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. 2021 Apr 21;99(6):skab124. doi: 10.1093/jas/skab124

Amino acid digestibility and digestible indispensable amino acid score-like values of black soldier fly larvae fed different forms and concentrations of calcium using the precision-fed cecectomized rooster assay

Sungho Do 1, Elizabeth A Koutsos 2, Pamela L Utterback 1, Carl M Parsons 1, Maria R C de Godoy 1,3, Kelly S Swanson 1,3,4,✉
PMCID: PMC8188813  PMID: 33880561

Abstract

Black soldier fly larvae (BSFL) are an alternative protein source for animals, including dogs and cats. Dietary calcium source is an essential nutrient for BSFL development in the pupal stage. Calcium carbonate (CaCO3) and calcium chloride (CaCl2) are common calcium sources but differ in solubility, acid-binding capacity, and calcium concentration. A high calcium concentration in BSFL may affect how well nitrogen and amino acids (AA) are digested by animals consuming them, thereby affecting feed conversion efficiency. Our objective was to determine the effects of dietary calcium form and concentration on nutrient composition, AA digestibility, and digestible indispensable amino acid score (DIAAS)-like values of BSFL intended for use in animal feeds using the precision-fed cecectomized rooster assay. All BSFL tested in this study were harvested at 18 d after hatch. Industry standard rearing conditions were maintained and a commercial layer ration was fed to all BSFL until 11 d post-hatch. From day 11 to 18, BSFL were fed a combination of distiller’s dried grains with solubles from a distillery, bakery byproduct meal, and varied calcium sources. All BSFL diets contained 0.2% calcium in the basal diet plus additional calcium in the following amounts and forms: BSFLA: 1.2% CaCl2, BSFLB: 1.2% CaCO3, BSFLC: 0.75% CaCO3, and BSFLD: 0.6% CaCO3 + 0.6% CaCl2. On day 18, BSFL were washed and frozen. Prior to the rooster assay, BSFL were lyophilized and ground. In total, 16 cecectomized roosters (4 roosters per substrate) were randomly assigned to test substrates. After 24 h of feed withdrawal, roosters were tube-fed 20 g of test substrates. Following crop intubation, excreta were collected for 48 h. Endogenous corrections for AA were made using five additional cecectomized roosters. All data were analyzed using a completely randomized design and the GLM procedure of SAS 9.4. Nutrient and AA digestibilities were not different among substrates. DIAAS-like values were calculated to determine protein quality according to the Association of American Feed Control Officials nutrient profiles and National Research Council recommended allowances for dogs and cats. Although AA digestibilities did not differ, those containing CaCO3 generally had higher DIAAS-like reference values than the diet containing CaCl2 alone (BSFLA). Aromatic AA (Phe + Tyr) and sulfur AA (Met + Cys) were often first-limiting AA. Our results suggest that calcium sources fed to BSFL did not affect AA digestibility and protein quality.

Keywords: amino acid digestion, canine nutrition, feline nutrition, insect protein, pet food

Introduction

Black soldier fly larvae (BSFL; Hermetia illucens) have been shown to efficiently convert various organic materials such as livestock manure, fruits, and vegetable waste into alternative protein and fat sources for swine (Newton et al., 1977, 2005), poultry (Cullere et al., 2016; Marono et al., 2017; Mwaniki et al., 2018), and fish species (Kroeckel et al., 2012; Lock et al., 2015). The environmental factors and housing conditions (e.g., temperature, humidity, and stocking density) used and dietary ingredient and nutrient composition fed to BSFL require consideration because they affect their growth rate, survival rate, and nutrient composition (Sheppard et al., 2002; Newton et al., 2005; Diener et al., 2009; Tomberlin et al., 2009; Holmes et al., 2012).

Chia et al. (2018), who tested nine temperatures (10 to 42 °C), demonstrated that housing temperature affected BSFL survival rate. Egg eclosion rate was highest at 30 °C (80%) and 35 °C (75%) and lowest at 15, 37, and 40 °C (below 11%). The survival rate of the larval and pupal stages was also highest at 30 °C (90% and 77%) and 35 °C (92% and 75%). Like temperature, relative humidity is also an essential factor affecting BSFL development and survival. Egg eclosion time (124.43 to 87.63 h) and mortality rate (62% to 3%) of the BSFL pupal stage decreased when the relative humidity increased from 25% to 70%. Also, adult longevity (5.17 to 7.94 d) and the emergence rate of BSF (16% to 93%) increased when they were reared on 40% and 70% of relative humidity compared with 25% (Holmes et al., 2012). Another aspect under consideration is stocking density. Barragan-Fonseca et al. (2018) placed 50, 100, 200, or 400 BSFL in plastic containers (15.5 × 10.5 × 6 cm), demonstrating that development time was shorter (13 vs. 15 d) at lower larval densities (50, 100, and 200) when raised on a diet with high nutrient concentrations (14.0% protein and 1.8% fat vs. 3.5% protein and 0.7% fat).

Dietary factors not only influence BSFL performance but also their nutrient composition. Oonincx et al. (2015) observed that when BSFL were fed a chicken feed diet (control diet), they had a shorter development time (20 d) than larvae fed animal manures (chicken: 144 d, pig: 144 d, and cow: 214 d). According to Nguyen et al. (2015), protein and fat concentrations of BSFL fed rendered fish (protein: 50% and fat: 36.2%) or pig liver (protein: 76.7% and fat: 12.8%) were higher than BSFL fed chicken feed (protein: 18% and fat: 2.52%). However, Bosch et al. (2014) reported that BSFL fed a broiler starter diet had protein (56.1%) and fat (12.8%) concentrations that were much higher than those fed a similar diet in the Nguyen et al. (2015) study. The mineral content of BSFL also depends on the diet fed. For instance, phosphorus and calcium concentrations of BSFL reared on poultry manure (1.5% and 7.8%) were higher than BSFL reared on swine manure (0.88% and 5.36%) or chicken feed (1.28% and 3.14%) (Newton et al., 2005; Dierenfeld and King, 2008; Finke, 2013).

