Methodological context
The determination of apparent digestibility coefficient (ADC) of amino acids was assessed by testing the effect of increasing inclusion levels of solvent-extracted soybean meal (SBM) upon digestibility of juvenile shrimp (Table 1). The dietary inclusion levels chosen are within the recommended range for use of SBM in compound shrimp feeds (Tacon et al., 2009). The four test diets with increasing levels of SBM (5, 10, 15 and 20%) were formulated to have similar levels of crude protein, ether extract and gross energy. For comparison, three other test diets were prepared by the conventionally applied replacement method for determination of ADC, when the target feed ingredient replaces the mash of a certain reference diet at a fixed proportion (test diet), and ADC of feed ingredient is calculated by the difference or not in ADC between test and reference diets (Cho et al., 1982; Bureau and Hua, 2006; NRC, 2011). Presently, SBM was tested replacing 10, 20 and 30% of a practical reference diet mash under this methodology. Therefore, the study focused on the comparison between a currently applied methodology (fixed replacement by target ingredient) and a new approach for the determination of ADC of amino acids in SBM for farmed shrimp.
Table 1
Proximate composition (%, as-is) of feed ingredients1 used in formulation of test diets for juvenile shrimp (Litopenaeus vannamei).
|
Moisture
|
Crude protein
|
Lipid
|
Energy (cal/g)
|
Ash
|
Wheat2
|
11.5
|
12.1
|
2.14
|
3,872
|
1.54
|
Soybean meal3
|
8.95
|
46.5
|
2.94
|
4,313
|
6.79
|
Fish meal (local)3
|
7.52
|
54.8
|
8.0
|
3,917
|
29.0
|
Poultry by-product meal3
|
5.55
|
59.5
|
14.4
|
4,919
|
16.5
|
Squid meal4
|
6.58
|
84.7
|
3.33
|
5,413
|
4.0
|
Fish hydrolysate5
|
-
|
24.5
|
10.0
|
-
|
-
|
Blood meal6
|
5.57
|
90.9
|
1.05
|
5,669
|
1.55
|
Dried yeast7
|
7.20
|
36.2
|
2.69
|
4,313
|
7.20
|
Fish oil3
|
-
|
0.32
|
99.4
|
-
|
-
|
Soy lecithin oil3
|
-
|
3.55
|
88.4
|
-
|
7.71
|
(-): not determined. |
1 Mean of duplicate analysis. |
2 Coamo, Campo Mourão, Brazil |
3 Guabi, Campinas, Brazil |
4 Polinutri, Osasco, Brazil |
5 Aquativ, Elven, France |
6 A&R, Maringá, Brazil |
7 ICC, São Paulo, Brazil |
Feed ingredients and experimental diets
Feed ingredients used in the present study were supplied by the local feed industry and are commonly employed in shrimp feeds. The composition of the feed ingredients was determined (Tables 1 and 2) and results followed typical values (Hertrampf and Piedad-Pascual, 2000; Tacon et al., 2009; NRC, 2011). Prior to pellet preparation, feed ingredients were milled to < 250 µm particle size in a hammer mill (MCS 350 Moinhos Vieira, Tatuí, Brazil), recommended for feeding juvenile shrimp (Terrazas-Fierro et al., 2010; Ye et al., 2012).
Table 2
Amino acid composition of soybean meal (%, as is) used in formulation of test diets for juvenile shrimp (Litopenaeus vannamei).
