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Chemical composition, true nutrient digestibility, and true metabolizable energy of chicken-based ingredients differing by processing method using the precision-fed cecectomized rooster assay

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Chicken-based ingredients are commonly used in pet food products, but vary greatly in nutrient composition and processing conditions that may affect their protein quality and digestibility. Testing the quality of protein sources undergoing different processing conditions provides important information to pet food producers. The objective of this study was to determine the chemical composition, nutrient digestibility, protein, and amino acid (AA) digestibility scores, and nitrogen-corrected true metabolizable energy (TMEn) of chicken-based ingredients that had undergone different processing conditions (i.e., chicken meal, raw chicken, retorted chicken, and steamed chicken) using the precision-fed cecectomized rooster assay. True nutrient digestibility was variable among the protein sources (60 to 76% of DM, 66 to 81% of OM, 83 to 90% of AHF, 48 to 59% of N, 50 to 95% of AA and 73 to 85% of TMEn/GE). In general, the chicken meal had a lower (P<0.05) nutrient digestibility than other ingredients tested, including DM, OM, and most indispensable and dispensable AA, with most having a true digestibility between 75 to 85%. The steamed chicken had the highest indispensable AA digestibilities, with all having a true digestibility greater than 88% and most being over 90%. TMEn value and digestible indispensable amino acid scores (DIAAS)-like values were higher (P < 0.0001) in the less processed chicken-based ingredients in comparison to chicken meal. Although animal proteins are often considered to be complete proteins, DIAAS-like values less than 100% suggest that ingredients like chicken meal may not provide all indispensable AA when included at levels to the meet minimal crude protein recommendation. Although raw protein sources are often touted as being the most digestible and of the highest quality, the steamed chicken had the highest (P < 0.0001) DIAAS-like values in this study. This study demonstrates the considerable variability exists, not only in the chemical composition but also in the true nutrient digestibility among chicken-based ingredients undergoing different processing conditions. These data justify more in vivo testing and the use of DIAAS-like values that consider AA profile, in vivo digestibility, and species-specific recommendations, to evaluate protein-based ingredients intended for use in dog and cat foods.
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1
Chemical composition, true nutrient digestibility, and true metabolizable energy of
chicken-based ingredients differing by processing method using the precision-fed
cecectomized rooster assay1
Patrícia M. Oba,, Pamela L. Utterback, Carl M. Parsons, Maria R.C. de Godoy,, and
Kelly S. Swanson,,,2
Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801; Division
of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana IL 61801; and Department of
Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana IL 61801
ABSTRACT: Chicken-based ingredients are
commonly used in pet food products, but vary
greatly in nutrient composition and processing
conditions that may affect their protein quality
and digestibility. Testing the quality of protein
sources undergoing different processing con-
ditions provides important information to pet
food producers. The objective of this study was
to determine the chemical composition, nutrient
digestibility, protein, and AA digestibility scores,
and nitrogen-corrected true metabolizable energy
(TMEn) of chicken-based ingredients that had
undergone different processing conditions (i.e.,
chicken meal, raw chicken, retorted chicken, and
steamed chicken) using the precision-fed cecect-
omized rooster assay. True nutrient digestibility
was variable among the protein sources (60% to
76% of DM, 66% to 81% of OM, 83% to 90%
of AHF, 50% to 95% of AA and 73% to 85% of
TMEn/GE). In general, the chicken meal had a
lower (P<0.05) nutrient digestibility than other
ingredients tested, including DM, OM, and
most indispensable and dispensable AA, with
most having a true digestibility between 75%
and 85%. The steamed chicken had the highest
indispensable AA digestibilities, with all having a
true digestibility greater than 88% and most being
over 90%. TMEn value and digestible indispensa-
ble AA scores (DIAAS)-like values were higher
(P<0.0001) in the less processed chicken-based
ingredients in comparison to chicken meal.
Although animal proteins are often considered to
be complete proteins, DIAAS-like values <100%
suggest that ingredients like chicken meal may
not provide all indispensable AA when included
at levels to the meet minimal crude protein rec-
ommendation. Although raw protein sources are
often touted as being the most digestible and of
the highest quality, the steamed chicken had the
highest (P < 0.0001) DIAAS-like values in this
study. This study demonstrates the considerable
variability that exists, not only in the chemical
composition but also in the true nutrient digest-
ibility among chicken-based ingredients under-
going different processing conditions. These
data justify more in vivo testing and the use of
DIAAS-like values that consider AA prole, in
vivo digestibility, and species-specic recommen-
dations, to evaluate protein-based ingredients
intended for use in dog and cat foods.
Key words: animal model, cat, dog, nutrient digestion, pet food, protein source
© The Author(s) 2018. Published by Oxford University Press on behalf of the American Society of
Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
J. Anim. Sci. 2018.XX:XX–XX
doi: 10.1093/jas/sky461
INTRODUCTION
Chicken-based ingredients are commonly used
in pet food products and are considered to have
high nutritional value (Faber et al., 2010; Deng
1Funding was provided by Freshpet, Bethlehem.
2Corresponding author: ksswanso@illinois.edu
Received September 19, 2018.
Accepted December 6, 2018.
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2Oba etal.
etal., 2016). Cooking temperature and processing
conditions may greatly affect the quality and digest-
ibility of protein-based ingredients (Kondos and
McClymont, 1974; Batterham etal., 1986; Johnson
etal., 1998). Protein denaturation in meat begins at
70°C, and at 100°C the oxidation of protein forms
aggregates that decrease enzyme activity, leading to
reduced AA digestibility (Santé-Lhoutellier etal.,
2008; Lund etal., 2011; Bax et al., 2012). Testing
the quality of protein sources undergoing different
processing conditions provides important infor-
mation to pet food manufacturers. Traditionally,
the pet food industry primarily used animal pro-
tein-based meals that had undergone the rendering
process in their formulations. Recently, there has
been an increased interest in less processed protein
sources to satisfy marketing and consumer demands
(Beaton, 2017; Food Processing, 2018; Wall, 2018a;
Wall, 2018b). Recently, it was shown that true AA
digestibility and digestible indispensable AA score
(DIAAS) values of beef topside steak was affected
by cooking conditions (Hodgkinson et al., 2018).
