Amino acid digestibility and protein quality of mealworm-based ingredients using the precision-fed cecectomized rooster assay

Abstract

Mealworms may serve as an alternative protein source for pet foods because of their high protein content and low environmental footprint. The amino acid (AA) content and protein quality of mealworm-based ingredients may vary depending on their composition and processing, however, so testing is required. Our objective was to measure the AA composition, AA digestibility, and protein quality of mealworm-based ingredients using the precision-fed cecectomized rooster assay. The University of Illinois Institutional Animal Care and Use Committee approved all animal procedures prior to experimentation. Sixteen cecectomized roosters (4 roosters per substrate) were randomly allotted to one of four test substrates: 1) whole lesser mealworm (A. diaperinus) meal (ADw); 2) defatted lesser mealworm (A. diaperinus) meal (ADd); 3) defatted yellow mealworm (T. molitor) meal (TMd); and 4) hydrolyzed T. molitor protein meal (TMh). Ingredients were provided by Ÿnsect, France. After 26 h of feed withdrawal, roosters were tube-fed test substrates. Following crop intubation, excreta samples were collected for 48 h. Endogenous loss corrections for AA were made by using five additional cecectomized roosters. All data were analyzed using SAS version 9.4. All substrates had high AA digestibilities, with all indispensable AA digestibilities being >90% with the exception of histidine (87.9% to 91.1%) and valine (77.9% to 79.7%). Amino acid digestibilities were not different among substrates (P > 0.05). Digestible indispensable AA score (DIAAS)-like values were calculated to determine protein quality according to Association of American Feed Control Officials (AAFCO) nutrient profiles, The European Pet Food Industry Nutritional Guidelines (FEDIAF) nutritional guidelines, National Research Council (NRC) recommended allowances for adult dogs, adult cats, growing puppies, and growing kittens, and NRC minimal requirements for growing puppies and growing kittens. In general, TMh had the highest and TMd had the lowest DIAAS-like values for most indispensable AA. Methionine (TMh; TMd; ADw) and phenylalanine (ADd) were the first-limiting AA. Our results demonstrate that mealworm-based ingredients are high-quality protein sources. Further research in dogs and cats is necessary to confirm sufficient palatability and digestibility, but these data suggest that they are valuable sources of protein for pet foods.

This study was conducted to measure the amino acid composition, amino acid digestibility, and protein quality of mealworm-based ingredients using the precision-fed cecectomized rooster assay. Results showed that all ingredients had high amino acid digestibilities and protein quality, with methionine and phenylalanine being the first-limiting amino acids. More research is necessary, but these data suggest that mealworm-based ingredients may serve as valuable protein sources for pet foods.

Introduction

The Food and Agriculture Organization predicts that by 2050 the world’s population will exceed 9 billion, meaning that food production must increase by approximately 70% to feed the entire population (FAO, 2009). A sustainable food system is imperative to meet the needs of everyone and maintain a society long-term. The ecological, social, and economic aspects must be balanced to support sustainability of the entire food system (Swanson et al., 2013). The pet food industry is largely based on byproducts and is intertwined with livestock production and the human food system. Although there is competition among these food systems, collaboration also exists. The use of byproducts and alternative ingredients by the pet food industry, especially when it comes to protein sources, helps support a sustainable food production industry as a whole. Insect proteins may contribute to sustainable food production because of their low greenhouse gas emissions, low water and land requirements, and high feed conversion efficiencies (Calvez and Gaudichon, 2021).

Yellow mealworms (Tenebrio molitor) and lesser mealworms, also called buffalo worms (Alphitobius diaperinus), may be considered alternative protein sources for pet foods because they are protein-rich and have a low ecological footprint (Grau et al., 2017). For instance, the water footprint of mealworms is estimated to be 4,341 m³ water/t of edible product, which is comparable to chicken meat and 3.5 times lower than that of beef (Grau et al., 2017). The energy required to produce 1 kg of mealworms is similar to that of beef, chicken, and pork, but requires less land (e.g., mealworms 0 m²; dairy and swine 60 m²; poultry 50 m²; and beef 250 m²; Oonincx and de Boer, 2012). Additionally, mealworm feed conversion efficiency is relatively similar to poultry, nitrogen usage is more efficient, and greenhouse gas emissions are lower than livestock (e.g., CO₂ production for T. molitor is 0.45 g/kg BM/d; for pigs is 2.03 g/kg BM/d; for beef cattle is 5.98 g/kg BM/d; Oonincx et al., 2010). Based on swine and poultry research, mealworms are generally thought to have a high nutritional value and digestibility, acceptable flavor, and presence of functional ingredients (Hong et al., 2020). For instance, their exoskeleton includes chitin, an indigestible fiber, located within the cuticle and is associated with positive effects on the immune system, making it a promising alternative for antibiotics currently used in livestock (FAO, 2013).

Although mealworms have been tested in some species, several product types exist, limited digestibility data exist in livestock species, and virtually nothing has been tested in companion animals. The cecectomized rooster assay is a common model used to measure nutrient and amino acid (AA) digestibility of individual feed ingredients or complete and balanced pet foods (Deng et al., 2016; Oba et al. 2019, 2020; Do et al. 2020, 2021) because the results have been shown to be similar to that of ileal-cannulated dogs (Johnson et al., 1998). Similar to ileal-cannulated dogs, cecectomized roosters allow for AA digestibility estimates with minimal interference from the bacterial fermentation of proteins in the hindgut. Additionally, the cecectomized rooster assay is more affordable, time efficient, and less labor intensive than the ileal-cannulated dog assay (Johnson et al., 1998; Faber et al., 2010). Thus, the cecectomized rooster assay is often the preferred model to evaluate the protein quality of novel ingredients for dogs and cats (Kerr et al., 2014; Oba et al., 2019).