Arg
|
His
|
Iso
|
Leu
|
Lys
|
Met
|
Phe
|
Thr
|
Try
|
Val
|
Tau
|
3.43
|
1.13
|
2.30
|
3.88
|
2.83
|
0.57
|
2.41
|
2.11
|
0.45
|
2.37
|
0.03
|
Asp
|
Glu
|
Ser
|
Gly
|
Ala
|
Pro
|
Tyr
|
Cys
|
|
|
|
2.88
|
7.30
|
2.25
|
2.23
|
2.37
|
2.61
|
1.67
|
0.85
|
|
|
|
A practical diet formulation (named T6), suitable in attending species nutrient requirements and previously proved efficient for juvenile L. vannamei, was chosen as the reference for preparation of diets designed for determination of ADC under the conventional methodology of replacing part of the reference diet mash by the target feed ingredient (Cho et al., 1982) (Table 3). Though the replacement methodology has been mostly applied to feed ingredients at 30:70 ingredient:reference diet ratio (presently diet T3), for further comparison, additional inclusion levels of SBM were also tested, at 10% (T1) and 20% (T2), as previously assessed with some feed ingredients assessed in shrimp diets (Merican and Shim, 1995; Divakaran et al., 2000; Smith et al., 2007; Rivas-Vega et al., 2009; Carvalho et al., 2016; Panini et al., 2017).
Table 3
Formulation (%) of test diets1 for juvenile shrimp (Litopenaeus vannamei).
|
T1
(10:90)2
|
T2
(20:80)2
|
T3
(30:70)2
|
T4
|
T5
|
T6
(reference)
|
T7
|
Soybean meal
|
23.5
|
32.0
|
40.5
|
5.0
|
10.0
|
15.0
|
20.0
|
Wheat
|
32.3
|
28.6
|
24.9
|
42.0
|
39.5
|
36.0
|
34.0
|
Fish meal
|
18.0
|
16.0
|
14.0
|
20.0
|
20.0
|
20.0
|
20.0
|
Poultry by-product meal
|
9.0
|
8.0
|
7.0
|
11.5
|
10.5
|
10.0
|
9.0
|
Blood meal
|
1.80
|
1.60
|
1.40
|
4.50
|
3.0
|
2.0
|
0.0
|
Squid meal
|
4.50
|
4.00
|
3.50
|
5.00
|
5.00
|
5.00
|
5.00
|
Fish hydrolysate
|
3.60
|
3.20
|
2.80
|
4.00
|
4.00
|
4.00
|
4.00
|
Dried yeast
|
2.70
|
2.40
|
2.10
|
3.00
|
3.00
|
3.00
|
3.00
|
Fish oil
|
1.80
|
1.60
|
1.40
|
2.00
|
2.00
|
2.00
|
2.00
|
Soy lecithin oil
|
0.90
|
0.80
|
0.70
|
1.00
|
1.00
|
1.00
|
1.00
|
Vit. and Min. premix3
|
0.90
|
0.80
|
0.70
|
1.00
|
1.00
|
1.00
|
1.00
|
Binder4
|
0.50
|
0.50
|
0.50
|
0.50
|
0.50
|
0.50
|
0.50
|
Chromic oxide5
|
0.50
|
0.50
|
0.50
|
0.50
|
0.50
|
0.50
|
0.50
|
1 Further details in Material and methods. |
2 Conventional replacement method, SBM inclusion at 10, 20 and 30% into the reference diet mash (T6). |
3 DSM, Mairinque, São Paulo, Brazil |
4 Guabi, Campinas, Brazil |
5 Dinâmica, Indaiatuba, Brazil |
A novel strategy to estimate ADC of nutrients in individual feed ingredients was presently assessed by testing diets containing SBM at 5, 10, 15 and 20%, corresponding to diets T4, T5, T6 and T7, respectively (Table 3). Thus, diet T6 played a double role as reference and test diet (at 15% SBM) under present methodological approach. To keep nutrient content, attractivity, palatability and nutritional consistency, dietary levels of fish meal, squid meal, fish hydrolysate, yeast, fish oil, lecithin, vitamin and mineral premix were kept constant in diets T4 to T7. Similar crude protein, lipid and gross energy levels among test diets T4 to T7 was intended by varying dietary levels of poultry by-product meal, blood meal and wheat in formulation of test diets. Chromic oxide was included at 0.5% in all diets as inert marker for determination of apparent digestibility.
