Experimental protocol and design
One hundred and eighty finishing pacu of 1,100 ± 10.3 g (initial body weight ± standard deviation) were obtained from a commercial fish farm. Before beginning the experiment, fish were acclimated to the laboratory conditions for one week. After the acclimation period, they were anesthetized with benzocaine (50 mg L− 1) (Sigma-Aldrich, Brazil) and then distributed in 18 tanks of 2,000 L (10 fish per tank) in a freshwater recirculation aquaculture system (RAS). The experiment consisted of six treatments with three randomly arranged replicates (tanks) per treatment. The photoperiod of 12 h light : 12 h dark was maintained during the experiment. Fish were fed according to the apparent satiation with diets containing six graded levels (16.3, 20.1, 23.8, 27.2, 31.5 and 34.8%) of digestible protein (DP) three times daily at 9:00, 12:00 and 17:00 h for 49 days.
Water quality of the rearing facility
During the present experiment, the water quality parameters including dissolved oxygen concentration (average, 5.74 ± 0.10 mg L− 1) and temperature (average, 28.6 ± 0.5°C) were measured daily by using YSI DO2200A (Yellow Springs Instruments, Ohio, USA) while pH (average, 7.20 ± 0.11) by YSI pH 100A (Yellow Springs Instruments, Ohio, USA) and ammonia concentration (average, 0.019 ± 0.004 mg L− 1) by colorimetric method, Golterman et al. 1978) were measured once a week.
The continuous aeration in experimental tanks was provided by a radial compressor. The water temperature was maintained at a constant level by using a heat exchange system. Moreover, the organic matter from the bottom of experimental tanks was removed by siphoning once a week.
Preparation of experimental diets
According to the procedure of Fisher and Morris (1970), two isoenergetic basal diets (one with high protein and the other with low protein content) were prepared (Table 1). An essential amino acid balance (ideal relationship among essential amino acids – EAAs) (Table 2) was maintained in the two basal diets by using the EAAs requirements recommended by Khan et al. (2020a) for pacu as a reference. The ratios of EAAs in the high protein and low protein diets were fixed according to the procedure of Khan et al. (2020b).
Table 1
Formulation (fed basis %) and analyzed composition (dry matter, %) of basal and experimental diets.
Ingredients
|
Low protein diet
|
High protein diet
|
Digestible protein levels (%)
|
16.3
|
20.1
|
23.8
|
27.2
|
31.5
|
34.8
|
Fish meal
|
4.71
|
17.64
|
7.21
|
9.27
|
11.33
|
13.39
|
15.45
|
17.51
|
Corn gluten
|
1.13
|
3.72
|
1.63
|
2.04
|
2.46
|
2.87
|
3.28
|
3.70
|
Soybean meal
|
12.43
|
44.10
|
18.55
|
23.60
|
28.64
|
33.69
|
38.74
|
43.78
|
Broken rice
|
16.27
|
0.00
|
13.12
|
10.53
|
7.94
|
5.35
|
2.76
|
0.16
|
Corn
|
15.15
|
25.87
|
17.22
|
18.93
|
20.64
|
22.35
|
24.06
|
25.76
|
Corn starch
|
25.50
|
0.00
|
20.57
|
16.51
|
12.44
|
8.38
|
4.32
|
0.26
|
Rice husk
|
7.50
|
0.00
|
6.05
|
4.86
|
3.66
|
2.47
|
1.27