In that study, DIAAS was greater for raw, boiled,
and pan-fried meat treatments (97% to 99%) than
for roasted meat (91%) or grilled meat (80%). To
our knowledge, the effects of processing on the
nutrient and AA composition, true nutrient and
AA digestibility, DIAAS, and energy content of
chicken-based ingredients have not been evaluated.
The cecectomized rooster assay (CRA) has
been used frequently as a model for measuring true
nutrient and AA digestibility of feed ingredients,
including those intended for pet foods (Parsons
etal., 1982). The CRA has been used to evaluate
animal-based ingredients (Johnson et al., 1998;
Folador etal., 2006; Faber etal., 2010; Deng etal.,
2016), plant-based ingredients (Parsons etal., 1982;
Knapp etal., 2008; de Godoy etal., 2009), and raw
diets (Kerr et al., 2013). Data collected from the
CRA has been shown to have similar AA digestibili-
ties and response patterns to that of ileal-cannulated
dogs (Johnson et al., 1998). Like ileal-cannulated
animals, the CRA accurately estimates AA digest-
ibility because it minimizes the bacterial fermenta-
tion of proteins in the hindgut that adds a source
of error in the calculations (Gross et al., 2000).
Another benet of the CRA is the exibility in
type of ingredients that can be tested, varying from
complete diets to most commonly using individual
ingredients; this differs from most canine and feline
studies that require the feeding of complete and
balanced diets for longer-periods of time. Nutrient
digestibility also impacts the overall energy con-
tent of an ingredient or diet, impacting food intake
of animals and feeding guidelines determined for
complete diets. Therefore, conventional roosters are
often used to determine the nitrogen-corrected true
metabolizable energy (TMEn) of novel ingredients
for use in human and pet foods (Knapp etal., 2008;
Deng etal., 2016).
Meat, meat and bone, pork, chicken, sh, cala-
mari, lamb, venison, duck, and alligator meals were
previously evaluated using the CRA (Folador etal.,
2006; Faber et al., 2010; Deng et al., 2016), but
chicken-based ingredients differing by processing
method have not been included. Given the popu-
larity of chicken-based ingredients and minimally
processed and raw diets, research is needed to deter-
mine how the temperature and processing method
affects nutrient digestibility and energy content.
Therefore, the purpose of this study was to deter-
mine the chemical composition, nutrient digestibil-
ity, TMEn and DIAAS-like values of chicken-based
ingredients that had undergone different processing
conditions (i.e., chicken meal, raw chicken, retorted
chicken, and steamed chicken) intended for use in
dog and cat foods using the precision-fed CRA. We
hypothesized that the digestibility would be highest
for raw chicken, followed by steamed and retorted
chicken, and lowest for chicken meal.
MATERIALS AND METHODS
Substrates
Four chicken-based ingredients, including
raw chicken (frozen), steamed chicken (cooked to
~93°C and held for 10min at ~93°C, cooled, and
frozen), retorted chicken (retorted at ~121 °C for
30min, cooled, and frozen), and rendered and dried
chicken meal, were evaluated in this study. These
ingredients were provided by Freshpet (Bethlehem,
PA) and intended for use in commercial dog and cat
foods. All ingredients were treated in a manner that
is consistent with their typical pet food processing
procedures. Before analysis, frozen ingredients were
lyophilized (Dura-Dry MP microprocessor-con-
trolled freeze-dryer; FTS Systems, Stone Ridge,
NY) and ground through a 2-mm screen (Wiley
mill model 4; Thomas Scientic, Swedesboro, NJ).
Cecectomized RoosterAssay
The protocol for the CRA, including all ani-
mal housing, handling, and surgical procedures,
was reviewed and approved by the Institutional
Animal Care and Use Committee at the University
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3
Processing affects ingredient digestibility
of Illinois at Urbana-Champaign prior to experi-
mentation. Two precision-fed rooster assays uti-
lizing cecectomized Single Comb White Leghorn
roosters were conducted as described by Parsons
(1985) to determine the true nutrient digestibility,
standardized AA digestibility, and TMEn content
of the four ingredients per diets tested. Prior to the
study, cecectomy was performed on roosters under
general anesthesia according to the procedures of
Parsons (1985).
In the rst rooster assay (to determine nutri-
ent and AA digestibility), 16 cecectomized roosters
were randomly assigned to the test ingredients (four
roosters per test substrate evaluated). In the second
rooster assay (to determine TMEn), 16 conventional
roosters were randomly assigned to the test ingredi-
ents (four roosters per test substrate evaluated). In
both assays, after 24h of feed withdrawal, roosters
were tube-fed 24g of the test substrates. 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 ana-
lysis. Endogenous corrections for AA were made
using ve additional cecectomized roosters that
had been fasted for 48h. Standardized nutrient and
AA digestibilities were calculated using the method
described by Sibbald (1979).
Chemical Analyses
The substrates and rooster excreta were ana-
lyzed for DM (105°C) and OM according to AOAC
(2006). N and CP were measured using a Leco
Nitrogen/Protein Determinator (Model FP-2000,
Leco Corporation, St. Joseph, MI) according to the
AOAC (2006; method 982.30E). Fat concentrations
were determined by acid hydrolysis according to
the AACC (1983) followed by diethyl ether extrac-
tion (Budde, 1952). Total dietary ber (TDF) was
determined according to Prosky etal. (1985). GE
was measured using a bomb calorimeter (Model
1261; Parr Instrument Com., Moline, IL). AA was
measured at the University of Missouri Experiment
Station Chemical Laboratories (Columbia, MO)
according to the AOAC (2006; method 982.30E).
DIAAS-Like Calculations
Calculation of DIAAS-like values was per-
formed according to Mathai et al. (2017). The
digestible indispensable AA reference ratios were
calculated for each ingredient using the following
equation (FAO, 2011):
Digestible indispensable AA reference
ratio= digestible indispensable AA content in 1g
protein of food (mg)/mg of the same dietary indis-
pensable AA in 1g of the reference protein.