The objective of this study was to measure the AA composition, AA digestibility, and protein quality of mealworm-based ingredients intended for use in pet foods using the precision-fed cecectomized rooster assay. We hypothesized that all substrates would have high AA digestibilities, but digestible indispensable amino acid score (DIAAS)-like values would differ among substrates.

Materials and Methods

Substrates

Four mealworm-based ingredients were tested in this study, including: whole lesser mealworm (A. diaperinus) meal (ADw); defatted lesser mealworm (A. diaperinus) meal (ADd); defatted yellow mealworm (T. molitor) meal (TMd); and hydrolyzed T. molitor protein meal (TMh). Ingredients were provided by Ÿnsect, France.

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 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). All roosters were given at least 8 wk to recover from surgery before being used in experiments.

Briefly, 16 cecectomized roosters (4 roosters per substrate) were randomly assigned to one of four test substrates. After 26 h of feed withdrawal, roosters were tube-fed (crop intubation) 12 to 13 g of test substrates + 12 to 13 g of corn. The test substrates were mixed with corn to enable the diets to be physically tube-fed. 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 loss corrections for AA were made by using five additional cecectomized roosters that had been fasted for 48 h. AA digestibilities were calculated using the method described by Engster et al. (1985). All birds were housed individually in cages (27.9 cm wide × 50.8 cm long × 53.3 cm high) with raised wire floors. They were kept in an environmentally controlled room (approximately 23.9 °C, 17 h light:7 h dark). Before the start of the experiment, feed and water were supplied ad libitum.

Chemical analyses

The substrates and rooster excreta were analyzed for dry matter (DM; 105 °C) and ash according to AOAC (2006) with organic matter (OM) being calculated (DM: method 934.01; OM: method 942.05). Nitrogen and crude protein (CP) were measured using a Leco Nitrogen/Protein Determinator (Model FP-2000, Leco Corporation, St. Joseph, MI) according to AOAC (2006; method 982.30E). AA were measured at the University of Missouri Experiment Station Chemical Laboratories (Columbia, MO) according to AOAC (2006; method 982.30E).

Amino acid digestibility calculations

As mentioned earlier, basal endogenous AA losses were determined using roosters that were fasted for 48 h and then standardized AA digestibility values were calculated by the method of Engster et al. (1985) using the equations below.

$${\displaystyle \begin{array}{l}{\displaystyle Aminoaciddigestibilityofdiet\left(\mathrm{\%}\right)}\\ =\left[\right(AAconsumed-AAexcretedbyfedbirds\\ +AAexcretedbyfastedbirds)/AAconsumed]\times 100\end{array}}$$

where AA consumed (g) = diet intake (g) × AA in diet (%); AA excreted by fed birds (g) = excreta output (g) × AA in excreta (%); AA excreted by fasted birds = excreta output (g) × AA in excreta (%). The AA digestibility values for test diets were then calculated by difference using the equation:

Amino acid digestibility of test ingredient (%) = AA digestibility of ground corn reference diet (determined previously) − [(standardized AA digestibility of ground corn reference diet − standardized AA digestibility of test diet mixture with corn)/proportion of test diet AA substituted into the test diet mixture with corn]

DIAAS-like calculations

Calculation of DIAAS-like values were performed 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, 2013): 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, 2022) nutrient profiles for adults at maintenance (dogs and cats) and growth and reproduction (puppies and kittens); National Research Council (NRC, 2006) recommended allowances for adults (dogs and cats), growing puppies (4 to 14 wk of age), and growing kittens; NRC minimal requirements for growing puppies (4 to 14 wk of age) and growing kittens; and The European Pet Food Industry (FEDIAF, 2021) nutritional guidelines. The DIAAS-like values were then calculated using the following equation adapted from (FAO, 2013): 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 analyses

All 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 as fixed effects. Tukey’s multiple comparison analysis were used to compare LS means and control for experiment-wise error. Differences were considered significant with P < 0.05.

Results

Chemical composition

The chemical composition of tested mealworm-based ingredients is presented in Table 1. Regarding chemical composition, OM and CP were highest in ADd (96.88% OM and 75.34% CP on DM basis) and lowest in ADw (87.15% OM and 58.86% CP on DM basis). Concentrations of indispensable and dispensable AA are presented in Table 2.

Table 1.

Analyzed chemical composition of mealworm-based ingredients

Item	ADw1	ADd	TMd	TMh
DM, %	86.32	95.89	89.98	91.59
OM, % DM	87.15	96.88	92.97	93.06
CP, % DM	58.86	75.34	72.90	71.71
CF, % DM	27.60	12.40	10.00	13.60
TDF, % DM	3.6	6.2	4.1	1.3

¹ADw, whole lesser mealworm (A. diaperinus) meal; ADd, defatted lesser mealworm (A. diaperinus) meal; TMd, defatted yellow mealworm (T. molitor) meal; TMh, hydrolyzed T. molitor protein meal; DM, dry matter; OM, organic matter; CP, crude protein; CF, crude fat; TDF, total dietary fiber.

Table 2.