|
0.08
|
Soybean oil
|
10.00
|
4.14
|
8.87
|
7.93
|
70.00
|
6.07
|
5.13
|
4.20
|
L-lysine
|
0.37
|
1.09
|
0.51
|
0.62
|
0.74
|
0.85
|
0.97
|
1.08
|
DL-methionine
|
0.04
|
0.19
|
0.07
|
0.09
|
0.12
|
0.14
|
0.16
|
0.18
|
L-threonine
|
0.05
|
0.15
|
0.07
|
0.08
|
0.10
|
0.12
|
0.13
|
0.15
|
Di-calcium phosphate
|
3.60
|
1.72
|
3.24
|
2.94
|
2.63
|
2.33
|
2.03
|
1.73
|
Limestone
|
2.40
|
0.54
|
2.04
|
1.74
|
1.45
|
1.15
|
0.85
|
0.56
|
Antifungal
|
0.30
|
0.30
|
0.30
|
0.30
|
0.30
|
0.30
|
0.30
|
0.30
|
Antioxidant BHT
|
0.05
|
0.05
|
0.05
|
0.05
|
0.05
|
0.05
|
0.05
|
0.05
|
Mineral and vitamin mixture1
|
0.50
|
0.50
|
0.50
|
0.50
|
0.50
|
0.50
|
0.50
|
0.50
|
Total
|
100.00
|
100.00
|
100.00
|
100.00
|
100.00
|
100.00
|
100.00
|
100.00
|
Analyzed composition
|
|
|
|
|
|
|
|
|
Crude protein (%)
|
14.70
|
40.98
|
18.50
|
22.71
|
26.80
|
30.56
|
35.32
|
38.97
|
Digestible protein (%)2
|
12.83
|
36.60
|
16.30
|
20.10
|
23.80
|
27.20
|
31.50
|
34.80
|
Crude fat (%)
|
12.68
|
9.01
|
11.78
|
11.24
|
10.60
|
9.96
|
9.45
|
8.86
|
Digestible fat (%)2
|
10.64
|
7.31
|
9.83
|
9.34
|
8.76
|
8.19
|
7.72
|
7.19
|
Carbohydrates (%)
|
52.50
|
32.67
|
48.67
|
45.51
|
42.35
|
39.19
|
36.03
|
32.87
|
Ash (%)
|
9.15
|
7.29
|
8.58
|
8.28
|
8.01
|
7.70
|
7.46
|
7.17
|
Gross energy (MJ kg− 1)
|
17.56
|
19.25
|
17.38
|
17.71
|
17.95
|
18.13
|
18.57
|
18.82
|
Digestible energy (MJ kg− 1)2
|
15.06
|
15.04
|
14.63
|
14.68
|
14.67
|
14.60
|
14.73
|
14.72
|
DP/DE (g MJ− 1)
|
---
|
---
|
1.11
|
1.37
|
1.62
|
1.86
|
2.13
|
2.36
|
Dry matter (%)
|
93.52
|
92.89
|
95.42
|
95.05
|
95.11
|
95.53
|
94.61
|
94.67
|
1Folic acid (1.00 mg kg− 1), pantothenic acid (20.00 mg kg− 1), antioxidant (125.00 mg kg− 1), choline (150.00 mg kg− 1), copper (10.00 mg kg− 1), iron (100.00 mg kg− 1), iodine (5.00 mg kg− 1), manganese (70.00 mg kg− 1), selenium (0.15 mg kg− 1), vitamin A (3.000.00 IU kg− 1), vitamin B (16.00 mg kg− 1), vitamin B12 (20.00 mg kg− 1), vitamin B2 (8.00 mg kg− 1), vitamin B6 (3.00 mg kg− 1), vitamin C (350.00 mg kg− 1), vitamin D3 (3000.00 IU kg− 1), vitamin E (200.00 IU kg− 1), vitamin K (6.00 mg kg− 1), zinc (150.00 mg kg− 1), niacin (100.00 mg kg− 1) and biotin (0.10 mg kg− 1). |
2Obtained by using the digestibility coefficients of Abimorad et al. (2008). |
Table 2
Essential amino acid (EAA) pattern maintained in the two basal and six experimental diets
|
Low P diet
|
High P diet
|
16.3
|
20.1
|
23.8
|
27.2
|
31.5
|
34.8
|
|
Crud
|
Digest
|
Crud
|
Digest
|
Crud
|
Digest
|
Crud
|
Digest
|
Crud
|
Digest
|
Crud
|
Digest
|
Crud
|
Digest
|
Crud
|
Digest
|
Methionine
|
0.26
|
0.23
|
0.78
|
0.71
|
0.36
|
0.32
|
0.44
|
0.40
|
0.53
|
0.48
|
0.61
|
0.55
|
0.69
|
0.63
|
0.77
|
0.71
|
Lysine
|
0.89
|
0.81
|
2.77
|
2.57
|
1.25
|
1.15
|
1.55
|
1.43
|
1.85
|
1.71
|
2.15
|
1.99
|
2.45
|
2.27
|
2.75
|
2.55
|
Threonine
|
0.49
|
0.42
|
1.48
|
1.31
|
0.68
|
0.59
|
0.84
|
0.73
|
0.99
|
0.88
|
1.15
|
1.02
|
1.31
|
1.16
|
1.47
|
1.30
|
Arginine
|
0.77
|
0.74
|
2.34
|
2.23