Diet preparation started with mixing the dry ingredients in a planetary mixer (ES-600, Hobart) for 10 min. Next, liquid ingredients, including boiled distilled water, were added to the ingredient mix to form the dough prior to pelleting. The resulting dough was cold pressed (ca. 45°C) and the pellets (3.0–5.0 mm length, 2.0 mm diameter) were dried in a forced air dryer overnight (35–45°C for 18 h). Pellets were stored in zip plastic bags at -15°C until used. The proximate and amino acid composition of the test diets are shown in Table 4. True protein levels refer to the sum of analyzed amino acids.
Table 4
Composition of experimental diets (%, as-is) for juvenile shrimp (Litopenaeus vannamei). Data expressed as mean for proximate composition (n = 2).
|
T1
(10/90)1
|
T2
(20/80)1
|
T3
(30/70)1
|
T4
|
T5
|
T6
(reference)
|
T7
|
Moisture
|
5.12
|
5.43
|
4.22
|
6.16
|
5.12
|
5.86
|
5.99
|
Crude protein
|
41.2
|
42.1
|
43.2
|
39.4
|
39.7
|
39.9
|
37.3
|
True protein2
|
37.2
|
38.7
|
39.3
|
35.5
|
36.4
|
35.9
|
34.0
|
Ether extract
|
6.42
|
6.76
|
5.97
|
7.55
|
7.66
|
8.41
|
7.27
|
Gross energy
|
4,581
|
4,557
|
4,603
|
4,552
|
4,573
|
4,508
|
4,646
|
Ash
|
10.1
|
9.64
|
9.39
|
10.1
|
10.4
|
10.2
|
7.71
|
Cr
|
0.320
|
0.328
|
0.314
|
0.305
|
0.314
|
0.323
|
0.353
|
Amino acids
|
|
|
|
|
|
|
|
Arg
|
2.50
|
2.65
|
2.77
|
2.25
|
2.37
|
2.37
|
2.32
|
His
|
0.90
|
0.95
|
0.97
|
0.89
|
0.89
|
0.86
|
0.81
|
Iso
|
1.54
|
1.63
|
1.75
|
1.31
|
1.39
|
1.44
|
1.56
|
Leu
|
2.79
|
3.01
|
3.19
|
2.81
|
2.82
|
2.76
|
2.65
|
Lys
|
2.21
|
2.33
|
2.43
|
2.14
|
2.13
|
2.16
|
1.94
|
Met
|
0.73
|
0.69
|
0.70
|
0.75
|
0.76
|
0.73
|
0.65
|
Phe
|
1.68
|
1.79
|
1.90
|
1.59
|
1.60
|
1.60
|
1.53
|
Thr
|
1.62
|
1.70
|
1.77
|
1.59
|
1.60
|
1.55
|
1.42
|
Try
|
0.27
|
0.29
|
0.30
|
0.19
|
0.21
|
0.22
|
0.24
|
Val
|
1.79
|
1.88
|
2.00
|
1.80
|
1.81
|
1.76
|
1.65
|
Tau
|
0.13
|
0.12
|
0.11
|
0.15
|
0.15
|
0.14
|
0.11
|
Asp
|
3.44
|
3.58
|
3.18
|
3.02
|
3.19
|
3.19
|
3.09
|
Glu
|
6.15
|
6.39
|
6.45
|
5.56
|
5.83
|
5.82
|
5.97
|
Ser
|
1.83
|
1.88
|
1.95
|
1.74
|
1.77
|
1.74
|
1.58
|
Gly
|
2.96
|
2.90
|
2.84
|
3.06
|
3.10
|
3.00
|
2.54
|
Ala
|
2.31
|
2.34
|
2.38
|
2.37
|
2.39
|
2.29
|
1.99
|
Pro
|
2.51
|
2.54
|
2.59
|
2.50
|
2.56
|
2.48
|
2.42
|
Tyr
|
1.13
|
1.20
|
1.29
|
1.01
|
1.07
|
1.07
|
1.03
|
Cys
|
0.71
|
0.84
|
0.76
|
0.74
|
0.71
|
0.69
|
0.49
|
T4, T5, T6 and T7: practical test diets with SBM inclusion at 5, 10, 15 and 20%, respectively |
1 Conventional replacement method, SBM inclusion at 10, 20 and 30% into the reference diet mash (T6). More details in Material and Methods. |
2 Sum of analyzed amino acids. |
Experimental shrimp
The study was carried out at the coastal Aquaculture Laboratory (LAM) from the University of São Paulo (USP) (Oceanographic Institute, Ubatuba, Brazil). Postlarval L. vannamei (PL 15, Speedline strain) was supplied by Aquatec hatchery (Barra do Cunhaú, Brazil) and was reared in clear water tank nursery system (2,000 L), including daily cleaning and partial water renewal until reaching approximately 0.2 g ind weight. Postlarvae were fed commercial crumbled feed (FlashShrimp, Polinutri, Brazil) continuously delivered by belt feeders (24h Baby belt feeder, Pentair, Brookfield, USA). The seawater (35 ppt salinity) was pumped from the sea (the sheltered Flamengo Cove, Ubatuba) following filtration through 25 µm and 5 µm cartridge filters. The nursery population was further stocked into a 12,000 L tank and raised in biofloc system, fed commercial pellets (Policamarão, Polinutri, Brazil) until stocking into the recirculated trial system at ca. 3.4 g ind shrimp weight. Water quality in the biofloc nursery system: dissolved oxygen (> 70% saturation), total ammonia-N (< 0.05 mg/L), nitrite (< 0.1 mg/L), pH (7.5–8.2) and alkalinity (> 120 mg CaCO3/L).
