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

Abstract

Commercial raw or minimally-processed diets, often referred to holistically as raw meat-based diets (RMBD) represent a small portion of the pet food market, but the growth of this sector has been significant in recent years. While traditionally, high-moisture, frozen options were the standard format of commercially available raw diets, freeze-dried raw diets have become more prevalent as of late. Despite the increasing popularity of these commercial raw diet formats, there is a dearth of literature describing their nutritional properties, particularly regarding freeze-dried diets. Therefore, the objective of this experiment was to determine and compare the standardized amino acid (AA) digestibilities and nitrogen-corrected true metabolizable energy (TME_n) of raw frozen and freeze-dried dog foods using precision-fed cecectomized and conventional rooster assays. Three formats of frozen or freeze-dried raw diets provided by Primal Pet Foods (Fairfield, CA, USA) were tested: traditional freeze-dried nuggets (T-FDN), hybrid freeze-dried nuggets (H-FDN), and frozen nuggets (FZN). Diets were fed to cecectomized roosters (4 roosters/diet) to determine AA digestibilities, while conventional roosters (4 roosters/diet) were used to determine TME_n. In both cases, after 26 h of feed withdrawal, roosters were tube-fed 12 to 13 g of test diets and 12 to 13 g of corn. Following crop intubation, excreta were collected for 48 h. Endogenous corrections for AA were made using five additional cecectomized roosters. All data were analyzed using the Mixed Models procedure of SAS version 9.4. There were no significant differences in standardized AA digestibilities among diets, with digestibilities being high for all diets tested. For most of the indispensable AA, digestibilities were greater than or equal to 90% for all diets. Histidine and lysine were the exceptions, with digestibilities ranging from 82% to 87% and 87% to 92%, respectively. Moreover, the reactive lysine:total lysine ratio, a measure of heat damage, ranged from 0.91 to 0.95. TME_n values were higher (P = 0.0127) in T-FDN (6.1 kcal/g) and FZN (5.9 kcal/g) than H-FDN (5.3 kcal/g) and were most similar to those estimated by Atwater factors. In general, all diets tested had high AA digestibilities and had TME_n values that were most similar to Atwater factors.

Precision-fed cecectomized and conventional rooster assays were used to determine the amino acid (AA) digestibilities and nitrogen-corrected true metabolizable energy values of raw frozen and freeze-dried dog foods. All diets had high AA digestibilities, with most indispensable AA digestibilities being >90%. Nitrogen-corrected true metabolizable energy values were most similar to those estimated by Atwater factors.

Introduction

The feeding of commercially available raw diets—often referred to as raw meat-based diets (RMBD) in the literature—to dogs and cats has become increasingly popular among pet owners in many developed countries. Of dog foods, raw diets are estimated to make up about 4% of sales and are growing at a rate of 14% annually (Altas Stackline, 2022; Nielson IQ Byzzer, 2023). These raw or minimally-processed diets contain primarily uncooked ingredients derived from domesticated or wild-caught food animal species, with lesser inclusions of vegetables, fruits, seeds, nuts, vegetable-based oils, grains, and/or micronutrient-enriched supplements rounding out the diet matrix, and are commercially available in fresh, frozen, freeze-dried, and dehydrated formats (Freeman et al., 2013). When compared with conventional processed diets (e.g., high-heat extruded kibble), raw diets are claimed to have higher palatability, lead to improved dental and skin health, and be associated with the prevention or control of disorders affecting many of the major body systems (Freeman et al., 2013). While not all of these claims have been proven by randomized studies, raw or minimally-processed diets have been shown to have high palatability, high macronutrient digestibility, and maintain adequate stool quality in dogs (Lefebvre et al., 2008; Beloshapka et al., 2012; Sandri et al., 2016; Kim et al., 2017; Schmidt et al., 2018) and cats (Kerr et al., 2012). Raw meat-based diets have also been tested using a cecectomized rooster model and have been shown to have high amino acid (AA) digestibilities (Kerr et al., 2013), which may be impacted by processing (Oba et al., 2019).