The references used included the feline and
canine nutrient recommendations suggested by
AAFCO (2018) for (i) adult maintenance and (ii)
growth and reproduction, and the recommended
allowances suggested by the National Research
Council (NRC, 2006) for (i) adult dogs at main-
tenance, (ii) adult cats at maintenance, (iii) grow-
ing puppies (4 to 14 wk old), and (iv) growing
kittens. The DIAAS-like values were then calcu-
lated using the following equation adapted from
FAO (2011):
DIAAS-like %=100× [(mg of digestible diet-
ary indispensable AA in 1g of the dietary protein) /
(mg of the minimum recommendation of the same
dietary indispensable AA in 1g of the minimum
protein recommendation)].
Nitrogen-Corrected TMEn Calculations
Calculation of TMEn was performed accord-
ing to Parsons etal. (1992). The TMEn values, cor-
rected for endogenous energy excretion using many
fasted birds over many years, were calculated using
the following equation:
TMEkcalg EI EE 822N
EE 822N
nfed fe
df
ed
fasted faste
/(.* )
(.*
()
=− ±
±± dd )/FI
In that equation, EIfed equals the GE intake of
the test substrate consumed; EEfed equals the energy
in the excreta collected from fed birds; 8.22 is the
correction factor for uric acid; Nfed equals the grams
of N retained by the fed birds; EEfasted equals the
energy in the excreta collected from the fasted birds
(16.74 kcal/g); Nfasted equals the g N retained by the
fasted birds (1.1256g); and FI equals the grams of
dry test substrate consumed Parsons etal. (1982).
Statistical Analyses
All data were analyzed as a completely ran-
domized design using the GLM procedure of
Statistical Analysis Systems 9.3 (SAS Inst., Cary,
NC). Substrates were considered to be a xed effect.
Tukey’s multiple comparison analysis was used to
separate the means when interaction effect was sig-
nicant according to the procedures of SAS (SAS
Inst. Inc., Cary, NC). Differences were considered
signicant with P<0.05.
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4Oba etal.
RESULTS
Chemical Composition
The chemical composition of tested ingre-
dients is presented in Table 1. Of note, the DM
content listed for the raw, steamed, and retorted
chicken ingredients were the values present after
the freeze-drying process, which was needed to
properly conduct the chemical analyses and dosing
for the rooster experiments. All other nutrients are
represented on a DM basis (DMB). Ash content
was fairly similar for raw chicken, steamed chicken,
and retorted chicken (5.84% to 7.62% DMB), but
higher in the chicken meal (16.29%DMB).
CP concentration increased as the processing
of chicken products increased, most likely due
to losses in fat. The chicken meal had the highest
CP (67.42% DMB) and AA of all chicken-based
ingredients, with raw chicken having the lowest,
and retorted and steamed chicken being interme-
diate. The retorted chicken had the lowest TDF
concentration (0.11% DMB), while chicken meal
had the highest (6.65% DMB). Raw chicken,
steamed chicken, and retorted chicken had simi-
lar acid-hydrolyzed fat (AHF) content (41.80% to
52.44% DMB) but was lower in the chicken meal
(15.73%DMB).
The chicken meal had the lowest GE (5.1 kcal/g
DM), with the raw, steamed, and retorted chick-
en-based ingredients being higher (6.6 to 7.0 kcal/g
DM) due to greater AHF and CP concentrations.
Concentrations of indispensable and dispensable
AA are presented in Table2. AA prole was similar
among the protein sources. The chicken meal had
higher AA concentrations, with the exception of
histidine, lysine, methionine, and tryptophan, and
raw chicken had lower AA composition. Retorted
and steamed chicken had similar and higher con-
centrations of histidine, lysine, methionine, and
tryptophan than raw chicken and chickenmeal.
Cecectomized RoosterAssay
The nutrient digestibility for DM and OM
was similar among raw chicken, steamed chicken,
and retorted chicken (73.49% to 76.46% DM and
77.78% to 80.56% OM), and greater (P < 0.01)
than chicken meal (60.05% DM and 65.87% OM;
Table1). TMEn values were higher for raw chicken,
steamed chicken, and retorted chicken (5.34 to
5.92 kcal/g) than chicken meal and were not sta-
tistically different from each other. The chicken
meal had the lowest caloric value (3.72 kcal/g) of
all chicken-based ingredients (P < 0.0001). The
TMEn expressed as a percentage of GE was higher
(P=0.0158) for raw chicken and retorted chicken
than chicken meal, with steamed chicken being
intermediate.
Standardized AA digestibility data are pre-
sented in Table 3. For all indispensable and dis-
pensable AA, the steamed chicken had the highest
digestibilities. In that ingredient, all indispensa-
ble AA had a digestibility greater than 88%. For
all indispensable AA and the majority of the dis-
pensable AA, raw and retorted chicken digestibil-
ities were similar to one another and higher than
that of chicken meal. Lysine, valine, histidine, and
Table1. Chemical composition (%, DM basis), true macronutrient digestibility and nitrogen-corrected true
ME (TMEn) of chicken-based ingredients using the precision-fed cecectomized rooster assay1
Item Chicken meal Retorted chicken Steamed chicken Raw chicken SEM P-value
Chemical composition
CP, % 67.42 55.56 52.97 41.72
N, % 10.79 8.89 8.48 6.68 — —
AHF, % 15.73 41.80 44.77 52.44
TDF, % 6.65 0.11 1.06 3.07
GE, kcal/g 5.09 6.68 6.59 6.98
Nutrient digestibility
DM, % 60.05b73.49a76.46a75.91a2.329 0.0012
OM, % 65.87b77.78a80.56a80.51a1.927 0.0006
AHF, % 90.34 83.45 86.46 88.25 2.185 0.2711
Nitrogen-corrected true ME
TMEn, kcal/g 3.72b5.52a5.34a5.92a0.126 <0.0001
TMEn/GE, % 73.14b82.68a81.05ab 84.83a1.880 0.0158
a,bMeans with different superscripts within a row differ (P<0.05).
