Standardized amino acid digestibility and nitrogen-corrected true metabolizable energy of frozen raw, freeze-dried raw, fresh, and extruded dog foods using precision-fed cecectomized and conventional rooster assays

Abstract

Processing conditions, particularly temperature and duration of heating, impact pet food digestibility. Various commercial pet food formats are now available, but few have been tested thoroughly. The objective of this study was to determine the amino acid (AA) digestibilities and nitrogen-corrected true metabolizable energy (TME_n) values of frozen raw, freeze-dried raw, fresh (mildly cooked), and extruded dog foods using the precision-fed cecectomized and conventional rooster assays. The diets tested were Chicken and Barley Recipe [Hill’s Science Diet, extruded diet (EXT)], Chicken and White Rice Recipe [Just Food for Dogs, fresh diet (FRSH)], Chicken Formula [Primal Pet Foods, frozen raw diet (FRZN)], Chicken and Sorghum Hybrid Freeze-dried Formula [Primal Pet Foods, hybrid freeze-dried raw diet (HFD)], and Chicken Dinner Patties [Stella & Chewy’s, freeze-dried raw diet (FD)]. Two precision-fed rooster assays utilizing Single Comb White Leghorn roosters were conducted. Cecectomized roosters (n = 4/treatment) and conventional roosters (n = 4/treatment) were used to determine standardized AA digestibilities and TME_n, respectively. All roosters were crop intubated with 12 g of test diet and 12 g of corn, with excreta collected for 48 h. In general, FD had the highest, while EXT had the lowest AA digestibilities; however, all diets performed relatively well and few differences in AA digestibility were detected among the diets. Lysine digestibility was higher (P < 0.05) in FD and FRZN than EXT, with other diets being intermediate. Threonine digestibility was higher (P < 0.05) in FD than EXT, with other diets being intermediate. Digestibilities of the other indispensable AA were not different among diets. The reactive lysine:total lysine ratios were 0.94, 0.96, 0.93, 0.93, and 0.95 for EXT, FRSH, FRZN, HFD, and FD, respectively. TME_n was higher (P < 0.05) in FRZN than FD, FRSH, and EXT, higher (P < 0.05) in HFD than FRSH and EXT, and higher (P < 0.05) in FD than EXT. In conclusion, our results support the notion that AA digestibilities are affected by diet processing, with FD, HFD, FRZN, and FRSH diets having higher AA digestibility coefficients and greater TME_n values, than the EXT diet; however, other factors such as ingredient inclusion and macronutrient composition may also have affected these results. More research in dogs is necessary to test the effects of format on diet palatability, digestibility, stool quality, and other physiologically relevant outcomes.

The current study expanded the research on different dietary formats of dog food, particularly raw formulations. It demonstrated that diets that have not been processed with heat, such as freeze-dried raw diets, have higher amino acid digestibilities than diets that undergo heat processing methods such as extruded diets.

Introduction

The list of alternatives to extruded and retorted pet foods continues to grow rapidly, with a variety of differently processed formats now available. Freeze-dried, dehydrated, and frozen dog foods only comprised 1.65%, 0.83%, and 1.43% of the market share in 2022, respectively, but are rapidly growing segments of the market, growth that is supported by an increasing demand for raw and minimally processed foods (Atlas Stackline, 2022; Nielsen IQ Byzzer, 2023). Raw pet food sales grew by 14.14% from 2021 to 2022 and comprised 3.91% of the market in 2022 (Atlas Stackline, 2022; Nielsen IQ Byzzer, 2023). Although these diet formats do not make up a major segment of the market, they are an important sector of the market to examine considering the rapid growth and popularity among consumers.

Many of the potential benefits, but also some risks, of raw foods are derived from the lack of heat processing. Because high-heat processing, such as temperature in excess of 120 °C, can reduce bioavailability of vitamins and denature proteins, manufacturing foods without heat treatment can minimize the processing loss of more volatile nutrients (Hendriks et al., 1999; Craig, 2019). Furthermore, heat treatment can cause the Maillard reaction, which reduces amino acid (AA) availability, especially lysine, and can cause the formation of advanced glycation end-products. Such compounds have been linked with disease in humans (Nowotny et al., 2018) and may contribute to health concerns in dogs. Because the concentrations of some advanced glycation end-products have been shown to be higher in pet diets than human diets, it is an area of active research (van Rooijen et al., 2014; Oba et al., 2022).

While processing using high temperatures, moisture content, and/or pressure can reduce nutrient digestibility by denaturing proteins, mild heat treatment may increase digestibility by breaking down collagen and allowing enzymes greater access to digest chemical linkages. Mildly cooked foods are heated at relatively low temperatures (75 to 95 °C) for short periods of time, enough to kill potential pathogens without a detrimental loss in digestibility or nutrients (Kerr et al., 2012; Algya et al., 2018; Oba et al., 2019; Do et al., 2021; Roberts et al., 2023). However, extruded foods, and particularly those that contain rendered protein meals, may be subjected to too much heat processing so that digestibility is reduced and harmful compounds from the Maillard reaction result. Processing with heat can also improve the digestibility of plant-based ingredients, by denaturing anti-nutritional factors, such as trypsin inhibitors, or inducing starch gelatinization; however, many commercial raw foods have low relative quantities of plant-based ingredients, so these factors would be less of a consideration.

Apparent total tract digestibility measurement overestimates AA digestibilities due to microbial fermentation of proteins in the hindgut (Holmes et al., 1974; Parsons et al., 1982b). To minimize the influence of gut microbiota, ileal–cannulated animals are typically used because they provide a more accurate assessment of AA digestibility (Hendriks et al., 2013). The precision-fed cecectomized rooster is a model for ileal-cannulated dogs and AA digestibilities between the two have been shown to be highly correlated (r = 0.87 to 0.92) (Johnson et al., 1998). Because the cecectomized rooster model also minimizes the effects of microbial fermentation, it has been used to obtain more accurate conclusions about the AA digestibilities of individual ingredients (Deng et al., 2016; Oba et al., 2019; Do et al., 2020; Smola et al., 2023), complete pet diets (Kerr et al., 2013; Oba et al., 2020, 2023; Roberts et al., 2023), and whole prey (Kerr et al., 2014) in recent years.