While traditionally, the standard format of commercially available raw diets was high-moisture frozen options, freeze-dried diets have become more prevalent as of late. Freeze-drying is a method of removing water from a substance by directly converting ice to vapor and avoiding the liquid state (Hua et al., 2010). The preservation of the biological, nutritional, and sensory properties of the dried product is one of its major advantages (Ratti, 2001; Assegehegn et al., 2019). Freezing the water in the material prior to lyophilization aids in the prevention of chemical, biochemical, and microbiological changes that would otherwise alter the product’s taste, aroma, and nutrient content (Nowak and Jakubczyk, 2020). Raw food materials typically contain a significant amount of water, and sublimating this water results in freeze-dried products with a highly porous structure, and after rehydration the freeze-dried product quickly regain their original size (Meda and Ratti, 2005; Jia et al., 2019). Freeze-drying technology allows pet food manufacturers to provide consumers with raw or minimally-processed diets that have extended shelf lives, are more convenient to feed, and present fewer storage challenges and health risks (Wall, 2022). In 2022, freeze-dried pet food sales accounted for only 1.2% of all pet food sales, but are growing by ~19% per year (Altas Stackline, 2022; Nielson IQ Byzzer, 2023).

Although frozen and freeze-dried diet popularity continues to increase, studies testing the effects of processing conditions on AA digestibility are lacking. Therefore, the objectives of this study were to determine and compare the standardized AA digestibilities and nitrogen-corrected true metabolizable energy (TME_n) of raw frozen and freeze-dried dog foods using precision-fed cecectomized and conventional rooster assays.

Materials and Methods

Diets

Three frozen or freeze-dried chicken-based complete and balanced foods for adult dogs were tested in this study. Diets included traditional freeze-dried nuggets (T-FDN), hybrid freeze-dried nuggets (H-FDN), and frozen nuggets (FZN) manufactured and provided by Primal Pet Foods (Primal Pet Group, Fairfield, CA). Dietary ingredient profiles are listed in Table 1. The processing steps for the diets tested were the following:

Table 1.

Chemical composition of the frozen and freeze-dried raw dog foods tested

Item	Traditional freeze-dried nuggets1	Hybrid freeze-dried nuggets2	Frozen nuggets3
Dry matter (DM), %	95.17	96.44	94.93
	-------------------- Dry matter basis --------------------
Ash, %	5.61	8.72	4.98
Crude protein, %	40.67	36.33	38.14
Acid-hydrolyzed fat, %	49.27	33.16	48.56
Total dietary fiber, %	5.40	5.71	5.62
Total insoluble fiber,%	4.83	4.86	4.44
Total soluble fiber,%	0.57	0.85	1.19
Nitrogen-free extract4, %	0.00	16.08	2.70
Gross energy, kcal/g	7.45	6.17	7.43

¹Ingredients: 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 sunflower oil, organic apple cider vinegar, montmorillonite clay, fish oil, organic quinoa, organic coconut oil, vitamin E supplement, organic rosemary extract, 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.

²Ingredients: 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 sulfate, ferrous sulfate, biotin, retinol palmitate, manganese sulfate, niacin, d-calcium pantothenate, cholecalciferol, sodium selenite, copper sulfate anhydrous, thiamin 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.

³Ingredients: 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.

⁴NFE = 100% − (% CP in DM + % AHF in DM + % TDF in DM + % Ash in DM).

• T-FDN: Raw ingredients were mixed, formed into nuggets, and frozen at −32 °C for 8 h. After freezing, it was placed under vacuum and warmed at −23 °C for 4 h, then the temperature was increased slowly over 11 h to reach an internal nugget temperature of 60 °C, thereby ensuring the product was fully dried.

• H-FDN: A portion of the raw chicken (35% of chicken) was steamed at 71 °C for 10 min and sorghum was cooked at a temperature and sufficient time to achieve starch conversion/gelatinization of at least 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 chicken and cooked sorghum were then blended with the remaining raw ingredients, formed into nuggets, and frozen at −32 °C for 8 h. After freezing, it was placed under vacuum and warmed at −23 °C for 3 h, then the temperature was increased slowly over 5 h to reach an internal nugget temperature of 48 °C, thereby ensuring the product was fully dried.

• FZN: Raw ingredients were mixed, formed into nuggets, and frozen at −32 °C.

Prior to testing, the frozen diet (FZN) was freeze-dried so that roosters could be dosed properly. The freeze-drying process was conducted as follows: once the diet was removed from −32 °C, it was kept at −23 °C for 24 h. The temperature was then increased slowly over the next 24 h to reach 0 °C, ensuring the product was fully dried. Internal temperature never exceeded freezing (0 °C) and was therefore extended over 48 h.