1n=4 roosters per treatment.
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5
Processing affects ingredient digestibility
threonine had digestibilities <80% for chicken
meal. For proline, the steamed chicken had a
higher (P = 0.0117) digestibility than that of raw
chicken and chicken meal, which were similar to
one another. For glycine, the raw chicken had a
lower (P=0.004) digestibility than all other chick-
en-based ingredients. For aspartic acid, chicken
meal and retorted chicken had a lower (P<0.0001)
digestibility than that of steamed chicken and raw
chicken. Raw chicken had a higher digestibility of
Table2. Concentrations (%, DM basis) of indispensable AA and select dispensable AA in chicken-based
ingredients intended for pet foods
Item Chicken meal Retorted chicken Steamed chicken Raw chicken
Indispensable AA
Arginine, % 4.19 3.31 3.41 2.22
Histidine, % 1.36 1.66 1.55 1.02
Isoleucine, % 2.33 2.34 2.35 1.75
Leucine, % 4.17 3.91 3.94 2.87
Lysine, % 3.94 4.20 4.29 2.94
Methionine, % 1.13 1.26 1.29 0.87
Phenylalanine, % 2.25 1.95 2.00 1.42
Threonine, % 2.30 2.11 2.16 1.59
Tryptophan, % 0.53 0.56 0.58 0.44
Valine, % 2.88 2.51 2.53 1.88
Selected dispensable AA
Alanine, % 4.23 3.12 3.08 2.01
Aspartic acid, % 4.86 4.40 4.49 3.22
Cysteine, % 0.69 0.42 0.52 0.36
Glutamic acid, % 7.68 6.89 6.88 4.57
Glycine, % 6.57 3.38 3.30 1.70
Proline, % 3.98 2.37 2.23 1.29
Serine, % 2.11 1.69 1.76 1.27
Tyrosine, % 1.41 2.09 1.89 1.24
Taurine, % 0.39 0.15 0.12 0.11
Table3. Standardized digestibility (%) of indispensable AA and select dispensable AA of chicken-based
ingredients using the precision-fed cecectomized rooster assay1
Item Chicken meal Retorted chicken Steamed chicken Raw chicken SEM P-value
Indispensable AA
Arginine, % 85.56b89.29ab 92.62a88.89ab 1.166 0.0120
Histidine, % 76.94c83.34ab 87.83a79.75bc 1.355 0.0013
Isoleucine, % 82.30b88.33a91.90a90.76a0.885 0.0001
Leucine, % 82.90b88.31a92.10a90.74a0.917 0.0004
Lysine, % 78.78b84.68ab 91.02a86.57ab 2.038 0.0133
Methionine, % 85.95c90.63b94.79a93.35ab 0.649 <0.0001
Phenylalanine, % 80.95b86.68a90.63a88.84a1.088 0.0016
Threonine, % 75.32b82.94a88.02a84.64a1.492 0.0011
Tryptophan, % 89.60b93.78a95.36a94.24a0.569 <0.0001
Valine, % 78.60b84.97a89.09a86.27a1.261 0.0017
Selected dispensable AA
Alanine, % 82.34b88.15a91.73a88.82a1.124 0.0023
Aspartic acid, % 66.14b73.30b90.36 a 88.85a2.038 <0.0001
Cysteine, % 52.44b52.90ab 68.37a50.22b3.443 0.0178
Glutamic acid, % 80.24b87.20a90.80a87.41a1.201 0.0006
Glycine, % 72.89a73.56a73.78a57.58b2.626 0.0040
Proline, % 77.09b85.61ab 86.66a77.64b1.853 0.0117
Serine, % 71.02b78.70ab 84.38a79.67ab 1.896 0.0059
Tyrosine, % 78.20c86.49ab 90.42a84.18b0.965 <0.0001
a–cMeans with different superscripts within a row differ (P<0.05).
1n=4 roosters per treatment.
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6Oba etal.
alanine (P = 0.0023), aspartic acid (P < 0.0001),
and glutamic acid (P= 0.0006), but lower digest-
ibility of cysteine (P=0.0178), glycine (P=0.004),
proline (P = 0.0117), and tyrosine (P < 0.0001)
compared to steamed chicken.
DIAAS-Like Calculations
DIAAS-like values for adult dogs and cats at
maintenance are presented in Tables4 and 6, respec-
tively. DIAAS-like values for growing puppies and
kittens are presented in Tables5 and 7, respectively.
Using these calculations, the rst-limiting AA for
adult dogs was methionine or tryptophan, depend-
ing on the ingredient.
Using the AAFCO recommended allowances
for adult dogs, chicken meal was the only protein
source that did not meet 100% DIAAS-like values
for all AA (methionine, tryptophan, and threo-
nine). Using the NRC recommended allowances for
adult dogs, all protein sources had some DIAAS-
like values <100%. Steamed chicken had the most
DIAAS-like values for indispensable AA over 100%
(arginine, histidine, isoleucine, leucine, and lysine),
followed by raw chicken (arginine, histidine, isoleu-
cine, and lysine), retorted chicken (arginine, histi-
dine, and lysine), and chicken meal (arginine and
lysine).
Using the AAFCO recommended allowances
for canine growth and reproduction and NRC rec-
ommended allowances for growing puppies, the
rst-limiting AA was threonine for almost all pro-
tein sources. The exception was chicken meal when
using the NRC recommended allowances a refer-
ence, where tryptophan was the rst-limiting AA.
Using AAFCO recommendations as a reference,
steamed chicken and retorted chicken had DIAAS-
like values below 100% for only threonine and
phenylalanine, while raw chicken had DIAAS-like
values below 100% for threonine, phenylalanine,
and histidine. Chicken meal, however, had DIAAS-
like values below 100% for seven indispensable
AA, with only arginine, lysine, and valine being
sufcient. Similar comparisons were observed
when using NRC recommended allowances as a
reference, with steamed chicken (threonine), raw
chicken (threonine; tryptophan), and retorted
chicken (threonine; tryptophan) having DIAAS-
like values <100% for only one or two indispensable
AA. Similar to using AAFCO references, chicken
meal had DIAAS-like values below 100% for seven
indispensable AA (only arginine, lysine, and valine
were sufcient) when using NRC as a reference.
