Nutrient profile, amino acid digestibility, true metabolizable energy, and indispensable amino acid scoring of whole hemp seeds for use in canine and feline diets

Abstract

The use of various hemp-derived products has been rapidly growing in the human nutrition industry and has sparked great interest in using these ingredients for companion animals as well. Thorough research is needed to determine the ingredient and safety standards required for AAFCO approval of hemp ingredients. In order to be effectively incorporated into pet food products, we must determine the nutrient content, quality, and utility of these ingredients in pet species. The objective of this study was to evaluate the nutrient composition of seeds from four different varieties of hemp, NWG 452, NWG 331, NWG 2730, X-59, and determine protein quality and true metabolizable energy using a cecectomized rooster model. The seeds were similar in macronutrient composition, with small variations in acid hydrolyzed fat, crude protein, total dietary fiber and gross energy content, as well as amino acid and long-chain fatty acid profiles. All essential amino acids were present in concentrations that exceeded the NRC (2006) recommended allowances for adult dogs and cats at maintenance with the exception of tryptophan. The long-chain fatty acid profile presented a favorable ratio of omega-6 to omega-3 fatty acids of close to 3.5:1. The results of the cecectomized rooster assay indicated no significant difference in the standardized amino acid digestibility of the indispensable amino acids among the seed varieties (P > 0.05). A significant difference in the true metabolizable energy corrected for nitrogen was observed among the seeds (P < 0.05), following the pattern of higher acid hydrolyzed fat and lower total dietary fiber content resulting in higher metabolizable energy. An adapted calculation of digestible indispensable amino acid score was made to determine protein quality of the hemp seeds using AAFCO nutrient profiles and NRC recommended allowances for adult dogs and cats at maintenance as reference points. The resulting scores determined tryptophan to be the first limiting amino acid and indicate that hemp seeds alone do not meet all the amino acid requirements for adult dogs and cats at maintenance, and would need a complimentary protein source for practical use in companion animal diets. The data from this study suggest that hemp seeds may provide a beneficial source of fat, protein, and dietary fiber, with consideration to differences in nutrient profile among seed varieties. However, further investigation in vivo is needed to determine the safety and efficacy of utilizing hemp in the diets of both canines and felines.

Macronutrient analysis and assessment of protein quality indicate that hemp seeds may provide a beneficial source of fat, protein, and dietary fiber to the pet food industry, with consideration to differences in nutrient profile among seed varieties.

Introduction

Hemp is a cultivar of the plant species, Cannibas sativa, which has been gaining interest since the enactment of the 2014 farm bill that more clearly defined this commodity and removed it from the list of Schedule I controlled substances under the Controlled Substances Act. The 2018 farm bill further distinguished hemp from marijuana, a different cultivar of Cannabis that contains higher levels of psychoactive compounds and remains a controlled substance under federal law. According to the Agriculture Improvement Act of 2018, in order to be considered hemp by definition, the Cannibas sativa plant, and any part of it, must not contain more than 0.3% ∆-9 tetrahydrocannabinol on a dry weight basis. Since this regulatory change, there has been an increase in the production of hemp-derived products for humans such as flours, oils, protein concentrates, and supplements including hemp extracts (Leonard et al., 2020). Different seed fractions and processing methods have been developed to maximize target nutrients such protein, fat, or dietary fiber, which comprise similar portions of whole hemp seeds (Leonard et al., 2020). However, hemp and products derived from it, have not been defined as ingredients by the Association of American feed Control Officials (AAFCO) and are not currently permissible as an ingredient in food products for companion animals according to the Food and Drug Administration.

The digestibility of protein-rich hemp-derived products have been evaluated in other species such as swine and rats that are commonly used to estimate protein quality and utility as a model for human nutrition. Standardized ileal digestibility of hemp seed cake, a byproduct that is produced after the majority of the oil fraction is expelled via cold-pressing, was greater than 80% for crude protein (CP) and all essential amino acids in pigs (Presto et al., 2011). A similar product, hempseed meal, was evaluated using a rat model along with whole and dehulled hemp seeds. All hemp products exhibited high-protein digestibility in rats ranging from 84% to 91% (House et al., 2010). Studies such as these aided in the FDA’s decision to grant “generally recognized as safe” status for hulled hemp seed, hemp seed protein powder, and hemp seed oil with the intended use as an ingredient in human food products.