Cecectomized and conventional rooster assays

The protocol for the cecectomized and conventional rooster assays, including all animal housing, handling, and surgical procedures, was reviewed and approved by the Institutional Animal Care and Use Committee at the University of Illinois at Urbana-Champaign prior to experimentation (IACUC #20131). First, a precision-fed rooster utilizing Single Comb White Leghorn roosters (1.5 to 2.5 yr old, 2.5 to 3 kg body weight) was conducted as described by Parsons (1985) to determine the standardized AA digestibility of the test diets. Prior to the study, cecectomy was performed on roosters under general anesthesia according to the procedures of Parsons (1985). All roosters were given at least 8 wk to recover from surgery before being used in experiments. In the first study, 12 cecectomized roosters were randomly assigned to test foods (n = 4 roosters/diet). In a second rooster assay, 12 conventional roosters were randomly assigned to the test diets (n = 4 roosters/diet) for TME_n calculations.

In both assays, after 26 h of feed withdrawal and ad libitum water, roosters were tube-fed 12 to 13 g of the test diets and 12 to 13 g of corn. Following crop intubation, excreta were collected for 48 h on plastic trays placed under each individual cage. Excreta samples then were lyophilized, weighed, and ground through a 0.25-mm screen prior to analysis. Endogenous corrections for AA were made using five additional cecectomized roosters (1.5 to 2.5 yr old, 2.5 to 3 kg body weight) that had been fasted for 48 h. Standardized AA digestibilities were calculated using the method described by Engster et al. (1985). All birds were housed individually in cages (27.9 cm wide × 50.8 cm long × 53.3 cm high) with raised wire floors. They were kept in an environmentally controlled room (~23.9 °C, 17:7 (L:D) h). Before the start of the experiment, feed and water were supplied for ad libitum consumption.

Chemical analyses

Before analysis, all diets were ground through a 2-mm screen (Wiley mill model 4; Thomas Scientific, Swedesboro, NJ). The diets and rooster excreta were analyzed for dry matter (DM; 105 °C) and ash according to AOAC (2006) with organic matter (OM) being calculated (DM: method 934.01; OM: method 942.05). Nitrogen was measured and crude protein (CP) was calculated using a Leco Nitrogen/Protein Determinator (Model FP-2000, Leco Corporation, St. Joseph, MI) according to the AOAC (2006; method 992.15). Acid-hydrolyzed fat (AHF) concentrations were determined by acid hydrolysis according to the AACC (1983; method 922.06), followed by diethyl ether extraction (Budde, 1952). Total dietary fiber (TDF) concentrations, including insoluble and soluble fractions, were determined according to Prosky et al. (1985). Gross energy (GE) was measured using a bomb calorimeter (Model 1261; Parr Instrument Co., Moline, IL). The AA were measured at the University of Missouri Experiment Station Chemical Laboratories (Columbia, MO) according to the AOAC (2006; method 982.30E).

Amino acid digestibility calculations

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

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

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

$${\displaystyle \begin{array}{l}{\displaystyle \begin{array}{l}\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{\%})\begin{array}{lllll}& {\displaystyle ={\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{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}}}& {\displaystyle \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}}& {\displaystyle \left(\mathrm{d}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{d}\mathrm{p}\mathrm{r}\mathrm{e}\mathrm{v}\mathrm{i}\mathrm{o}\mathrm{u}\mathrm{s}\mathrm{l}\mathrm{y}\right)}& {\displaystyle -\left[\frac{\begin{array}{lllll}& {\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{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}}& {\displaystyle \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}}& {\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{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}}& {\displaystyle \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}}{\begin{array}{llll}& {\displaystyle \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}}& {\displaystyle \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}}& {\displaystyle \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}\end{array}}\end{array}}$$

Nitrogen-corrected true metabolizable energy (TME_n) calculations

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

$${\displaystyle \begin{array}{l}{\displaystyle \begin{array}{l}\mathrm{T}\mathrm{M}{\mathrm{E}}_{\mathrm{n}}\mathrm{o}\mathrm{f}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}(\mathrm{k}\mathrm{c}\mathrm{a}\mathrm{l}/\mathrm{g})={\displaystyle \frac{\left[\begin{array}{llllll}& {\displaystyle \mathrm{G}\mathrm{E}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{s}\mathrm{u}\mathrm{m}\mathrm{e}\mathrm{d}}& {\displaystyle -(\mathrm{G}\mathrm{E}\mathrm{e}\mathrm{x}\mathrm{c}\mathrm{r}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{b}\mathrm{y}\mathrm{f}\mathrm{e}\mathrm{d}\mathrm{b}\mathrm{i}\mathrm{r}\mathrm{d}\mathrm{s}}& {\displaystyle +8.22\times \mathrm{n}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{r}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{d}}& {\displaystyle \mathrm{b}\mathrm{y}\mathrm{f}\mathrm{e}\mathrm{d}\mathrm{b}\mathrm{i}\mathrm{r}\mathrm{d}\mathrm{s})+}& {\displaystyle \begin{array}{llll}& {\displaystyle (\mathrm{G}\mathrm{E}\mathrm{e}\mathrm{x}\mathrm{c}\mathrm{r}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{b}\mathrm{y}\mathrm{f}\mathrm{a}\mathrm{s}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{b}\mathrm{i}\mathrm{r}\mathrm{d}\mathrm{s}}& {\displaystyle +8.22\times \mathrm{n}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{r}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{d}}& {\displaystyle \mathrm{b}\mathrm{y}\mathrm{f}\mathrm{a}\mathrm{s}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{b}\mathrm{i}\mathrm{r}\mathrm{d}\mathrm{s})}\end{array}}\end{array}\right]}{\mathrm{f}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}(\mathrm{g})}}\end{array}}\end{array}}$$

