Chemical analysis of food and experimental diets
To formulate the experimental diets, analysis of corn, soybean meal, corn starch and soy oil were performed (Table 1). The crude protein (CP) of the food was determined by the standard method [13], and the percentage of nitrogen was obtained by the Kjeldahl method after acid digestion. Gross energy (GE) content was measured by combustion in a Parr bomb calorimeter (model: ECO-CAL2K). The analysis of the total amino acid (TAA) content of corn and soybean meal was performed by high performance liquid chromatography (HPLC). To obtain the digestible essential amino acids (DAA), the values of TAA were converted using the digestibility coefficients of corn and soybean meal proposed by [14] for tambaqui.
Table 1
Composition of the main ingredients used in experimental diets (on the natural matter bases)
Nutrientes | Corn | Soybean meal | Corn starch | Soy oil |
---|
Dry matter | 93.90 | 95.20 | 88.90 | 99.61 |
Crude protein (%) | 7.62 | 46.65 | --- | --- |
Gross energy (kcal kg− 1) | 3941.00 | 4092.00 | 3821.00 | 9333.00 |
Amino acids (%) | TAA1 | DAA2 | TAA1 | DAA2 | TAA1 | DAA2 | TAA1 | DAA2 |
Lysine | 0.27 | 0.25 | 2.76 | 2.61 | --- | --- | --- | --- |
Methionine | 0.17 | 0.15 | 0.59 | 0.55 | --- | --- | --- | --- |
Threonine | 0.26 | 0.25 | 1.71 | 1.68 | --- | --- | --- | --- |
Tryptophan | 0.02 | 0.02 | 0.78 | 0.72 | --- | --- | --- | --- |
Valine | 0.39 | 0.38 | 2.28 | 2.20 | --- | --- | --- | --- |
Histidine | 0.24 | 0.22 | 1.25 | 1.20 | --- | --- | --- | --- |
Leucine | 0.75 | 0.74 | 3.41 | 3.31 | --- | --- | --- | --- |
Isoleucine | 0.27 | 0.26 | 2.20 | 2.13 | --- | --- | --- | --- |
Arginine | 0.42 | 0.41 | 3.42 | 3.39 | --- | --- | --- | --- |
Phenylalanine | 0.33 | 0.32 | 2.37 | 2.33 | --- | --- | --- | --- |
Cystine | 0.13 | 0.12 | 0.68 | 0.61 | --- | --- | --- | --- |
1Total amino acids; 2Digestible amino acids. |
The experimental diets were formulated using the “dilution” technique [15]. Initially, a reference diet (RD) was formulated containing 18.06% crude protein (CP) and 0.901% digestible lysine, based on corn and soybean meal (Table 2). Subsequently, this was diluted with another protein-free diets (PFD) based on corn starch, containing the same levels of energy, vitamins, and minerals, enabling levels of 0.225, 0.450, 0.675 and 0.901% digestible lysine (Table 2). After preparing the experimental diets, the total amino acids were analyzed and their contents confirmed with the formulation targets.
To confirm that lysine was the first limiting nutrient in experimental diets, a fifth treatment (CD: control diet) was added, with five replications, to which synthetic lysine was added at the first level (0.225%) until reaching the second level (0.450%). Thus, it is possible to confirm that the response obtained in the study occurred due to the variation in lysine and not due to the variation between crude proteins (4.51–18.06%) of the experimental diets.
In experimental diets, the ratios of methionine plus cystine / lysine, threonine / lysine and tryptophan / lysine were maintained above the ideal protein ratio proposed by [16˗18], respectively, for tambaqui (Table 2), in order to prevent another amino acid from becoming limited for each level of digestible lysine evaluated.
