of A. japonicus.

Item	PS no. (protein/sea mud)				ANOVA (p value)
Item	PS1(10/30)	PS3(10/60)	PS4(13/30)	PS6(13/60)	Protein	Sea mud	Protein × sea mud
ACE	276.55 ± 28.53	323.05 ± 19.12	532.17 ± 153.18	447.06 ± 32.58	0.045	0.816	0.435
Chao	276.38 ± 28.52	322.80 ± 18.91	532.18 ± 153.11	446.80 ± 32.56	0.045	0.814	0.435
Shannon	2.99 ± 0.68	3.54 ± 0.09	4.62 ± 0.69	4.38 ± 0.12	0.036	0.758	0.442
Coverage	0.99	0.99	0.99	0.99

Table 7.

Effects of different dietary protein-to-sea mud ratio on gut microbial diversity of A.japonicus.