3. 1. Growth performance
The inclusion of defatted Tenebrio molitor larvae meal had a significant effect (P < 0.05) on body weight and total length of juvenile European perch throughout biometry time-series (Fig. 1). Feeding perch with dietary TM did not affect survival rate (P = 0.729) and condition factor (P = 0.479) after 105-day trial, but, at substantial inclusion levels (TM50 and TM75), significantly compromised weight gain and SGR (P < 0.001), and increased FCR (P < 0.001) compared with TM0 (Table 3). The negative correlation with increasing TM level and PER (P < 0.001) was also detected (Table 3).
Table 3
Production performance, feed efficiency, organo-somatic indices, and serum biochemistry of European perch (Perca fluviatilis) fed experimental diets for 105 days
Item | Experimental diets | SEM1 | P-value |
TM0 | TM25 | TM50 | TM75 |
Production parameters | | | | | | |
Survival (%) | 99.09 | 99.09 | 98.48 | 98.78 | 0.21 | 0.729 |
CF | 1.53 | 1.54 | 1.52 | 1.46 | 0.02 | 0.497 |
WG (g) | 57.56a | 57.14a | 46.48b | 30.59c | 2.90 | < 0.001 |
SGR (%/day) | 1.26a | 1.26a | 1.12b | 0.86c | 0.04 | < 0.001 |
FCR | 1.15a | 1.19ab | 1.33b | 1.77c | 0.07 | < 0.001 |
PER | 1.85a | 1.72ab | 1.60b | 1.21c | 0.06 | < 0.001 |
DFR (% BW/day) | 1.26a | 1.31ab | 1.33ab | 1.41b | 0.02 | 0.021 |
Fillet yield (%) | 38.50 | 39.79 | 38.22 | 37.42 | 0.40 | 0.209 |
Somatic indies | | | | | | |
VSI (%) | 14.23 | 13.65 | 14.14 | 14.27 | 0.26 | 0.959 |
HSI (%) | 1.53 | 1.53 | 1.65 | 1.58 | 0.05 | 0.868 |
MFI (%) | 9.27 | 8.89 | 8.87 | 8.75 | 0.22 | 0.880 |
ISI (%) | 0.75 | 0.73 | 0.84 | 0.87 | 0.03 | 0.420 |
RGL | 0.58 | 0.61 | 0.60 | 0.56 | 0.01 | 0.139 |
SSI (%) | 0.08 | 0.09 | 0.09 | 0.10 | 0.00 | 0.459 |
Serum biochemistry | | | | | | |
ALT (ukat/L) | 0.29 | 0.33 | 0.30 | 0.36 | 0.01 | 0.249 |
AST (ukat/L) | 0.62a | 1.29ab | 1.48ab | 2.04b | 0.19 | 0.044 |
Glucose (mmol/L) | 5.90 | 4.75 | 4.65 | 6.94 | 0.42 | 0.161 |
Cholesterol (mmol/L) | 5.66 | 4.61 | 4.55 | 4.61 | 0.21 | 0.182 |
Triglycerides (mmol/L) | 9.75 | 7.79 | 7.97 | 9.05 | 0.44 | 0.384 |
Total protein (g/L) | 40.70 | 32.64 | 43.29 | 42.25 | 2.54 | 0.476 |
ALP (ukat/L) | 0.43 | 0.37 | 0.40 | 0.49 | 0.02 | 0.256 |
CF = condition factor; WG = weight gain; SGR = specific growth rate; FCR = feed conversion ratio; PER = protein efficiency ratio; DFR = daily feeding rate; VSI = visceral somatic index; HSI = hepatic somatic index; MFI = mesenteric fat index; ISI = intestine somatic index; RGL = relative gut length; SSI = spleen somatic index; ALT = alanine aminotransferase; AST = aspartate aminotransferase; ALP = alkaline phosphatase. |
1 Standard error of the mean (pooled). Means with the same superscript letter in a same row are not significantly different (P > 0.05). |
Feeding dietary defatted TM appeared to be satisfied by European perch in terms of palatability as indicated by DFR values, which were significantly higher in TM75 than TM0 (P = 0.021). Fish displayed an excellent response to TM25 as those growth indices were comparable with TM0 (P > 0.05). Fillet yield of perch, ranged 37.42–39.79%, was TM-inclusion independent (P = 0.209). A similar pattern was observed for all organo-somatic indies (P > 0.05) (Table 3).