For adult cat AAFCO and NRC recommended
allowance references, threonine from the chicken
meal was the only DIAAS-like value <100%.
Using the AAFCO recommended allowances for
feline growth and reproduction, methionine was
the rst-limiting AA and had a DIAAS-like value
<100% for chicken meal, retorted chicken, and
raw chicken. When using AAFCO as a reference,
tryptophan also had a DIAAS-like value <100%
for chicken meal. Using NRC recommended allow-
ances of growing kittens as a reference, methionine
was the rst-limiting AA and had a DIAAS-like
value <100% for chicken meal. When using NRC as
Table 4. Digestible indispensable AA scores1 values of chicken-based ingredients for adult dogs at
maintenance2
AAFCO NRC
Item
Chicken
meal
Retorted
chicken
Steamed
chicken
Raw
chicken SEM
Chicken
meal
Retorted
chicken
Steamed
chicken
Raw
chicken SEM
Arginine, % 187.66b187.75b210.44a166.94c2.500 151.92b151.99b170.36a135.14c2.023
Histidine, % 147.03c235.90a243.47a184.71b3.255 81.68c131.06a135.26a102.62b1.808
Isoleucine, % 134.73c176.21b193.13a180.34b1.645 74.85c97.89b107.30a100.19b0.914
Leucine, % 135.73c164.50b181.35a165.24b1.636 75.41c91.39b100.75a91.80b0.909
Lysine, % 131.54c182.90b210.62a174.30b4.143 131.54c182.90b210.62a174.30b4.143
Methionine, % 78.58d112.10b125.91a106.17c0.721 43.65d62.28b69.95a58.98c0.401
Phenylalanine, % 108.06c121.69b136.87a120.94b1.518 60.03c67.61b76.04a67.19b0.843
Threonine, % 96.36c118.12b134.59a120.97b2.083 59.76c73.25b83.47a75.01b1.292
Tryptophan, % 79.24d106.34c117.46a111.82b0.633 50.31d67.51c74.58a70.99b0.401
Valine, % 123.34c141.01b156.32a142.81b2.082 68.52c78.34b86.84a79.34b1.157
a–dMeans with different superscripts within a row and guidelines (AAFCO or NRC) differ (P<0.05); n=4 roosters per treatment.
1DIAAS-like %=100× [(mg of digestible dietary indispensable AA in 1g of the dietary protein)/(mg of the minimum recommendation of the
same dietary indispensable AA in 1g of the minimum protein recommendation)].
2DIAAS-like values were calculated from the ileal digestibility of AA in cecectomized roosters and Association of American Feed Control
Ofcials (AAFCO, 2018) recommended allowances and National Research Council (NRC, 2006) minimal requirements of AA for adult dogs at
maintenance. The indispensable AA reference patterns are expressed as gram AA per kilogram DM.
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7
Processing affects ingredient digestibility
a reference, threonine also had a DIAAS-like value
<100% for chicken meal. All other DIAAS-like val-
ues were above100%.
In general, steamed chicken had the high-
est (P< 0.0001) and chicken meal had the lowest
(P<0.0001) DIAAS-like values for all indispensa-
ble AA, while retorted chicken and raw chicken had
intermediate values. The one exception was argin-
ine, whereby raw chicken typically had the lowest
(P<0.0001) DIAAS-like value, but was still above
100% in all protein sources.
DISCUSSION
Pet owners have become more interested in raw
and less-processed ingredients and diets recently
(Schlesinger and Joffe, 2011; Freeman etal., 2013;
Parr and Remillard, 2014; Wall, 2018b; Wall,
2018a). As a result, pet food companies have started
the commercialization of raw foods and diets using
mild processing methods (Parr and Remillard,
2014). It is known that ash content and processing
temperature can affect AA digestibility (Kondos and
Table5. Digestible indispensable AA scores1 values of chicken-based ingredients for growing puppies after
weaning2
AAFCO NRC
Item Chicken meal Retorted chicken Steamed chicken
Raw
chicken SEM
Chicken
meal
Retorted
chicken
Steamed
chicken
Raw
chicken SEM
Arginine, % 119.65b119.70b134.17a106.43c1.595 151.44b151.51b169.82a134.72c2.018
Histidine, % 79.35c127.30a131.39a99.67b1.757 89.55c143.69a148.30a112.50b1.983
Isoleucine, % 90.12c117.87b129.19a120.63b1.102 98.46c128.77b141.14a131.78b1.203
Leucine, % 89.44c108.40b119.50a108.89b1.078 89.44c108.40b119.50a108.89b1.078
Lysine, % 115.09c160.04b184.29a152.51b3.625 117.71c163.68b188.49a155.98b3.709
Methionine, % 92.58d132.09b148.35a125.10c0.849 98.20d140.10b157.35a132.69c0.901
Phenylalanine, % 73.23c82.46b92.76a81.96b1.029 93.51c105.30b118.44a104.66b1.314
Threonine, % 55.59c68.15b77.65a69.79b1.202 71.38c87.50b99.70a89.60b1.543
Tryptophan, % 79.24d106.34c117.46a111.82b0.633 68.92d92.49c102.16a97.25b0.550
Valine, % 111.10c127.02b140.81a128.64b1.876 111.10c127.02b140.81a128.64b1.876
a–dMeans with different superscripts within a row and guidelines (AAFCO or NRC) differ (P<0.05); n=4 roosters per treatment.
1DIAAS-like %=100× [(mg of digestible dietary indispensable AA in 1g of the dietary protein)/(mg of the minimum recommendation of the
same dietary indispensable AA in 1g of the minimum protein recommendation)].
2DIAAS-like values were calculated from the ileal digestibility of AA in cecectomized roosters and Association of American Feed Control
Ofcials (AAFCO, 2018) recommended allowances of AA for canine growth and reproduction and National Research Council (NRC, 2006) mini-
mal requirements of AA for growing puppies after weaning. The indispensable AA reference patterns are expressed as gram AA per kilogram DM.