The nutrient digestibility of a diet also impacts its metabolizable energy (ME) content. Metabolizable energy may be estimated using various equations. The human food industry generally uses Atwater factors to estimate ME, assuming 4 kcal/g for proteins and digestible carbohydrates and 9 kcal/g for fats (Atwater, 1902). The Association of American Feed Control Officials (AAFCO) uses modified Atwater values, assuming 3.5 kcal/g for proteins and digestible carbohydrates and 8.5 kcal/g for fats (AAFCO, 2023). These lower, modified values are used to estimate the ME values present on pet food labels. The National Research Council (NRC, 2006) developed equations to more accurately estimate the ME of pet foods, but recent evidence suggests these NRC equations still underestimate the ME content of most premium pet foods (Oba et al., 2020, 2023; Roberts et al., 2023).

Given the minimal studies that have tested fresh, frozen raw, and freeze-dried raw dog foods, the objectives of this experiment were to determine the AA digestibilities and TME_n values of frozen raw, freeze-dried raw, mildly cooked/fresh, and extruded dog diets. We hypothesized that the raw diets would have greater AA digestibilities than the mildly cooked diet, which would be higher than the extruded diet due to the differing levels of heat processing. We also hypothesized that the TME_n would be highest for the frozen diet, followed by the freeze-dried diets, and then the mildly cooked and lastly extruded diets, due to the macronutrient composition, specifically the fat concentrations. Lastly, we hypothesized that the TME_n values determined would be higher than the ME estimates using modified and traditional Atwater factors.

Materials and Methods

All animal care procedures were approved by the University of Illinois Urbana-Champaign Institutional Animal Care and Use Committee prior to the start of the experiment.

Diets

An extruded kibble diet [Hill’s Science Diet Adult Chicken & Barley Recipe; Hills’s Pet Nutrition, Topeka, KS (EXT)], a human-grade mildly cooked/fresh diet [Chicken & White Rice Recipe; Just Food For Dogs, Irvine, CA (FRSH)], a frozen raw diet [Primal Frozen Nuggets Chicken Formula; Primal Pet Foods, Fairfield, CA (FRZN)], a hybrid freeze-dried raw diet [Primal Hybrid Freeze-Dried Chicken & Sorghum Formula; Primal Pet Foods, Fairfield, CA (HFD)], and a freeze-dried raw diet [Chicken Dinner Patties; Stella & Chewy’s, Milwaukee, WI (FD)] were tested in this study. The EXT diet was produced via extrusion, but the specific parameters were unknown. The FRSH diet was cooked in skillets with a target temperature between 76 and 82 °C for 20 to 40 min and frozen until thawed to feed. The FRZN diet’s raw ingredients were mixed, formed into nugget shapes, and then frozen at −32 °C until thawed to feed. To process the HFD diet, 35% of the included chicken was steamed at 71 °C for 10 min, while the rest of the chicken remained raw. The sorghum was cooked at a sufficient temperature and time combination to achieve starch gelatinization over 90%. More specifically, sorghum undergoes preconditioning at temperatures up to 176 °C for 1 to 3 min, followed by steam exposure at pressures of 120 to 220 psi and temperatures of 260 to 315 °C for 1 to 5 min. The steamed fraction of the chicken and the cooked sorghum were mixed with the raw ingredients, shaped, and frozen at −32 °C for 8 h. The diet was then placed under vacuum while the temperature was raised to −23 °C for 3 h. To ensure the product was fully dried, the temperature was increased slowly to reach an internal temperature of 48 °C over 5 h. The FD diet was produced by freeze-drying raw ingredients, but the specific conditions were unknown. All diets were formulated to meet/exceed AAFCO (2023) nutrient profiles for adult dogs at maintenance (Table 1).

Table 1.

Analyzed chemical analysis and ingredients of dog diets tested

Item	EXT1	FRSH2	FRZN3	HFD4	FD5
Dry matter, %	92.89	30.82	22.69	95.43	94.76
	DM basis
Protein, %	24.61	31.06	46.70	35.17	56.75
Acid-hydrolyzed fat, %	15.41	15.30	33.83	30.51	30.71
Total dietary fiber, %	9.58	2.21	3.48	3.88	1.90
Ash, %	5.21	6.51	7.40	9.23	10.13
Nitrogen-free extract6, %	45.19	44.92	8.59	21.21	0.51
Gross energy, kcal/kg	4.97	5.01	6.40	5.76	6.01
Metabolizable energy7, kcal/g	4.18	4.42	5.26	5.00	5.05
Metabolizable energy8, kcal/g	3.75	3.96	4.81	4.57	4.61
Reactive lysine: total lysine ratio	0.94	0.96	0.93	0.93	0.95

¹Extruded diet (EXT); Chicken and Barley Recipe (Hill’s Pet Nutrition, Topeka, KS); ingredients: chicken, whole grain wheat, cracked pearled barley, whole grain sorghum, whole grain corn, corn gluten meal, chicken meal, chicken fat, chicken liver flavor, dried beet pulp, soybean oil, pork flavor, lactic acid, flaxseed, potassium chloride, choline chloride, iodized salt, calcium carbonate, vitamins (vitamin E supplement, L-ascorbyl-2-polyphosphate (source of vitamin C), niacin supplement, thiamine mononitrate, vitamin A supplement, calcium pantothenate, riboflavin supplement, biotin, vitamin B12 supplement, pyridoxine hydrochloride, folic acid, vitamin D3 supplement), minerals (ferrous sulphate, zinc oxide, copper sulphate, manganous oxide, calcium iodate, sodium selenite), taurine, oat fibre, mixed tocopherols for freshness, natural flavors, beta-carotene, apples, broccoli, carrots, cranberries, green peas.