The current literature lacks thorough research that is needed to determine the ingredient and safety standards required for AAFCO approval or “generally recognized as safe” status of hemp ingredients for companion animal species. In order to be effectively incorporated into pet food products, we must determine the nutrient content, quality, and utility of these ingredients in pet species, as well as potential nutritional discrepancies among different varieties of hemp that may be used as feed ingredients. The objective of this study was to evaluate the nutrient composition of seeds from four different varieties of hemp, and determine the protein quality using a cecectomized rooster model. It was hypothesized that small compositional differences would be present among the evaluated hemp seed varieties, with amino acids being well-digested in the cecectomized rooster model.

Materials and Methods

All protocols were approved by the Institutional Animal Care and Use Committee at the University of Illinois at Urbana-Champaign, and were in accordance with the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Sample preparation and chemical analysis

Whole seed samples from four varieties of industrial hemp were obtained from the Department of Crop Sciences at the University of Illinois at Urbana-Champaign. Three of the tested varieties, NWG 452, NWG 331, and NWG 2730, were produced by New West Genetics (Fort Collins, CO), and one variety, X-59, was produced by Legacy Hemp, LLC (Prescott, WI). Whole seeds were ground to 2 mm particle size using a Wiley mill (model 4; Thomas Scientific, Swedesboro, NJ) for analysis. All chemical composition analyses were completed in duplicate. Dry matter (DM), organic matter (OM), and ash content of the seed samples were analyzed according to AOAC (2006; methods 934.01 and 942.05). The CP content of the seeds was analyzed using LECO (TruMac N, Leco Corporation, St. Joseph, MI), and AOAC method 992.15 (2006) was used to determine total nitrogen content. The fat content of the seeds was determined by measuring acid hydrolyzed fat (AHF) according to AACC (1983) and Budde (1952). Bomb calorimetry (Model 6200, Parr Instruments Co., Moline, IL) was used to determine gross energy. Total dietary fiber (TDF) content was evaluated according to Prosky et al. (1992), and soluble and insoluble fiber fractions were determined according to AOAC (2006; Methods 985.29 and 991.43). Dispensable and indispensable amino acid profile of the seeds was evaluated according to AOAC (2006; 982.30 and 988.15).

Long-chain fatty acid (LCFA) profile of the seeds was analyzed using gas chromatography with methods modified from Lepage and Roy (1986) and Masood et al. (2005). Nonadecanoic acid (19:0; Nuchek Prep, Elysian, MN) was used as the internal standard as well as external fatty acid methyl ester standards (Supelco 37 Component FAME Mix, Sigma-Aldrich, St. Louis, MO). The internal standard was dissolved at a concentration of 1 mg/mL in a solution of BHT and methanol (50 µg BHT/mL methanol) before being added to the seed samples in duplicate. A mixture of methanol-hexane (4:1, v/v) was then added before mixing and cooling the samples over ice. Acetyl chloride was added drop-wise and samples were capped under nitrogen gas. Samples were then heated to 100 °C for 1 h before being cooled over ice and neutralized using a solution of 6% Na₂CO₃. Centrifugation was used to separate the sample phases, and the organic phases were extracted and combined before being evaporated under nitrogen gas to a volume of 300 µl. This solution was analyzed for fatty acid methyl ether using a Gas Chromatograph (TRACE 1300, Thermo Scientific) and flame ionization detector. One µl of sample was injected at 250 °C and compounds were separated using a fused silica SP-2560 capillary column (100 m length, 0.25 mm diameter) with a film thickness of 0.2 µm. Helium was used as the carrier gas at a split-ratio of 100:1 and a flow rate of 20 cm/sec. The temperature was held at 140 °C for 5 min and then increased at 4 °C per min. When the temperature reached 240 °C, it was held constant for 15 min. Internal and external FAME standards were used to compare retention times and identify LCFA peaks.

Precision-fed rooster assay

Standardized amino acid digestibility (SAAD) of the hemp seeds and true metabolizable energy corrected for nitrogen retention (TMEn) were determined using a precision-fed rooster assay (Parsons, 1985). Sixteen cecectomized, single-comb White Leghorn roosters, 4 roosters per hemp seed variety, were used in this analysis. The animals were housed in a temperature-controlled environment in individual wire-bottomed cages with a 16 h light and 8 h dark cycle. After a 26 h fast, the roosters were crop intubated with 25 g of ground hemp seeds. Excreta was collected for the following 48 h before freeze-drying and grinding to homogenous particle size. The dry, ground excreta was evaluated for complete amino acid profile (AOAC, 2007). Energy content was measured using bomb calorimetry, and nitrogen content was determined using LECO, similarly to the hemp seed samples mentioned previously.