Serum biochemistry indices of perch did not differ among diet groups (P > 0.05), except AST activity which was significantly higher in perch fed TM75 than did TM0 (P = 0.044) (Table 3).
3. 2. Apparent digestibility of experimental diets
As depicted in Table 4, the nutrient digestibility of European perch was significantly affected by increasing dietary defatted TM (P < 0.05), except ash (P = 0.05). Digestibility of fatty acids were significantly influenced by dietary defatted TM (P < 0.05), except C14:0 (P = 0.19) and C16:1 (P = 0.39).
3. 3. Proximate composition perch fillet
Feeding defatted TM levels did not alter the proximate composition and fatty acid profile of the perch fillets (P > 0.05) (Table 5). DHA, ranged from 14.95% (TM50) to 18.18% (TM75) of the total fatty acids, was the second-largest constitute in perch fillets following oleic acid, and unaffected by administration of defatted TM (P = 0.48). The EPA share was meager (2.47–3.08 % of the total fatty acids) and was consistent among diet groups (P = 0.13) (Table 5).
3. 4. Microbiota analysis
After sequencing and quality filtering, 334,095 reads were obtained and used for further analysis with an average value of 12,661 reads/sample. Analysis of the rarefactions and estimated sample coverage indicated a satisfactory coverage of all samples (median coverage value of 98%). No significant difference in alpha diversity indices of Shannon (P = 0.557), observed OTUs (P = 0.523), and Chao1 (P = 0.741) (Fig. 2).
The bacterial community in the perch intestine was mainly dominated by phyla Firmicutes, Actinobacteria and Fusobacteria (Fig. 3A). The microbiota composition at the genus level (Fig. 3B) was mainly represented by Clostridium (52%, 87%, 50 and 63% in TM0, TM25, TM50 and TM75, respectively), Mycobacterium (33, 4, 31 and 9%), Lactobacillus (4, 3, 1 and 1%) and Cetobacterium (1, 0.1, 9 and 22%). A minor OTUs fraction (below 0.2%) was composed of Enterobacteriaceae, Streptococcus, Candidatus, Chlamydia, Clavibacter, Bacillus, Parachlamydiaceae, and Solirubrobacterales (Fig. 3B).
The principal component analysis based on OTUs abundance showed no clear separation across diet groups (Fig. 4).
A significant change in microbial composition as a result of the inclusion of defatted TM was observed. By considering the significant difference in the OTUs among diets (Fig. 5), the inclusion of 20.3% or 75% FM replacement by defatted TM significantly reduced the abundance of Lactobacillus (P = 0.02) and Streptococcus (P = 0.038) genera compared with the control group.
3. 5. Solid waste output and environmental impacts
Dietary defatted TM significantly affect environmental impacts associated with TSW (P < 0.001), SNW (P < 0.001) and SPW (P < 0.001) (Table 6). TM75 showed a significant reduction in TSW (P = 0.031), whereas feeding perch with TM25 inverted the pattern (P = 0.005) compared with the control diet. SPW was not different among TM0, TM50 and TM75 (P > 0.05), but was significantly higher in TM25 (P < 0.001). Dietary defatted TM significantly increased SNW (< 0.001) (Table 6).
The eFIFO, ranged 0.99–1.45, was significantly reduced with increasing levels of defatted TM (P < 0.001). Among the TM-containing groups, the ratio was less than one for the TM75 diet, whereas those of TM25, and TM50 were greater than 1 (1.19 and 1.07, respectively) (Table 6).
