Standardized amino acid digestibility and protein quality in extruded canine diets containing hydrolyzed protein using a precision-fed rooster assay

Abstract

Protein hydrolysate has become a choice of alternative protein source in canine diets as it showed greater digestibility, lower allergenic responses, and various functional properties when compared with intact proteins. The objective of the study was to determine the effect of hydrolyzed protein inclusion on amino acid digestibility and protein quality in extruded canine diets when compared with a traditional protein source for adult dogs. Five treatment diets were formulated to have similar compositions except for the main protein source. The control diet was formulated with chicken meal (CM) as the primary protein source. Test hydrolyzed proteins, chicken liver and heart hydrolysate (CLH) and chicken hydrolysate (CH) were used to partially or completely substitute CM. The diets were: CONd: CM (30%) diet; 5%CLHd: 5% CLH with 25% CM diet; CLHd: CLH (30%) diet; 5%CHd: 5% CH with 25% CM diet; CHd: CH (30%) diet. A precision-fed rooster assay was used to determine standardized amino acid digestibility for the ingredients and diets. In addition, Digestible Indispensable Amino Acid Score (DIAAS)-like values were calculated for the protein ingredients. All protein ingredients had higher than 80% of digestibility for all indispensable amino acids with no difference among sources (P > 0.05). From the DIAAS-like values referencing AAFCO nutrient profile for adult dogs, CLH and CH did not have any limiting amino acid; on the other hand, CM has a lower DIAAS-like value (93.3%) than CLH and CH (P < 0.05) with tryptophan being the first-limiting amino acid. The DIAAS-like values were often lower when the amino acid combinations methionine + cysteine and phenylalanine + tyrosine were included in the calculation. When referencing NRC recommended allowances and minimal requirements, methionine was the first-limiting amino acid for all protein sources. Amino acid digestibility was mostly above 80% and comparable among the treatment diets. Regarding the digestible indispensable amino acid concentrations in the diets, all of them met the AAFCO nutrient profile for adult dogs at maintenance. In conclusion, both protein hydrolysates were highly digestible, high-quality protein sources, and a full substitution from CM to protein hydrolysate could result in greater protein quality, according to the DIAAS-like values of the ingredients, when compared with CM in extruded canine diets.

Hydrolyzed proteins from poultry origins could serve as valuable protein sources in extruded canine and an effective method to upcycle and add value to chicken by-products.

Introduction

Traditionally, animal protein such as poultry meal has been used as the main protein source in pet foods. With the diversification of the pet industry, however, alternative protein sources have been sought out to substitute or replace traditional protein sources for various reasons and hydrolyzed protein is one of them. According to the Association of American Feed Control Officials (AAFCO; 2022), hydrolyzed protein from chicken liver, heart, and meat falls into the category of animal digest, a material that results from chemical and/or enzymatic hydrolysis of clean and undecomposed animal tissue. To manufacture hydrolysates, selected protein ­ingredients go through acid, alkaline, or enzymatic hydrolysis, forming smaller molecular-weight products (Pasupuleti and Braun, 2010). The hydrolyzed protein can then be incorporated into the diet formulation in the hopes of increasing digestibility, decreasing allergenic responses, and/or providing additional benefits as bioactive peptides (Cave, 2006; Hou et al., 2017). Lower molecular weight resulting from hydrolysis could result in greater digestibility since the partially denatured structure would be easier to break down by enzymes in the small intestine of the animal (Zhao et al., 1997). The benefit of higher digestibility after hydrolysis is of particular interest for utilizing poorly digested intact protein sources (Eugenio et al., 2022). As food allergen hypersensitivity heavily relies on mast cell reaction, several studies have proposed a lower molecular-weight protein source can ameliorate allergic responses by decreasing immunoglobulin E binding (Lehrer et al., 1996; Hartmann et al., 2007; Cavatorta et al., 2010; Olivry et al., 2017; Nutten et al., 2020). Some studies fed hydrolyzed protein from various sources to animals for the nutraceutical properties, such as antioxidant, antimicrobial, antihypertension, and immunomodulatory activities (Haque et al., 2009; Lasekan et al., 2013; Bhat et al., 2015; Jo et al., 2017; Chai et al., 2021).

Protein hydrolysate could be produced from a wide variety of origins, such as marine, poultry, egg, milk, plant, and byproducts. Hence, the properties and functions of the protein hydrolysate that have been used in animal feed also vary greatly (Cave, 2006; Hou et al., 2017). New protein hydrolysate ingredients should be tested for their nutrient profiles and protein quality before being incorporated into pet food diets. Furthermore, in diets, the processing and interaction with other food matrices could also impact the protein properties. The hypothesis was the protein hydrolysates would be highly digestible and be of greater protein quality than the traditional protein source to serve as alternative protein sources in premium or specialty canine diets. The objectives of the current study were to determine the chemical composition, amino acid digestibility, and protein quality of two types of poultry hydrolysate sources compared with the traditional CM, as well as to examine the effect of different inclusion rates of protein hydrolysates on digestibility and protein quality in canine diets, using a precision-fed rooster assay. The current study could provide the fundamental information about the quality of the test protein hydrolysate ingredients so that future canine studies could proceed to determine their additional physiological health benefits.