Shrimp trial system for simultaneous determination of performance and digestibility
The trial was conducted in a clear water system composed of 21 x 500 L semi-conical tanks connected in full recirculation including mechanical and biological filtration (Sweetwater bead filter with UV treatment 4 cubic feet, model 930087, Pentair, Brookfield, USA), and a flow heater (10 kW, Globalmar, São Paulo, Brazil). Feeding was provided by belt feeders (Baby belt feeder, Pentair, Brookfield, USA) individually installed upon experimental tanks that continuously delivered feed pellets over the trial (20–22 h/day). Each tank contained a settling column for feces and solids accumulation and removal with trans lucid Falcon tubes threaded to receive residual solids (feces and eventual pellet leftovers), allowing tube cleaning and exchange. The layout and operational efficiency of the recirculated system and settling columns were previously detailed in Carvalho et al. (2013).
Seawater was filtered (25 and 5 µm cartridge filters) and disinfected by UV before entering the recirculated tank system. A flow rate of 4 L min− 1 into each tank allowed water vortex for feces removal. The trial system continually delivered small amounts of pellets so that pellet apprehension time by shrimp did not exceed one minute. An air stone installed at 30 cm above each tank bottom further sustained dissolved oxygen level and avoided particle suspension.
Shrimp was stocked in tanks after individual weighing in seawater at 34 shrimp per tank that correspond to 85 individuals/m3. Shrimp was acclimated to test diets five days prior starting collection of feces samples. Feed ration was initially set based on tank population biomass, mean individual weight and temperature (Forster et al., 2003). During the trial, daily ration was adjusted according to presence or not of pellet leftovers in the Falcon tube attached to the settling columns overnight. Eighty percent of daily feed ration was continuously delivered by belt feeders and the remaining 20% were divided in three hand-fed meals at 9 am, 11 am and 2 pm. Under this continuous feeding regime, nutrient leaching is minimized once average time for pellet apprehension by shrimp was 30s and 5 min for total pellet intake (visual and camera observations). The trial comprised 55 days and freshly released feces samples were collected 5 days per week, 4 to 6 times per day, morning and afternoon. Daily sample collection started 1h after cleaning of settling tubes and columns (shrimp feces settled during the night were discarded). Maximum exposure time of sampled feces in water (Falcon tube collector) was 1.5h. Collected feces samples were gently rinsed with distilled water during vacuum filtration to remove excess salt. Pooled feces were accumulated during the trial and kept frozen (-15 oC). Feces samples were lyophilized, milled, and stored at -18°C until analysis. Daily monitored water parameters were dissolved oxygen, salinity, and temperature (YSI Pro 2030, Yellow Springs, USA), weekly determination for total ammonia, nitrite, and alkalinity (Alfakit, Florianópolis, Brazil). During the trial, water quality parameters were [mean (s.d.)]: dissolved oxygen 5.60 (0.18) mg/L, 86.5 (3.22) % of saturation; temperature 29.1 (1.10) oC; salinity 35.2 (0.41) ppt; total ammonia 0.30 (0.46) mg/L; nitrite 0.10 (0.08) mg/L; pH 8.0, and alkalinity 106.7 (10.3) mg/L CaCO3.