As far as the environmental impacts associated with one kg farmed perch production are concerned, TM25 was comparable with TM0 for the global warming potential, acidification, and land use (P > 0.05). TM50 and TM75 exerted heavier burdens than the control diet on all the impact categories (P < 0.05).
Table 4 Nutrient digestibility (%) of European perch (Perca fluviatilis) fed experimental diets for 105 days
Nutrients
|
Experimental diets
|
SEM1
|
P-value
|
TM0
|
TM25
|
TM50
|
TM75
|
Dry matter
|
78.95a
|
77.01c
|
77.58b
|
76.52d
|
0.28
|
<0.001
|
Crude protein
|
92.72a
|
90.99b
|
90.42b
|
87.48c
|
0.58
|
<0.001
|
Crude lipid
|
92.62a
|
91.96a
|
92.95a
|
89.14b
|
0.49
|
<0.001
|
Phosphorus
|
45.93a
|
36.26b
|
38.24b
|
37.76b
|
1.46
|
0.041
|
Ash
|
41.98
|
38.14
|
40.11
|
38.30
|
0.61
|
0.058
|
Fatty acids
|
|
|
|
|
|
|
C14:0
|
98.13
|
97.91
|
98.05
|
97.97
|
0.04
|
0.192
|
C16:0
|
96.81a
|
96.09c
|
96.10bc
|
96.33c
|
0.10
|
0.002
|
C16:1
|
98.53
|
98.53
|
98.62
|
98.40
|
0.04
|
0.390
|
C18:1n9
|
97.90ab
|
97.79bc
|
97.93a
|
97.71c
|
0.03
|
<0.001
|
C18:2n6
|
98.40a
|
98.10b
|
98.21ab
|
98.21ab
|
0.04
|
<0.001
|
C18:3n3
|
98.90a
|
98.70b
|
98.84ab
|
98.15c
|
0.09
|
<0.001
|
C20:1n9
|
97.05a
|
96.81a
|
95.68b
|
77.40c
|
2.50
|
<0.001
|
SFA
|
96.71a
|
95.91b
|
95.89b
|
96.13b
|
0.11
|
<0.001
|
MUFA
|
97.76b
|
97.77b
|
97.88a
|
97.60c
|
0.03
|
<0.001
|
PUFA
|
98.34a
|
97.97b
|
98.07b
|
98.08b
|
0.04
|
<0.001
|
Ʃn3
|
98.99a
|
98.70c
|
98.83b
|
98.65c
|
0.04
|
<0.001
|
Ʃn6
|
98.43a
|
98.14b
|
98.22b
|
98.21b
|
0.04
|
<0.001
|
DHA
|
98.93a
|
98.49b
|
98.61b
|
98.91a
|
0.06
|
<0.001
|
EPA
|
99.38a
|
99.16b
|
99.24b
|
99.37a
|
0.03
|
<0.001
|
EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid; SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids.
1 Standard error of the mean (pooled). Means with the same superscript letter in a same row are not significantly different (P > 0.05).