Materials and Methods

All animal procedures were approved by the University of Illinois Institutional Animal Care and Use Committee. All methods were performed in accordance with the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Test ingredients and diets

Five treatment diets were formulated to have similar ingredient compositions except for the main protein source (Table 1). The control diet was formulated with low ash chicken meal (CM) as the primary protein source and rice as the primary carbohydrate source. CM was chosen as the control protein source because it is a high-quality protein and is commonly used in the pet food industry. The current study aimed to determine if the hydrolyzed proteins, as alternative protein sources, would be comparable with or be of higher quality than a traditional high-quality protein ingredient. Test enzymatic hydrolyzed proteins, PROSURANCE CHX Liver.HD (CLH) and PROSURANCE CHX.HD (CH; Kemin Industries, Des Moines, IA), were used to partially or completely substitute CM for the manufacturing of extruded diets for adult dogs. The ingredient CLH was hydrolyzed from chicken liver and heart; CH was hydrolyzed from mechanically separated chicken. The diets were as follows, 1) CONd: CM (30%) diet; 2) 5%CLHd: 5% chicken liver and heart hydrolysate with 25% CM diet; 3) CLHd: chicken liver and heart hydrolysate (30%) diet; 4) 5%CHd: 5% CH with 25% CM diet; 5) CHd: CH (30%) diet. The partial substitution rate was chosen at 5% as this was a more economical choice that could potentially still demonstrate health benefits that may be examined in future canine studies. Due to the higher cost of hydrolyzed proteins in contrast with CM, full substitution would be a viable solution for value-added and specialty pet foods. All diets were formulated to meet or exceed the AAFCO (2022) recommendation for adult dog maintenance and were extruded at Wenger Pilot Plant in Sabetha, KS.

Table 1.

Ingredient inclusion rates of treatment diets containing different sources of protein

Ingredient, % as-is	Treatment1
	CONd	5% CLHd	CLHd	5% CHd	CHd
Brewer’s rice	40.5	40.5	34.8	40.5	36.8
Chicken meal, low ash	29.7	24.2	—	24.2	—
Chicken liver and heart hydrolysate2	—	5.5	32.3	—	—
Chicken hydrolysate3	—	—	—	5.5	31.6
Corn	11.0	11.0	10.8	11.0	10.5
Whole green pea	5.5	5.5	5.4	5.5	5.3
Cellulose	4.4	4.4	5.4	4.4	5.3
Beet pulp	2.2	2.2	2.2	2.2	2.1
Eggs dried 45%	2.2	2.2	2.2	2.2	2.1
Whole flaxseed	2.2	2.2	2.2	2.2	2.1
Salt	0.44	0.44	0.43	0.44	0.42
Potassium chloride	0.49	0.49	0.48	0.49	0.47
Calcium carbonate	—	—	1.61	—	1.05
Dicalcium phosphate	—	—	1.29	—	1.37
l-Lysine	0.44	0.44	0.22	0.44	—
dl-Methionine	0.33	0.33	0.32	0.33	0.32
Mineral premix4	0.24	0.24	0.24	0.24	0.23
Vitamin premix5	0.15	0.15	0.15	0.15	0.15
Choline chloride	0.11	0.11	0.11	0.11	0.11
Taurine	0.11	0.11	0.11	0.11	0.11

¹CONd: chicken meal diet; 5% CLHd: 5% substitution of chicken liver and heart hydrolysate of chicken meal diet; CLHd: chicken liver and heart hydrolysate diet; 5% CHd: 5% substitution of chicken hydrolysate of chicken meal diet; CHd: chicken hydrolysate diet.

²PROSURANCE CHX Liver.HD (Kemin Industries).

³PROSURANCE CHX.HD (Kemin Industries).

⁴Iron (10.0%), copper (1.5%), manganese (5.5%), zinc (20.0%), iodine (1500.0 mg/kg), selenium (200 mg/kg), cobalt (1000.0 mg/kg).

⁵Vitamin A (17,163,000.0 IU/kg), vitamin D₃ (920,000.0 IU/kg), vitamin E (79,887.0 IU/kg), thiamin (14,252.0 mg/kg), riboflavin (4,719.0 mg/kg), d-pantothenic acid (12,186.0 mg/kg), niacin (64,736.0 mg/kg), vitamin B₆ (5,537.0 mg/kg), folic acid (720.0 mg/kg), biotin (70.0 mg/kg), vitamin B₁₂ (22.0 mg/kg).