²Fresh diet (FRSH); Chicken and White Rice Recipe (Just Food for Dogs, Irvine, CA); ingredients: chicken thigh, long grain white rice, spinach, carrots, apples, chicken gizzard, chicken liver, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), dicalcium phosphate dihydrate, calcium, sodium chloride, choline bitartrate, dried seaweed meal, zinc oxide, magnesium amino acid chelate, vitamin E (as a-tocopherol succinate), ferrous amino acid chelate, copper amino acid chelate, vitamin D3 (as cholecalciferol), vitamin B5 (as calcium d-pantothenate), riboflavin, vitamin B12 (as cyanocobalamin).

³Raw frozen diet (FRZN); Chicken Formula (Primal Pet Group, Fairfield, CA); ingredients: chicken (with ground bone), chicken livers, organic carrots, organic squash, organic kale, organic apples, organic pumpkin seeds, organic sunflower seeds, organic broccoli, organic blueberries, organic cranberries, organic parsley, organic apple cider vinegar, montmorillonite clay, fish oil, organic quinoa, organic coconut oil, vitamin E supplement, organic ground alfalfa, dried organic kelp, zinc sulfate, liquid Lactobacillus acidophilus fermentation product, liquid Lactobacillus casei fermentation product, liquid Lactobacillus reuteri fermentation product, liquid Bifidobacterium animalis fermentation product.

⁴Hybrid freeze-dried raw diet (HFD); Chicken and Sorghum Hybrid Freeze-Dried Formula (Primal Pet Group, Fairfield, CA); ingredients: chicken, sorghum, chicken livers, chicken fat, inulin, apple pomace, dicalcium phosphate, potassium chloride, salt, salmon oil, choline chloride, vitamin and mineral premix (sodium chloride, dl-alpha-tocopheryl acetate, zinc sulphate, ferrous sulphate, biotin, retinol palmitate, manganese sulphate, niacin, d-calcium pantothenate, cholecalciferol, sodium selenite, copper sulphate anhydrous, thiamine mononitrate, riboflavin, cyanocobalamin, pyridoxine hydrochloride, potassium iodide, folic acid), vegetable oil, liquid Lactobacillus acidophilus fermentation product, liquid Lactobacillus casei fermentation product, liquid Lactobacillus reuteri fermentation product, liquid Bifidobacterium animalis fermentation product, rosemary extract.

⁵Freeze-dried diet (FD); Chicken Dinner Patties (Stella & Chewy’s, Milwaukee, WI); ingredients: chicken with ground bone, chicken liver, chicken gizzard, pumpkin seed, organic cranberries, organic spinach, organic broccoli, organic beets, organic carrots, organic squash, organic blueberries, fenugreek seed, potassium chloride, dried kelp, sodium phosphate, tocopherols (preservative), choline chloride, dried Pediococcus acidilactici fermentation product, dried Lactobacillus acidophilus fermentation product, dried Bifidobacterium longum fermentation product, dried Bacillus coagulans fermentation product, zinc proteinate, iron proteinate, taurine, calcium carbonate, vitamin E supplement, thiamine mononitrate, copper proteinate, manganese proteinate, sodium selenite, niacin supplement, D-calcium pantothenate, riboflavin supplement, vitamin A supplement, vitamin D3 supplement, vitamin B12 supplement, pyridoxine hydrochloride, folic acid.

⁶Nitrogen-free extract, % = 100 – (acid-hydrolyzed fat {%} + crude protein {%} + ash {%} + TDF {%}).

⁷Metabolizable energy estimated using Atwater factors (4 kcal/g for protein and nitrogen-free extract; 9 kcal/g for fat).

⁸Metabolizable energy estimated using modified Atwater factors (3.5 kcal/g for protein and nitrogen-free extract; 8.5 kcal/g for fat).

Animals, Housing, and Experimental Timeline

Prior to feeding, wet diets (FRSH and FRZN) were freeze dried (Dura-Dry MP microprocessor-controlled freeze-dryer; FTS Systems, Stone Ridge, NY), and then all diets were ground through a 2-mm screen (Wiley mill model 4, Thomas Scientific, Swedesboro, NJ). 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 (~23.9 °C, 17 h light: 7 h dark). Two precision-fed rooster assays utilizing Single Comb White Leghorn (1.5- to 2.5-yr old, 2.5 to 3 kg body weight) were conducted as described by Parsons (1985) to determine the standardized AA digestibility and TME_n content of the five pet foods. In the first rooster assay (to determine AA digestibility), 20 cecectomized roosters were randomly assigned to the five test foods (n = 4 roosters per test diet evaluated). Prior to the study, cecectomy was performed on roosters under general anesthesia according to the procedures of Parsons (1985). Roosters were given at least eight weeks to recover from surgery before being used in the experiments. In the second rooster assay (to determine TME_n), 20 conventional roosters were randomly assigned to the five test foods (n = 4 roosters per test diet evaluated). Before the start of the experiment, feed and water were supplied for ad libitum consumption. In both assays, after 26 h of feed withdrawal but ad libitum water, roosters were tube-fed 12 g of the test substrates combined with 12 g of corn.

Sample Collection and Analysis

Following crop intubation, excreta was collected for 48 h on plastic trays placed under each individual cage. Excreta samples were lyophilized, weighed, and ground through a 0.25-mm screen prior to analysis. Endogenous corrections for AA were made using five additional cecectomized roosters that had been fasted for 48 h. Standardized AA digestibilities were calculated using the method described by Engster et al. (1985).