Experimental diets were tested in triplicate tanks. At trial finishing, harvested shrimp was counted and individually weighted for determination of survival, growth and feed conversion ratio considering total feed delivered during trial.
Feed Conversion Rate (FCR) = total feed consumed (g) divided by total weight gain (g);
Daily feed intake (g/ind) = total feed consumed divided by stock number considering survival rate and days of culture.
Ingredient and diet analysis
Moisture, crude protein, lipid and ash contents of diets, feces and shrimp were determined following AOAC (2005). Moisture was analyzed after oven drying at 105°C until constant weight - dried samples were used for further analysis. Crude protein was analyzed by Kjeldahl (N x 6.25) using copper sulphate as catalyst in the acid digestion. Lipid was determined by ether extraction (ST255, Soxtectm, FOSS). Gross energy content of samples was analyzed with a calorimetric bomb (IKA 5000, Staufen, Germany). Amino acids were determined by HPLC (White et al., 1986; Hagen et al., 1989) after acid and alkaline digestion by the ionic exchange method (Kwanyuen and Burton, 2010), with tryptophan analyzed separately (Lucas and Sotelo, 1980). Feces amino acid content was the only analysis performed in samples sourced from pools of replicate tanks. Chromium in diets and feces samples was determined by atomic absorption spectrometry according to standard method no. 0968.08 (AOAC, 2005).
Calculation of apparent digestibility coefficients: conventional and novel approaches for feed ingredients
Apparent digestibility coefficients (ADC) of dry matter, energy and nutrients (%) in test diets were calculated as follows:
ADC (dry matter, %) = 100–100 (% Cr in diet/% Cr in feces)
ADC (nutrient or energy) = 100–100 [(% Cr in diet/% Cr in feces) x (Nutrient or energy in feces/Nutrient or energy in diet)]
The partial replacement of a reference diet mix by the target ingredient as dietary strategy, following the comparison of the ADC of this test diet with ADC of the reference diet using mathematical calculations (formula), has been the most used method in digestibility studies assessing feed ingredients for shrimp species (Akiyama et al., 1989; Davis and Arnold, 1993; Ezquerra et al., 1997; Cruz-Suárez et al., 2000; Nieto-López et al., 2011; Davis et al., 2002; Cruz-Suárez et al., 2007; Lemos et al., 2009; Niu et al., 2011; Liu et al., 2013; Carvalho et al., 2016). The present study aimed to compare this rather consensual method for ADC determination in a feed ingredient with a novel approach, considering the target ingredient (presently SBM) at different inclusion levels in practical test diets and using the determined ADC of these test diets as reference for estimation of ingredient ADC (Hernández et al., 2008; Yang et al., 2015).
The calculation for ADC of energy and nutrients (%) of SBM under the conventional methodology (SBM presently tested at 10, 20, and 30% inclusion into the reference diet - diets T1, T2 and T3) followed the mathematical approach of weighing ADC of test and reference diets with nutrient or energy content in diet and ingredient:
ADCtest ingredient = ADCtest diet + [(ADCtest diet – ADCref. diet) x (0.7 or 0.8 or 0.9 x Dref/0.3 or 0.2 or 0.1 X Dingr)]
Where, Dref is % nutrient or energy in reference diet mash and Dingr is % nutrient or energy in test ingredient (Bureau and Hua, 2006; Terrazas-Fierro et al., 2010; Hernández et al., 2011; NRC, 2011; Carvalho et al., 2016; Panini et al., 2017; Qiu et al., 2018).