Table 2
Diets formulated by dilution method with increasing digestible lysine levels for tambaqui with different weights
Ingredients (%) | Digestible lysine levels (%) |
---|
PFD | 0.225 | 0.450 | 0.675 | 0.901 (RD) | CD |
---|
Soybean | 0.00 | 7.13 | 14.26 | 21.39 | 28.53 | 7.13 |
Corn | 0.00 | 15.60 | 31.20 | 46.80 | 62.40 | 15.60 |
Corn starch | 81.80 | 61.35 | 40.90 | 20.45 | 0.00 | 61.35 |
Soy oil | 9.85 | 8.36 | 6.88 | 5.39 | 3.91 | 8.36 |
Cellulose | 2.90 | 2.17 | 1.45 | 0.72 | 0.00 | 2.17 |
L-lysine HCl (78.4%) | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.29 |
DL-Methionine (99%) | 0.00 | 0.03 | 0.06 | 0.09 | 0.11 | 0.03 |
L-Threonine (98.5%) | 0.00 | 0.03 | 0.05 | 0.08 | 0.11 | 0.03 |
Calcitic limestone | 1.05 | 1.10 | 1.16 | 1.21 | 1.26 | 1.10 |
Dicalcium phosphate | 3.28 | 3.11 | 2.94 | 2.77 | 2.60 | 3.11 |
Premix Vit. and Min.1 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 |
Vitamin C2 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 |
Salt | 0.55 | 0.54 | 0.53 | 0.52 | 0.51 | 0.54 |
Antioxidant (BHT) | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 |
Diets | Dilution (%) |
PFD | 100.00 | 75.00 | 50.00 | 25.00 | — | 75.00 |
RD | 0.00 | 25.00 | 50.00 | 75.00 | 100.00 | 25.00 |
Total | 100.00 | 100.00 | 100.00 | 100.00 | 100.00 | 100.00 |
Nutritional Composition (%) |
Dry matter | 89.40 | 90.73 | 91.71 | 91.90 | 92.26 | 90.86 |
Crude protein | 0.00 | 4.51 | 9.03 | 13.54 | 18.06 | 4.51 |
Gross energy (kcal kg− 1) | 4044.88 | 4030.98 | 4018.01 | 4004.11 | 3991.51 | 4030.98 |
Digestible energy (kcal kg− 1)3 | 3000.00 | 3000.00 | 3000.00 | 3000.00 | 3000.00 | 3000.00 |
Crude fiber | 2.81 | 2.81 | 2.81 | 2.81 | 2.861 | 2.81 |
Available P4 | 0.60 | 0.60 | 0.60 | 0.60 | 0.60 | 0.60 |
Total Ca4 | 1.20 | 1.20 | 1.20 | 1.20 | 1.20 | 1.20 |
Total lysine | 0.00 | 0.239 | 0.478 | 0.717 | 0.956 | 0.466 |
Digestible lysine5 | 0.00 | 0.225 | 0.450 | 0.675 | 0.901 | 0.450 |
Total methionine + cystine | 0.00 | 0.167 | 0.334 | 0.501 | 0.658 | 0.167 |
Digestible methionine + cystine5 | 0.00 | 0.153 | 0.306 | 0.466 | 0.612 | 0.153 |
Total threonine | 0.00 | 0.192 | 0.374 | 0.566 | 0.758 | 0.192 |
Digestible threonine5 | 0.00 | 0.185 | 0.369 | 0.561 | 0.739 | 0.185 |
Total tryptophan | 0.00 | 0.059 | 0.117 | 0.176 | 0.235 | 0.059 |
Digestible tryptophan5 | 0.00 | 0.043 | 0.086 | 0.129 | 0.173 | 0.043 |
Relation based on the ideal protein concept |
Methionine + Cys/digestible lysine | 0.00 | 68 | 68 | 68 | 68 | 34 |
Threonine/digestible lysine | 0.00 | 82 | 82 | 82 | 82 | 41 |
Tryptophan/digestible lysine | 0.00 | 24 | 24 | 24 | 24 | 10 |
1Vitamin and mineral supplement, amounts supplied per kg of diet: Vit. A, 6,000 IU; Vit. D3, 1,000 IU; Vit. E, 60.0 mg; Vit. K3, 12.0 mg; Vit. B1, 24.00 mg; Vit. B2, 24.00 mg; Vit. B6, 24.00 mg; Vit. B12, 24.00 mg; Vit. C, 24.00 mg; folic acid, 6.00 mg; Ca pantothenate, 60.00 mg; biotin, 0.24 mg; choline chloride, 108 g; niacin, 100.00 mg; Fe, 250.00 mg; Cu, 15.0 mg; Mn, 100.00 mg; Zn, 150.00 mg; I, 0.5 mg; Co, 0.05 mg; Se, 0.5 mg. 2Vit. C: Calcium L-Ascorbic acid 2-monophosphate, 42% of active ingredient. 3Values calculated based on the digestibility coefficients determined by [19, 20]. 4Values calculated based by [21]. 5Values calculated based on the digestibility coefficients determined by [14]. |
For the extrusion process of experimental diets, the ingredients of PDF and RD were finely ground (Trf 60, Trapp®) and weighed individually and mixed (Horizontal Mixer 300 Kg, Branorte®). To obtain intermediate diets (0.225, 0.450 and 0.675) mixtures were performed by dilution (Table 2). Subsequently, they were pelleted in equipment with a 4–5 mm sieve (Extruder model MX 40, Inbramaq®, Laboratório de Nutrição e Alimentação de Organismos Aquáticos do Maranhão, Chapadinha, in Brazil).
The experimental diets were provided daily in four meals (08:00, 11:00, 14:00 and 17:00 h), until apparent satiation. At each meal, they were supplied in small quantities with successive passes, allowing maximum intake.
Fish, rearing conditions and experimental design
Three experimental trials, using different tambaqui body weights, were conducted at the Laboratory of Nutrition and Food of Aquatic Organisms of Maranhão, located at the Center for Agricultural and Environmental Sciences of the Federal University of Maranhão, Chapadinha, in Brazil (03°44′33″S, 43°21′21″W; altitude 105 m). The experimental procedures were approved by Animal Use Ethics Committee of the Federal University of Maranhão (Protocol Nº 23115.005476/2017-00).
Each experimental trial lasted 20 days, and five days before the beginning of the experimental period, the fish were acclimated to the experimental conditions.