Chemical Analyses

The FRSH and FRZN diets were first freeze dried. All diets were then ground in a Wiley mill (model 4, Thomas Scientific) through a 2-mm screen. Diets and rooster excreta were analyzed for dry matter (DM) and ash according to the Association of Official Analytical Chemists (AOAC, 2006; methods 934.01 and 942.05), with organic matter being calculated for diets and excreta from cecectomized birds. Crude protein for diets and excreta was calculated from a Leco Nitrogen/Protein Determinator (Model FP2000, Leco Corporation, St. Joseph, MI) and total nitrogen values according to AOAC (2006; method 992.15). Gross energy content of diets and excreta from conventional roosters was measured using an oxygen bomb calorimeter (model 6200, Parr Instruments, Moline, IL). For excreta from cecectomized roosters, AA were measured at the University of Missouri Experimental Station Chemical Laboratories (Columbia, MO) according to AOAC (2006; method 982.30E). For diets only, total lipid content (acid-hydrolyzed fat) was determined according to the methods of the American Association of Cereal Chemists (AACC, 1983) and Budde (1952). Total dietary fiber content of the diets was determined according to Prosky et al. (1992) by Eurofins (Eurofins Nutrition Analysis Center, Des Moines, IA).

AA Digestibility Calculations

Basal endogenous AA concentrations 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 following equation:

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{A}\mathrm{A}\mathrm{d}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{o}\mathrm{f}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}(\mathrm{\%})=\left[\frac{\begin{array}{c}\mathrm{A}\mathrm{A}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{s}\mathrm{u}\mathrm{m}\mathrm{e}\mathrm{d}-\mathrm{A}\mathrm{A}\mathrm{e}\mathrm{x}\mathrm{c}\mathrm{r}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{b}\mathrm{y}\mathrm{f}\mathrm{e}\mathrm{d}\mathrm{b}\mathrm{i}\mathrm{r}\mathrm{d}\mathrm{s}+\mathrm{A}\mathrm{A}\mathrm{e}\mathrm{x}\mathrm{c}\mathrm{r}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{b}\mathrm{y}\mathrm{f}\mathrm{a}\mathrm{s}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{b}\mathrm{i}\mathrm{r}\mathrm{d}\mathrm{s}\end{array}}{\mathrm{A}\mathrm{A}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{s}\mathrm{u}\mathrm{m}\mathrm{e}\mathrm{d}}\right]\times 100}\end{array}}$$

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 following equation:

$${\displaystyle \begin{array}{lll}{\displaystyle \mathrm{A}\mathrm{A}\mathrm{d}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}}& \mathrm{o}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}(\mathrm{\%})=& \mathrm{A}\mathrm{A}\mathrm{d}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{o}\mathrm{f}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{n}\mathrm{r}\mathrm{e}\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle \left[\frac{\left(\begin{array}{c}\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{r}\mathrm{d}\mathrm{i}\mathrm{z}\mathrm{e}\mathrm{d}\mathrm{A}\mathrm{A}\mathrm{d}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{o}\mathrm{f}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{n}\mathrm{r}\mathrm{e}\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}\text{-}\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{r}\mathrm{d}\mathrm{i}\mathrm{z}\mathrm{e}\mathrm{d}\mathrm{A}\mathrm{A}\mathrm{d}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}\mathrm{m}\mathrm{i}\mathrm{x}\mathrm{t}\mathrm{u}\mathrm{r}\mathrm{e}\mathrm{w}\mathrm{i}\mathrm{t}\mathrm{h}\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{n}\end{array}\right)}{\begin{array}{c}\mathrm{p}\mathrm{r}\mathrm{o}\mathrm{p}\mathrm{o}\mathrm{r}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}\mathrm{A}\mathrm{A}\mathrm{s}\mathrm{u}\mathrm{b}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{t}\mathrm{u}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}\mathrm{m}\mathrm{i}\mathrm{x}\mathrm{t}\mathrm{u}\mathrm{r}\mathrm{e}\mathrm{w}\mathrm{i}\mathrm{t}\mathrm{h}\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{n}\end{array}}\right]}\end{array}}$$

Nitrogen-Corrected True Metabolizable Energy (TME_n) Calculations

The calculation of TME_n was performed according to Parsons et al. (1982a). Correction for endogenous energy excretion was done using data from many fasted birds over several years. The TME_n values were calculated using the following equation:

$$\text{Misplaced \&}$$

where GE consumed (kcal) = diet intake (g) × GE of diet (kcal/g), GE excreted by fed or fasted birds (kcal) = excreta output (g) × GE of excreta (kcal/g), 8.22 = GE (kcal) of uric acid per g of nitrogen (Hill and Anderson, 1958), nitrogen retained by fed or fasted birds (g) = diet intake (g) × diet nitrogen (%) – excreta output (g) × excreta nitrogen (%). The TME_n values were then calculated by difference as

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{T}\mathrm{M}{\mathrm{E}}_n\left(\frac{\mathrm{k}\mathrm{c}\mathrm{a}\mathrm{l}}{\mathrm{g}}\right)=\mathrm{T}\mathrm{M}{\mathrm{E}}_n\mathrm{o}\mathrm{f}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{n}\mathrm{r}\mathrm{e}\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle \left[\frac{\mathrm{T}\mathrm{M}{\mathrm{E}}_n\mathrm{o}\mathrm{f}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{n}\mathrm{r}\mathrm{e}\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}\mathrm{T}\mathrm{M}{\mathrm{E}}_n\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}}{\mathrm{p}\mathrm{r}\mathrm{o}\mathrm{p}\mathrm{o}\mathrm{r}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}\mathrm{s}\mathrm{u}\mathrm{b}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{t}\mathrm{u}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{n}\mathrm{r}\mathrm{e}\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}}\right]}\end{array}}$$

The AA digestibility data derived from the current study were used to estimate the digestible indispensable AA concentrations of the diets tested on a weight basis (g digestible AA/100 g diet). The AA digestibility data and TME_n data derived from the current study were used to estimate the digestible indispensable AA concentrations of the diets tested on a caloric basis (g digestible AA/1,000 kcal ME).