Indispensable and dispensable amino acid (AA) concentrations (% DM) of mealworm-based ingredients

Item	ADw1	ADd	TMd	TMh
Indispensable AA
Arginine	3.64	4.35	4.17	4.48
Histidine	2.33	2.40	2.52	2.27
Isoleucine	3.19	3.86	3.99	4.27
Leucine	4.58	5.79	6.27	6.55
Lysine	4.67	5.88	4.93	5.96
Methionine	1.00	1.32	1.09	1.47
Phenylalanine	2.87	4.21	3.06	3.47
Threonine	2.69	3.31	3.23	3.54
Tryptophan	0.77	0.97	0.99	1.30
Valine	4.63	4.75	6.23	5.07
Selected dispensable AA
Alanine	4.86	4.39	6.31	4.25
Aspartic acid	5.62	7.33	6.66	7.85
Cysteine	0.65	0.78	0.71	0.90
Glutamic acid	7.91	9.65	8.82	10.40
Glycine	3.26	3.56	4.38	3.63
Proline	4.02	4.09	4.79	4.42
Serine	2.52	2.83	3.27	3.08
Tyrosine	3.60	5.84	4.71	3.79

¹ADw, whole lesser mealworm (A. diaperinus) meal; ADd, defatted lesser mealworm (A. diaperinus) meal; TMd, defatted yellow mealworm (T. molitor) meal; TMh, hydrolyzed T. molitor protein meal.

Cecectomized rooster assay

AA digestibility of mealworm-based ingredients are presented in Table 3. All substrates had high AA digestibilities, with all indispensable AA digestibilities being >90% with the exception of histidine (87.9% for TMd; 88.8% for ADw) and valine (77.9% for TMd; 79.5% for TMh; 79.7% for ADw). AA digestibilities were not different among substrates (P > 0.05).

Table 3.

Amino acid (AA) digestibility (%) of mealworm-based ingredients using the precision-fed cecectomized rooster assay

Item	ADw1	ADd	TMd	TMh	SEM	P-value
Indispensable AA
Arginine	95.3	94.3	96.5	95.7	1.16	0.6305
Histidine	88.8	90.5	87.9	91.1	1.36	0.3671
Isoleucine	91.1	93.0	92.5	93.1	1.50	0.7680
Leucine	91.6	93.3	93.5	95.9	2.02	0.5304
Lysine	90.9	92.1	91.0	92.3	1.38	0.8517
Methionine	92.5	91.6	93.2	93.0	1.45	0.8696
Phenylalanine	91.9	92.0	94.2	93.5	1.64	0.7014
Threonine	91.0	91.8	91.3	91.5	1.92	0.9916
Tryptophan	95.7	91.8	98.7	97.4	1.02	0.3849
Valine	79.7	91.8	77.9	79.5	6.51	0.4337
Selected dispensable AA
Alanine	90.5	90.2	92.8	91.3	1.81	0.7469
Aspartic acid	91.1	87.9	90.1	89.6	1.86	0.6835
Cysteine	76.3	81.7	86.7	81.8	4.04	0.3879
Glutamic acid	91.7	91.9	92.6	90.7	1.54	0.8522
Glycine	83.7	81.7	83.3	81.3	3.21	0.9382
Proline	90.0	92.0	92.4	93.1	1.63	0.5821
Serine	89.1	89.2	90.8	91.7	2.41	0.8312
Tyrosine	93.2	90.8	97.1	95.8	1.59	0.0657

¹ADw, whole lesser mealworm (A. diaperinus) meal; ADd, defatted lesser mealworm (A. diaperinus) meal; TMd, defatted yellow mealworm (T. molitor) meal; TMh, hydrolyzed T. molitor protein meal.

DIAAS-like calculations

DIAAS-like values for adult dogs are presented in Tables 4–6. Based on the AAFCO nutrient profiles for adult dogs, ADw, TMh, and TMd had DIAAS-like values above 100% for all AA except methionine, which was the limiting AA. All ADd DIAAS-like values were above 100% for AA all except phenylalaine, which was the limiting AA when using AAFCO nutrient profiles. According to the NRC recommended allowances, all mealworm-based ingredients had several DIAAS-like values below 100%, with methionine being the limiting AA for ADw, TMh, and TMd, and phenylalanine being the limiting AA for ADd. Using the FEDIAF nutritional guidelines, ADw, TMh, and TMd had DIAAS-like values above 100% for all AA except methionine, which was the limiting AA. For ADd, all DIAAS-like values were above 100% except for phenylalanine when using FEDIAF nutritional guidelines.

Table 4.

Digestible indispensable AA score (DIAAS)-like values of mealworm-based ingredients for adult dogs based on AAFCO nutrient profiles

	AAFCO, 2022
Item	ADw1	ADd	TMd	TMh	SEM	P-value
Arginine	192a	195a	185b	141c	1.50	<0.0001
Histidine	305a	251c	271b	151d	3.23	<0.0001
Isoleucine	216b	244a	228b	189c	3.17	<0.0001
Leucine	175b	215a	203a	204a	3.83	<0.0001
Lysine	190a	203a	167b	204a	3.72	<0.0001
Methionine	78.8c	95.6b	71.8c	225a	3.28	<0.0001
Phenylalanine	165a	167a	150b	87.0c	1.83	<0.0001
Threonine	144c	157b	144c	191a	2.86	<0.0001
Tryptophan	130b	185a	143b	203a	4.55	<0.0001
Valine	204b	183b	223a,b	269a	14.9	0.0096
Methionine-cystine	121b,c	148a	117c	133b	3.91	0.0005
Phenylalanine-tyrosine	458b,c	432c	472b	592a	9.34	<0.0001

¹ADw, whole lesser mealworm (A. diaperinus) meal; ADd, defatted lesser mealworm (A. diaperinus) meal; TMd, defatted yellow mealworm (T. molitor) meal; TMh, hydrolyzed T. molitor protein meal.

^a,b,c,dWithin a row, means lacking a common superscript differ (P < 0.05); N = 4 roosters per treatment.