|
1.08
|
1.03
|
1.33
|
1.27
|
1.58
|
1.50
|
1.83
|
1.74
|
2.08
|
1.98
|
2.33
|
2.22
|
Isoleucine
|
0.49
|
0.42
|
1.48
|
1.30
|
0.68
|
0.59
|
0.83
|
0.73
|
0.99
|
0.87
|
1.15
|
1.01
|
1.31
|
1.15
|
1.47
|
1.29
|
Leucine
|
0.98
|
0.89
|
2.86
|
2.66
|
1.34
|
1.23
|
1.64
|
1.51
|
1.94
|
1.79
|
2.24
|
2.08
|
2.54
|
2.36
|
2.84
|
2.64
|
Valine
|
0.56
|
0.48
|
1.65
|
1.45
|
0.77
|
0.66
|
0.95
|
0.82
|
1.12
|
0.97
|
1.29
|
1.13
|
1.47
|
1.28
|
1.64
|
1.44
|
Histidine
|
0.28
|
0.26
|
0.83
|
0.78
|
0.39
|
0.36
|
0.47
|
0.44
|
0.56
|
0.53
|
0.65
|
0.61
|
0.73
|
0.69
|
0.82
|
0.77
|
Phenylalanine
|
0.57
|
0.52
|
1.65
|
1.45
|
0.78
|
0.72
|
0.96
|
0.89
|
1.13
|
1.05
|
1.31
|
1.22
|
1.49
|
1.39
|
1.66
|
1.55
|
Tryptophan
|
0.13
|
0.12
|
0.39
|
0.35
|
0.18
|
0.16
|
0.22
|
0.20
|
0.26
|
0.24
|
0.30
|
0.27
|
0.34
|
0.31
|
0.39
|
0.35
|
Note: Low P diet = Low protein diet; High P diet = High protein diet; Crud = crude (the analyzed values); Digest = digestible (the calculated values obtained by using the digestibility coefficients of Abimorad et al. (2008). |
The unprocessed feed ingredients required for each basal diet (high protein and low protein) were weighed, ground and mixed. The six experimental diets containing 16.3, 20.1, 23.8, 27.2, 31.5 and 34.8% DP were prepared by the serial dilution of the high protein diet with the low protein diet (Table 1). The EAAs balance being provided to the two basal diets was maintained in the six experimental diets as well as the proportions of EAAs gradually increased with the increasing dietary protein content (Table 2).
After the addition of water and oil, each experimental diet was extruded through a micro laboratory extruder (Ex-Micro, Exteec, Ribeirão Preto, Brazil). The feed pellets were dried in a forced-air-oven at 55°C for 12 h, then cooled at room temperature, packed into plastic bags and stored in a cold chamber (− 12°C) until use. The nutritional composition of the basal and experimental diets was analyzed according to the standard procedures of AOAC International (2016) (Table 1, 2).
Sample collection
At the end of the 49-day experiment, 24 h after the fasting period, fish were weighed and blood and tissue samples were collected. Before the sampling procedure, fish were anesthetized with benzocaine (50 mg L− 1). Blood samples from the caudal vessel of three pacu per replicate (tank) (totaling nine fish per treatment) were collected through syringes. Some aliquots were destined for plasma separation (15 µL potassium fluoride + EDTA, Gold Analisa Diagnóstica, Belo Horizonte, Brazil) and the remaining amount was used to obtain serum. The separation of blood aliquots was performed by centrifugation (Allegra x- 30R Centrifuge, Beckman Coulter, Brea, California, USA) of blood (10 min, 827 g, 10°C) immediately after sampling for plasma separation and 1 h after sampling at room temperature for serum separation. The samples collected were stored at − 18°C for subsequent physio-biochemical analyzes.