Statistical Analyses

Data were analyzed using the mixed models procedure of SAS (version 9.4, SAS Institute, Inc., Cary, NC), with the experimental unit being individual roosters. The fixed effect of treatment was tested, and roosters were considered to be a random effect. Data were tested for normality using the UNIVARIATE procedure of SAS. Differences between treatments were determined using a Fisher-protected least significant difference with a Tukey adjustment to control for experiment-wise error. P < 0.05 was accepted as statistically significant. Reported pooled standard errors of the mean was determined according to the mixed models procedure of SAS.

Results

The analyzed nutrient composition of the diets is shown in Table 1. On a DM basis, the FD diet was the highest in protein, with over half (56.75%) of the diet being comprised of protein. The FRZN diet was ~10 percentage units lower, containing 46.70% (DM) protein. The EXT, FRSH, and HFD diets had more moderate protein concentrations at 24.61%, 31.06%, and 35.17% DM, respectively. The EXT diet (15.41% DM) and FRSH diet (15.30% DM) had moderate fat concentrations, while the other three diets contained about twice as much (33.83%, 30.51%, and 30.71% DM for the FRZN, HFD, and FD diets, respectively). The disparities in protein and fat inclusion largely contributed to the sizable differences in NFE content and ME estimates. For ME estimates, the EXT diet was the lowest (4.18 kcal/g DM; 3.75 kcal/g DM), with increased caloric density values for the FRSH diet (4.42 kcal/g DM; 3.96 kcal/g DM), the HFD diet (5.00 kcal/g DM; 4.57 kcal/g DM), the FD diet (5.05 kcal/g DM; 4.61 kcal/g DM), and the FRZN diet (5.26 kcal/g DM; 4.81 kcal/g DM). The reactive lysine:total lysine ratios were 0.94, 0.96, 0.93, 0.93, and 0.95 for the EXT, FRSH, FRZN, HFD, and FD diets, respectively.

Dietary indispensable and dispensable AA concentrations and AA concentrations recommended for adult dogs at maintenance by AAFCO (2023) are presented in Table 2. All five diets had dietary AA concentrations well above those recommended by AAFCO, both on a weight (% AA, DM basis) and caloric (g/1,000 kcal ME) basis.

Table 2.

Indispensable and dispensable amino acid (AA) concentrations of dog foods tested

	% dry matter						g/1,000 kcal
Item	AAFCO1	EXT2	FRSH	FRZN	HFD	FD	AAFCO	EXT	FRSH	FRZN	HFD	FD
Indispensable AA
Arginine	0.51	1.15	2.01	3.11	2.21	3.73	1.28	3.07	5.08	6.47	4.84	8.09
Histidine	0.19	0.55	0.83	1.19	1.00	1.64	0.48	1.47	2.10	2.47	2.19	3.56
Isoleucine	0.38	1.02	1.48	2.15	1.76	2.62	0.95	2.72	3.74	4.47	3.85	5.68
Leucine	0.68	2.67	2.40	3.54	2.79	4.18	1.70	7.12	6.06	7.36	6.11	9.07
Lysine	0.63	0.92	2.33	3.38	2.56	4.39	1.58	2.45	5.88	7.03	5.60	9.52
Methionine	0.33	0.50	0.79	1.12	0.82	1.36	0.83	1.33	1.99	2.33	1.79	2.95
Methionine + cystine	0.65	0.91	1.17	1.65	1.25	1.94	1.63	2.43	2.95	3.43	2.74	4.21
Phenylalanine	0.45	1.26	1.30	1.92	1.52	2.20	1.13	3.36	3.28	3.99	3.33	4.77
Phenylalanine + tyrosine	0.74	2.19	2.52	3.59	2.82	4.64	1.85	5.84	6.36	7.46	6.17	10.07
Threonine	0.48	0.82	1.23	1.88	1.47	2.26	1.20	2.19	3.11	3.91	3.22	4.90
Tryptophan	0.16	0.20	0.36	0.39	0.34	0.55	0.40	0.53	0.91	0.81	0.74	1.19
Valine	0.49	1.17	1.60	2.42	1.89	2.82	1.23	3.12	4.04	5.03	4.14	6.12
Dispensable AA
Alanine	—	1.63	1.73	2.77	1.99	3.33	—	4.35	4.37	5.76	4.35	7.22
Aspartic acid	—	1.72	2.78	4.06	3.06	4.93	—	4.59	7.02	8.44	6.70	10.69
Cysteine	—	0.41	0.38	0.53	0.43	0.58	—	1.09	0.96	1.10	0.94	1.26
Glutamic acid	—	4.66	4.56	5.96	5.10	7.50	—	12.43	11.52	12.39	11.16	16.27
Glycine	—	1.10	1.56	2.93	1.83	3.55	—	2.93	3.94	6.09	4.00	7.70
Proline	—	1.87	1.23	2.15	1.61	2.51	—	4.99	3.11	4.47	3.52	5.44
Serine	—	0.97	1.06	1.63	1.21	1.87	—	2.59	2.68	3.39	2.65	4.06
Tyrosine	—	0.93	1.22	1.67	1.30	2.44	—	2.48	3.08	3.47	2.84	5.29
Taurine	—	0.36	0.37	0.25	0.20	0.18	—	0.96	0.93	0.52	0.44	0.39

¹Association of American Feed Control Officials (AAFCO, 2023) for adult dogs at maintenance.

²EXT: Chicken and Barley Recipe (Hill’s Pet Nutrition, Topeka, KS); FRSH: Chicken and White Rice Recipe (Just Food for Dogs, Irvine, CA); FRZN: Chicken Formula (Primal Pet Group, Fairfield, CA); HFD: Chicken and Sorghum Hybrid Freeze-Dried Formula (Primal Pet Group, Fairfield, CA); FD: Chicken Dinner Patties (Stella & Chewy’s, Milwaukee, WI).