Table6. Digestible indispensable amino acid scores1 values of chicken-based ingredients for adult cats at
maintenance2
AAFCO NRC
Item
Chicken
meal
Retorted
chicken
Steamed
chicken
Raw
chicken SEM
Chicken
meal
Retorted
chicken
Steamed
chicken
Raw
chicken SEM
Arginine, % 132.92b132.99b149.07a118.24c1.772 138.11b138.17b154.87a122.85c1.840
Histidine, % 130.17c208.84a215.55a163.52b2.882 119.38c191.54a197.69a149.98b2.642
Isoleucine, % 142.21c186.00b203.86a190.36b1.737 132.29c173.02b189.64a177.08b1.616
Leucine, % 107.52c130.30b143.64a130.89b1.296 100.54c121.85b134.33a122.40b1.212
Lysine, % 144.21c200.53b230.92a191.10b4.543 270.81c376.57b433.64a358.85b8.530
Methionine, % 187.28d267.18b300.09a253.06c1.718 169.48d241.79b271.57a229.01c1.554
Phenylalanine, % 175.59c197.74b222.41a196.54b2.467 135.07c152.11b171.09a151.18b1.898
Threonine, % 91.52c112.18b127.83a114.89b1.978 98.83c121.15b138.04a124.07b2.136
Tryptophan, % 114.45d153.60c169.67a161.51b0.913 108.36d145.42c160.63a152.91b0.865
Valine, % 140.80c160.98b178.45a163.03b2.377 131.67c150.54b166.88a152.45b2.223
a–dMeans with different superscripts within a row and guidelines (AAFCO or NRC) differ (P<0.05); n=4 roosters per treatment.
1DIAAS-like %=100× [(mg of digestible dietary indispensable AA in 1g of the dietary protein)/(mg of the minimum recommendation of the
same dietary indispensable AA in 1g of the minimum protein recommendation)].
2DIAAS-like values were calculated from the ileal digestibility of AA in cecectomized roosters and Association of American Feed Control
Ofcials (AAFCO, 2018) recommended allowances and National Research Council (NRC, 2006) minimal requirements of AA for adult cats at
maintenance. The indispensable AA reference patterns are expressed as gram AA per kilogram DM.
Downloaded from https://academic.oup.com/jas/advance-article-abstract/doi/10.1093/jas/sky461/5238082 by guest on 17 January 2019
8Oba etal.
McClymont, 1974; Batterham etal., 1986; Johnson
et al., 1998; Hodgkinson etal., 2018). Processing
temperature has been shown to greatly affect AA
digestibility in animal meals, with high tempera-
tures (≥150°C) reducing digestibility (Kondos and
McClymont, 1974; Batterham et al., 1986; Wang
et al., 1997; Johnson et al., 1998; Hodgkinson
et al., 2018). Protein denaturation in meat begins
at 70°C and, at 100 °C the oxidation of protein
forms aggregates, leading to reduced AA digestibil-
ity (Bax etal., 2012). This occurs because the myo-
brillar proteins bound with the AA form carbonyl
groups on the side chains of arginine, lysine, and
proline (Santé-Lhoutellier etal., 2008; Lund etal.,
2011), disulde cross-linkages in S-containing AA
(cysteine and methionine), and di-tyrosine cross
linkages (Lund et al., 2011). These changes are
thought to have negative effects on enzyme action,
decreasing AA digestibility. Additionally, cooking
meat at 90°C for 30min was shown to have lower
true ileal digestibility of protein than cooking at
55°C for 5min (90.1% vs. 94.1%; P=0.08) (Oberli
et al., 2015), which demonstrates that even with
lower cooking temperatures (<150°C), it is possible
to have losses in protein digestibility. The response
to ash content appears to be more variable; how-
ever, it did not have a negative effect on AA digest-
ibility in roosters (Johnson etal., 1998) or protein
digestibility in pigs (Partanen, 1994). Because of
differences in raw materials and processing method,
the AA composition may be variable among animal
protein sources, within and across animal species
and ingredient categories.
In the current study, the chicken meal had the
highest ash, CP, and TDF content and the lowest
AHF. This was probably due to the processing of
this ingredient, where the fat is extracted to be sold
as chicken fat. Compared with the chicken meal
evaluated by Deng etal. (2016), this protein source
had a similar chemical composition, but the ash
content was higher in the present study (16.29%
vs. 11.8% DMB), perhaps because the animal esh
of the chicken meal in the present study had more
pieces of bone compared with that tested in the
Deng study. Additionally, while the nutrient digest-
ibility was similar, DM (64.5% vs. 60.05%) and
OM (75.7% vs. 65.87%) digestibilities were slightly
higher in the Deng etal. (2016) study. The chemical
composition of chicken breast evaluated by Faber
etal. (2010) had lower ash and AHF, but higher CP
than all of the chicken-based products tested in the
present study. However, it is important to note that
the chicken breast used on Faber etal. (2010) was
a prime meat cut, used for human grade, and the
ingredients used in the present study may add other
parts of the chicken carcass or meat cuts besides the
chicken breast, with could explain the differences in
the chemical composition.
The TMEn value of the chicken meal (3.72
kcal/g) in the present study was similar to that of the
chicken meal (3.49 kcal/g) reported by Deng etal.
(2016) and low-ash poultry byproduct meal (PBP)
(3.805 kcal/g) reported by Johnson et al. (1998).
Furthermore, the retorted chicken (5.52 kcal/g) in
the present study had similar TMEn values to the
ground chicken (5.53 kcal/g) reported by Kerr etal.