SAAD was calculated according to Sibbald (1979) with the following equation in which FAA is the total amino acids fed; EAA is the total amino acids in the excreta of roosters fed hemp seeds, and EndAA is the total amino acids in the excreta of fasted roosters to account for endogenous losses:

$${\displaystyle \begin{array}{ll}{\displaystyle \mathrm{S}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{r}\mathrm{d}\mathrm{i}\mathrm{z}\mathrm{e}\mathrm{d}\mathrm{A}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{o}}& \mathrm{A}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{D}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\left(\mathrm{\%}\right)\\ & =\frac{\mathrm{F}\mathrm{A}\mathrm{A}\text{~}\text{-}(\mathrm{E}\mathrm{A}\mathrm{A}\text{-}\mathrm{E}\mathrm{n}\mathrm{d}\mathrm{A}\mathrm{A})}{\mathrm{F}\mathrm{A}\mathrm{A}}\times 100.\end{array}}$$

The TMEn was calculated according to Parsons et al. (1982) with the following equation in which FE_fed is the gross energy (kcal) that was fed; EE represents the total energy in the excreta from both fed and fasted animals; N represents the nitrogen (g) retained by fed and fasted animals; 8.22 is used as the gross energy per g of nitrogen in uric acid; and FI is the feed intake (g) of the roosters fed the hemp seeds.

$${\displaystyle \begin{array}{ll}& \mathrm{T}\mathrm{M}\mathrm{E}\mathrm{n}\left(\mathrm{k}\mathrm{c}\mathrm{a}\mathrm{l}/\mathrm{g}\right)\\ & =\frac{\mathrm{F}{\mathrm{E}}_{\mathrm{f}\mathrm{e}\mathrm{d}}\text{-}\left(\mathrm{E}{\mathrm{E}}_{\mathrm{f}\mathrm{e}\mathrm{d}}+8.22\left({\mathrm{N}}_{\mathrm{f}\mathrm{e}\mathrm{d}}\right)\right)+\left(\mathrm{E}{\mathrm{E}}_{\mathrm{f}\mathrm{a}\mathrm{s}\mathrm{t}\mathrm{e}\mathrm{d}}+8.22\left({\mathrm{N}}_{\mathrm{f}\mathrm{a}\mathrm{s}\mathrm{t}\mathrm{e}\mathrm{d}}\right)\right)}{\mathrm{F}\mathrm{I}}.\end{array}}$$

DIAAS-like values

A modified calculation of digestible indispensable amino acid score (DIAAS-like) was evaluated using SAAD of cecectomized roosters to calculate the amount of each indispensable amino acid (mg) available in 1 g of protein from the hemp seeds. Four different reference protein (mg/g) values were evaluated, including the AAFCO and National Research Council (NRC) requirements for both adult canine and felines at maintenance to determine the amount of each indispensable amino acid (mg) present in 1 g of required protein. The DIAAS-like value was calculated according to Mathai et al. (2017) using the following equation:

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

The calculation was completed for each indispensable amino acid, and the final DIAAS-like value was determined by the first limiting amino acid. Values greater than 100% indicate that the protein meets all the amino acid requirements according to the reference.

Statistical analysis

Data was analyzed using Mixed Model procedure in SAS, version 9.4 with rooster as the random effect and treatment as the fixed effect. Data are reported as LS means with Tukey-adjusted P-values. Significance was reported at P < 0.05. Standard errors of the mean (SEM) were calculated.

Results and Discussion

Chemical composition of hemp seeds

The DM and OM content were similar across the four hemp seed varieties, ranging from 92.8% to 93.3%, and 94.5% to 94.9%, respectively (Table 1). CP and AHF content also had little variation, with only 5% unit difference among the hemp seeds (19.7% to 24.7% and 29.9% to 34.1%, respectively). As expected with similar macronutrient composition, the gross energy (GE) content (Table 1) of the hemp seeds also fell within a narrow range (5.4 to 5.6 kcal/g DM) and was similar to the energy content reported by Callaway (2004; 5.25 kcal/g). Vonapartis et al. (2015) evaluated the composition of 10 hemp seed varieties and reported a slightly higher range of CP content (23.8% to 28.0%) and slightly lower oil content (26.9% to 30.6%). Rahemi et al. (2021) similarly evaluated seven hemp seed varieties and reported similar CP (22.3% to 25.8%) and lower crude fat (19.5% to 24.9%).