Standardized AA digestibilities are presented in Table 3. In general, the FD diet had the highest AA digestibilities and the EXT diet had lowest AA digestibilities. Amino acid digestibilities were very high for the FD diet, with all indispensable AA having coefficients > 90%. The FRZN diet also had high AA digestibilities, with all indispensable AA having coefficients > 89%. For the HFD and FRSH diets, digestibility coefficients were > 83% for all indispensable AA. Lastly, the EXT diet had AA digestibility coefficients > 76% for all indispensable AA. Standardized lysine digestibility was greater (P < 0.05) in both raw diets [FD (92.18%) and FRZN (90.37%)] than the EXT diet (78.77%), with the other two diets being intermediate. Standardized threonine digestibility was greater (P < 0.05) in the FD diet (94.51%) than the EXT diet (76.90%), with the other diets being intermediate. The TME_n value of the FRZN diet (5.86 kcal/g DM) was higher (P < 0.05) than that of the FD (4.97 kcal/g DM), FRSH (4.62 kcal/g DM), and EXT (4.26 kcal/g DM) diets. The TME_n value was also higher (P < 0.05) in the HFD diet (5.40 kcal/g DM) than that of the FRSH and EXT diets, and higher (P < 0.05) in the FD diet than the EXT diet.

Table 3.

Amino acid (AA) digestibility and nitrogen-corrected true metabolizable energy (TME_n) of dog diets using cecectomized and conventional rooster assays

Item	EXT1	FRSH	FRZN	HFD	FD	SEM	P-value
Indispensable AA
Arginine2	89.09	92.11	93.47	89.88	95.29	2.5425	0.0442
Histidine	86.04	89.68	89.26	86.58	91.11	2.6030	0.6147
Isoleucine2	86.94	87.35	91.70	86.49	95.06	2.4857	0.0439
Leucine	92.39	88.00	92.33	87.47	95.68	2.5105	0.0732
Lysine	78.77b	89.60ab	90.37a	86.95ab	92.18a	2.5836	0.0183
Methionine2	91.29	89.34	93.90	90.55	96.55	1.5935	0.0492
Phenylalanine	90.01	86.99	90.98	86.25	94.59	2.7512	0.0784
Threonine	76.90b	86.34ab	91.48ab	83.22ab	94.51a	3.9509	0.0491
Tryptophan	98.13	99.50	98.57	95.87	99.53	1.0515	0.1420
Valine2	84.83	83.71	89.78	83.41	94.00	3.4465	0.0469
Dispensable AA
Alanine	89.02	85.22	90.16	84.97	94.31	2.6498	0.0537
Aspartic acid	78.61b	86.49ab	90.90ab	82.00b	93.96a	2.6983	0.0067
Cysteine	79.82	72.11	77.16	67.96	89.10	6.8924	0.2918
Glutamic acid	89.48	88.28	90.94	88.60	95.00	2.1111	0.2063
Proline	86.10	83.17	86.00	82.20	92.27	4.3772	0.5407
Serine	79.75	83.01	88.41	78.79	93.25	5.0812	0.2680
Tyrosine	84.38	85.27	88.22	83.74	92.31	2.4287	0.0766
TMEn	4.26d	4.62cd	5.86a	5.40ab	4.97bc	0.1207	<0.0001

¹EXT: Chicken and Barley Recipe (Hill’s Pet Nutrition, Topeka, KS); FRSH: Chicken and White Rice Recipe (Just Food for Dogs, Irvine, CA); FRZN: Chicken Formula (Primal Pet Group, Fairfield, CA); HFD: Chicken and Sorghum Hybrid Freeze-Dried Formula (Primal Pet Group, Fairfield, CA); FD: Chicken Dinner Patties (Stella & Chewy’s, Milwaukee, WI).

²Significant differences among treatments, but not after multiple comparisons.

^abcdWithin a row, means lacking a common superscript differ (P < 0.05).

Using the AA digestibility data and the TME_n data determined from the present study, estimates of digestible AA on a weight (g AA/100 g DM diet) and caloric (g AA/1,000 kcal ME) basis were calculated and are presented in Table 4. On a weight basis, the FD diet had the highest digestible AA concentrations, while the FRZN diet had the second highest. The HFD diet had the third highest digestible AA concentrations, excluding leucine (EXT) and tryptophan (FRSH). The FRSH diet had the fourth highest digestible AA concentrations, with the EXT diet having the lowest digestible AA concentrations with the exception of leucine and tryptophan that were lowest in the HFD diet. Not surprisingly, these results correlated largely with the dietary protein concentrations.

Table 4.

Digestible indispensable amino acid concentrations of dog foods tested¹

	g/100 g DM of diet					g/1,000 kcal of diet
Item	EXT2	FRSH	FRZN	HFD	FD	EXT	FRSH	FRZN	HFD	FD
Arginine	1.02	1.85	2.91	1.99	3.55	2.41	4.01	4.96	3.68	7.15
Histidine	0.47	0.74	1.06	0.87	1.49	1.11	1.61	1.81	1.60	3.01
Isoleucine	0.89	1.29	1.97	1.52	2.49	2.08	2.80	3.36	2.82	5.01
Leucine	2.47	2.11	3.27	2.44	4.00	5.79	4.57	5.58	4.52	8.05
Lysine	0.72	2.09	3.05	2.23	4.05	1.70	4.52	5.21	4.12	8.14
Methionine	0.46	0.71	1.05	0.74	1.31	1.07	1.53	1.79	1.38	2.64
Methionine + cystine	0.78	0.98	1.46	1.03	1.83	1.84	2.12	2.49	1.92	3.68
Phenylalanine	1.13	1.13	1.75	1.31	2.08	2.66	2.45	2.98	2.43	4.19
Phenylalanine + tyrosine	1.92	2.17	3.22	2.40	4.33	4.50	4.70	5.50	4.44	8.72
Threonine	0.63	1.06	1.72	1.22	2.14	1.48	2.30	2.93	2.27	4.30
Tryptophan	0.20	0.36	0.38	0.33	0.55	0.46	0.78	0.66	0.60	1.10
Valine	0.99	1.34	2.17	1.58	2.65	2.33	2.90	3.71	2.92	5.33

¹Using the AA digestibility and TME_n data from the current study.