BSFL contain high calcium concentrations and greater calcium, magnesium, and potassium concentrations than other insects, such as tebo worms, Turkestan cockroaches, and house flies (Finke, 2013). The reason that BSFL contain high calcium content is because their exoskeleton layer is rich in CaCO3 (Johannsen, 1922). Dietary calcium sources for BSFL may impact the development of their epidermis in the prepupal to pupal stages. There are many dietary calcium options when it comes to feeding BSFL, with variance in acid-binding capacity, water solubility, and calcium concentration. To our knowledge, no scientific research has been conducted to test how dietary calcium form and concentration affect the growth rate of BSFL and their protein quality for use in animal feed. For this reason, our objective was to determine the effects of dietary calcium form and concentration on nutrient composition, amino acid (AA) digestibility, and digestible indispensable amino acid score (DIAAS)-like values of BSFL intended for use in animal feeds using the precision-fed cecectomized rooster assay. We hypothesized that the different forms and concentrations of dietary calcium sources used in this study would affect the nutrient composition of BSFL. However, we hypothesized that the protein quality of BSFL would not be changed.

Materials and Methods

Substrates

All BSFL tested in this study were harvested at 18 d after hatch. Industry standard rearing conditions were maintained (Sheppard et al., 2002) and a commercial layer ration was fed to all BSFL until 11 d post-hatch. From day 11 to 18, they were fed a combination of distiller’s dried grains with solubles from a distillery, bakery byproduct meal, and calcium sources (CaCl2 and CaCO3). Treatment groups included the following:

  1. BSFLA (0.2% calcium in basal diet + 1.2% Ca from CaCl2)

  2. BSFLB (0.2% calcium in basal diet + 1.2% Ca from CaCO3)

  3. BSFLC (0.2% calcium in basal diet + 0.75% Ca from CaCO3)

  4. BSFLD (0.2% calcium in basal diet + 0.6% Ca from CaCO3 + 0.6 % Ca from CaCl2)

Although CaCl2 is the typical calcium source used to raise BSFL in production, it is expensive, so a more economical option was tested at various inclusion levels. Because calcium sources differ in solubility, calcium content, and acid-binding capacities, numerous options exist. In this initial study, it was of interest to test whether 50% or total replacement of the 1.2% CaCl2 was possible without detrimental effects. On day 18, larvae were washed and frozen. All BSFL were then lyophilized and ground through a 2-mm screen with dry ice prior to chemical analysis and feeding to cecectomized roosters.

Cecectomized rooster assay

The protocol for the cecectomized rooster assay, including all animal housing, handling, and surgical procedures, was reviewed and approved by the Institutional Animal Care and Use Committee at the University of Illinois at Urbana-Champaign prior to experimentation. A precision-fed rooster assay using cecectomized Single Comb White Leghorn roosters was conducted as described by Parsons (1985) to determine the dry matter (DM), organic matter (OM), acid-hydrolyzed fat (AHF), and AA digestibility of the substrates listed above. Prior to the study, the cecectomy surgery was performed on roosters under general anesthesia according to the procedures of Parsons (1985).

Briefly, 16 cecectomized roosters were randomly assigned to the test substrates (4 roosters per test substrate evaluated). After 24 h of feed withdrawal, roosters were tube-fed 20 g of test substrates. Following crop intubation, excreta (urine and feces) were collected for 48 h on plastic trays placed under each individual cage. Excreta samples then were lyophilized, weighed, and ground through a 0.25-mm screen prior to analysis. Endogenous corrections for AA were made using five additional cecectomized roosters that had been fasted for 48 h. Nutrient and AA digestibilities were calculated using the method described by Sibbald (1979).

Chemical analyses

The substrates and rooster excreta were analyzed for DM (105 °C) and ash (OM was calculated based on ash) according to AOAC (2006; DM: method 934.01; OM: method 942.05). Nitrogen and crude protein (CP) were determined using a Leco Nitrogen/Protein Determinator (Model FP-2000, Leco Corporation, St. Joseph, MI) according to AOAC (2006; method 982.30E). Fat concentrations were measured by acid hydrolysis according to AACC (1983) followed by diethyl ether extraction (Budde, 1952). Gross energy (GE) was measured using a bomb calorimeter (Model 1261; Parr Instrument Co., Moline, IL). AA and calcium were measured at the University of Missouri Experiment Station Chemical Laboratories (Columbia, MO) according to the AOAC (2006) method 982.30 E [a, b, c] and method 968.08.

DIAAS-like calculations

The calculation of DIAAS-like values was followed according to Mathai et al. (2017) and Oba et al. (2019). The digestible indispensable AA reference ratios were calculated for each ingredient using the following equation (FAO, 2011): Digestible indispensable AA reference ratio = digestible indispensable AA content in 1 g protein of food (mg)/mg of the same dietary indispensable AA in 1 g of the reference protein.

The references used were the Association of American Feed Control Officials (AAFCO, 2019) nutrient profiles for adults at maintenance (dogs and cats) and growth and reproduction (dogs and cats) and National Research Council (NRC, 2006) recommended allowances for adults (dogs and cats), growing puppies (4 to 14 wk of age), and growing kittens.

The DIAAS-like values were then calculated using the following equation adapted from the Food and Agriculture Organization (FAO, 2011): DIAAS-like % = 100 × [(mg of digestible dietary indispensable AA in 1 g of the dietary protein)/(mg of the minimum recommendation of the same dietary indispensable AA in 1 g of the minimum protein recommendation)].

Statistical analysis

All rooster data were analyzed as a completely randomized design using the GLM procedure of Statistical Analysis Systems 9.4 (SAS Inst., Cary, NC). Substrates were considered to be a fixed effect. Tukey’s multiple comparison analysis was used to compare least squares means and control for experiment-wise error. Differences were considered significant with P < 0.05.