Table 1.

Chemical composition of select hemp seed varieties

	Hemp seed varieties
Item	NWG 452	X-59	NWG 331	NWG 2730
Dry matter, %	93.3	92.8	93.1	92.9
	% DM basis
Organic matter	94.5	94.9	94.6	94.8
Ash	5.6	5.1	5.4	5.2
Acid hydrolyzed fat	34.1	30.6	32.1	29.9
Crude protein	22.0	24.7	19.7	21.8
Total dietary fiber	34.2	37.2	38.7	42.0
“Soluble dietary fiber”	0.0	1.1	0.0	0.0
“Insoluble dietary fiber”	34.2	36.1	38.7	42.0
Gross energy, kcal/g	5.6	5.4	5.5	5.4

More variation was observed in TDF content among the hemp seeds, ranging from 34.2% to 42.0% (Table 1). Further analysis revealed that, with the exception of X-59, the fiber fractions were comprised entirely of insoluble dietary fiber. Although soluble dietary fiber was found in X-59, the content remained low at 1.1%. Few studies have evaluated the fiber fraction of whole hemp seed. One study reported lower TDF content (27.6%) in the Finola hemp seed variety with 5.4% soluble fiber and 22.2% insoluble fiber (Callaway, 2004). However, it is unclear which analytical methods were used to evaluate these fiber fractions from this source, and could impact quantification.

The essential amino acid composition of the hemp seeds was also found to be similar among the varieties (Table 2). All essential amino acids were present in concentrations that exceeded the NRC (2006) recommended allowances for adult dogs and cats at maintenance with the exception of tryptophan. The tryptophan content of the seeds ranged from 0.11% to 0.12%, which fell below the recommended allowances for dogs and cats, 0.14% and 0.13%, respectively (NRC, 2006). For the majority of the seeds, the tryptophan content just reached the minimum requirement for adult dogs in maintenance (0.11%), and no minimum requirement has been reported for this amino acid in adult felines (NRC, 2006). Calloway (2004) and House et al. (2010) reported similar amino acid profiles among various hemp seed varieties. However, both reported higher concentrations of ­tryptophan, 0.20% and 0.24%, respectively, which exceed the recommended allowances for both adult dogs and cats at maintenance (NRC, 2006).

Table 2.

Amino acid profile of select hemp seed varieties

	Hemp seed varieties
Amino acid (% DM)	NWG 452	X-59	NWG 331	NWG 2730
“Indispensable amino acids”
Arginine	2.57	2.48	3.07	2.64
Histidine	0.58	0.58	0.67	0.61
Isoleucine	0.90	0.88	1.04	0.95
Leucine	1.46	1.40	1.71	1.50
Lysine	0.87	0.85	1.00	0.87
Methionine	0.52	0.50	0.57	0.54
Phenylalanine	1.03	0.98	1.19	1.05
Threonine	0.77	0.74	0.87	0.77
Tryptophan	0.11	0.12	0.11	0.11
Valine	1.11	1.08	1.31	1.15
Dispensable amino acids
Alanine	0.95	0.92	1.09	0.97
Aspartate	2.34	2.22	2.68	2.37
Cysteine	0.38	0.36	0.40	0.38
Glutamate	3.51	3.38	4.09	3.52
Glycine	0.97	0.97	1.12	1.00
Proline	0.87	0.82	0.99	0.93
Serine	0.96	0.93	1.07	0.97
Tyrosine	0.69	0.67	0.85	0.70