²EXT: Chicken and Barley Recipe (Hill’s Pet Nutrition, Topeka, KS); FRSH: Chicken and White Rice Recipe (Just Food for Dogs, Irvine, CA); FRZN: Chicken Formula (Primal Pet Group, Fairfield, CA); HFD: Chicken and Sorghum Hybrid Freeze-Dried Formula (Primal Pet Group, Fairfield, CA); FD: Chicken Dinner Patties (Stella & Chewy’s, Milwaukee, WI).

On a caloric basis, the FD diet had the highest digestible AA concentrations. The FRZN diet had the second highest digestible AA concentrations, with the exception of leucine and tryptophan that were the third highest. The FRSH diet had the third highest digestible AA concentrations, with the exception of tryptophan (second highest), isoleucine (fourth highest), and leucine (fourth highest). The HFD diet had the fourth highest digestible AA concentrations, with exceptions of isoleucine and valine that were the third highest and leucine, phenylalanine, and phenylalanine-tyrosine that had the lowest. Generally, EXT had the lowest digestible AA concentrations, with the exception of leucine (second highest), phenylalanine (fourth highest), and phenylalanine-tyrosine (fourth highest).

Discussion

With the vast majority of dog owners considering their pets to be family members, there is a desire to feed dogs food that is as health promoting as the food they feed themselves (Tesform and Birch 2010; AVMA 2018). However, there is much ambiguity surrounding what comprises a “healthy” food for dogs. Diets that have been deemed “healthy” may differ in regard to ingredients, nutrient concentrations, processing methods used for production, and format. This uncertainty in the optimal food for dogs has led to the development of several different philosophies (such as science based, “natural/wild” based, human based) for pet food, with a wide variety of products available on the market. In several cases, innovation has become incorporated into pet foods without sufficient scientific basis supporting their merit (example.g., human-grade, grain-free, and vegan dog foods), Therefore, it is critical to research all novel and emerging pet food formats to ensure safety and investigate any structure-function or health benefits associated with the diet. One key aspect of pet food that should be examined is macronutrient digestibility because dogs may not fully reap the nutritional benefits of a diet unless it is adequately digestible.

Each protein source has a different profile of AA, which may need to be combined carefully together to ensure the minimum requirements of every indispensable AA are met or exceeded. All diets in this study had AA concentrations in excess of the AAFCO minimum recommendations for adult dogs at maintenance. Although mild heat treatment can improve protein digestibility by denaturing protein structure, allowing enzymes greater access chemical bonds, and reducing trypsin inhibitor activity, heat generally decreases digestibility of animal proteins due to aggregation and coagulation (López-Pedrouso et al., 2019). Increasing heat treatment is known to decrease the digestibility of most AA (Hendriks et al., 1999), with lysine being the most affected (Almeida et al., 2013). Heat processing can also promote the Maillard reaction, which is a non-enzymatic glycation reaction that occurs between AA and reducing sugars. Because of its free amino group, lysine reacts readily with reducing sugars, which can reduce lysine bioavailability in the diet (Williams et al., 2006). A reduction in lysine digestibility was demonstrated in the current study, with the lowest lysine digestibility occurring in the diet processed with the highest level of heat. Although EXT had digestible lysine levels that exceeded AAFCO recommendations, its lysine digestibility was numerically lower than all other diets tested.

The extent of the Maillard reaction depends on numerous factors such as the chemical composition of the diet, the presence of transition metals as well as anti- or pro-oxidants, moisture levels, cooking time, dietary pH, maximum processing temperature reached, and the processing method used (Poulsen et al., 2013). The reactive lysine:total lysine ratio is used as an indicator of heat damage during processing, with a score close to one indicating minimal heat damage. All diets tested in the current study were above 0.93, indicating nominal heat damage. Lysine reactivity, however, does not necessarily correlate with its digestibility/absorption, and it is not always a good indicator of Maillard reaction and advanced glycation end-product production (van Rooijen et al., 2014). The measurement of advanced glycation end-products may have provided a more accurate assessment of heat damage of the diets.

In addition to needing to ensure the diet contains AA concentrations that exceed minimum recommendations, it is imperative to verify that the AA are highly digestible. Although apparent total tract digestibility studies in dogs provide an estimation of protein digestibility, measuring AA concentrations in the feces is not accurate because of the alterations by bacteria in the hindgut (Hendriks and Sritharan, 2002). Therefore, in vivo models that can provide an estimation of AA digestibility while bypassing the hindgut contribution are utilized. Although ileal-cannulated dogs were historically the gold standard for assessing AA digestibility (Hendriks et al., 2013), ethical concerns and research restrictions with cannulation procedures have made the cecectomized rooster a popular model. Although there is a surgery involved, roosters can be fed and maintain health the same as intact roosters after recovery.