Results

Chemical composition

The chemical composition of the tested BSFL is presented in Table 1. The chemical composition of all four BSFL treatments was similar, containing 87.8% to 89.5% OM, 40.0% to 41.7% CP, 31.1% to 33.5% AHF, and 2.87% to 3.47% calcium, and GE content between 5.61 and 5.85 kcal/g. Concentrations of indispensable and dispensable AA are presented in Table 2. AA concentrations and patterns of all BSFL were similar.

Table 1.

Chemical composition and nutrient digestibility of BSFL fed different forms and concentrations of calcium using the precision-fed cecectomized rooster assay

Item BSFLA1 BSFLB2 BSFLC3 BSFLD4 SEM P-value
Chemical composition
 DM, % 93.34 94.09 93.16 93.56 — —
 OM, % DM 88.07 88.06 89.49 87.84 — —
 CP, % DM 41.68 40.15 41.58 41.04 — —
 AHF, % DM 31.15 33.45 33.05 32.73 — —
 GE, kcal/g DM 5.63 5.84 5.85 5.61 — —
 Ca, % DM 3.20 3.42 2.87 3.47 — —
Nutrient digestibility
 DM, % 62.30 60.39 62.55 59.77 1.403 0.8926
 OM, % 74.19 71.02 74.19 68.63 1.173 0.3548
 AHF, % 85.30 82.94 85.91 83.21 0.929 0.6373
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1BSFLA: 0.2% calcium in basal diet + 1.2% CaCl2.

2BSFLB: 0.2% calcium in basal + 1.2% CaCO3.

3BSFLC: 0.2% calcium in basal diet + 0.75% CaCO3.

4BSFLD: 0.2% calcium in basal diet + 0.6% CaCO3 + 0.6% CaCl2.

Table 2.

Indispensable and dispensable AA concentrations (% DM) of BSFL fed different forms and concentrations of calcium

Item BSFLA1 BSFLB2 BSFLC3 BSFLD4
Indispensable AA
 Arginine 1.92 1.99 2.02 2.00
 Histidine 1.24 1.27 1.31 1.29
 Isoleucine 1.71 1.85 1.90 1.78
 Leucine 2.60 2.76 2.81 2.69
 Lysine 2.78 2.91 2.89 2.89
 Methionine 0.69 0.72 0.73 0.73
 Phenylalanine 1.71 1.81 1.83 1.78
 Threonine 1.52 1.61 1.63 1.58
 Tryptophan 0.58 0.60 0.60 0.58
 Valine 2.60 2.91 3.01 2.81
Selected dispensable AA
 Alanine 2.45 2.76 2.74 2.63
 Aspartic acid 3.54 3.69 3.74 3.65
 Cysteine 0.33 0.36 0.34 0.35
 Glutamic acid 3.79 3.95 3.89 3.88
 Glycine 1.97 2.14 2.19 2.06
 Proline 2.27 2.41 2.47 2.36
 Serine 1.43 1.55 1.56 1.48
 Tyrosine 2.58 2.69 2.57 2.73
 Taurine 0.08 0.07 0.06 0.07
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1BSFLA: 0.2% calcium in basal diet + 1.2% CaCl2.

2BSFLB: 0.2% calcium in basal + 1.2% CaCO3.

3BSFLC: 0.2% calcium in basal diet + 0.75% CaCO3.

4BSFLD: 0.2% calcium in basal diet + 0.6% CaCO3 + 0.6% CaCl2.

Cecectomized rooster assay

The digestibilities of DM, OM, and AHF were not different among BSFL tested (Table 1). There were no differences in indispensable and dispensable AA digestibilities for all BSFL tested (Table 3). All indispensable AA digestibilities were higher than 90%, with the exception of valine (81.29% to 84.03%). For most of the dispensable AA, digestibilities were greater than 90%. The exceptions were for cysteine (76.88% to 81.95%), glycine (79.1% to 86.38%), and serine (87.92% to 91.18%).

Table 3.

AA digestibilities (%) of BSFL fed different forms and concentrations of calcium using the precision-fed cecectomized rooster assay

Item BSFLA1 BSFLB2 BSFLC3 BSFLD4 SEM P-value
Indispensable AA
 Arginine 95.05 93.54 94.58 95.77 0.600 0.6539
 Histidine 91.85 90.79 91.43 91.32 0.409 0.8653
 Isoleucine 92.53 91.48 92.25 92.75 0.531 0.8740
 Leucine 93.06 92.42 92.52 93.47 0.576 0.9271
 Lysine 92.28 91.15 91.15 92.25 0.570 0.8438
 Methionine 93.71 92.50 93.25 93.52 0.500 0.8515
 Phenylalanine 92.80 91.91 92.00 92.47 0.523 0.9397
 Threonine 91.62 90.56 90.76 92.11 0.738 0.8910
 Tryptophan 95.50 94.40 94.44 95.48 0.411 0.6832
 Valine 81.29 83.22 82.00 84.03 1.165 0.8704
Selected dispensable AA
 Alanine 92.69 91.73 92.62 93.29 0.530 0.8137
 Aspartic acid 93.68 92.49 93.37 93.57 0.459 0.8278
 Cysteine 80.38 76.88 78.08 81.95 1.914 0.8220
 Glutamic acid 91.60 89.51 91.44 91.57 0.615 0.6088
 Glycine 81.08 79.10 84.72 86.38 1.466 0.2947
 Proline 90.31 90.62 91.07 91.67 0.543 0.8576
 Serine 89.52 87.92 89.37 91.18 0.939 0.7240
 Tyrosine 93.66 93.26 92.64 93.85 0.395 0.7535
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1BSFLA: 0.2% calcium in basal diet + 1.2% CaCl2.

2BSFLB: 0.2% calcium in basal + 1.2% CaCO3.