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 shown below.

$${\displaystyle \begin{array}{l}{\displaystyle \begin{array}{l}\mathrm{T}\mathrm{M}{\mathrm{E}}_{\mathrm{n}}(\mathrm{k}\mathrm{c}\mathrm{a}\mathrm{l}/\mathrm{g})\begin{array}{llll}{\displaystyle =}& {\displaystyle \mathrm{T}\mathrm{M}{\mathrm{E}}_{\mathrm{n}}\mathrm{o}\mathrm{f}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}}& {\displaystyle \mathrm{c}\mathrm{o}\mathrm{r}\mathrm{n}\mathrm{r}\mathrm{e}\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}}& {\displaystyle -\left[\frac{\begin{array}{llll}& {\displaystyle (\mathrm{T}\mathrm{M}{\mathrm{E}}_{\mathrm{n}}\mathrm{o}\mathrm{f}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}}& {\displaystyle \mathrm{c}\mathrm{o}\mathrm{r}\mathrm{n}\mathrm{r}\mathrm{e}\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}}& {\displaystyle -\mathrm{T}\mathrm{M}{\mathrm{E}}_{\mathrm{n}}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t})}\end{array}}{\begin{array}{llll}& {\displaystyle \mathrm{p}\mathrm{r}\mathrm{o}\mathrm{p}\mathrm{o}\mathrm{r}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}}& {\displaystyle \mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}\mathrm{s}\mathrm{u}\mathrm{b}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{t}\mathrm{u}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{o}}& {\displaystyle \mathrm{t}\mathrm{h}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{n}\mathrm{r}\mathrm{e}\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}}\end{array}}\right]}\end{array}\end{array}}\end{array}}$$

Metabolizable energy calculations

To compare against TME_n data, metabolizable energy (ME) estimates were performed according to modified NRC (2006) calculations [using TDF instead of crude fiber (CF) values], Atwater factors (Atwater, 1902), and modified Atwater factors. Nitrogen-free extract (NFE) values (using TDF instead of CF values) and ME were calculated using the following equations:

1. a) NFE (%) = 100% − (% CP in DM + % AHF in DM + % TDF in DM + % ash in DM)

2. b) ME (Atwater values, kcal/g) = [(4 × CP in DM) + (9 × AHF in DM) + (4 × NFE in DM)] / 100

3. c) ME (modif ied Atwater values, kcal/g) = [(3.5 × CP in DM) + (8.5 × AHF in DM) + (3.5 × NFE in DM)] / 100

4. d) ME (NRC, 2006 equation; kcal/g) =

   1. GE (kcal) = (5.7 × % CP in DM) + (9.4 × % AHF in DM) + [4.1 × (% NFE + % TDF in DM)]

   2. Energy digestibility (%): 96.6 − (0.95 × % TDF in DM)

   3. Digestible energy (DE): (kcal GE × energy digestibility)/100

   4. ME (NRC) (kcal/g) = [kcal DE − (1.04 × % CP in DM)]/100

Because the CF assay does not accurately measure fiber, it should not be used for estimating the fiber content in pet foods (Fahey et al., 2019; Traughber et al., 2021). Therefore, the TDF assay, which allows the measurement of both soluble and insoluble fiber fractions and is a much better fiber estimate, was used in the equations above. Using TDF to estimate NFE has been shown to have a high correlation with starch content in dog foods (de-Oliveira et al., 2012).

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

All data were analyzed using the Mixed Models procedure of SAS (version 9.4; SAS Institute, Cary, NC). Diets were considered to be a fixed effect and roosters were considered to be a random effect. Tukey’s multiple comparison analysis was used to compare LS means for experiment-wise error. Differences were considered significant with P < 0.05.