Chemical analysis

All treatment diets were ground with a Wiley mini-mill (Thomas Scientific, Swedesboro, NJ) through a 2-mm screen. All experimental diets, ingredients, and dried excreta were analyzed for dry matter (DM), ash, crude protein (CP), and complete amino acid profile. Experimental diets and ingredients were also analyzed for organic matter (OM), acid hydrolyzed fat (AHF), CP, total dietary fiber (TDF), and gross energy (GE). DM and ash content were determined according to AOAC (2006; methods 934.01 and 942.05). CP was calculated from Leco (TruMac N, Leco Corporation, St. Joseph, MI) with total nitrogen values determined referencing AOAC (2006; method 992.15). AHF was analyzed according to AACC (1983) and Budde (1952). Gross energy was determined through bomb calorimetry (Model 6200, Parr Instruments Co., Moline, IL). TDF was analyzed according to Prosky et al. (1992). The complete amino acid profile was determined according to AOAC (2007).

Precision-fed rooster assay

A precision-fed rooster assay was conducted according to Parsons (1985) to determine the standardized amino acid digestibility for the three protein ingredients and five treatment diets. Digestible Indispensable Amino Acid Score (DIAAS)-like values were also calculated for the protein ingredients. The precision-fed rooster assay has been validated as an adequate model of canine in vivo digestibility, as it has shown similar results to ileal-cannulated dogs for ileal digestibility (Johnson et al., 1998). Since using ileal-cannulated dogs was no longer a feasible option, a precision-fed rooster assay was chosen to be used as a model. The study included 32 cecectomized single-comb White Leghorn roosters in total with four roosters per treatment. The roosters were housed in a temperature-controlled room with a 16:8 (L:D) h cycle individually in cages with wire floors. The roosters were fed either 30 g of ground diet or a mixture of 15 g of ground corn and 15 g of ingredient after 26 h of fasting. The excreta were collected for 48 h after feeding, freeze-dried, and ground for analysis. The protein sources were mixed in a 1:1 ratio with corn to improve ingredient flow during the feeding procedure. The addition of corn also ensured that all of the test ­ingredients were deposited into the crop of the rooster. Apparent amino acid digestibility was measured and corrected for endogenous losses to report standardized amino acid digestibility. The complete amino acid profile and endogenous value were previously determined with roosters that were fasted for 48 h and could, therefore, be accounted for in the equation to obtain the standardized digestibility of only the protein ingredients (Supplementary Table S1).

Standardized amino acid digestibility was calculated according to Sibbald (1979):

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{S}\mathrm{t}\mathrm{e}\mathrm{p}1:\mathrm{F}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{A}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{o}\mathrm{A}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{D}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\left(\mathrm{\%}\right)=\frac{\mathrm{F}\mathrm{A}\mathrm{A}-\mathrm{E}\mathrm{A}\mathrm{A}+\mathrm{E}\mathrm{n}\mathrm{d}\mathrm{o}\mathrm{A}\mathrm{A}}{\mathrm{F}\mathrm{A}\mathrm{A}}\times 100}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{S}\mathrm{t}\mathrm{e}\mathrm{p}2:\mathrm{I}\mathrm{n}\mathrm{g}\mathrm{r}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{A}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{o}\mathrm{A}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{D}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\left(\mathrm{\%}\right)=\mathrm{A}\mathrm{A}{\mathrm{D}}_c-\frac{\mathrm{A}\mathrm{A}{\mathrm{D}}_c-\mathrm{A}\mathrm{A}{\mathrm{D}}_f}{\mathrm{F}\mathrm{A}\mathrm{A}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}}\times 100}\end{array}}$$

The ingredients needed a two-step calculation since they were mixed with corn whereas the digestibility of diets was only calculated through the first step. In the above equations, FAA is the total amino acid fed; EAA is the total amino acid excreted after feeding; EndoAA is the endogenous loss of amino acid from fasted roosters; AAD_c is the amino acid digestibility of corn; AAD_f is the calculated amino acid digestibility of the feed from step 1; FAA ratio is the amino acid ratio of the amino acid in the ingredient to the total amino acid in the mixed feed.

The DIAAS-like values for each ingredient were calculated according to Mathai et al. (2017) to determine the protein quality. The reference protein was calculated from the AAFCO nutrient profile, the National Research Council (NRC) recommended allowances, and NRC minimum requirements for adult canine at maintenance.

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{D}\mathrm{I}\mathrm{A}\mathrm{A}\mathrm{S}\text{-}\mathrm{l}\mathrm{i}\mathrm{k}\mathrm{e}\left(\mathrm{\%}\right)=\frac{\mathrm{m}\mathrm{g}\mathrm{o}\mathrm{f}\mathrm{d}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{o}\mathrm{a}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{i}\mathrm{n}1\mathrm{g}\mathrm{o}\mathrm{f}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{r}\mathrm{y}\mathrm{p}\mathrm{r}\mathrm{o}\mathrm{t}\mathrm{e}\mathrm{i}\mathrm{n}}{\mathrm{m}\mathrm{g}\mathrm{o}\mathrm{f}\mathrm{a}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{o}\mathrm{a}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{i}\mathrm{n}1\mathrm{g}\mathrm{o}\mathrm{f}\mathrm{r}\mathrm{e}\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{p}\mathrm{r}\mathrm{o}\mathrm{t}\mathrm{e}\mathrm{i}\mathrm{n}}\times 100}\end{array}}$$

A DIAAS-like value greater than 100% is considered to be of “high” quality, a value between 75% and 100% is considered to be of “good” quality, and a value below 75% is considered to be of moderate or poor quality (Mathai et al., 2017; Reilly et al., 2020a; Reilly et al., 2020b). The DIAAS-like value for each ingredient and diet was determined by the first-limiting amino acid, the amino acid with the lowest DIAAS-like value.