Assessments
Blood metabolites
The circulating glucose (GOD-Trinder Method, Labtest Diagnostic SA, Lagoa Santa, Minas Gerais, Brazil) was analyzed in the plasma while the total triglycerides (Enzymatic colorimetric method, Labtest Diagnostics SA, Lagoa Santa, Minas Gerais, Brazil), total protein (Biuret Method, Cain and Skilleter 1987) and cholesterol (Enzymatic colorimetric method, Bioclin/Quibasa Quımica Basica Ltda, Belo Horizonte, MG, Brazil) were analyzed in the serum. The circulating ammonia concentrations were determined in the serum according to the method of Verdouw et al. (1978) with some modifications. A total of 20 µL trichloroacetic acid (15%) was added to a 60 µL aliquot. After centrifugation (9,184 g, 3 min), the supernatant was separated for subsequent analysis of ammonia concentration. The reaction was initiated by the addition of sodium nitroprusside (0.01 mM), sodium hypochlorite (0.32%), sodium hydroxide (64.5 mM), sodium citrate (87.1 mM) and sodium salicylate (161.2 mM). After homogenization, the samples were incubated for 2 h in dark, with further reading in a spectrophotometer at 540 nm. The serial dilutions of an ammonium chloride solution were used to establish a standard curve for the determination of ammonia concentration in the serum samples.
Hepatic enzyme activities
The liver samples obtained from the three fish per replicate (tank) were homogenized and centrifuged (15 min, 30,000 g, 10°C). The activities of enzymes associated with amino acid metabolism (alanine aminotransferase [ALT; EC 2.6.1.2] and aspartate aminotransferase [AST; EC 2.6.1.1]) were determined in the homogenate of the liver tissue and were measured by using commercial kits (Gold Analyzes Diagnostics, Belo Horizonte, Brazil), followed by the kinetic reading in a microplate spectrophotometer (Multiskan GO Microplate Spectrophotometer, Thermo Fisher Scientific Inc., Madison, Wisconsin, USA).
The activities of hepatic enzymes associated with glycolytic pathway including hexokinase (HK; EC 2.7.1.1), glucokinase (GK; EC 2.7.1.2) and pyruvate kinase (PK; EC 2.7.1.40) were also determined in the liver homogenates. The activities of hexokinase (HK) and glucokinase (GK) enzymes were determined according to the method of Vijayan et al. (1990) while pyruvate kinase (PK) activity was determined by the method followed by Morales et al. (1990).
Energy reserves
Three fish per replicate (tank) used in the blood sampling were later on subjected to the laparotomy for the removal of visceral fat and liver tissue. After removal from the abdominal cavity of fish, the visceral fat and liver tissue were immediately weighed for the calculation of somatic indices including mesenteric fat index {MFI = [100 x (visceral fat content weight / live weight)]} and hepatosomatic index {HSI = [100 x (liver tissue weight / live weight)]}.
The liver samples, after the weighing procedure, were immediately frozen in liquid nitrogen for the subsequent analysis of total lipid content. The hepatic total lipid content was determined by the solvent extraction method according to Bligh and Dyer (1959).
Growth performance
At the end of the 49-day experiment, the growth performance parameters including food intake, feeding rate and absolute weight gain (WG) were evaluated according to the following formulas:
-
Food intake (g): total amount of food (g) offered to the fish over the 49-day feeding period
-
Feeding rate (%BW day− 1): 100 × food intake ÷ {feeding days × [(final body weight + initial body weight) ÷ 2]}
-
Absolute weight gain per fish (g): (final body weight − initial body weight)
Statistical analysis
The metabolic and production data obtained over the 49-day experiment were tested for normality (Cramer Von Mises test) and homoscedasticity (Brown-Forsythe test) and then analyzed by the one-way analysis of variance (ANOVA). The means obtained for different treatments were compared by the Tukey's multiple range test and the significance level was set at 5% (p < 0.05). All statistical analyses were performed by using the SAS 9.0 (SAS Inst. Inc., Raleigh, North Carolina, USA) software.