In each experiment, 100 tambaqui with different initial mean weights of 121 ± 1.35; 235 ± 1.23 and 596 ± 47.57 g were used, distributed in a completely randomized design, consisting of four treatments (0.225; 0.450; 0.675 and 0.901% of digestible lysine in the diets) and five replicates, with 5 fish per experimental unit. A fifth treatment (CD: control diet) was added, with five replications, to confirm that the response obtained was due to the variation of digestible lysine and not to the variation between crude proteins (4.51 - 18.06%).
During the experimental period, the fish were kept in polyethylene boxes, with a capacity of 1000 L each, equipped with individual water supplies, drainage systems, and aeration systems, the water supply for the boxes was derived from an artesian well, with flow rate of 40 L h-1 per box.
Water quality parameters
The water temperature was measured daily at 7:00 and 16:00 h with a mercury bulb thermometer graduated from 0 to 50 °C. Controls for pH and the content of dissolved oxygen and ammonia in the water were measured every three days using a pH meter (HI 8424, Hanna®), oximeter (HI 9146, Hanna®) and commercial kit (Arcor®) for toxic ammonia test, respectively.
Carcass preparation and analysis
The comparative slaughter technique was used to study maintenance requirements and efficiency of utilization of lysine of tambaqui. At the beginning of each experiment, 15 fish from the same population used in each experiment were euthanized (benzocaine 100 mg L-1, after fasting for 24 h) and frozen in an ultra-freezer with temperature -70 °C for later determination of the initial body protein content. At the end of the study, after a 24 h fast, all fish in each experimental unit were weighed, euthanized (benzocaine 100 mg L-1) and frozen in an ultra-freezer with temperature -70 ° C to determine the final body protein content.
Subsequently, the initial and final samples were lyophilized for 72 h (-50 °C, -80 kPa) in L108 freeze-drying equipment (LIOTOP®) and then processed in an analytical mill (IKA® A11 basic).
The crude protein of whole body tambaqui was determined by the standard method [13], and the percentage of nitrogen was obtained by the Kjeldahl method after acid digestion.
The analysis of lysine content of whole body of tambaqui was performed using high performance liquid chromatography (HPLC) at the CBO laboratory (Valinhos, Brazil).
Evaluated variables
The variables evaluated were feed intake (FI; g fish-1 day-1), digestible lysine intake (DLI; mg kg-0.7 day-1), final weight (FW; g), weight gain (WG; %), body protein retention (BPR; mg kg-0.7 day-1), and body lysine retention (BLR; mg kg-0.7 day-1).
For the calculate weight gain (%) the following formula was used:
- WG (%) = (Final Weight (g) - Initial Weight (g)) / Initial Weight (g) × 100.
For the calculate body protein retention (BPR) were used analyzes of the initial body protein content (initial fish samples) and analyzes of the final body protein content, calculated using the following formula:
- BPR (mg kg-0.7 day-1) = (final body protein (%) × final weight (g)) - (initial body protein (%) × initial weight (g) / experimental period / final weight (kg-0.7).
For the calculate body lysine retention (BLR) (mg kg-0.7day-1), lysine analysis was performed, which corresponds to 7.41, 6.55 and 6.35% of whole body protein of tambaqui, for body weight 121, 235 and 596, respectively, and calculated by the following formula:
- BLR (mg kg-0.7 day-1) = {[(body protein retention (%) × protein lysine content (%)) / 100] / final weight (kg-0.7)}.
Maintenance lysine requirements were determination based on digestible lysine intake (mg kg-0.7day-1) that provided zero protein retention (mg kg-0.7day-1).The efficiencies for using lysine for growth were obtained by the slope coefficient of the line between the digestible lysine consumption (mg kg-0.7 day-1) as a function of body lysine retention (mg kg-0.7day-1) [8, 12].
Statistical analysis
The data obtained, in each experimental trial, were submitted to an analysis of variance. For those with significant effects as a function of digestible lysine levels, the means of each treatment were compared using the Duncan test. Linear regression analysis of protein and body lysine deposition as a function of digestible lysine consumption was also performed.
Additionally, to compare the responses of the different body weights, the body protein retention (BPR) and body lysine retention (BLR) data obtained in the experimental tests were compared using the parallelism test [22] between equations two by two. The weight was a categorical variable and DLI (mg kg-0.7 day-1) was a covariate in the model:
- BPR / BLRij = β0 + BWi + β1*DLIij + Σiβ2i (BW* DLI)ij + eij;
Where BPR / BLRij = body protein retention / body lysine retention corresponding to the j body weight observation i;
BWi = effect of body weight;
DLIij = digestible lysine intake;
β0, β1, and β2i = regression parameters;
(BW*DLI)ij = effect of the interaction between the categorical variable and covariate;
eij = random error associated with observation j of body weight i.
In this case, the hypotheses tested were:
a) H0: Gi = 0 for all i, there is no effect on body weight;
H1: Gi ≠ 0 for at least one i; there is an effect of body weight;
b) H0: β1 = 0, general slope is zero, non-significant regression;
H1: β1 ≠ 0, the overall slope differs from zero, significant regression;
c) H0: β2i = 0, the inclination of body weight i does not differ from the average inclination.
All analyses were performed with the aid of SAS software (Statistical Analysis System, version 9.0) considering a significance level of up to 5%.