The LCFA profile (Table 3) was similar across the evaluated hemp seeds, and concentrations of individual LCFA fell within the ranges previously reported in other studies (Callaway, 2004; Vonapartis et al., 2015; Irakli et al., 2019; Xin et al., 2022). Six LCFA made up the largest portions of the total LCFA concentration, palmitic acid (6.73% to 7.75%), stearic acid (2.26% to 2.69%), oleic acid (12.77% to 13.98%), linoleic acid (LA; 53.82% to 56.55%), γ-linolenic acid (GLA; 1.83% to 5.45%), and α-linolenic acid (ALA; 14.60% to 16.84%). Small quantities of other LCFA such as arachidic, behenic, and lignoceric acids were also detected, but each represented less than 1% of the total LCFA content. Identification of ingredients that can act as rich sources of omega-3 fatty acids such as ALA and omega-6 fatty acids such as LA and GLA is important for companion animal nutrition, as dogs and cats cannot synthesize these compounds de novo. Because of this, they must be supplied in the animal’s diet in order to maintain their roles in sustaining proper immune function and skin health (Campbell, 1990).

Table 3.

Long-chain fatty acid composition of select hemp seed varieties

	Hemp seed varieties
LCFA Concentration1 (% of Total LCFA)	NWG 452	X-59	NWG 331	NWG 2730
Palmitic (C16:0)	6.99	7.75	6.73	7.30
Stearic (C18:0)	2.69	2.58	2.66	2.26
Oleic (C18:1n9c)	13.73	13.37	13.98	12.77
Linoleic (C18:2n6c)	56.55	53.82	56.40	56.45
γ-linolenic (C18:3n6)	2.18	5.45	1.83	2.01
α-linolenic (C18:3n3)	15.71	14.60	16.21	16.84

¹Other LCFA that were evaluated but not listed here accounted for less than 1% of the total LCFA content when combined.

The concentration of γ-linolenic acid had the highest variability in the LCFA profile of the hemp seeds evaluated. This is also reflected across the literature, with Xin et al. (2022) reporting γ-linolenic acid making up only 0.67% of the fatty acid composition while Callaway (2004) and Irakli et al. (2019) reported similar ranges (1.0% to 4.0% and 1.9% to 5.0%, respectively). Although the concentration of ALA was consistent across our evaluated hemp seed samples, the proportion of this LCFA in other hemp seeds across the literature was much more variable. Vonapartis et al. (2015) reported a similar proportion of ALA (14.69% to 17.27%), while Irakli et al. (2019) reported ALA at a lower concentration (10.5% to 15.3%), and Callaway (2004) reported higher values (21% to 22%).

Utilizing hemp seed varieties with higher concentrations of these essential fatty acids may provide additional functional benefits in companion animal nutrition. Research has been conducted to evaluate the potential anti-inflammatory effects of GLA and ALA, and several studies have implicated that supplementation of these LCFA in the diet may help to improve the condition of animals suffering from skin conditions such as atopic dermatitis (Campbell, 1990; Lloyd and Thomsett, 1990; Bond and Lloyd, 1992; Harvey, 1993; Abba et al., 2005; Xin et al., 2022).

In addition to total concentration, the ratio of omega-6 to omega-3 LCFA is an important factor to consider. According to the NRC (2006) guidelines, a maximum ratio of 30:1 should be used in companion animal diets. Studies have indicated that ratios closer to 10:1 to 3:1 are ideal for promoting healthy skin and immune function (Vaughn et al., 1994; Scott et al., 1997; Mooney et al., 1998; Glos et al., 2008). We observed a ratio close to 3.5:1 for all evaluated varieties, further indicating that hemp seeds have the potential to be utilized as a functional ingredient supplying essential fatty acids in diets formulated to promote skin and coat health. Although there are no established minimum requirements, there are recommendations for adequate intake of other longer-chain omega-6 and omega-3 fatty acids such as arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid that could not be supplied by the fatty acid content observed in hemp seeds (NRC, 2006). Complementary fat sources supplying these would be necessary to formulate a complete and balanced diet, especially for cats.

The current data suggest that there are measurable variations in macronutrient composition among different hemp seed varieties. However, differences in analytical methods may also lead to variation in reported values across the literature. Similarly to other plant-derived products, environmental conditions and management practices can impact overall nutritional quality (Rahemi et al., 2021). In addition to these factors, research also suggests that the genotypic variation among hemp seed varieties has a strong influence on nutrient profiles (Irakli et al., 2019).