Table 5 Fillet composition (% as wet basis) and fatty acid profile (% of total FA) of European perch (Perca fluviatilis) fed experimental diets
|
Experimental diets
|
SEM1
|
P-value
|
TM0
|
TM25
|
TM50
|
TM75
|
Moisture
|
79.04
|
77.70
|
77.93
|
76.99
|
0.35
|
0.239
|
Crude protein
|
22.25
|
22.74
|
22.55
|
22.0
|
0.25
|
0.784
|
Crude lipid
|
1.45
|
1.30
|
1.21
|
1.28
|
0.04
|
0.193
|
Ash
|
1.11
|
1.11
|
1.12
|
1.11
|
0.01
|
0.99
|
Fatty acid profile2
|
|
C14:0
|
1.59
|
1.62
|
1.37
|
1.53
|
0.05
|
0.378
|
C16:0
|
16.97
|
18.35
|
15.94
|
17.20
|
0.55
|
0.527
|
C16:1
|
4.54
|
5.09
|
4.50
|
4.87
|
0.18
|
0.647
|
C18:0
|
2.51
|
2.68
|
2.37
|
2.73
|
0.10
|
0.585
|
C18:1n9
|
32.58
|
31.44
|
27.39
|
29.69
|
1.16
|
0.499
|
C18:2n6
|
12.34
|
12.07
|
10.58
|
12.24
|
0.43
|
0.477
|
C18:3n3
|
2.84
|
2.66
|
2.15
|
2.37
|
0.11
|
0.144
|
C20:1n9
|
1.56
|
1.42
|
1.18
|
1.36
|
0.05
|
0.055
|
C20:5n3 (EPA)
|
3.06
|
3.08
|
2.47
|
3.03
|
0.12
|
0.13
|
C22:6n3 (DHA)
|
17.00
|
16.92
|
14.95
|
18.18
|
0.71
|
0.483
|
ƩSFA
|
22.34
|
23.87
|
20.76
|
22.67
|
0.71
|
0.531
|
ƩMUFA
|
39.97
|
38.92
|
34.34
|
38.66
|
1.33
|
0.496
|
ƩPUFA
|
37.61
|
37.13
|
44.83
|
38.47
|
1.98
|
0.525
|
1 Standard error of the mean (pooled).
2 Fatty acids with less than 1% total fatty acids in experimental diets (C10:0, C12:0, C13:0, C14:1, C15:0, C15:1, C17:0, C17:1, C16:3, C18:1n9 trans, C18:1n7, C18:2n6 trans, C18:3n6, C20:0, C21:0, C20:3n6, C20:3n3, C20:4n6, C22:0, C24:0, C24:1n9, C22:5n6) were not presented in the table but included in fatty acids group calculation. EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid; SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids.
Means with the same superscript letter in a same row are not significantly different (P > 0.05).
Table 6 Solid waste output and environmental impacts associated with 1 kg production of European perch (Perca fluviatilis) fed experimental diets for 105 days
|
Experimental diets
|
SEM1
|
P-value
|
|
TM0
|
TM25
|
TM50
|
TM75
|
Solid waste output
|
|
|
|
|
|
|
TSW (g)
|
1115b
|
1268a
|
1104b
|
1004c
|
52
|
0.0002
|
SNW (g)
|
385c
|
497ab
|
471b
|
535a
|
30
|
0.0001
|
SPW (g)
|
2860b
|
3514a
|
3038b
|
2660b
|
178
|
0.0008
|
Environmental impacts associated with 1 kg perch production
|
GWP (kg CO2 eq.)
|
3.00c
|
3.24c
|
4.01b
|
5.58a
|
0.27
|
<0.001
|
Energy use (kg oil eq.)
|
0.35d
|
2.45c
|
5.29b
|
10.19a
|
0.96
|
<0.001
|
Acidification (kg SO2 eq.)
|
10.71c
|
11.94c
|
15.17b
|
21.65a
|
1.11
|
<0.001
|
Eutrophication (kg P eq.)
|
0.36d
|
1.54c
|
3.14b
|
5.94a
|
0.54
|
<0.001
|
Land use (m2a)
|
2.40c
|
2.76c
|
3.60b
|
5.23a
|
0.29
|
<0.001
|
Water use (m3)
|
0.03d
|
0.37c
|
0.82b
|
1.61a
|
0.15
|
<0.001
|
Economic fish-in fish-out
|
eFIFO
|
1.45a
|
1.19b
|
1.07bc
|
0.99c
|
0.05
|
<0.001
|
1 Standard error of the mean (pooled).
TSW = total solid waste; SNW = solid nitrogen waste; SPW = solid phosphorus waste; GWP = global warming potential, eFIFO = economic fish-in fish-out.