Statistical analysis

All data were analyzed with MIXED model procedures of SAS version 9.4 (SAS Institute Inc., Cary, NC). Dietary treatment was the fixed effect and animal was considered the random effect. Data normality was checked with the UNIVARIATE procedure. Tukey adjustment was used and the alpha was set at 0.05 for statistical significance.

Results

Chemical composition of protein ingredients and diets

The chemical composition of protein ingredients is shown in Table 2. The DM content of the ingredients ranged from 93.4% for CM to 96.8% for CLH. The OM content on a DM basis (DMB) was higher for both hydrolyzed protein sources; 95.2 and 95.5% for CLH and CH, respectively, in contrast with 89.2% for CM. CP concentration was highest for CM at 69.3%, followed by CLH (67.8%) and CH (53.9%) on DMB. AHF concentration was highest in CH (42.4%), followed by CLH (25.5%), and CM was the lowest at 16.6%. The GE was highest in CH at 7.0 kcal/g compared with CLH and CM (5.9 and 5.6 kcal/g, respectively). The complete amino acid profile (Table 2) showed that all three protein sources had comparable essential amino acid contents and these were all well above the minimum requirements established by NRC (2006) or the nutrient profile suggested by AAFCO (2022) for adult dogs.

Table 2.

Chemical composition of different sources of protein ingredients

	Ingredient1
	CM	CLH	CH
Dry matter, %	93.4	96.8	94.1
	% DM basis
Organic matter	89.2	95.2	95.5
Ash	10.8	4.6	4.5
Acid hydrolyzed fat	16.6	25.5	42.4
Crude protein	69.3	67.8	53.9
Gross energy, kcal/g	5.6	5.9	7.0
Indispensable amino acids
Arginine	4.56	3.67	3.53
Histidine	1.40	1.33	1.45
Isoleucine	2.71	2.80	2.35
Leucine	4.71	5.00	4.05
Lysine	4.45	4.61	4.53
Methionine	1.43	1.39	1.40
Phenylalanine	2.63	2.54	2.12
Threonine	2.62	2.75	2.34
Tryptophan	0.61	0.72	0.58
Valine	3.51	3.49	2.56
Dispensable amino acids
Alanine	4.65	3.53	3.43
Aspartic acid	5.59	5.33	4.92
Cysteine	0.82	0.67	0.52
Glutamic acid	9.07	6.81	8.03
Proline	4.44	2.64	2.74
Serine	2.67	2.42	2.17
Tyrosine	2.05	2.22	2.22

¹CM: chicken meal; CLH: chicken liver and heart hydrolysate; CH: chicken hydrolysate.

Chemical composition was similar among all five dietary treatments (Table 3). On average, DM content was approximately 91%, OM concentration was around 95% on DMB, AHF content ranged from 15.2% to 18.9%, CP concentration ranged from 25.2% to 29.4%, TDF content was around 14%, and GE was 5.1 to 5.2 kcal/g. The slight differences in CP and AHF concentration were reflective of the select ingredients used as the primary protein sources in these diets. The complete amino acid profile of the five extruded diets is also depicted in Table 3. All diets had similar concentrations of indispensable and dispensable amino acids. All indispensable amino acids were at higher concentrations in contrast with the minimum requirements established by NRC (2006) or the nutrient profile suggested by AAFCO (2022) for adult dogs.

Table 3.

Chemical composition of treatment diets containing different sources of protein

	Treatment1
	CONd	5% CLHd	CLHd	5% CHd	CHd
Dry matter, %	90.5	90.8	91.4	90.3	91.4
	% DM basis
Organic matter	95.1	95.2	94.2	95.2	94.7
Ash	4.9	4.8	5.8	4.8	5.3
Acid hydrolyzed fat	15.2	15.2	15.8	16.2	18.9
Crude protein	29.4	28.8	26.8	28.1	25.2
Total dietary fiber	14.3	14.2	14.0	14.7	13.4
Soluble dietary fiber	4.4	4.4	4.5	4.6	4.4
Insoluble dietary fiber	9.9	9.7	9.5	10.1	9.0
Gross energy, kcal/g	5.2	5.2	5.1	5.2	5.2
Indispensable amino acids
Arginine	2.04	1.91	1.61	1.88	1.63
Histidine	0.61	0.59	0.57	0.59	0.62
Isoleucine	1.12	1.12	1.15	1.08	1.08
Leucine	2.05	2.03	2.11	1.98	1.88
Lysine	2.06	2.08	1.93	2.04	1.80
Methionine	0.83	0.87	0.87	0.84	0.85
Phenylalanine	1.20	1.16	1.15	1.14	1.02
Threonine	1.09	1.07	1.08	1.05	0.99
Tryptophan	0.27	0.27	0.27	0.26	0.28
Valine	1.38	1.37	1.44	1.32	1.23
Dispensable amino acids
Alanine	1.73	1.69	1.49	1.68	1.46
Aspartic acid	2.48	2.40	2.30	2.35	2.21
Cysteine	0.40	0.37	0.36	0.36	0.31
Glutamic acid	4.06	3.83	3.44	3.85	3.55
Proline	1.65	1.59	1.21	1.57	1.22
Serine	1.17	1.11	1.01	1.05	0.90
Tyrosine	0.96	0.93	0.90	0.91	0.90