Precision-fed Rooster Assay

A precision-fed rooster assay using cecectomized animals was completed to determine the SAAD of the hemp seeds (Table 4). In a study by Johnson et al. (1998), this method was determined to be a comparable model to evaluating in vivo digestibility from ileal-cannulated dogs. However, no direct comparison has been established for modeling digestibility in felines. No significant difference in the SAAD of the indispensable amino acids was observed among the evaluated hemp seeds. Additionally, almost all indispensable amino acids had a SAAD greater than 80%, indicating that they were well-digested by the animal. The SAAD of lysine fell just below this threshold for NWG 2730 (79.0%), but exceeded 80% digestibility in the rest of the samples (80.9% to 85.4%). Tryptophan had the lowest digestibility across all varieties and ranged from 77.54% to 80.78%. This value is important to note as tryptophan is a common limiting nutrient in pet food. No differences were observed among the SAAD of dispensable amino acids (P > 0.05; data not shown).

Table 4.

Standardized amino acid digestibility of select hemp seed varieties determined by precision-fed rooster assay

	Hemp seed varieties
Standardized digestibility (%DM basis)	NWG 452	X-59	NWG 331	NWG 2730
					SEM	P-value
“Indispensable amino acids”
Arginine	95.1	96.6	95.6	94.2	0.61	0.09
Histidine	87.7	90.2	87.1	86.8	1.52	0.39
Isoleucine	87.4	90.3	88.5	87.2	1.39	0.42
Leucine	85.6	89.8	86.8	85.5	1.60	0.25
Lysine	80.9	85.4	82.7	79.0	1.90	0.16
Methionine	90.0	92.5	90.9	90.6	1.01	0.39
Phenylalanine	85.2	89.5	85.6	84.5	1.68	0.21
Threonine	81.5	86.6	87.1	83.0	2.04	0.20
Tryptophan	77.5	78.8	80.8	77.9	2.16	0.72
Valine	83.0	86.7	85.6	81.7	1.86	0.26

N = 4 roosters per treatment.

The same model was also used to evaluate TMEn of the hemp seeds (Figure 1). The calculations described by Parsons et al. (1982) take into account the endogenous losses of energy and nitrogen from fasted roosters in order to accurately evaluate true metabolizable energy. The TMEn values for NWG 452 and X-59, 4.56 and 4.46 kcal/g, respectively, were significantly higher (P < 0.05) than NWG 2730 (4.01 kcal/g). The differences in TMEn among the hemp seeds can likely be explained by the macronutrient composition. The highest TMEn was observed in NWG 452, which also had the highest AHF content (34.1%) and lowest TDF content (34.2%). The lowest TMEn value was observed in NWG 2730, which had the lowest AHF content (29.9%) and highest TDF content (42.0%).

Figure 1.

True metabolizable energy corrected for nitrogen (TMEn) of select hemp seed varieties. ^a,b,cSuperscripts with different letters in a row represent statistical differences (P < 0.05).

DIAAS-like values

In human nutrition, DIAAS calculations have been utilized to help determine protein quality using SAAD from an ileal-cannulated swine model and reference requirements for 2 to 5 yr old children (Marinangeli and House, 2017). A modified version of this calculation, DIAAS-like value, has recently been utilized in several studies as a tool to evaluate the quality of various protein ingredients for use in pet foods (Oba et al., 2019; Reilly et al. 2019, 2021; Do et al., 2020). The DIAAS-like values were calculated using both AAFCO nutrient profiles and NRC recommended allowances for adult dogs (Tables 5 and 6) and cats (Tables 7 and 8). It is important to note when interpreting these results that AAFCO nutrient profiles and NRC recommended allowances as references are not based solely on the animals’ physiological requirements, but are adjusted values that include a safety margin for nutrients with uncertain bioavailability (NRC, 2006). Calculation of DIAAS-like values using minimum requirement or recommended allowance values will result in slightly different scores, however, recommended allowance values were chosen in this study, for a few reasons: 1) they are used as reference values to generate nutrient guidelines by AAFCO, 2) commercial pet foods are not formulated based on minimum nutrient requirements, 3) there is a lack of established minimum requirement values for several indispensable amino acids for adult cats, except for methionine, methionine + cystine, lysine, and taurine (NRC, 2006). Herein, DIAAS-like values are provided for all indispensable amino acids, however the lowest score indicates the first limiting amino acid and determines the DIAAS-like score of a protein source. Across all the hemp seeds and references, tryptophan was the limiting amino acid. Because values greater than 100% indicate that the maintenance requirement was fully met, this discussion will focus on the individual amino acids that fell below this threshold.