Table7. Digestible indispensable AA scores values1 of chicken-based ingredients for growing kittens after
weaning2
AAFCO NRC
Item
Chicken
meal
Retorted
chicken
Steamed
chicken
Raw
chicken SEM
Chicken
meal
Retorted
chicken
Steamed
chicken
Raw
chicken SEM
Arginine, % 128.65b128.71b144.27a114.44c1.714 138.47b138.53b155.28a123.17c1.845
Histidine, % 141.09c226.37a233.63a177.25b3.123 117.57c188.64a194.70a147.70b2.602
Isoleucine, % 152.35c199.25b218.38a203.92b1.861 131.68c172.22b188.76a176.26b1.609
Leucine, % 120.17c145.64b160.55a146.30b1.448 100.15c121.38b133.80a121.92b1.206
Lysine, % 115.09c160.04b184.29a152.51b3.625 135.40c188.28b216.81a179.43b4.266
Methionine, % 69.69d99.43b111.68a94.17c0.640 81.85d116.78b131.15a110.60c0.750
Phenylalanine, % 155.88c175.54b197.45a174.48b2.189 135.07c152.11b171.09a151.18b1.898
Threonine, % 105.61c129.46b147.52a132.58b2.283 98.83c121.15b138.04a124.07b2.136
Tryptophan, % 84.52d113.43c125.30a119.27b0.675 110.05d147.69c163.14a155.30b0.878
Valine, % 157.41c179.97b199.50a182.26b2.658 131.15c149.95b166.22a151.86b2.214
a–dMeans with different superscripts within a row and guidelines (AAFCO or NRC) differ (P<0.05); n=4 roosters per treatment.
1DIAAS-like %=100× [(mg of digestible dietary indispensable AA in 1g of the dietary protein)/(mg of the minimum recommendation of the
same dietary indispensable AA in 1g of the minimum protein recommendation)].
2DIAAS-like values were calculated from the ileal digestibility of AA in cecectomized roosters and Association of American Feed Control
Ofcials (AAFCO, 2018) recommended allowances of AA for feline growth and reproduction and National Research Council (NRC, 2006) mini-
mal requirements of AA for growing kittens after weaning. The indispensable AA reference patterns are expressed as gram AA per kilogram DM.
Downloaded from https://academic.oup.com/jas/advance-article-abstract/doi/10.1093/jas/sky461/5238082 by guest on 17 January 2019
9
Processing affects ingredient digestibility
(2014). The variation in energy content among pro-
tein sources may result from the processing pro-
cedures of producing meals, including cooking,
pressing, drying, and milling (Deng et al., 2016).
High-energy and high-quality protein sources are
important when formulating diets because it allows
for a lower amount of protein compared to those
containing low energy and/or AA concentrations.
Compared with the concentrations of all
indispensable AA reported by Deng et al. (2016)
for chicken meal and by Johnson etal. (1998) for
high- and low-ash PBP, the chicken meal tested in
the present study was similar. However, the chicken
breast from the Faber etal. (2010) study had higher
indispensable AA concentrations than all of the
chicken-based ingredients tested in the present
study. The ground chicken reported by Kerr et al.
(2014) had similar concentrations of indispensable
AA with the chicken meal, retorted chicken, and
steamed chicken tested in the present study. For the
dispensable AA, the chicken meal tested by Deng
etal. (2016) had similar concentrations of aspartic
acid, glutamic acid, and glycine to the chicken meal
tested in the present study. The PBP with high-ash
content tested by Johnson etal. (1998) study had a
similar concentration of alanine, glycine, and tyros-
ine to the chicken meal tested in the present study,
and the low-ash PBP had similar concentrations of
alanine and proline compared to the chicken meal
tested in the present study. The chicken breast tested
by Faber etal., (2010) had higher concentrations
of the majority of the dispensable indispensable
AA, but lower taurine than all of the chicken-based
ingredients tested in the present study. Additionally,
glycine and proline concentrations were similar to
the retorted and steamed chicken, but lower than
the chicken meal tested in the presentstudy.
Donadelli et al. (2018) used chicks and the
protein efciency ratio (PER) assay, which ranks
protein source based on AA composition (Cramer
etal., 2007), to evaluate protein-based ingredients
intended for pet foods. In that study, diets con-
tained 10% CP from a novel protein source and
demonstrated that spray-dried egg (SDEG), spray-
dried inedible whole egg and low-temperature and
pressure uid bed dried chicken (LTPC) had the
highest PER, low-temperature uid bed air-dried
chicken (LTCK) and spray-dried chicken (SDCK)
were intermediate, and the chicken by-product
meal (CBPM) and chicken meal (CKML) were the
lowest. Additionally, CBPM had lower methionine
than SDEG and CKML had lower tryptophan.
Phenylalanine was the limiting AA for LTCK and
SDCK, and while valine was limiting for LTPC
(Donadelli et al., 2018). Similar to our study,
chicken meal had the lowest performance of all
animal protein sources tested, which suggests that
the processing by which the chicken meal is sub-
jected to negatively affects the protein quality of
this ingredient.
Nutrient composition does not correlate with
in vivo digestibility (Moughan, 1999; Ravindran
and Bryden, 1999; Butts etal., 2012), stressing the
need for animal testing. Testing nutrient digestibil-
ity of protein sources is needed to verify adequacy
and improve diet quality. The CRA or ileal-cannu-
lated dogs may be used to evaluate the quality of
proteins without the inuence of gut microbiota in
the large intestine. Ileal cannulation in cats is not
recommended due to complications with cannu-
lation, including displacement and leakage, with
subsequent abscess and skin inammation, and dif-
culty in obtaining sufcient sample size (0.5mL
sample of ileal uid requires ~3h) (Mawby etal.,
1999). Because of the issues with cannulation in
cats and the cost and animal welfare concerns per-
taining to ileal-cannulated dogs, the cecectomized
rooster is a popular model and appropriate alterna-
tive. Additionally, a previous study reported a high
correlation between cecectomized rooster and ile-
al-cannulated dog data (Johnson etal., 1998).
For all AA in the current study, the digestibil-
ity was highest for steamed chicken. Each AA had
a digestibility >90% in steamed chicken, except
for two dispensable AA (cysteine and glycine).
Therefore, it appears that steamed chicken was the
most easily hydrolyzed and absorbed. In contrast,
standardized indispensable AA digestibility data
were lowest for chickenmeal.