Table 5.

Digestible indispensable AA score (DIAAS)-like values¹ of mealworm-based ingredients for adult dogs based on NRC recommended allowances

	NRC, 2006
Item	ADw2	ADd	TMd	TMh	SEM	P-value
Arginine	156a	89.5c	150b	158a	1.23	<0.0001
Histidine	171a	93.5d	152b	141c	1.94	<0.0001
Isoleucine	120b	105c	127b	135a	1.80	<0.0001
Leucine	97.0c	204a	112b	120b	2.79	<0.0001
Lysine	191b	114d	167c	203a	2.63	<0.0001
Methionine	44.1c	125a	40.1c	53.5b	1.83	<0.0001
Phenylalanine	92.2a	48.6c	83.5b	93.1a	1.02	<0.0001
Threonine	89.4b	155a	89.5b	97.5b	1.99	<0.0001
Tryptophan	82.5b	118a	91.0b	117a	2.68	<0.0001
Valine	118a,b	151a	129a,b	106b	8.66	0.0202
Methionine-cystine	67.4b	74.2a,b	65.0b	82.4a	2.18	0.0005
Phenylalanine-tyrosine	255b,c	328a	262b	240c	5.17	<0.0001

¹DIAAS-like values were calculated from the digestibility of AA in cecectomized roosters.

²ADw, whole lesser mealworm (A. diaperinus) meal; ADd, defatted lesser mealworm (A. diaperinus) meal; TMd, defatted yellow mealworm (T. molitor) meal; TMh, hydrolyzed T. molitor protein meal.

^a,b,c,dWithin a row, means lacking a common superscript differ (P < 0.05); N = 4 roosters per treatment.

Table 6.

Digestible indispensable AA score (DIAAS)-like values¹ of mealworm-based ingredients for adult dogs based on FEDIAF nutritional guidelines

	FEDIAF, 2021
Item	ADw2	ADd	TMd	TMh	SEM	P-value
Arginine	191a	132c	184b	194a	1.52	<0.0001
Histidine	252a	141d	225b	208c	2.90	<0.0001
Isoleucine	181b	158c	191b	204a	2.71	<0.0001
Leucine	146c	327a	169b	189b	4.35	<0.0001
Lysine	305b	171d	267c	325a	4.09	<0.0001
Methionine	66.4c	188a	60.5c	80.6b	2.75	<0.0001
Phenylalanine	138a	73.2c	125b	140a	1.53	<0.0001
Threonine	135c	190a	135c	147b	2.71	<0.0001
Tryptophan	121b	178a	134b	172a	4.04	<0.0001
Valine	179a,b	223a	196a,b	161b	18.5	0.0331
Methionine-cystine	105b,c	115a,b	101c	128a	3.14	0.0002
Phenylalanine-tyrosine	384b,c	495a	395b	362c	7.80	<0.0001

¹DIAAS-like values were calculated from the digestibility of AA in cecectomized roosters.

²ADw, whole lesser mealworm (A. diaperinus) meal; ADd, defatted lesser mealworm (A. diaperinus) meal; TMd, defatted yellow mealworm (T. molitor) meal; TMh, hydrolyzed T. molitor protein meal.

^a,b,c,dWithin a row, means lacking a common superscript differ (P < 0.05); N = 4 roosters per treatment.

DIAAS-like values for adult cats are presented in Tables 7–9. Based on the AAFCO nutrient profiles, NRC recommended allowances, and FEDIAF nutritional guidelines for adult cats, all mealworm-based ingredients had DIAAS-like values above 100%.

Table 7.

Digestible indispensable AA score (DIAAS)-like values¹ of mealworm-based ingredients for adult cats based on AAFCO nutrient profiles

	AAFCO, 2022
Item	ADw2	ADd	TMd	TMh	SEM	P-value
Arginine	136b	204a	131c	138b	1.20	<0.0001
Histidine	271a	143d	241b	223c	3.02	<0.0001
Isoleucine	228c	150d	241b	257a	2.84	<0.0001
Leucine	138c	224a	160b	170b	3.48	<0.0001
Lysine	209b	317a	183c	222b	5.38	<0.0001
Methionine	189b	238a	172c	229a	3.78	<0.0001
Phenylalanine	257	208	233	259	27.6	0.6250
Threonine	137b	136b	137b	149a	2.42	0.0061
Tryptophan	188c	144d	207b	267a	3.80	<0.0001
Valine	145b	239a	158b	130b	10.8	<0.0001
Methionine-cystine	285b	314a,b	275b	348a	9.21	0.0005
Phenylalanine-tyrosine	257a	208c	233b	259a	3.52	<0.0001

¹DIAAS-like values were calculated from the digestibility of AA in cecectomized roosters.

²ADw, whole lesser mealworm (A. diaperinus) meal; ADd, defatted lesser mealworm (A. diaperinus) meal; TMd, defatted yellow mealworm (T. molitor) meal; TMh, hydrolyzed T. molitor protein meal.

^a,b,c,dWithin a row, means lacking a common superscript differ (P < 0.05); N = 4 roosters per treatment.

Table 8.