RESULTS

Chemical composition

The chemical composition of the test diets is presented in Table 1. Of note, the DM contents listed for the foods were the values present after the freeze-drying process (including for the FZN diet), which was needed to properly conduct the chemical analyses and dosing for the rooster experiments. All other nutrients are represented on a dry matter basis (DMB). Regarding chemical composition, all diets were rich in protein (>35% DMB) and fat (>30% DMB), low in TDF (<6% DMB), and high in energy density (>6 kcal/g GE DMB; >5 kcal/g TME_n DMB). Concentrations of indispensable and dispensable AA, and reactive lysine:total lysine ratios are presented in Table 2. The reactive: total lysine ratios were above 0.9 for all diets tested.

Table 2.

Indispensable and dispensable amino acid (AA) concentrations (% DM) of frozen and freeze-dried raw dog foods

Amino acid	AAFCO1	Traditional freeze-dried nuggets	Hybrid freeze-dried nuggets	Frozen nuggets
Indispensable AA
Arginine	0.51	2.61	2.42	2.35
Histidine	0.19	1.01	1.05	0.87
Isoleucine	0.38	1.75	1.81	1.47
Leucine	0.68	3.31	2.91	2.81
Total lysine	0.63	3.05	3.00	2.64
Reactive lysine	—	2.76	2.77	2.52
Methionine	0.33	0.91	0.87	0.75
Phenylalanine	0.45	1.96	1.58	1.57
Threonine	0.48	1.66	1.58	1.44
Tryptophan	0.16	0.37	0.43	0.44
Valine	0.49	2.21	1.92	1.82
Selected dispensable AA
Alanine	—	2.46	2.04	2.14
Aspartic acid	—	3.48	3.29	3.02
Cysteine	—	0.54	0.47	0.48
Glutamic acid	—	5.18	5.40	4.49
Glycine	—	2.60	1.84	2.50
Proline	—	2.02	1.67	1.82
Serine	—	1.49	1.35	1.33
Tyrosine	—	1.39	1.38	1.15
Taurine	—	0.16	0.17	0.14
Reactive lysine:total lysine	—	0.91	0.93	0.95

¹Association of American Feed Control Officials (AAFCO, 2022) nutrient profiles for adult dogs at maintenance.

AA digestibilities

Standardized AA digestibility data are presented in Table 3. All diets had statistically similar digestibility coefficients for all AA tested. For the majority of the indispensable AA, digestibilities were higher or equal to 90%, with the exception of histidine for all diets (82% to 87%), and lysine for T-FDN (87%). For the majority of the dispensable AA, digestibilities were higher than 80%, with the exception of glycine for T-FDN and H-FDN (74% to 79%) and cysteine for T-FDN and H-FDN (74% to 78%).

Table 3.

Standardized amino acid (AA) digestibilities (%) of frozen and freeze-dried raw dog foods using the precision-fed cecectomized rooster assay¹

Amino acid	Traditional freeze-dried nuggets	Hybrid freeze-dried nuggets	Frozen nuggets	SEM2	P-value
Indispensable AA
Arginine	94.13	94.24	95.62	1.87	0.8243
Histidine	81.70	85.49	86.59	3.04	0.5168
Isoleucine	91.86	91.22	93.10	2.14	0.8216
Leucine	93.68	92.43	95.28	2.03	0.6259
Lysine	86.67	91.32	91.70	2.14	0.2337
Methionine	93.76	92.75	94.92	1.42	0.5753
Phenylalanine	92.44	90.76	93.58	2.18	0.6693
Threonine	91.27	89.61	91.72	3.25	0.8910
Tryptophan	96.16	98.59	97.79	1.40	0.4885
Valine	91.92	90.15	92.91	2.50	0.7387
Selected dispensable AA
Alanine	91.58	89.74	92.59	2.01	0.6129
Aspartic acid	91.96	87.76	92.29	2.11	0.2884
Cysteine	77.68	73.92	84.03	6.98	0.6028
Glutamic acid	92.36	91.22	92.77	2.12	0.8675
Glycine	73.51	79.42	79.59	6.12	0.7337
Proline	87.17	86.67	89.84	3.41	0.7840
Serine	90.12	88.65	91.96	3.78	0.8288
Tyrosine	89.74	90.10	90.33	2.56	0.9868

¹ n = 4 roosters per treatment.

²Pooled standard error of the mean.