3BSFLC: 0.2% calcium in basal diet + 0.75% CaCO3.

4BSFLD: 0.2% calcium in basal diet + 0.6% CaCO3 + 0.6% CaCl2.

DIAAS-like calculations

DIAAS-like reference values for growing puppies and kittens are presented in Table 4 and Table 5, respectively. DIAAS-like reference values for adult dogs and cats at maintenance are presented in Table 6 and Table 7, respectively. With the exception of threonine and sulfur AA (methionine + cysteine), most of the BSFL tested had DIAAS-like values over 100%. For BSFLA, DIAAS-like reference values for arginine, sulfur AA (methionine + cysteine), and threonine were less than 100% when using the AAFCO recommended allowances for growing puppies. Based on the NRC recommended allowances for growing puppies, all BSFL had DIAAS-like reference values above 100%, with the exception of threonine and sulfur AA (methionine + cysteine). According to the AAFCO and NRC recommended allowances for growing kittens, all BSFL had DIAAS-like reference values above 100% with the exception of sulfur AA (methionine + cysteine) and aromatic AA (phenylalanine + tyrosine).

Table 4.

DIAAS-like reference values1 of BSFL fed different forms and concentrations of calcium for growing and reproducing dogs and growing puppies2

AAFCO (2019)3 NRC (2006)3
Item BSFLA BSFLB BSFLC BSFLD SEM BSFLA BSFLB BSFLC BSFLD SEM
Indispensable AA
 Arginine 98.51b 104.32ab 103.39ab 105.03a 0.906 124.70b 132.05ab 130.88ab 132.95a 1.147
 Histidine 139.73b 146.86a 147.31a 146.79a 1.030 157.65b 165.69a 166.20a 165.61a 1.162
 Isoleucine 120.30a 133.59a 133.60a 127.50a 1.600 131.41b 145.92a 145.93a 139.27a 1.748
 Leucine 101.25b 110.81a 109.07a 106.87ab 1.148 101.25b 110.81a 109.07a 106.87ab 1.148
 Lysine 153.87b 165.17a 158.41ab 162.42ab 1.460 157.37b 168.93a 162.01ab 166.11ab 1.493
 Methionine 99.72b 106.59a 105.25a 106.94a 0.930 105.77b 113.05a 111.63a 113.43a 0.986
 Met + Cys 49.89 51.38 51.51 53.24 0.751 49.89 51.38 51.51 53.24 0.752
 Phenylalanine 103.20b 112.33a 109.77a 108.74ab 1.063 131.78b 143.44a 140.17a 138.85ab 1.357
 Phe + Tyr 121.01b 126.07ab 122.91ab 127.13a 0.867 121.01b 126.07ab 122.91ab 127.13a 0.867
 Threonine 72.28b 78.57a 76.98ab 76.73ab 0.862 92.80b 100.88a 98.84ab 98.52ab 1.106
 Tryptophan 149.50b 158.72a 153.32ab 151.82b 1.085 130.00b 138.02a 133.32ab 132.01b 0.944
 Valine 167.77b 199.59a 196.42a 190.39ab 4.174 167.77b 199.59a 196.42a 190.39ab 4.174
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1DIAAS-like reference values were calculated from the true digestibility of AA in cecectomized roosters.

2DIAAS-like reference values were calculated using the AAFCO (2019) nutrient profiles of AA for growth and reproduction of dogs and NRC (2006) recommended allowances of AA for growing puppies (4 to 14 wk of age).

3BSFLA: 0.2% calcium in basal diet + 1.2% CaCl2; BSFLB: 0.2% calcium in basal + 1.2% CaCO3; BSFLC: 0.2% calcium in basal diet + 0.75% CaCO3; BSFLD: 0.2% calcium in basal diet + 0.6% CaCO3 + 0.6% CaCl2.

a,bWithin a row, means lacking a common superscript letter differ (P < 0.05); n, 4 roosters per treatment.

Table 5.

DIAAS-like reference values1 of BSFL fed different forms and concentrations of calcium for growing and reproducing cats and growing kittens2

AAFCO (2019)3 NRC (2006)3
Item BSFLA BSFLB BSFLC BSFLD SEM BSFLA BSFLB BSFLC BSFLD SEM
Indispensable AA
 Arginine 105.93b 112.17ab 111.18ab 112.93a 0.974 114.02b 120.74ab 119.67ab 121.56a 1.049
 Histidine 248.41b 261.09a 261.89a 260.96a 1.831 207.01b 217.58a 218.24a 217.47a 1.526
 Isoleucine 203.37b 225.83a 225.85a 215.53a 2.705 175.75b 195.17a 195.18a 186.26a 2.337
 Leucine 136.05b 148.91a 146.57a 143.61ab 1.542 113.38b 124.09a 122.14a 119.67ab 1.285
 Lysine 153.87b 165.17a 158.41ab 162.42ab 1.460 181.03b 194.32a 186.36ab 191.09ab 1.718
 Methionine 75.06b 80.23a 79.22a 80.50a 0.700 88.14b 94.21b 93.03a 94.52a 0.822
 Met + Cys 42.33 43.60 43.70 45.18 0.638 44.10 45.41 45.52 47.06 0.664
 Phenylalanine 219.63b 239.06a 233.62a 231.42ab 2.261 190.35b 207.19a 202.47a 200.56ab 1.960
 Phe + Tyr 109.25b 113.81ab 110.96ab 114.77a 0.782 91.52b 95.34ab 92.95ab 96.15a 0.655
 Threonine 137.30b 149.25a 146.23ab 145.75ab 1.637 128.50b 139.68a 136.86ab 136.41ab 1.532
 Tryptophan 159.46b 169.30a 163.54ab 161.94b 1.158 207.63b 220.44a 212.95ab 210.86b 1.507
 Valine 237.67b 282.75a 278.27a 269.72ab 5.913 198.06b 235.63a 231.89a 224.76ab 4.928
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1DIAAS-like reference values were calculated from the true digestibility of AA in cecectomized roosters.