Digestible indispensable AA score (DIAAS)-like values¹ of mealworm-based ingredients for adult cats based on NRC recommended allowances

	NRC, 2006
Item	ADw2	ADd	TMd	TMh	SEM	P-value
Arginine	142b	193a	136c	144b	1.23	<0.0001
Histidine	250a	155d	223b	206c	3.04	<0.0001
Isoleucine	212c	140d	224b	239a	2.65	<0.0001
Leucine	129c	421a	150b,c	159b	4.99	<0.0001
Lysine	393b	256d	345c	418a	5.65	<0.0001
Methionine	171b	221a	156c	208a	3.50	<0.0001
Phenylalanine	207a	189b	188b	210a	3.06	0.0003
Threonine	148b	141b	148b	161a	2.59	0.0011
Tryptophan	178c	226b	196c	253a	5.24	<0.0001
Valine	227	221	248	204	16.5	0.3383
Methionine-cystine	258b	284a	249b	315a	8.33	0.0005
Phenylalanine-tyrosine	246b,c	318a	253b	232c	5.00	<0.0001

¹DIAAS-like values were calculated from the digestibility of AA in cecectomized roosters.

²ADw, whole lesser mealworm (A. diaperinus) meal; ADd, defatted lesser mealworm (A. diaperinus) meal; TMd, defatted yellow mealworm (T. molitor) meal; TMh, hydrolyzed T. molitor protein meal.

^a,b,c,dWithin a row, means lacking a common superscript differ (P < 0.05); N = 4 roosters per treatment.

Table 9.

Digestible indispensable AA score (DIAAS)-like values¹ of mealworm-based ingredients for adult cats based on FEDIAF nutritional guidelines

	FEDIAF, 2021
Item	ADw2	ADd	TMd	TMh	SEM	P-value
Arginine	140b	245a	134c	142b	1.24	<0.0001
Histidine	309a	194d	275b	255c	3.80	<0.0001
Isoleucine	267c	175d	281b	300a	3.32	<0.0001
Leucine	162c	529a	187b	199b	6.27	<0.0001
Lysine	495b	322d	434c	526a	7.10	<0.0001
Methionine	210c	278a	192d	255b	4.38	<0.0001
Phenylalanine	261a	232b	236b	263a	3.79	<0.0001
Threonine	185b	139c	186b	202a	3.08	<0.0001
Tryptophan	226c	282b	249c	321a	6.56	<0.0001
Valine	284	273	310	255	20.6	0.3293
Methionine-cystine	324b,c	357a,b	313c	396a	10.49	0.0005
Phenylalanine-tyrosine	308b,c	397a	317b	290c	6.25	<0.0001

¹DIAAS-like values were calculated from the digestibility of AA in cecectomized roosters.

²ADw, whole lesser mealworm (A. diaperinus) meal; ADd, defatted lesser mealworm (A. diaperinus) meal; TMd, defatted yellow mealworm (T. molitor) meal; TMh, hydrolyzed T. molitor protein meal.

^a,b,c,dWithin a row, means lacking a common superscript differ (P < 0.05); N = 4 roosters per treatment.

DIAAS-like values for growing puppies are presented in Tables 10–12. Based on the AAFCO nutrient profiles for growing puppies, ADw and TMd had DIAAS-like values above 100% for all AA except methionine and threonine, with threonine being the limiting AA. Based on AAFCO nutrient profiles, ADd had DIAAS-like values above 100% for all AA except histidine, and TMh had DIAAS-like values above 100% for all AA except threonine. According to the NRC minimal requirments and recommended allowances, all ingredients had DIAAS-like values above 100% for all AA except for methionine for TMd and ADw. Because the relationship between each individual AA and CP is consistent between minimal requirements and recommended allowances of NRC, the DIAAS-like values were the same for both calculations. Using the FEDIAF nutritional guidelines, all mealworm-based ingredients all had DIAAS-like values above 100% except methionine for TMd.

Table 10.

Digestible indispensable AA score (DIAAS)-like values¹ of mealworm-based ingredients for growing puppies based on AAFCO nutrient profiles

	AAFCO, 2022
Item	ADw2	ADd	TMd	TMh	SEM	P-value
Arginine	123b	141a	118c	125b	1.03	<0.0001
Histidine	166a	87.1d	148b	140c	1.85	<0.0001
Isoleucine	144b	125c	152b	163a	2.14	<0.0001
Leucine	115c	179a	133b	142b	2.84	<0.0001
Lysine	167a	139b	147b	178a	2.75	<0.0001
Methionine	93.0b	111a	84.7c	113a	1.55	<0.0001
Phenylalanine	112a	103b	102b	113a	1.66	0.0003
Threonine	83.2c	122a	83.4c	90.8b	1.71	<0.0001
Tryptophan	130b	191a	143b	185a	4.33	<0.0001
Valine	192a,b	147b	210a	172a,b	13.8	0.0392
Methionine-cystine	141b,c	155a,b	136c	172a	4.56	0.0005
Phenylalanine-tyrosine	327b,c	421a	336b	308c	6.63	<0.0001

¹DIAAS-like values were calculated from the digestibility of AA in cecectomized roosters.

²ADw, whole lesser mealworm (A. diaperinus) meal; ADd, defatted lesser mealworm (A. diaperinus) meal; TMd, defatted yellow mealworm (T. molitor) meal; TMh, hydrolyzed T. molitor protein meal.

^a,b,c,dWithin a row, means lacking a common superscript differ (P < 0.05); N = 4 roosters per treatment.

Table 11.

Digestible indispensable AA score (DIAAS)-like values¹ of mealworm-based ingredients for growing puppies based on NRC minimal requirements and recommended allowances

	NRC, 2006 2
Item	ADw3	ADd	TMd	TMh	SEM	P-value
Arginine	155a	136c	150b	158a	1.25	<0.0001
Histidine	188a	111d	168b	155c	2.23	<0.0001
Isoleucine	158b	125c	167b	178a	2.21	<0.0001
Leucine	116c	184a	134b	142b	2.87	<0.0001
Lysine	172a	177a	151b	183a	3.26	<0.0001
Methionine	93.8c	165a	85.0c	113b	2.49	<0.0001
Phenylalanine	144a	103c	130b	145a	1.81	<0.0001
Threonine	107c	156a	107c	116b	2.04	<0.0001
Tryptophan	116c	192a	127c	164b	4.25	<0.0001
Valine	193	167	211	174	13.8	0.1540
Methionine-cystine	141b,c	155a,b	136c	172a	4.55	0.0005
Phenylalanine-tyrosine	326b,c	421a	336b	308c	6.63	<0.0001

¹DIAAS-like values were calculated from the digestibility of AA in cecectomized roosters.