The presently proposed new approach for estimating amino acid ADC in individual feed ingredients (presently focused on SBM) is based on the premise that the value of apparent true protein digestibility (ATPD) of a certain diet represents the average of each individual amino acid digestibilities composing this diet (NRC, 2011). Accordingly, the apparent digestibility value of individual amino acids in the diet would range above and below the mean value found for diet ATPD. Under the present new proposal and dietary experimental strategy, the calculation (mathematical) of apparent individual amino acid digestibility (AIAAD) in a feed ingredient requires: 1. Estimation of the ATPD value of the target ingredient included in test diets (presently SBM); and 2. Comparison of ATPD and AIAAD in each test diet (here composed by different inclusion levels of SBM, diets T4, T5 T6 and T7) that represent the effect of increasing inclusion levels of the target ingredient upon AIAAD of test diet.
The estimation of the ATPD value to be assigned for SBM was based on the combination of different criteria: a. the additive effect of increasing dietary inclusion level of SBM upon ATPD of test diets (presently between 5 and 20%) - i.e., if higher dietary inclusion levels of SBM results in increasing ATPD of test diets, the ATPD value of SBM as feed ingredient is therefore higher than the ATPD of teste diets – b. literature values of ADC in SBM (Akiyama et al., 1989; Divakaran et al., 2000; Yang et al., 2009; Zhu et al., 2013; Fang et al., 2016; Qiu et al., 2018) and author´s experience under currently tested SBM dietary inclusion levels (Lemos et al., 2004; Lemos et al., 2009; Carvalho et al., 2016); and c. the consistency of total dietary protein supplied in test diets (5–20% SBM), i.e. the sum of digestible protein sourced from each relevant proteinaceous feed ingredient in formulation was equivalent to determined (in vivo) ATPD value in the diet, considering the additivity of ingredient protein supply and the estimated ATPD of each feed ingredient using literature data (Ezquerra et al., 1997; Nieto-López et al., 2011; Yang et al., 2009; Ye et al., 2012; Liu et al., 2013; Villarreal-Cavazos et al., 2014; Zhao et al., 2017) and author´s experience for the ingredients fish meal, wheat, poultry by-product meal, blood meal, squid meal, fish hydrolysate, dried yeast and soy lecithin. It was also considered ATPD of test diets may be affected by rather elevated inclusion level of the feed ingredients blood meal (diets T4 and T5) and poultry by-product meal (T4, T5 and T6) used in the present study (Tacon et al., 2009).
The true protein ADC value of SBM assigned for test diets was then used for calculation of individual amino acid digestibility of SBM, so that:
ADCAA SBM = Mean [(ADCAA diet (T4, T5, T6 or T7)/ ADCprotein diet (T4, T5, T6 or T7)) x ADCprotein SBM]
Where:
ADCAA SBM : apparent digestibility coefficient of individual amino acid;
ADCAA diet (T4, T5, T6 or T7) : apparent digestibility coefficient of individual amino acid in the test diet;
ADCprotein diet T4, T5, T6 or T7 : apparent digestibility coefficient of true protein in test diet;
ADCprotein SBM : apparent digestibility coefficient of true protein assigned to SBM (as above described).
Example
- ADC of arginine in test diets = 72, 76, 77 and 80% with SBM inclusion at 5, 10, 15 and 20%, respectively;
-
ADC of true protein in test diets = 69, 73, 74 and 77% with SBM inclusion at 5, 10, 15 and 20%, respectively;
-
ADC of true protein assigned to SBM = 85%;
Thus, ADC of arginine in SBM (%) = Mean [(72/69) x 85]; [(76/73) x 85]; [(77/74) x 85]; [(80/77)] x 85 = 88.5%.
Statistical analysis
Data was submitted to normality and homoscedasticity test prior to application of analysis of variance (ANOVA) or Kruskall-Wallis ANOVA to compare diet performance. Differences between means were analyzed by post-hoc Tukey HSD or Dunn tests. Pearson analysis was applied to test for the statistical significance of the correlations and coefficient of determination (R2). Differences and correlations were considered significant at P < 0.05 (Zar, 1984).