2DIAAS-like reference values were calculated using the AAFCO (2019) nutrient profiles of AA for growth and reproduction of cats and NRC (2006) recommended allowances of AA for growing kittens.

3BSFLA: 0.2% calcium in basal diet + 1.2% CaCl2; BSFLB: 0.2% calcium in basal + 1.2% CaCO3; BSFLC: 0.2% calcium in basal diet + 0.75% CaCO3; BSFLD: 0.2% calcium in basal diet + 0.6% CaCO3 + 0.6% CaCl2.

a,bWithin a row, means lacking a common superscript letter differ (P < 0.05); n, 4 roosters per treatment.

Table 6.

DIAAS-like reference values1 of BSFL fed different forms and concentrations of calcium for adult dogs2

AAFCO (2019)3 NRC (2006)3
Item BSFLA BSFLB BSFLC BSFLD SEM BSFLA BSFLB BSFLC BSFLD SEM
Indispensable AA
 Arginine 154.53b 163.64ab 162.19ab 164.75a 1.422 125.09b 132.48ab 131.29ab 133.37a 1.151
 Histidine 258.87b 272.08a 272.92a 271.95a 1.901 143.81b 151.16a 151.62a 151.08a 1.060
 Isoleucine 179.82b 199.69a 199.70a 190.57a 2.392 99.90b 110.94a 110.94a 105.87a 1.329
 Leucine 153.66b 168.18a 165.53a 162.19ab 1.742 85.37b 93.43a 91.96a 90.11ab 0.968
 Lysine 175.85b 188.77a 181.04ab 185.63ab 1.669 175.85b 188.77a 181.04ab 185.63ab 1.669
 Methionine 84.61b 90.44a 89.31a 90.74a 0.789 47.01b 50.24a 49.61a 50.41a 0.438
 Met + Cys 42.98 44.27 44.37 45.87 0.648 23.88 24.59 24.65 25.48 0.360
 Phenylalanine 152.28b 165.75a 161.98a 160.45ab 1.568 84.60b 92.08a 89.99a 89.14ab 0.871
 Phe + Tyr 170.07b 177.18ab 172.74ab 178.68a 1.218 94.49b 98.43ab 95.96ab 99.26a 0.677
 Threonine 125.28b 136.19a 133.44ab 133.00ab 1.494 77.70b 84.46a 82.75ab 82.48ab 0.926
 Tryptophan 149.50b 158.72a 153.32ab 151.82b 1.085 94.92b 100.77a 97.35ab 96.39b 0.689
 Valine 186.26b 221.59a 218.07a 211.37ab 4.634 103.48b 123.10a 121.15a 117.43ab 2.574
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1DIAAS-like reference values were calculated from the true digestibility of AA in cecectomized roosters.

2DIAAS-like reference values were calculated using the AAFCO (2019) nutrient profiles and NRC (2006) recommended allowances of AA for adult dogs at maintenance.

3BSFLA: 0.2% calcium in basal diet + 1.2% CaCl2; BSFLB: 0.2% calcium in basal + 1.2% CaCO3; BSFLC: 0.2% calcium in basal diet + 0.75% CaCO3; BSFLD: 0.2% calcium in basal diet + 0.6% CaCO3 + 0.6% CaCl2.

a,bWithin a row, means lacking a common superscript letter differ (P < 0.05); n, 4 roosters per treatment.

Table 7.

DIAAS-like reference values1 of BSFL fed different forms and concentrations of calcium for adult cats2

AAFCO (2019)3 NRC (2006)3
Item BSFLA BSFLB BSFLC BSFLD SEM BSFLA BSFLB BSFLC BSFLD SEM
Indispensable AA
 Arginine 109.46b 115.91ab 114.88ab 116.70a 1.001 113.72b 120.43ab 119.36ab 121.24a 1.046
 Histidine 229.17b 240.88a 241.61a 240.76a 1.689 210.19b 220.92a 221.60a 220.82a 1.549
 Isoleucine 189.81b 210.78a 210.79a 201.16a 2.524 176.57b 196.07a 196.09a 187.13a 2.348
 Leucine 121.72b 133.22a 131.12a 128.47ab 1.380 113.82b 124.58a 122.62a 120.14ab 1.290
 Lysine 192.80b 206.96a 198.48ab 203.52ab 1.829 362.05b 388.64a 372.72ab 382.17ab 3.435
 Methionine 201.67b 215.54a 212.84a 216.27a 1.880 182.50b 195.06a 192.62a 195.71a 1.701
 Met + Cys 100.89 103.91 104.16 107.67 1.520 91.31 94.03 94.26 97.44 1.375
 Phenylalanine 247.45b 269.34a 263.21a 260.73ab 2.548 190.35b 207.19a 202.47a 200.56ab 1.960
 Phe + Tyr 118.82b 123.78ab 120.68ab 124.83a 0.851 91.40b 95.22ab 92.83ab 96.02a 0.655
 Threonine 118.99b 129.35a 126.73ab 126.32ab 1.419 128.50b 139.68a 136.86ab 136.41ab 1.532
 Tryptophan 215.94b 229.26a 221.46ab 219.29b 1.568 204.44b 217.05a 209.67ab 207.61b 1.484
 Valine 212.63b 252.96a 248.94a 241.29ab 5.290 198.84b 236.55a 232.80a 225.65ab 4.947
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1DIAAS-like reference values were calculated from the true digestibility of AA in cecectomized roosters.

2DIAAS-like reference values were calculated using the AAFCO (2019) nutrient profiles and NRC (2006) recommended allowances of AA for adult cats at maintenance.