²The relationship between each individual AA and CP is consistent between minimal requirements and recommended allowances so DIAAS-like values are the same for both calculations.

³ADw, whole lesser mealworm (A. diaperinus) meal; ADd, defatted lesser mealworm (A. diaperinus) meal; TMd, defatted yellow mealworm (T. molitor) meal; TMh, hydrolyzed T. molitor protein meal.

^a,b,c,dWithin a row, means lacking a common superscript differ (P < 0.05); N = 4 roosters per treatment.

Table 12.

Digestible indispensable AA score (DIAAS)-like values¹ of mealworm-based ingredients for growing puppies based on FEDIAF nutritional guidelines

	FEDIAF, 2021
Item	ADw2	ADd	TMd	TMh	SEM	P-value
Arginine	166a	136c	160b	169a	1.34	<0.0001
Histidine	208a	124d	185b	171c	2.48	<0.0001
Isoleucine	175b	139c	185b	198a	2.45	<0.0001
Leucine	128c	203a	148b	158b	3.19	<0.0001
Lysine	190a	197a	166b	202a	3.63	<0.0001
Methionine	104c	183a	94.6c	126b	2.77	<0.0001
Phenylalanine	160a	115c	144b	161a	2.04	<0.0001
Threonine	119c	166a	119c	129b	2.09	<0.0001
Tryptophan	125c	212a	138c	178b	4.73	<0.0001
Valine	213	184	233	191	15.1	0.1471
Methionine-cystine	156b,c	172a,b	151c	191a	5.06	0.0005
Phenylalanine-tyrosine	362b,c	468a	373b	342c	6.40	<0.0001

¹DIAAS-like values were calculated from the digestibility of AA in cecectomized roosters.

²ADw, whole lesser mealworm (A. diaperinus) meal; ADd, defatted lesser mealworm (A. diaperinus) meal; TMd, defatted yellow mealworm (T. molitor) meal; TMh, hydrolyzed T. molitor protein meal.

^a,b,c,dWithin a row, means lacking a common superscript differ (P < 0.05); N = 4 roosters per treatment.

DIAAS-like values for growing kittens are presented in Tables 13–15. Based on the AAFCO nutrient profiles and NRC minimal requirments and recommended allowances for growing kittens, ADw, TMh, and TMd had DIAAS-like values above 100% for all AA except for methionine, which was the limiting AA. Based on AAFCO nutrient profiles and NRC minimal requirements and recommended allowances, ADd had DIAAS-like values above 100% for all AA except for phenylalanine, which was the limiting AA. Because the relationship between each individual AA and CP is consistent between minimal requirements and recommended allowances of NRC, the DIAAS-like values were the same for both calculations. Using the FEDIAF nutritional guidelines, all mealworm-based ingredients had DIAAS-like values above 100% except methionine for ADw and TMd.

Table 13.

Digestible indispensable AA score (DIAAS)-like values¹ of mealworm-based ingredients for growing kittens based on AAFCO nutrient profiles

	AAFCO, 2022
Item	ADw2	ADd	TMd	TMh	SEM	P-value
Arginine	132b	149a	127c	134b	1.10	<0.0001
Histidine	293a	165d	261b	242c	3.38	<0.0001
Isoleucine	244c	168d	258b	275a	3.12	<0.0001
Leucine	155b	179a	179a	190a	3.44	<0.0001
Lysine	167b, c	295a	146c	178b	4.91	<0.0001
Methionine	70.3b, c	255a	64.1c	85.4b	3.69	<0.0001
Phenylalanine	239a	77.6c	217b	242a	2.29	<0.0001
Threonine	158b	131c	158b	172a	2.67	<0.0001
Tryptophan	137c	279a	152c	195b	6.12	<0.0001
Valine	280	259	306	252	20.3	0.2729
Methionine-cystine	119b,c	132a,b	115c	146a	3.86	0.0005
Phenylalanine-tyrosine	294b,c	389a	303b	278c	5.98	<0.0001

¹DIAAS-like values were calculated from the digestibility of AA in cecectomized roosters.

²ADw, whole lesser mealworm (A. diaperinus) meal; ADd, defatted lesser mealworm (A. diaperinus) meal; TMd, defatted yellow mealworm (T. molitor) meal; TMh, hydrolyzed T. molitor protein meal.

^a,b,c,dWithin a row, means lacking a common superscript differ (P < 0.05); N = 4 roosters per treatment.

Table 15.

Digestible indispensable AA score (DIAAS)-like values¹ of mealworm-based ingredients for growing kittens based on FEDIAF nutritional guidelines

	FEDIAF, 2021
Item	ADw2	ADd	TMd	TMh	SEM	P-value
Arginine	143b	220a	137c	145b	1.27	<0.0001
Histidine	275a	173d	245b	227c	3.39	<0.0001
Isoleucine	237c	156d	250b	267a	2.96	<0.0001
Leucine	144c	236a	167b	178b	3.64	<0.0001
Lysine	220b	287a	193c	234b	5.00	<0.0001
Methionine	92.6c	246a	84.3c	112b	3.61	<0.0001
Phenylalanine	232a	102c	210b	235a	2.40	<0.0001
Threonine	166b	142c	166b	181a	2.83	<0.0001
Tryptophan	202c	252b	223c	288a	5.86	<0.0001
Valine	254	243	277	228	18.4	0.3270
Methionine-cystine	139b,c	154a,b	134c	170a	4.20	0.0002
Phenylalanine-tyrosine	345b,c	445a	355b	325c	7.01	<0.0001

¹DIAAS-like values were calculated from the digestibility of AA in cecectomized roosters.