¹CONd: chicken meal diet; 5% CLHd: 5% substitution of chicken liver and heart hydrolysate of chicken meal diet; CLHd: chicken liver and heart hydrolysate diet; 5% CHd: 5% substitution of chicken hydrolysate of chicken meal diet; CHd: chicken hydrolysate diet.

Precision-fed rooster assay

The standardized amino acid digestibility for all indispensable amino acids among the three protein sources tested was higher than 80% and no difference (P > 0.05) among sources was observed (Table 4). The standardized cysteine digestibility was highest in CM (67.6%), followed by CH (63.6%), and CLH (47.9) was the lowest (P < 0.05). For tyrosine, the standardized digestibility was all above 80% for the ingredients, ranging from 84.8% in CM to 93.5% in CH. The standardized amino acid digestibility of the five extruded diets (Table 5) was higher than 80% for most indispensable amino acids, except for histidine (79.1%) in the 5% CLHd. The standardized digestibility of arginine, lysine, methionine, and tryptophan did not differ (P > 0.05) among roosters fed the extruded diets. For histidine, digestibility was greatest for the CONd (87.1%) in comparison with all treatments containing the hydrolyzed protein sources, ranging from 79.1% to 85.9%. As for leucine, phenylalanine, threonine, and valine, 5% CLHd resulted in a lower (P < 0.05) digestibility (84.8%, 83.4%, 79.9%, and 80.3%, respectively) compared with CLHd (90.3%, 88.5%, 88.6%, and 87.9%, respectively). However, the digestibility of leucine, phenylalanine, threonine, and valine in 5% CLHd was comparable to CONd (P > 0.05). Cysteine digestibility was lower than 80% for all diets, ranging from 61.7% to 73.7%, and there was no difference among treatments (P > 0.05). Tyrosine digestibility was higher than 80% in all diets with CHd being the highest at 91.4% and 5% CLHd being the lowest at 83.4% (P < 0.05).

Table 4.

Standardized amino acid digestibility of different sources of protein ingredients

Amino acid, %	Ingredient1			SEM2
	CM	CLH	CH
Indispensable amino acid
Arginine	90.7	90.4	91.8	1.21
Histidine	84.1	81.1	84.4	2.88
Isoleucine	86.8	88.8	88.4	1.49
Leucine	87.3	89.3	89.0	1.38
Lysine	85.4	86.0	87.9	1.71
Methionine	89.6	90.3	90.2	1.05
Phenylalanine	86.7	86.2	87.6	1.53
Threonine	82.5	84.7	85.7	1.87
Tryptophan	94.1	93.9	94.2	1.03
Valine	85.0	87.4	85.2	1.57
Selected dispensable amino acid
Cysteine	67.6a	47.9b	63.6ab	4.92
Tyrosine	84.8b	87.7ab	93.5a	1.70

¹CM: chicken meal; CLH: chicken liver and heart hydrolysate; CH: chicken hydrolysate.

²Standard error of the mean.

^a,bMeans in the same row with different superscript letters are different (P < 0.05).

Table 5.

Standardized amino acid digestibility of treatment diets containing different sources of protein

Amino acid, %	Treatment1					SEM2
	CONd	5% CLHd	CLHd	5% CHd	CHd
Indispensable amino acid
Arginine	91.7	88.7	91.8	92.4	91.8	0.88
Histidine	87.1a	79.1b	81.9b	81.4b	85.9b	1.76
Isoleucine	86.4	83.4	88.5	88.5	88.2	1.20
Leucine	88.0ab	84.8b	90.3a	89.9a	89.2ab	1.11
Lysine	87.7	82.8	87.6	87.3	86.7	1.17
Methionine	92.4	91.0	93.5	92.7	92.1	0.70
Phenylalanine	87.3ab	83.4b	88.5a	88.7a	87.2ab	1.15
Threonine	84.2ab	79.9b	88.6a	87.4ab	86.4ab	1.79
Tryptophan	92.5	89.9	92.0	92.3	93.5	0.83
Valine	84.0ab	80.3b	87.9a	86.9a	85.4ab	1.28
Selected dispensable amino acid
Cysteine	69.4	61.7	71.5	73.7	71.8	3.49
Tyrosine	87.5ab	83.4b	89.8ab	88.1ab	91.4a	1.46

¹CONd: chicken meal diet; 5% CLHd: 5% substitution of chicken liver and heart hydrolysate of chicken meal diet; CLHd: chicken liver and heart hydrolysate diet; 5% CHd: 5% substitution of chicken hydrolysate of chicken meal diet; CHd: chicken hydrolysate diet.