Table 5.

Digestible indispensable amino acid score (DIAAS)-like value¹ for select hemp seed varieties compared to AAFCO recommended values for adult dogs at maintenance

	Hemp seed varieties
Indispensable amino acid (%, DMB)	NWG 452	X-59	NWG 331	NWG 2730	SEM	P-value
Arginine	401.9b	422.7a	423.1a	390.5c	2.58	<0.001
Histidine	228.2a,b	229.5a,b	240.4a	215.8b	3.97	0.008
Isoleucine	179.0a,b	180.3a,b	187.2a	170.7b	2.76	0.009
Leucine	154.9a,b	164.8a	163.3a,b	151.5b	2.89	0.018
Lysine	91.3b	98.6a,b	101.7a	89.8b	2.17	0.006
Methionine	120.0a,b	115.9b	125.1a	117.2b	1.32	0.002
Phenylalanine	162.3	171.9	169.6	159.1	3.20	0. 063
Threonine	107.1b	114.4a,b	122.7a	110.0b	2.70	0.008
Tryptophan	43.7b	39.5b	55.4a	44.2b	1.20	<0.001
Valine	159.1a,b	168.5a	171.7a	152.3b	3.53	0.008

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

N = 4 roosters per treatment.

^a,b,cSuperscripts with different letters in a row represent statistical differences (P < 0.05).

Table 6.

Digestible indispensable amino acid score (DIAAS)-like value¹ for select hemp seed varieties compared to NRC recommended allowances² for adult dogs at maintenance

	Hemp seed varieties
Indispensable Amino Acid (%, DMB)	NWG 452	X-59	NWG 331	NWG 2730	SEM	P-value
Arginine	326.6b	343.5a	343.8a	317.3c	2.10	<0.001
Histidine	128.2a,b	128.9a,b	135.0a	121.2b	2.23	0.008
Isoleucine	99.4a,b	100.1a,b	104.0a	94.8b	1.53	0.009
Leucine	86.0a,b	91.5a	90.7a,b	84.2b	1.60	0.018
Lysine	91.6b	98.9a,b	102.0a	90.1b	2.18	0.006
Methionine	67.1a,b	64.8b	69.9a	65.5b	0.74	0.002
Phenylalanine	90.5	95.9	94.6	88.8	1.78	0.063
Threonine	66.5b	71.0a,b	76.1a	68.2b	1.68	0.008
Tryptophan	27.7b	25.1b	35.2a	28.1b	0.76	<0.001
Valine	88.7a,b	94.0a	95.8a	85.0b	1.97	0.008

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

N = 4 roosters per treatment.

²The relationship between individual AA and CP is consistent between minimum requirements and recommended allowances, therefore the DIAAS-like values are the same regardless of reference value.

^a,b,cSuperscripts with different letters in a row represent statistical differences (P < 0.05).

Table 7.

Digestible indispensable amino acid score (DIAAS)-like value¹ for select hemp seed varieties compared to AAFCO recommended values for adult cats at maintenance

	Hemp seed varieties
Indispensable amino acid (%, DMB)	NWG 452	X-59	NWG 331	NWG 2730	SEM	P-value
Arginine	285.7b	300.5a	300.8a	277.6c	1.84	<0.001
Histidine	202.9a,b	204.0a,b	213.8a	191.9b	3.53	0.008
Isoleucine	188.9a,b	190.3a,b	197.6a	180.2b	2.91	0.009
Leucine	122.7a,b	130.5a	129.3a,b	120.1b	2.29	0.018
Lysine	100.2b	108.2a,b	111.6a	98.6b	2.38	0.006
Methionine	287.8a,b	277.9b	300.0a	281.1b	3.16	0.002
Phenylalanine	252.3	267.3	263.7	247.3	4.97	0.063
Threonine	101.5b	108.4a,b	116.2a	104.2b	2.56	<0.001
Tryptophan	63.2b	57.2b	80.0a	63.9b	1.74	<0.001
Valine	182.3a,b	193.1a	196.8a	174.5b	4.05	0.008

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

N = 4 roosters per treatment.

^a,b,cSuperscripts with different letters in a row represent statistical differences (P < 0.05).

Table 8.