Metabolizable energy and digestible AA

The TME_n values were higher (P = 0.0127) for T-FDN (6.1 kcal/g) and FZN (5.9 kcal/g) than H-FDN (5.3 kcal/g) (Table 4). However, the TME_n expressed as a percentage of GE did not differ statistically among diets (80% to 86%). The modified Atwater values produced the lowest ME estimates, ranging from 4.7 to 5.6 kcal/g DMB, whereas the Atwater values resulted in slightly higher ME estimates ranging from 5.1 to 6.1 kcal/g DMB. Both underestimated the true energy content of the diets tested, with the exception for FZN (6.0 vs. 5.9 kcal/g DMB). The NRC equations yielded the highest ME estimates, ranging from 5.5 to 6.5 kcal/g. The Atwater values produced ME estimates that were closest to the TME_n values (below or above 1%) for T-FDN (0.99) and FZN (1.01). ME estimates based on Atwater ­values (0.96) and NRC equations (1.04) were 4% either below or above the TME_n value for H-FDN. The amount of digestible AA available from all diets (g/100 g; g/1,000 kcal) is presented in Table 5.

Table 4.

Nitrogen-corrected true metabolizable energy (TME_n) values and metabolizable energy (ME) estimates of frozen and freeze-dried raw dog foods using conventional roosters

Item	Traditional freeze-dried nuggets	Hybrid freeze-dried nuggets	Frozen nuggets	SEM1	P-value
TMEn, kcal/g	6.11a	5.31b	5.92a	0.15	0.0127
ME (Atwater values)2, kcal/g	6.06	5.08	6.00
ME (modified Atwater values)3, kcal/g	5.61	4.65	5.56
ME (NRC equation)4, kcal/g	6.52	5.51	6.42
TMEn/GE, %	82.07	86.06	79.74	2.03	0.1282

¹Pooled standard error of the mean.

²ME (Atwater values; kcal/g) = [(4 × CP in DM) + (9 × AHF in DM) + (4 × NFE in DM)]/100.

³ME (modified Atwater values; kcal/g) = [(3.5 × CP in DM) + (8.5 × AHF in DM) + (3.5 × NFE in DM)]/100.

⁴ME (NRC, 2006 equation): Gross energy (GE) (kcal) = (5.7 × % CP in DM) + (9.4 × % AHF in DM) + [4.1 × (% NFE + % TDF in DM)]; Energy digestibility (ED) (%): 96.6 – (0.95 × % TDF in DM); Digestible energy (DE): (kcal GE × ED)/100; ME (NRC) (kcal/g) = [kcal DE – (1.04 × % CP in DM)]/100.

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

Table 5.

Digestible amino acid (AA) concentrations of frozen and freeze-dried raw dog foods¹

	g/100g of diet			g/1,000 kcal of diet
Amino acid	T-FDN2	H-FDN2	FZN2	T-FDN	H-FDN	FZN
Indispensable AA
Arginine	2.45	2.28	2.25	4.01	5.15	3.82
Histidine	0.82	0.90	0.76	1.32	2.02	1.27
Isoleucine	1.61	1.66	1.37	2.62	3.74	2.33
Leucine	3.10	2.69	2.68	5.06	6.11	4.56
Lysine	2.64	2.74	2.42	3.93	5.82	4.00
Methionine	0.86	0.81	0.71	1.40	1.83	1.21
Phenylalanine	1.82	1.43	1.47	2.95	3.24	2.49
Threonine	1.52	1.41	1.32	2.47	3.25	2.27
Tryptophan	0.35	0.42	0.43	0.55	0.90	0.70
Valine	2.03	1.73	1.69	3.36	4.00	2.93
Selected dispensable AA
Alanine	2.25	1.83	1.98	3.68	4.18	3.38
Aspartic acid	3.20	2.88	2.79	5.24	6.59	4.77
Cysteine	0.42	0.34	0.41	0.67	0.81	0.69
Glutamic acid	4.78	4.93	4.16	7.82	11.20	7.11
Glycine	1.91	1.46	1.99	3.40	3.67	3.60
Proline	1.76	1.45	1.64	2.89	3.38	2.83
Serine	1.34	1.19	1.22	2.21	2.78	2.12
Tyrosine	1.24	1.24	1.04	2.02	2.80	1.76
Methionine–cysteine	1.27	1.15	1.12	2.07	2.64	1.91
Phenylalanine–tyrosine	3.06	2.67	2.51	4.97	6.04	4.25

¹Values are based on the measured AA digestibilities and TME_n of the dog foods tested.

²T-FDN: traditional freeze-dried nuggets, H-FDN: hybrid freeze-dried nuggets; FZN: frozen nuggets.