3BSFLA: 0.2% calcium in basal diet + 1.2% CaCl2; BSFLB: 0.2% calcium in basal + 1.2% CaCO3; BSFLC: 0.2% calcium in basal diet + 0.75% CaCO3; BSFLD: 0.2% calcium in basal diet + 0.6% CaCO3 + 0.6% CaCl2.

a,bWithin a row, means lacking a common superscript letter differ (P < 0.05); n, 4 roosters per treatment.

All BSFL ingredients had DIAAS-like reference values over 100%, with the exception of methionine and sulfur AA (methionine + cysteine), when using the AAFCO recommended allowances for adult dogs. Based on the NRC recommended allowances for an adult dog at maintenance, however, DIAAS-like reference values were lower than 100% for leucine, sulfur AA (methionine + cysteine), aromatic AA (phenylalanine + tyrosine), threonine, and tryptophan for most of the BSFL tested. According to the AAFCO and NRC recommended allowances for adult cats, all BSFL tested had DIAAS-like reference values over 100% with the exception of sulfur AA (methionine + cysteine) and aromatic AA (phenylalanine + tyrosine).

The first-limiting AA based on DIAAS-like reference values from AAFCO (2019) nutrient profiles for dogs and cats (growth and reproduction; adults at maintenance) are provided in Table 8. Sulfur AA (methionine + cysteine) was the first-limiting AA for all BSFL when calculating DIAAS-like reference values using AAFCO (2019) nutrient profiles for growing and reproducing dogs. Sulfur AA (methionine + cysteine) was also the first-limiting AA for all BSFL when calculating DIAAS-like reference values using AAFCO (2019) nutrient profiles for adult dogs and cats at maintenance. For these categories, all of the DIAAS-like reference values for limiting AA were less than 100, suggesting insufficiency if a diet was formulated with only that protein source and at an inclusion level to meet the nutrient profile. Sulfur AA (methionine + cysteine) was the first-limiting AA when calculating DIAAS-like reference values using the AAFCO (2019) nutrient profiles for adult cats at maintenance, with all values being greater than 100, suggesting sufficiency.

Table 8.

First-limiting AA based on DIAAS-like reference values1 of BSFL fed different forms and concentrations of calcium from AAFCO (2019) nutrient profiles2

Growth and reproduction Adults
Item3 Dogs Cats Dogs Cats
BSFLA 50 (Met + Cys) 42 (Met + Cys) 43 (Met + Cys) 101 (Met + Cys)
BSFLB 51 (Met + Cys) 44 (Met + Cys) 44 (Met + Cys) 104 (Met + Cys)
BSFLC 51 (Met + Cys) 44 (Met + Cys) 44 (Met + Cys) 104 (Met + Cys)
BSFLD 53 (Met + Cys) 45 (Met + Cys) 45 (Met + Cys) 108 (Met + Cys)
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1DIAAS-like values were calculated from the true digestibility of AA in cecectomized roosters.

2DIAAS-like values were calculated using the AAFCO (2019) nutrient profiles of AA for dogs and cats.

3BSFLA: 0.2% calcium in basal diet + 1.2% CaCl2; BSFLB: 0.2% calcium in basal + 1.2% CaCO3; BSFLC: 0.2% calcium in basal diet + 0.75% CaCO3; BSFLD: 0.2% calcium in basal diet + 0.6% CaCO3 + 0.6% CaCl2.

The first-limiting AA based on DIAAS-like reference values from NRC (2006) recommended allowances for growing puppies (4 to 14 wk of age), growing kittens, and adult dogs and cats at maintenance are provided in Table 9. Sulfur AA (methionine + cysteine) was the first-limiting AA for all BSFL for growing puppies and kittens and adult dogs and cats at maintenance, whereas aromatic AA (phenylalanine + tyrosine) was the first-limiting AA for BSFLC and BSFLD for adult cats at maintenance. For these categories, most of the DIAAS-like values for limiting AA were less than 100, suggesting insufficiency if a diet was formulated with only that protein source and at an inclusion level to meet the nutrient profile.

Table 9.

First-limiting AA based on DIAAS-like reference values1 of BSFL fed different forms and concentrations of calcium from NRC (2006) recommended allowances2

Adult
Item3 Puppies (4 to 14 wk of age) Kittens Dogs Cats
BSFLA 50 (Met + Cys) 44 (Met + Cys) 24 (Met + Cys) 91 (Met + Cys)
BSFLB 51 (Met + Cys) 45 (Met + Cys) 25 (Met + Cys) 94 (Met + Cys)
BSFLC 52 (Met + Cys) 46 (Met + Cys) 25 (Met + Cys) 93 (Phe + Tyr)
BSFLD 53 (Met + Cys) 47 (Met + Cys) 25 (Met + Cys) 96 (Phe + Tyr)
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1DIAAS-like values were calculated from the true digestibility of AA in cecectomized roosters.

2DIAAS-like values were calculated using the NRC (2006) recommended allowances of AA for dogs and cats.

3BSFLA: 0.2% calcium in basal diet + 1.2% CaCl2; BSFLB: 0.2% calcium in basal + 1.2% CaCO3; BSFLC: 0.2% calcium in basal diet + 0.75% CaCO3; BSFLD: 0.2% calcium in basal diet + 0.6% CaCO3 + 0.6% CaCl2.

Discussion

BSFL have been of substantial interest over the past couple of decades as a means for organic waste management as well as their ability to convert these food sources into a high-quality nutrient source for livestock feeds and pet foods (Newton et al., 1977, 2005; Sheppard et al., 2002; Tomberlin et al., 2009; Holmes et al., 2012; Kroeckel et al., 2012; Cullere et al., 2016; Marono et al., 2017; Barragan-Fonseca et al., 2018; Chia et al., 2018; Mwaniki et al., 2018; Do et al., 2020; Freel et al., 2021). Similar to mammals, BSFL must be fed a sufficient amount of essential nutrients to meet their nutrient and metabolic requirements (Cohen, 2003). Carbohydrates can be used as an energy source and building block for BSFL tissues. Essential AA such as arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine are important for the production of BSFL tissues, hormones, transport proteins, and energy (Cohen, 2003). Lipids also play several roles in these organisms, such as serving as a rich energy source, as a structural component of cell membranes, and as chemical messengers (Cohen, 2003). Micronutrients are indispensable for BSFL development, but limited information is available in regard to how dietary micronutrients affect BSFL development (Cohen, 2003).