Digestible indispensable amino acid score (DIAAS)-like value¹ for select hemp seed varieties compared to NRC recommended allowances² for adult cats at maintenance

	Hemp seed varieties
Indispensable amino acid (%, DMB)	NWG 452	X-59	NWG 331	NWG 2730	SEM	P-value
Arginine	296.9b	312.2a	312.5a	288.4c	1.91	<0.001
Histidine	187.3a,b	188.3a,b	197.3a	177.2b	3.26	0.008
Isoleucine	175.8a,b	177.0a,b	183.8a	167.6b	2.71	0.009
Leucine	114.7a.b	122.1a	120.9a,b	112.3b	2.13	0.018
Lysine	188.6b	203.6a,b	210.0a	185.5b	4.49	0.006
Methionine	260.4a,b	251.4b	271.4a	254.4b	2.86	0.002
Phenylalanine	203.7	215.8	212.9	199.7	4.02	0.063
Threonine	109.9b	117.4a,b	125.8a	112.8b	2.77	0.008
Tryptophan	59.8b	54.1b	75.7a	60.5b	1.64	<0.001
Valine	170.5a,b	180.6a	184.1a	163.2b	3.79	0.008

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

N = 4 roosters per treatment.

²The relationship between individual AA and CP is consistent between minimum requirements and recommended allowances, therefore the DIAAS-like values are the same regardless of reference value.

^a,b,cSuperscripts with different letters in a row represent statistical differences (P < 0.05).

Lysine and tryptophan were the only amino acids with DIAAS-like values below 100% for dogs compared to AAFCO (2018) nutrient profiles. The value for lysine only reached this threshold for NWG 331 (101.7%), which was significantly higher (P < 0.05) than NWG 452 and NWG 2730 (91.3% and 89.8%, respectively). All hemp seeds were below this threshold for tryptophan, with NWG 331 (55.4%) having a higher value (P < 0.05) than all others (39.5% to 44.2%). When comparing these scores to the NRC (2006) recommended allowances for dogs, the majority of the individual amino acid scores fall below 100% due to the lower overall protein requirement from this reference. Tryptophan remained the lowest scoring amino acid, with NWG 331 (35.2%) having a higher value (P < 0.05) than all others (25.1% to 28.1%).

The DIAAS-like values for adult cats compared to AAFCO nutrient profiles indicate that the hemp seeds meet maintenance requirements for all amino acids with the exception of lysine in NWG 2730 (98.6%) and tryptophan in all seeds. The value for tryptophan was significantly higher (P < 0.05) in NWG 331 (80.0%) compared to all others (57.2% to 63.2%). Similar trends were observed when comparing these values to NRC (2006) recommended allowances for cats. Only tryptophan fell below the maintenance requirement with NWG 331 (75.7%) having a significantly higher (P < 0.05) value than all others (54.1% to 60.5%).

Based on these values, none of the hemp seeds meet all the requirements for adult dogs and cats at maintenance. This indicates that hemp seeds would not be adequate as a single protein source in companion animal diets. However, in combination with other complementary protein sources, specifically ingredients higher in tryptophan and lysine, hemp seeds could provide a beneficial source of several indispensable amino acids.

Implications

The data from this study suggest that hemp seeds may provide a beneficial source of fat, protein, and insoluble dietary fiber when included in companion animal diets. The LCFA profile of the hemp seeds can promote an ideal ratio of omega-6 to omega-3 fatty acids, which could be of particular benefit to formulations promoting skin and coat health. The SAAD and DIAAS-like values indicate that hemp seeds have moderate protein quality, and could easily be combined with other protein sources to eliminate potentially limiting nutrients. Variations in nutrient profile were observed across the evaluated varieties, and must be considered when utilizing this ingredient in formulation for companion animals. Future in vivo research is needed in both canine and feline species to determine the safety and efficacy of including hemp seeds in the diets of these animals.

Glossary

Abbreviations

AHF

acid-hydrolyzed fat

ALA

α-linolenic acid

CP

crude protein

DM

dry matter

DIAAS-like

digestible indispensable amino acid score-like

GLA

γ-linolenic acid

LA

linoleic acid

LCFA

long-chain fatty acid

OM

organic matter

SAAD

standardized amino acid digestibility

SEM

standard error of the mean

TDF

total dietary fiber

TMEn

true metabolizable energy corrected for nitrogen

Conflict of Interest Statement

The authors declare no real or perceived conflicts of interest.