Discussion

Extensive industrial processing has the potential to reduce raw ingredient digestibility, initiate oxidation processes, and cause partial degradation (Van Rooijen et al., 2013; Ribeiro et al., 2019). Furthermore, it can influence the release and absorption of food constituents during digestion (Parada and Aguilera, 2007; Dalmau et al., 2017), potentially affecting AA and bioactive compound digestibility. Drying is an important preservation method in the industry, but depending on the thermal treatment and processing conditions used, it can cause chemical changes in AA that alter the structural, digestible, and functional properties of proteins (Santé-Lhoutellier et al., 2008; Reed and Park, 2011; Dima et al., 2012; Oliveira et al., 2013; Deng et al., 2014; Oba et al., 2019). Cooking raw beef for 30 min has been shown to reduce tryptophan, tyrosine, and phenylalanine concentrations, while also causing incomplete digestion of small molecular weight peptides (Kaur et al., 2014). Changes in meat protein digestibility are caused primarily by changes in protein structure (Kazemi et al., 2011). Myosin, which accounts for more than half of the myofibrillar fractions in muscle tissues (Reed and Park, 2011), is heat-sensitive and denatures at temperatures ranging from 43 to 58°C (Deng et al., 2014). Furthermore, AA and peptides are directly responsible for flavor, but can be destroyed by thermal treatment (70 to 100 °C for 8 to 15 min) (Dima et al., 2012). In the current study, T-FDN had an internal nugget temperature of 60 °C by the end of freeze-drying, and H-FDN used steam to cook part of the chicken (71 °C for 10 min) and had an internal nugget temperature of 48 °C by the end of freeze-drying. Therefore, these diets would be expected to have a greater thermal denaturation of proteins than FZN.

Freeze-drying is an alternative to traditional drying techniques for preserving the nutrients, color, flavor, and texture of the original products, while causing minimal structural changes (Nowak and Jakubczyk, 2020). It entails dehydrating frozen products by sublimating them (Hua et al., 2010). Because liquid water is not present and the process requires low temperatures, most deterioration and microbiological reactions are effectively stopped, resulting in a high-quality end product (Ratti, 2001). The types of AA present and their ability to be digested determine the protein quality of meat products (Millward et al., 2008), and a balanced diet must include all indispensable AA in appropriate concentrations and ratios to support normal growth, metabolic functions, and overall animal well-being (Case et al., 2010). With the growing popularity of RMBD among pet owners seeking to avoid over-processing, preservatives, and additives (Freeman et al., 2013; Morgan et al., 2017), it is critical for the pet industry to employ effective preservation methods that prevent protein degradation while maintaining food quality and safety.

In the present study, the impact of ingredient composition, ingredient processing, and complete diet processing of chicken-based RMBD on the AA digestibility and TME_n were tested. For the T-FDN and FZN diets, the same ingredients were used, all of which were raw, but the former diet was freeze-dried while the latter was frozen. For the H-FDN diet, ~35% of the raw chicken was replaced with steamed chicken, and pre-gelatinized sorghum was added at ~15% inclusion. Moreover, the freeze-drying methods differed between the T-FDN and H-FDN, as differing internal nugget temperatures were achieved at the end of the process. All of the diets tested had high concentrations of protein (over 35%) and fat (over 33%), low concentrations of fiber (less than 6%), as well as high energy (TME_n) content (over 5 kcal/g) and AA digestibilities (over 80% for all indispensable AA). Previously, a cooked high-protein, high-fat, low-fiber diet (31% CP in DMB, 46% AHF in DMB, and 4% TDF in DMB) was reported to have high values for both TME_n (5.8 kcal/g in DMB) and AA digestibilities (more than 80% for all indispensable AA) (Oba et al., 2020), which was consistent with the current study. Furthermore, fresh, mildly cooked, human-grade vegan diets were recently reported to have high AA digestibilities, with the majority of the indispensable AA having digestibility values >80% (Roberts et al., 2023).

Earlier research indicated that cooking chicken by steaming it at ~93 °C for 10 min, then cooling and freezing it, improved the digestibility of certain AA (e.g., histidine, cysteine, glycine, proline, and tyrosine) compared with frozen raw chicken (Oba et al., 2019). However, the digestibilities of DM, OM, AHF, and TME_n values in that study were found to be statistically similar to that of raw chicken. In the present study, the AA digestibilities were consistent across all three diets. Similarly, a study that compared the impact of various cooking methods (e.g., raw, boiled, grilled, pan-fried, roasted) determined that the AA digestibilities between raw and boiled (80°C for 15 min) beef were comparable (Hodgkinson et al., 2018). The TME_n values were greater in the frozen and freeze-dried diets when compared with the hybrid diet. The lower TME_n observed in H-FDN diet tested in the current study was primarily due to the addition of sorghum, which resulted in a lower fat content.