BSFL can be a good source of calcium, but their calcium concentration is very responsive to diet and contain as little as 0.12% Ca (DM basis) up to 6.61% (Dierenfeld and King, 2008; Tschirner and Simon, 2015; Spranghers et al., 2016). If the diet was formulated with 20% of BSFL, the minimal requirement of calcium for kittens and puppies (NRC, 2006) would be met easily (providing 250% and 230% of the requirement). BSFL also have a calcium to phosphorus ratio (2.6:1) that is relatively easy to use in dietary formulations (Finke, 2013). Conversely, tebo worms (11%), Turkestan cockroaches (33%), and house flies (66%) contain low calcium concentrations and calcium and phosphorus ratios (1:18, 1:4.6, and 1:4.9) that are not easy to use in dietary formulas (Finke, 2013). Compared with other insects, BSFL can accumulate high calcium concentrations because they have a mineralized exoskeleton. Therefore, like face flies (Musca autumnalis), dietary calcium is positively linked with calcium accumulation in BSFL due to the formation of the cuticle layer during pupation (Roseland et al., 1985; Tomberlin et al., 2002; Finke, 2013). For those reasons, dietary calcium source may be an important consideration for BSFL development.

The gastrointestinal tract of BSFL is composed of three parts, including the foregut, midgut, and hindgut, with the midgut serving as the most important section for nutrient absorption (Kim et al., 2011). The Malpighian tubules located in the midgut and hindgut junction are essential for the excretion of nitrogenous products and other metabolites and maintaining nutrient balance (Murakami and Shiotsuki, 2001; Chapman, 2013; Gold et al., 2018). According to Grodowitz et al. (1987) and Krueger et al. (1988), dietary calcium in face flies is transported from the Malpighian tubules to the cuticle via hemolymph. They have mineralization of the epidermis during the pupal stage because mineralized granules, which are composed of calcium, magnesium, phosphate, and carbonate, accumulate in the lumen of the larval Malpighian tubules. Therefore, as with face flies, BSFL may have a similar mechanism of dietary calcium absorption.

There are many potential calcium sources in animal feeds, such as organic salts (tricalcium citrate, calcium lactate, calcium lactate gluconate, and calcium gluconate) and inorganic salts (calcium chloride, calcium carbonate, and calcium phosphate; Trailokya et al., 2017). In the current study, we compared CaCO3 and CaCl2 as dietary calcium sources for BSFL and they have different solubilities (insoluble vs. soluble; pH range between 3 and 6), calcium concentrations (40% vs. 27%), and acid-binding capacities (244 vs. 2.4 mEq/kg to reach pH of 3) (Weaver, 1998; Hamdi et al., 2015). Several studies have determined that calcium citrate (soluble) has a higher absorption rate than CaCO3 (insoluble) due to its solubility in water (Nicar and Pak, 1985; Heller et al., 1999). Heaney et al. (1990), however, tested seven different calcium salts (calcium oxalate, hydroxyapatite, tricalcium phosphate, calcium citrate, calcium citrate malate, bisglycinocalcium, and CaCO3) to evaluate their relationship between solubility and absorption and were unable to identify a significant correlation. Another in vitro study conducted by Goss et al. (2007) reported that while CaCO3 (3.6 mg mL−1) had a greater solubility in the gastrointestinal tract than calcium citrate (0.2 mg mL−1) at a pH of 6, CaCO3 (0.12 mg mL−1) had a lower solubility than calcium citrate (0.24 mg mL−1) at a pH of 7.5. The solubility of dietary calcium in water is affected by pH because it impacts its form (anion vs. cation). Secretion of hydrochloric acid and sodium bicarbonate from the stomach and intestines affects the pH in the gastrointestinal tract. Thus, the solubility values determined from the gastrointestinal tract would be a key factor to understand the effect of the solubility on calcium absorption rather than simple aqueous solubility. Dietary calcium sources used in the current study have different pH-dependent solubilities and may have different absorption rates in the midgut of BSFL. The pH of the BSFL midgut changes according to region (anterior midgut: pH = 7, mid-midgut: pH = 2, and posterior midgut: pH = 6.3 to 9.3; Espinoza-Fuentes and Terra, 1987; Overend et al., 2016; Gold et al., 2018). Although CaCO3 has a higher calcium content than CaCl2, CaCl2 may be more bioavailable to BSFL because of its higher solubility in the midgut, and, in this trial, the combination of both forms resulted in the highest numerical concentration of BSFL calcium.

The nutrient concentrations, including indispensable AA, were similar for all BSFL tested in the current study. Shumo et al. (2019) reared BSFL on three different organic wastes, including chicken manure (high ash: 20% and low fat: 2.7%), brewer’s spent grain (high neutral detergent fiber [NDF]: 50% and high acid detergent fiber [ADF]: 39%), and kitchen waste (high protein: 20%). BSFL fed kitchen waste had higher DM (87.5%) and fat (34.3%) concentrations and lower CP (33%) concentrations than BSFL fed chicken manure or brewer’s spent grain. Also, BSFL reared on brewer’s spent grain had higher ADF (15%) and NDF (28.6%) concentrations than larvae fed chicken manure (ADF: 12.6% and NDF: 21.9%) or kitchen waste (ADF: 13.2% and NDF: 20.4%). Barragan-Fonseca et al. (2019) reported that BSFL CP concentration increased when larvae were fed a diet containing 10% CP concentration compared with a high-protein diet (17%). Similarly, Tschirner and Simon (2015) reported that larvae fed a high-fiber diet containing only 8.5% CP had a higher CP content (52.3%) than larvae (44.6%) fed a high-protein diet (31.2% CP).