In a study using cecectomized roosters, AA digestibilities of raw chicken, steamed chicken, retorted chicken, and rendered chicken meal was assessed (Oba et al., 2019). Chicken meal, which is an ingredient used in the EXT diet and not incorporated into any other diet in this study, had indispensable AA digestibilities ranging from 75.32% to 89.60%. In the present study, the EXT had eight indispensable AA with digestibilities over 80%, with four being above 90%. The AA digestibilities measured in the EXT diet of the current study are likely higher than those of the chicken meal assessed in the previous study because chicken meal was only the 7th most abundant ingredient of EXT. Additionally, EXT contained numerous other ingredients that are protein sources, such as chicken (first ingredient), barley, sorghum, corn, and more. The steamed chicken tested by Oba et al. (2019), which was processed (~93 °C for 10 min) using a method similar to how the chicken in the FRSH diet (76 to 82 °C for 20 to 40 min) of the current study, had indispensable AA digestibilities ranging from 87.83% to 95.36%. All indispensable AA digestibilities were over 80% and seven were over 90%. The FRSH diet in the current study performed similarly (indispensable AA digestibilities ranged from 83.7% to 99.5%), but somewhat lower than the steamed chicken of the previous study, with all 10 indispensable AA digestibilities being over 80% and 2 being over 90%. The slight disparity can be attributed to the incorporation of other ingredients, including other protein sources, in the FRSH diet besides chicken. Raw chicken from the study of Oba et al. (2019) had indispensable AA digestibilities ranging from 79.75% to 94.24%. All indispensable AA were ~80% digestible, with nine of those being over 90% (Oba et al., 2019). In this case, the raw diets (FRZN and FD) of the current study performed much better (indispensable AA digestibilities ranged from 89.26% to 99.53%; FRZN had 8 AA digestibilities over 90%; FD had all AA digestibilities over 90%) than the raw chicken of the previous study. These findings may be attributable to the parts of the chicken used between studies, the processing of other ingredients present in the diets, or other unknown factors.

In another recent study, AA digestibilities were assessed in three commercial raw diets, two of which had identical formulas (but different batches) to the diets tested in the current study (FRZN and HFD; Oba et al., 2023). Digestibilities of all indispensable AA were above 80% for all three diets (frozen raw, hybrid freeze-dried raw, and freeze-dried raw) in that study. The frozen raw diet had digestibilities of all indispensable AA above 90%, except for histidine. Those results are comparable to that of the current study, with all AA above 90% except histidine and valine. The hybrid freeze-dried raw diet in that study had indispensable AA digestibilities above 90%, with the exception of histidine and threonine. The results for that diet are higher than that of the current study, where only two of the indispensable AA were above 90%. The freeze-dried raw diet in that study had indispensable AA digestibilities above 90%, except for histidine and lysine. The freeze-dried diet in the present study performed moderately better than the diet tested in the previous study and had all indispensable AA above 90% (Oba et al., 2023). This disparity in results may be explained by the freeze-dried diet having different ingredients and/or macronutrient composition.

The feeding guidelines for commercial dog foods are based on the estimated ME content using modified Atwater values (e.g., 3.5 kcal/g for protein and digestible carbohydrates and 8.5 kcal/g for fats) (AAFCO, 2023). These were developed to account for the reduced digestibility of ingredients traditionally used to make pet foods so that ME was not overestimated. Those values derive from digestibility coefficients of 85% for digestible carbohydrates, 80% for protein, and 90% for fat that were suggested by the NRC (1985). The ME of human foods is determined by Atwater factors (e.g., 4 kcal/g for protein and digestible carbohydrates and 9 kcal/g for fats). Those factors are determined by gross energy (the heat of combustion), with corrections for losses that occur with digestion, absorption, and urinary excretion. Those values derive from digestibility coefficients of 91% for digestible carbohydrates and 96% for proteins and fats (Atwater, 1902). As recent studies have demonstrated, the use of these modified factors often results in underestimating the energy content of premium pet foods (Oba et al., 2020, 2023; Roberts et al., 2023). In the current study, the ME estimates using modified Atwater and Atwater values both underestimated the ME of the EXT, FRSH, FRZN, and HFD diets. The ME using Atwater factors (5.05 kcal/g) was a close predictor of TME_n (4.97 kcal/g) for the FD diet, however. The FD diet is likely predicted well with the Atwater factors because it has such low fiber concentrations while having relatively high fat and protein concentrations. The TME_n values determined in this study were higher than the modified Atwater estimates by 14%, 17%, 22%, 18%, and 8% for EXT, FRSH, FRZN, HFD, and FD diets, respectively. The TME_n values were much closer to the Atwater estimates, being only 2%, 5%, 11%, 8%, and −1.6% higher, respectively. Because these equations have so many assumptions built into their calculations, it is much more accurate to conduct an in vivo study to determine ME. TME_n provides a better estimate of the ME than any of the equations, without the expense and time required for conducting an entire canine study.

A limitation of this study was the variation among diets. While the main protein source of all the diets was chicken, the other ingredients, processing methods, and macronutrient concentrations all varied. For instance, dietary protein, fat, ash, and fiber concentrations varied greatly among diets. Higher fiber and ash content usually reduce AA digestibilities. In contrast, high protein concentrations often increase digestibility calculations because the proportion of nitrogen loss coming from endogenous sources decreases with increasing dietary protein. While particle size of the diets was made consistent by grinding prior to feeding, the heat processing of some ingredients and complete diets may have formed unique chemical bonds that negatively impact digestibility. These differences among diets make drawing conclusions related to what effect each of these factors has upon AA digestibility alone rather challenging.

In conclusion, all diets tested in this study met the indispensable AA concentrations for adult dogs at maintenance according to AAFCO (2023) recommendations. Reactive: total lysine ratios were all above 0.9. Indispensable AA digestibilities were high for all diets, with almost all being above 80%. The TME_n values ranged from 4.26 to 5.86 kcal/g. The Atwater factors predicted the ME content of all diets better than the modified Atwater values, but still underestimated it in four out of five diets. Future research should assess the stool quality, nutrient digestibility, and other health outcomes from such diets in dogs.

Glossary

Abbreviations

AA

amino acid

AAFCO

Association of American Feed Control Officials

AOAC

Association of Official Analytical Chemists

DM

dry matter

EXT

extruded diet

FD

freeze-dried raw diet

FRSH

fresh diet

FRZN

frozen raw diet

HFD

hybrid freeze-dried raw diet

ME

metabolizable energy

NRC

National Research Council

TME_n

nitrogen-corrected true metabolizable energy

Conflict of Interest Statement

J.R.T. is an employee of Primal Pet Group. All other authors have no conflicts of interest.