²ADw, whole lesser mealworm (A. diaperinus) meal; ADd, defatted lesser mealworm (A. diaperinus) meal; TMd, defatted yellow mealworm (T. molitor) meal; TMh, hydrolyzed T. molitor protein meal.

^a,b,c,dWithin a row, means lacking a common superscript differ (P < 0.05); N = 4 roosters per treatment.

Table 14.

Digestible indispensable AA score (DIAAS)-like values¹ of mealworm-based ingredients for growing kittens based on NRC minimal requirements and recommended allowances

	NRC, 2006 2
Item	ADw3	ADd	TMd	TMh	SEM	P-value
Arginine	128b	173a	123c	130b	1.11	<0.0001
Histidine	225a	139d	200b	185c	2.72	<0.0001
Isoleucine	191d	126c	202b	215a	2.37	<0.0001
Leucine	117c	189a	135b	144b	2.93	<0.0001
Lysine	177b	231a	155c	188b	4.02	<0.0001
Methionine	74.8c	199a	68.0c	90.8b	2.90	<0.0001
Phenylalanine	187a	82.5c	169b	189a	1.93	<0.0001
Threonine	133b	127b	133b	145a	2.33	0.0012
Tryptophan	160c	204b	177c	227a	4.71	<0.0001
Valine	205	199	224	184	14.8	0.3268
Methionine-cystine	113b	124a,b	109b	138a	3.62	0.0005
Phenylalanine-tyrosine	222b	286a	228b	209b	4.51	<0.0001

¹DIAAS-like values were calculated from the digestibility of AA in cecectomized roosters.

²The relationship between each individual AA and CP is consistent between minimal requirements and recommended allowances so DIAAS-like values are the same for both calculations.

³ADw, whole lesser mealworm (A. diaperinus) meal; ADd, defatted lesser mealworm (A. diaperinus) meal; TMd, defatted yellow mealworm (T. molitor) meal; TMh, hydrolyzed T. molitor protein meal.

^a,b,c,dWithin a row, means lacking a common superscript differ (P < 0.05); N = 4 roosters per treatment.

Discussion

The world population is expected to exceed 9 billion in 2050 (FAO, 2009) and one of the greatest world challenges will be feeding everyone with a safe, sustainable, and nutritious food source (FAO, 2013). Insects have been proposed as environmentally friendly alternative protein source in terms of a high feed conversion efficiency, lower greenhouse gas emissions, and reduced resources needed for large-scale production (Veldkamp et al., 2022). However, the European Commission (EU) is currently having supply-demand challenges (e.g., insect farming is currently not meeting the requisite volumes with a constant quality). Additionally, insect protein products are not cost competitive with traditional protein sources such as soybean meal at this time (Veldkamp et al., 2022). Nevertheless, the demands for animal-derived protein sources will continue to increase competition between the pet food industry and the human food system, requiring alternative protein sources. Thus, investigating how insects may serve as an alternative protein source now and in the future is justified.

Evaluating the protein quality of novel feed ingredients is necessary, including the measurement of AA and macronutrient composition and nutrient digestibility. Knowing the digestibility and bioavailability of AA in feedstuffs is important to ensure that all AA requirements are met and optimal performance may be achieved (Engster et al., 1985). Studies have concluded that mealworm proteins are high-quality protein sources for broiler chickens, swine, and aquaculture (Sedgh-Gooya et al., 2021; Ji et al., 2016; Rema et al., 2019; Yoo et al., 2019; Basto et al., 2020). A study conducted by Sedgh-Gooya et al. (2021) compared diets containing 2.5% or 5% T. molitor larvae meal (TM) against a control corn-soybean meal diet in broiler chickens. Those researchers reported that broilers fed the 2.5% TM diet had a significantly higher body weight gain than controls from 1 to 10 d of age (starter period; 186 vs. 169 g). While the feed conversion ratio over the entire experiment was not affected by treatment, it was significantly lower and more efficient in broilers fed the 2.5% TM diet than controls during the starter period. Also, cecal contents indicated that Escherichia coli counts significantly decreased linearly with increasing dietary TM concentrations in that study.

A separate study by Yoo et al. (2019) tested T. molitor larvae protein in growing (3.5 mo old; 24.1 kg) pigs. In that study, a corn-vegetable byproduct basal diet was tested against a basal diet containing 9.95% dried T. molitor larvae and three animal protein byproduct-containing diets (fish meal; meat meal; poultry meal). Results showed that T. molitor larvae contained the highest percentage of indispensable AA. The apparent ileal digestibility (AID) of pigs fed T. molitor was significantly higher in lysine (89.65%), histidine (89.68%), and arginine (89.74%) compared with fish meal (histidine = 85.51%; arginine = 87.37%), and meat meal (histidine = 86.68%; arginine = 87.66%). Lastly, the standardized ileal digestibility (SID) of cysteine was significantly lower in those fed the fish meal (83.62%) compared with those fed the poultry meal (88.35%) and T. molitor (90.21%).