BSFL crude fat concentration was shown to increase as dietary carbohydrate concentration increased (Barragan-Fonseca et al., 2019). Compared with AA concentrations of BSFL reported by Shumo et al. (2019), BSFL fed kitchen waste had higher concentrations of indispensable AA (arginine and phenylalanine) and dispensable AA (proline, hydro-proline, and tyrosine) than BSFL fed chicken manure or brewer’s spent grain. Based on the previous studies, the nutrient composition (DM, CP, fat, ADF, and NDF) of the substrates affected the chemical composition and AA concentration of BSFL. In the current study, different forms and concentrations of dietary calcium sources influenced the calcium concentrations of BSFL, but there was no significant difference in the nutrient composition of BSFL among treatments.

The precision-fed cecectomized rooster assay is often used to test the protein quality of pet food ingredients because the AA digestibilities and response patterns have been shown to be similar to that of ileal-cannulated dogs (r = 0.87 to 0.92; Johnson et al., 1998; Faber et al., 2010; Kerr et al., 2014; Oba et al., 2019). Other methods of testing AA digestibilities have drawbacks when compared with the cecectomized rooster assay. For example, the total collection method that uses fecal analysis is heavily influenced by the fermentative activity of the large intestinal microbiota and is a time-consuming, labor-intensive, and expensive method (Hendriks and Sritharan, 2002; Hendriks et al., 2013). Intestinal cannulas have been used to estimate nutrient digestion at specific points in the gastrointestinal tract for many years. Cannulation, however, also has many disadvantages, such as leakage of chyme from the cannula, skin ulceration and infection, and the discomfort of animals (Hill et al., 1996). Therefore, the cecectomized rooster assay is an appropriate model to evaluate the protein quality of ingredients because it minimizes the influence of microbes in the hindgut.

Because BSFL have a high calcium concentration due to their mineralized exoskeleton (Grodowitz and Broce, 1983; Krueger et al., 1988) and high dietary calcium concentrations may influence the digestibility of nitrogen and AA (Selle et al., 2009; Wilkinson et al., 2014), the current study was conducted to test whether dietary calcium source or concentration affected nutrient digestibility. It was interesting to note that nutrient and AA digestibilities were similar among all BSFL tested in the current study. According to McDonald and Solvyns (1964), increasing dietary calcium (CaCO3) concentrations from 9 to 25 g/kg resulted in a greater small intestinal pH from 5.6 to 6.0 and a reduction in weight gain of chickens from 77 to 63.5 g/d. Wilkinson et al. (2014) demonstrated that increasing dietary calcium concentration (CaCO3) from 2.5 to 10 g/kg led to a reduction in the apparent ileal digestibility of DM, nitrogen, and AA in broiler chickens. The possible reason for these results is that the increased intestinal pH resulting from the high dietary calcium concentrations reduced the action of pepsin. In the current study, the calcium form and concentration fed to BSFL had no adverse effects on BSFL protein quality or calcium concentrations, which may have been due to a low acid-binding capacity, impact on intestinal pH, and consequent nutrient absorption.

DIAAS-like reference values are an indicator of protein quality, providing a more accurate measure than the protein digestibility-corrected AA score because it uses ileal rather than fecal digestibility in its calculation (FAO, 2011; Mathai et al., 2017; Oba et al., 2019). Based on the DIAAS-like reference values in the current study, all BSFL tested seem to provide sufficient AA for growing puppies and kittens except for threonine and sulfur AA (methionine + cysteine). Because the AAFCO and NRC recommended allowances are lower for adult dogs and cats, more AA would be insufficient for those life stages if diets were only formulated using BSFL as the sole protein source and at a rate to meet the recommended CP concentrations.

This study had a couple of limitations that should be discussed briefly. First, although the precision-fed cecectomized rooster assay is a highly repeatable method that has been used to study many novel proteins in recent years (Kerr et al., 2013, 2014; Deng et al., 2016; Oba et al., 2019; Do et al., 2020), the number of replications in this study (4 to 5 roosters per treatment) was low. This replication number may have provided us with lower than ideal statistical power and limited our ability to detect differences among treatments. Also, the DIAAS-like values used to estimate protein quality for dog and cat foods are based on the assumption that diets would be formulated with that single protein source and contain an inclusion level to meet the CP recommendation. Dog or cat foods formulated using a different strategy would need to keep this in mind when assessing the value of BSFL.

In conclusion, our data show that BSFL contain a relatively high concentration of calcium and that BSFLD fed CaCl2 and CaCO3 accumulated more calcium than the other BSFL. Despite their differences in nutrient composition, nutrient and AA digestibilities among all BSFL sources were similar when tested in the precision-fed cecectomized rooster assay. Sulfur AA (methionine + cysteine) and aromatic AA (phenylalanine + tyrosine) were estimated to be the first-limiting AA of BSFL based on DIAAS-like reference values for dogs and cats. All BSFL ingredients, however, showed very high AA digestibilities, with most exceeding 90%. Therefore, although dietary calcium form and concentration may affect the calcium concentrations of BSFL, they all serve as high-quality protein sources for use in livestock feeds and pet foods.

Acknowledgment

The funding for this study was provided by EnviroFlight.

Glossary

Abbreviations

AA

amino acids

AAFCO

Association of American Feed Control Officials

ADF

acid detergent fiber

AHF

acid-hydrolyzed fat

BSFL

black soldier fly larvae

CP

crude protein

DM

dry matter

FAO

Food and Agriculture Organization

GE

gross energy

NDF

neutral detergent fiber

NRC

National Research Council

OM

organic matter

Conflict of interest statement

L.K. is employed by EnviroFlight. All other authors have no conflicts of interest.

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