²Standard error of the mean.

^a,bMeans in the same row with different superscript letters are different (P < 0.05).

DIAAS-like values

Traditional DIAAS are calculated using standardized ileal amino acid digestibility from ileal-cannulated pigs with the estimated average requirement of 2- to 5-year-old children as reference (Marinangeli and House, 2017). In this study, the DIAAS-like values were calculated using standardized amino acid digestibility from cecectomized roosters with AAFCO nutrient profile, NRC recommended allowances, and NRC minimal requirements for adult dogs at maintenance as the reference protein. This is to determine the first-limiting amino acid for the three protein sources tested (Table 6). Results from DIAAS-like values for the three protein sources tested revealed that CLH and CH were most limited in methionine + cysteine (with DIAAS-like values of 72.5% and 81.6%, respectively) when referencing the AAFCO nutrient profile for adult dogs. In contrast, phenylalanine + tyrosine was the first-limiting amino acid for CM with a DIAAS-like value of 61.1%, which was significantly lower than the two protein hydrolysates (P < 0.05). Overall, DIAAS-like values for isoleucine, leucine, lysine, methionine, threonine, tryptophan, and phenylalanine + tyrosine were greater (P < 0.05) for CLH and CH in contrast with CM. When NRC recommended allowances and minimal requirements for adult dogs were used as the reference protein, methionine + cysteine was the first-limiting amino acid for CLH and CH; on the other hand, phenylalanine + tyrosine was the first-limiting amino acid for CM. The final ­DIAAS-like values for the three protein ingredients, which were determined by the first-limiting amino acid of each ingredient, are shown in Figure 1.

Table 6.

Digestible indispensable amino acid score-like values of different sources of protein ingredients using reference protein for canine adult maintenance

Indispensable amino acid	Reference1	Ingredient2			SEM3
		CM	CLH	CH
Arginine	AAFCO	209.9a	194.3b	211.3a	2.78
	NRC RA	170.6a	157.9b	170.6a	2.26
	NRC MR	170.6a	157.9b	170.6a	2.26
Histidine	AAFCO	159.3b	168.3b	212.6a	5.95
	NRC RA	89.5b	94.5b	119.4a	3.34
	NRC MR	90.7b	95.7b	121.0a	3.39
Isoleucine	AAFCO	161.0c	196.1a	182.4b	2.88
	NRC RA	89.4c	109.0a	101.4b	1.60
	NRC MR	90.6c	110.4a	102.7b	1.62
Leucine	AAFCO	157.2c	196.4a	177.0b	2.60
	NRC RA	87.3c	109.1a	98.3b	1.44
	NRC MR	88.0c	110.0a	99.1b	1.46
Lysine	AAFCO	156.2c	187.7b	210.2a	3.53
	NRC RA	156.7c	188.3b	210.8a	3.54
	NRC MR	156.7c	188.3b	210.8a	3.54
Methionine	AAFCO	100.3c	113.5b	127.0a	1.23
	NRC RA	56.0c	63.4b	71.0a	0.68
	NRC MR	56.9c	64.4b	72.1a	0.70
Phenylalanine	AAFCO	131.2b	144.9a	137.1ab	2.36
	NRC RA	73.2b	80.8a	76.5ab	1.31
	NRC MR	73.2b	80.8a	76.5ab	1.31
Threonine	AAFCO	117.0b	145.3a	139.4a	2.80
	NRC RA	72.5b	90.1a	86.5a	1.74
	NRC MR	73.4b	91.2a	87.5a	1.76
Tryptophan	AAFCO	93.3c	126.3a	113.9b	1.18
	NRC RA	59.2c	80.2a	73.3b	0.75
	NRC MR	60.3c	81.6a	73.7b	0.76
Valine	AAFCO	157.6b	185.4a	148.0b	2.94
	NRC RA	87.9b	103.4a	82.6b	1.64
	NRC MR	88.4b	103.9a	83.0b	1.65
Methionine + cystine	AAFCO	73.2b	72.5b	81.6a	2.14
	NRC RA	40.8b	40.4b	45.5a	1.19
	NRC MR	40.8b	40.4b	45.5a	1.19
Phenylalanine + tyrosine	AAFCO	61.1c	78.8b	93.6a	1.32
	NRC RA	33.9c	43.9b	52.0a	0.74
	NRC MR	34.1c	43.9b	52.2a	0.74

¹AAFCO: AAFCO nutrient profile; NRC RA: NRC recommended allowance; NRC MR: NRC minimal requirement.

²CM: chicken meal; CLH: chicken liver and heart hydrolysate; CH: chicken hydrolysate.

³Standard error of the mean.

^a,b,scMeans in the same row with different superscript letters are different (P < 0.05).

Figure 1.