Similar results were reported by Ji et al. (2016), who demonstrated that insect powders had high apparent AA digestibilities and lower diarrhea rates in early-weaned piglets without affecting growth performance. In that study, piglets were weaned at 14 d of age and had an average body weight of 4.74 kg. Diets included a maize-soybean meal-based control diet containing 5% plasma protein powder and diets supplemented with 5% T. molitor, Musca domestica (housefly) larvae, or Zophobas morio (superworm) in place of the plasma protein powder. There were no significant differences in nutrient AID among diets, but diarrhea rates for pigs in all insect-fed groups were significantly lower from days 15 to 28 than those in the control group. On day 28, plasma ammonia concentrations were significantly lower in all insect-fed groups (T. molitor = 23.82 μmol/L; Musca domestica larvae = 24.68 μmol/L; Zophobas morio = 22.48 μmol/L) than those in the control group (27.05 μmol/L).

Although mealworm-based ingredients have been tested in livestock and aquaculture species, minimal research evaluating their use in companion animals exists (Matin et al., 2021). Testing AA and macronutrient composition and digestibility are imperative for novel ingredient evaluation. Cats do not have high flow rates of digesta and there are difficulties in cannulating small animals, so ileal cannulation in cats is not practical (Engster et al., 1985; Kerr et al., 2014; Deng et al., 2016). Ileal-cannulated dogs, however, were used to test the AA digestibility of protein-based ingredients for many years (Hill et al., 1996; Muir et al., 1996; Yamka et al. 2003, 2004; 2005; Faber et al., 2010). The cannulation procedure is no longer used in dogs due to animal welfare concerns. Although there are species differences in regard to anatomy and metabolism, the cecectomized rooster assay is a good model for measuring nutrient and AA digestibility of pet food ingredients. AA digestibility results of the cecectomized rooster assay and ileal-cannulated dogs were shown to be highly correlated (r = 0.87 to 0.92) by Johnson et al. (1998). In that study, 6 ileal-cannulated dogs were used in a 6 × 6 Latin square design and 24 cecectomized roosters were used to test 6 animal byproduct-based dog diets.

Based on the results of Johnson et al. (1998) and other studies testing novel proteins for use in pet foods in the past few years (Kerr et al., 2014; Deng et al., 2016; Oba et al., 2019), we used the cecectomized rooster assay to evaluate mealworm-based ingredients. The products used in this study are commercially available ingredients based on mealworm from two different species: A. diaperinus (lesser mealworm) and T. molitor (yellow mealworm). Both species are close relatives and members of the Tenebrionidae family and are similar in terms of nutrient composition. Their differences reside mostly on their rearing conditions. The defatted mealworm ingredients tested were produced by a thermomechanical process to increase the protein concentration of the final products. The remaining fat content of these ingredients is in the range of 10% to 12.4%. An enzymatically-hydrolyzed T. molitor protein meal was also tested. Differently than the other ingredients, it does not contain the exoskeleton fraction of the insect. All substrates tested were shown to have high AA digestibilities, with all indispensable AA being >90% apart from histidine and valine. Similar results were reported by Yoo et al. (2019), who reported that T. molitor-based diet AA AID and SID were high in growing pigs (all indispensable AA > 89%). Furthermore, a study conducted by Ji et al. (2016) reported high AA AID for a T. molitor-based diet fed to early-weaned pigs (all indispensable AA > 84%).

The DIAAS procedure is used to estimate protein quality of ingredients and diets for humans (Mathai et al., 2017) and is an appropriate method to assess AA content and determine protein quality of ingredients used in pet foods. Using the DIAAS-like values, our data shows that TMh had the highest DIAAS-like values for most indispensable AA, whereas TMd had the lowest. All protein sources performed well, however. Methionine and phenylalanine were the first limiting AA when references for adult dogs and growing kittens were used. The limiting AA were slightly different when references for growing puppies were used (methionine, threonine, histidine), but the results demonstrate that mealworm-based ingredients are high-quality protein sources. Based on the DIAAS-like values generated in the current study, which assumes that diets would be formulated with only mealworm-based ingredients and would contain the recommended CP concentration, synthetic AA supplementation may be required to meet the requirements of all indispensable AA in diets of growing or adult dogs and cats. If used in combination with other protein sources, as is usually done in commercial pet foods, our results suggest that mealworm-based proteins may serve as a valuable protein source that may be used with a variety of complementary proteins.

In conclusion, the current experiment demonstrated high AA digestibilities and DIAAS-like values for mealworm-based ingredients intended for use in dog and cat foods. There were no AA digestibility differences among substrates, with digestibilities of all indispensable AA being >90% except for histidine and valine. The first-limiting AA were methionine, phenylalanine, or threonine, depending on life stage and species of animal (dog; cat). Collectively, our data suggest that mealworm-based ingredients may serve as high-quality protein sources for pet foods. Future research in dogs and cats is necessary to confirm sufficient palatability, digestibility, and product safety.

Glossary

Abbreviations

AA

amino acid

AAFCO

Association of American Feed Control Officials

ADd

defatted lesser mealworm (Alphitobius diaperinus) flour

ADw

whole lesser mealworm (Alphitobius diaperinus) flour

AID

apparent ileal digestibility

CP

crude protein

DIAAS

digestible indispensable amino acid score

DM

dry matter

FEDIAF

The European Pet Food Industry Nutritional Guidelines

NRC

National Research Council

OM

organic matter

SID

standardized ileal digestibility

TM

Tenebrio molitor larvae meal

TMd

defatted yellow mealworm (Tenebrio molitor) flour

TMh

hydrolyzed Tenebrio molitor proteins

Conflict of Interest Statement

The authors have no conflicts of interest.