Studies have demonstrated that processing of commercial pet foods can lead to a reduction in the amount of reactive lysine present, potentially due to the occurrence of the Maillard reaction (van Rooijen et al., 2014). It should be noted that the total amount of lysine in the food does not necessarily equate to the amount of bioavailable (reactive) lysine. Consequently, depending on the specific processing techniques employed, the ratio of reactive lysine: total lysine in the final pet food product may be diminished. The ratio of reactive lysine: total lysine in 19 commercially available extruded diets for canine maintenance was reported to be 0.85 ± 0.16 (Williams et al., 2006). Similarly, the ratio of reactive lysine: total lysine was determined for four extruded diets, four pelleted diets, and seven dinners, resulting in ratios of 0.88 ± 0.05, 0.80 ± 0.09, and 0.83 ± 0.10, respectively (Tran et al., 2007). The high reactive lysine: total lysine ratios (> 0.90) in the diets tested in this study suggest that there was minimal damage.

Metabolizable energy estimation is a point of debate among pet food manufacturers, especially those who produce highly digestible foods. Based on high digestibility coefficients (>90%) for fat, carbohydrate, and protein (Harris, 1966), the Atwater factors of 4, 9, and 4 kcal/g for digestible carbohydrates, fats, and proteins, respectively, are commonly used to estimate ME in human foods (Atwater, 1902). In pets, modified Atwater factors were introduced by the National Research Council (NRC) nearly 40 yr ago (NRC, 1985) to compensate for the lower digestibilities of pet foods at that time, using the factors 3.5, 8.5, and 3.5 kcal/g for proteins, fats, and digestible carbohydrates, respectively. The NRC now suggests using Atwater factors for estimating the ME of highly digestible pet foods (NRC, 2006). The use of the modified Atwater factors is still the current recommendation by the Association of American Feed Control Officials (AAFCO, 2023), but these factors are known to underestimate energy content of many commercially available pet foods. In the present study, ME estimates using Atwater factors (−4% to −1%) and modified Atwater factors (−12% to −6%) underestimated the energy content for all diets tested, with the exception of the frozen diet that was similar to Atwater factors (+1%). Furthermore, the ME estimates based on the NRC equations overestimated the energy content for all diets tested in the present study (4% to 8%).

In recent studies, modified Atwater factors were shown to underestimate the energy content of fresh, mildly cooked, human-grade vegan diets and animal-based human-grade dog foods (Oba et al., 2020; Roberts et al., 2023). The TME_n values determined for the vegan diets were ~15% higher (Roberts et al., 2023), while those for most human-grade animal-based diets were over 20% higher (Oba et al., 2020) than the estimates determined by modified Atwater factors. The one exception was a high-protein, high-fat, low-fiber diet that had a TME_n value only 5% higher than that derived from the modified Atwater factors (Oba et al., 2020). The current findings and those from this study suggest that the Atwater factors are the most accurate ME estimates for minimally-processed or raw diets containing high-protein and fat concentrations and low fiber concentrations.

In summary, all diets tested in this study performed well, having digestibilities for most indispensable AA above 90%. Histidine, lysine, and threonine digestibilities did not meet that threshold, but were still found to be highly digestible. The reactive lysine:total lysine ratio was 0.91, 0.93, and 0.95 for T-FDN, H-FDN, and FZN, respectively, showing that little heat damage had occurred. TME_n values of the traditional freeze-dried and frozen diets were found to be higher than that of the hybrid freeze-dried diet (5.9 to 6.1 kcal/g DM vs. 5.3 kcal/g DM) due to the addition of sorghum and consequent lower fat content of the latter diet. The Atwater factors appeared to be the best available method for estimating the ME of the diets tested. The frozen diet had higher numerical AA digestibilities and the greatest reactive lysine:total lysine ratio, suggesting the least amount of heat damage, but all diets performed well overall. While these findings fill a gap in our knowledge, more in vivo research in dogs consuming minimally-processed is required.

Glossary

Abbreviations:

AA

amino acid

AAFCO

Association of American Feed Control Officials

AHF

acid-hydrolyzed fat

CF

crude fiber

CP

crude protein

DM

dry matter

DMB

dry matter basis

FZN

frozen nuggets

GE

gross energy

H-FDN

hybrid freeze-dried nuggets

ME

metabolizable energy

NRC

National Research Council

NFE

nitrogen-free extract

RMBD

raw meat-based diets

TDF

total dietary fiber

T-FDN

traditional freeze-dried nuggets

TME_n

nitrogen-corrected true metabolizable energy

Conflicts of interest statement

J.R.T. is employed by Primal Pet Foods. All other authors have no conflicts of interest.