Digestible indispensable amino acid score-like values of different sources of protein ingredients according to the first-limiting amino acid using AAFCO nutrient profile, NRC recommended allowance, and NRC minimum requirement for canine adult maintenance as reference. CM: chicken meal; CLH: chicken liver and heart hydrolysate; CH: chicken hydrolysate. AAFCO: AAFCO nutrient profile; NRC RA: NRC recommended allowance; NRC MR: NRC minimal requirement. ^{A,B,C, a,b,c, α,β,γ}: means with different superscripts within the same reference source differ (P < 0.05). Methionine + cysteine was the first-limiting amino acid for CLH and CH. Phenylalanine + tyrosine was the first-limiting amino acid for CM.

The digestible indispensable amino acid concentrations in the treatment diets are shown in Table 7 by percentage DM and per 1,000 kcal of estimated metabolizable energy, calculated based on NRC (2006). The digestible indispensable amino acid concentrations of all the diets exceeded the AAFCO nutrient profile for adult canine at maintenance.

Table 7.

Digestible indispensable amino acid concentration in treatment diets containing different sources of protein

Indispensable amino acid	Treatment1, % DM					Treatment, g/1,000 kcal ME2
	CONd	5% CLHd	CLHd	5% CHd	CHd	CONd	5% CLHd	CLHd	5% CHd	CHd
Arginine	1.87	1.69	1.48	1.74	1.50	5.21	4.71	4.11	4.79	3.95
Histidine	0.53	0.47	0.47	0.48	0.53	1.48	1.30	1.30	1.32	1.40
Isoleucine	0.97	0.93	1.02	0.96	0.95	2.70	2.60	2.83	2.63	2.51
Leucine	1.80	1.72	1.91	1.78	1.68	5.03	4.79	5.30	4.91	4.42
Lysine	1.81	1.72	1.69	1.78	1.56	5.04	4.79	4.70	4.91	4.12
Methionine	0.77	0.79	0.81	0.78	0.78	2.14	2.20	2.26	2.15	2.07
Phenylalanine	1.05	0.97	1.02	1.01	0.89	2.92	2.69	2.83	2.79	2.35
Threonine	0.92	0.85	0.96	0.92	0.86	2.56	2.38	2.66	2.53	2.26
Tryptophan	0.25	0.24	0.25	0.24	0.26	0.70	0.68	0.69	0.66	0.69
Valine	1.16	1.10	1.27	1.15	1.05	3.23	3.06	3.52	3.16	2.77
Methionine + cysteine	1.04	1.02	1.07	1.04	1.01	2.91	2.84	2.98	2.88	2.65
Phenylalanine + tyrosine	1.89	1.74	1.83	1.81	1.71	5.26	4.85	5.08	5.00	4.52

¹CONd: chicken meal diet; 5% CLHd: 5% substitution of chicken liver and heart hydrolysate of chicken meal diet; CLHd: chicken liver and heart hydrolysate diet; 5% CHd: 5% substitution of chicken hydrolysate of chicken meal diet; CHd: chicken hydrolysate diet.

²ME: estimated metabolizable energy based on NRC calculations and modified Atwater factors.

Discussion

In general, the test hydrolysates from chicken liver and heart as well as mechanically separated chicken demonstrated higher protein quality than the traditional CM. The chemical composition and amino acid profiles of the CM were comparable to chicken or poultry meals from previous studies, except for a higher OM content than some since the present study used a low ash CM (Deng et al., 2016; Tjernsbekk et al., 2017; Oba et al., 2019). As for CH, it was closer in overall nutrient content and amino acid profile to retorted chicken and steamed chicken from Oba et al. (2019). The amino acid profile of CH was also comparable to the raw mechanically separated chicken from Tjernsbekk et al. (2017) and chicken breast hydrolysate from Sun et al. (2012). This similarity was expected as CH was produced from mechanically separated chicken hydrolysis. Previous research has been done in examining the composition of chicken liver and chicken byproduct (Li et al., 2011; Chen et al., 2020); however, the results were not fully compatible as CLH from the current study was hydrolyzed from only chicken liver and heart. Overall, extruded diets using either CM, CH, or CLH all contained higher amino acid concentrations than required or recommended by AAFCO and NRC; therefore, both CLH and CH could serve as the main protein sources in complete and balanced canine diets since their nutrient profiles exceeded the amino acid concentrations from AAFCO and NRC.

In the current study, the two test protein hydrolysates were compared with CM, which is a highly digestible protein source shown in previous research with 75% to 90% standardized digestibility of indispensable amino acids (Deng et al., 2016; Oba et al., 2019). Therefore, it was not surprising that all three protein sources tested in the study were comparable and had higher than 80% digestibility for all ­indispensable amino acids. After the protein sources were incorporated into diets, the amino acid digestibility was still comparable among all diets and mostly above 80%. Only 5% CLHd had slightly lower histidine and threonine digestibility (79.1% and 79.9%, respectively). This finding corresponded to previous studies showing various poultry protein sources, such as CM, duck meal, poultry meal, and raw chicken, resulted in lower than 80% of histidine and/or threonine digestibility (Deng et al., 2016; Tjernsbekk et al., 2017; Oba et al., 2019). The lower cysteine digestibility was correspondent to previous studies in roosters; research showed lower than 80% of standardized cysteine digestibility in both plant and animal proteins (Douglas et al., 1997; Wang and Parsons, 1998; Yadav et al., 2022).

A standard reference protein for canine DIAAS-like values has yet to be established. Therefore, the current study used the AAFCO nutrient profile (the commercial guideline for pet food manufacturing), NRC recommended allowances (closest to the AAFCO nutrient profile and the basis of establishing the nutrient profile), and also NRC minimal requirements (values assuming the ingredients were highly digestible). Since NRC recommended allowances were established based on minimal requirements with the consideration of digestibility, both the protein concentration and individual amino acid concentrations were proportionally increased from the minimal requirements. This proportional change indicated that the percentage of amino acid per gram of protein would still remain similar. Hence, DIAAS-like values calculated from NRC recommended allowances and minimal requirements as the reference were identical or next to no difference. When comparing AAFCO and NRC being the reference for DIAAS-like values, since AAFCO suggested a higher protein concentration than NRC, the percentage of amino acid per gram of protein would be lower when using AAFCO than NRC. The lower percentages of amino acid per gram of protein from AAFCO would then result in higher DIAAS-like values of the test protein. This was shown in the results of the present study.

Several recent studies examined the protein quality of alternative protein sources for pet foods using DIAAS-like values referencing AAFCO and NRC for adult dogs at maintenance (Oba et al., 2019; Do et al., 2020; Reilly et al., 2020a; 2020b). Pulses of different sources had 30.5 to 64.8% of DIAAS-like values according to AAFCO or 17.1% to 36.2% according to NRC (Reilly et al., 2020a). The DIAAS-like values of soy protein concentrate were 74.1% and 41.4%, referencing AAFCO and NRC respectively (Reilly et al., 2020b). In the same study, the DIAAS-like values of dried yeast were 99.9% and 55.9%, referencing AAFCO and NRC, respectively (Reilly et al., 2020b). Black soldier fly larvae, depending on life stage, had DIAAS-like values ranging from 72.7% to 92.8% or 40.4% to 51.6% using either AAFCO or NRC as reference protein, respectively (Do et al., 2020). The study from Oba et al. (2019) showed above 100% of DIAAS-like values for retorted, steamed, and raw chicken while CM had a 79.2% value with tryptophan being the first-limiting amino acid when referencing AAFCO. On the other hand, when using NRC as the reference, all chicken-based proteins had lower than 75% of DIAAS-like values (ranging from 43.7% to 70.0%) with methionine as the first-limiting amino acid (Oba et al., 2019). Comparing the results of the current study with the literature, CM was comparable to the dried yeast, black soldier fly larvae, and CM in previous studies when considering individual amino acids (Oba et al., 2019; Do et al., 2020; Reilly et al., 2020b). Both protein hydrolysates (CLH and CH) in the present study had higher DIAAS-like values than CM and were comparable to retorted, steamed, and raw chicken from Oba et al. (2019). Regarding individual amino acids, the first-limiting amino acids for CM, CLH, and CH also corresponded with findings from Oba et al. (2019). However, these studies did not include the combination of methionine + cysteine and phenylalanine + tyrosine. In the present study, these combination of amino acids were the most limiting and, therefore, could be something to consider when determining protein quality of ingredients. The use of hydrolyzed chicken liver and heart could also be a method of upcycling chicken byproducts that manufacturers tend to avoid putting on labels for tag appeal. Therefore, protein hydrolysates would not only serve as high-quality proteins, but also result in greater market value for byproducts to be incorporated into pet foods with appealing ingredient labels.

Since the AAFCO nutrient profile already puts digestibility into consideration, commercial diets have been formulated according to the chemical composition of the ingredients (including both the indigestible and digestible portions) and not the digestible nutrient concentrations. In the current study, as designed, all test diets met or exceeded the AAFCO nutrient profile for adult dogs at maintenance. Moreover, if only considering the digestible amino acids of the diets calculated from the standardized amino acid digestibility, the digestible amino acid concentrations still exceeded the AAFCO nutrient profile. This indicated that the experimental diets were of good protein quality that can meet the animals’ requirements even with only the digestible amino acid portion.

In conclusion, both test protein hydrolysate ingredients were highly digestible, with comparable protein quality of minimally processed chicken-based ingredients reported in the literature, and of better protein quality than CM. Hydrolyzed proteins from chicken liver and heart or mechanically separated chicken can be effectively incorporated into canine extruded diets as the main protein source to develop high-quality protein diets.

Glossary

Abbreviations:

AAFCO

Association of American Feed Control Officials

AHF

acid hydrolyzed fat

CP

crude protein

DIAAS

Digestible Indispensable Amino Acid Score

DM

dry matter

DMB

dry matter basis

GE

gross energy

NRC

National Research Council

OM

organic matter

TDF

total dietary fiber

Conflict of Interest Statement

CH, PLU, CMP, and MRCG have no conflict of interest to declare. FM and RG are employed by Kemin Industries, Inc.