When compared with the digestibility data
of chicken meal reported by Deng et al. (2016),
the chicken meal in the current study had similar
responses, but higher isoleucine, lysine, methionine,
tryptophan, and glutamic acid digestibilities. The
ground chicken reported by Kerr etal. (2014) had
similar AA digestibilities to the steamed chicken
in the present study, with the exceptions of histi-
dine and threonine that were more digestible in the
ground chicken. Compared with the high- and low-
ash PBP reported by Johnson etal. (1998), all AA
digestibilities with the exception of cysteine, gly-
cine, and serine were higher in chicken meal from
the present study, but similar to PBP with high-ash
content. It was reported that increased ash con-
tent had a negative effect on AA digestibility and
protein efcient ratio (Cramer et al., 2007). In
agreement with that study, the chicken meal in the
current study, which had the greatest ash content
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10 Oba etal.
among all ingredients, had the lowest indispensable
and dispensable AA digestibilities. The variability
of digestibility among different chicken meals can
be due to the different tissues included in the meals,
processing methods, variation in analytical proce-
dures, experimental design, and animal models.
The digestibility of the chicken breast reported
by Faber etal. (2010) was similar to the retorted
and raw chicken digestibility of the present study.
However, cysteine had a much lower digestibility
value in the present study (50.2% raw chicken and
52.9% retorted chicken) compared to that of Faber
et al. (2010; 82.0% chicken breast). Cysteine is a
component of keratin and other brous proteins,
and typically has a lower digestibility (Kerr etal.,
2014). One must consider the low digestibility of
cysteine (range from 50.22% to 68.37% in present
study) and low methionine DIAAS-like value for
chicken meal (78.10) when formulating diets, espe-
cially when using meals that have undergone ren-
dering and drying processes due to the fact that
cysteine is supported by the metabolism of methio-
nine (Weichselbaum et al., 1932). If an ingredient
has a low cysteine digestibility plus a methionine
DIAAS-like value that does not meet 100%, sup-
plementation of methionine or cysteine may be
required to prevent deciency.
Recent studies have supported the use of
DIAAS-like values to estimate protein qual-
ity of ingredients and diets for humans (Mathai
et al., (2017) and we believe that this an appro-
priate method to score AA and determine protein
quality of ingredients used in pet foods. Using
the DIAAS-like values, our data suggest that if
chicken meal is used as the only source of protein
in a diet formulation, it may not provide sufcient
methionine (DIAAS-like value= 78.58%), trypto-
phan (DIAAS-like value=79.24%), and threonine
(DIAAS-like value=96.36%) when adult dogs, and
threonine (DIAAS-like value= 91.52%) for adult
cats if diets are formulated to meet the AAFCO rec-
ommendations. If NRC recommendations are used
as a reference, chicken meal only provides sufcient
arginine (DIAAS-like value=151.92%) and lysine
(DIAAS-like value=131.54%) for adult dogs, and
does not provide sufcient threonine (DIAAS-like
value=98.83%) for adult cats. For puppies, chicken
meal was also the lowest quality protein source, only
providing sufcient (over 100% DIAAS-like value)
for arginine (DIAAS-like value = 119.65% and
151.44% for AAFCO and NRC references, respec-
tively), lysine (DIAAS-like value = 115.09% and
117.71% for AAFCO and NRC references, respec-
tively), and valine (DIAAS-like value = 111.10%
for AAFCO and NRC). Similar data were observed
when using growing kittens as a reference, with
chicken meal having DIAAS-like values below 100%
for methionine (81.85%) and threonine (98.83%)
when compared to NRC recommendations. When
compared to AAFCO recommendations, chicken
meal (69.69%), raw chicken (94.17%), and retorted
chicken (99.43%) did not meet sufcient (DIAAS-
like values of at least 100%) for methionine, and
chicken meal did not meet 100% for tryptophan
(DIAAS-like value=84.52%). Stated another way,
our data indicate that if the chicken meal has a low
digestibility, it may not meet the minimal recom-
mendations for indispensable AA without supple-
mentation, especially if the diet is formulated to
meet the minimal protein requirement for the dog
or cat. Although many consider animal-based pro-
teins to be complete proteins not requiring add-
itional supplementation, our data demonstrate that
supplementation may be required when formulat-
ing with meals that have undergone extensive heat
processing.
Our results corroborate those of a recent study
evaluating how cooking conditions affect the pro-
tein quality of beef topside steak, as indicated by
true ileal digestible AA and DIAAS determined
using a pig model (Hodgkinson et al., 2018).
Similar to our study, the less processed protein
(boiled: ≤80 °C intermediate cooking duration;
pain-fried: 186°C and shortest duration compared
with the other meat protein sources) had greater
ileal digestible AA concentrations than the more
processed proteins (roasted: 160 °C and longest
cooking duration; grilled: 225 °C and intermedi-
ate cooking duration). In that study, valine was the
limiting AA, with DIAAS values for boiled (99%),
pan-fried (98%) and raw meat (97%) being higher
than roasted meat (91%) and grilled meat(80%).
Steamed chicken appears to be the best option
of those tested in the current study, followed by raw
chicken and retorted chicken. This evidence suggests
that cooking provides benets over raw chicken
because of greater AA availability. Even though the
raw chicken tested had intermediate digestibilities,
it was a good source of indispensableAA.
In conclusion, this study provides the true
nutrient digestibility data of four chicken-based
ingredients intended for use in dog and cat foods.
To our knowledge, it is also the rst study to use
DIAAS-like values to predict protein quality for
use in pet food. The protein sources varied greatly
in ash, CP, N, and fat content. According to our
data, the chicken meal has the lowest nutrient and
AA digestibilities and may not be sufcient if used
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11
Processing affects ingredient digestibility
as the sole protein source, when the cat or dog is
fed to meet the minimum protein recommendations
of AAFCO or NRC. In contrast, AA digestibility
was highest for steamed chicken, out-performing
all other protein sources tested. AA digestibili-
ties of raw and retorted chicken-based proteins
were slightly lower than that of steamed chicken,
but still high-quality proteins. This study demon-
strates the importance of in vivo testing to evalu-
ate protein-based ingredients, as raw material and
processing methods can greatly affect their protein
quality and energy content. Our study also justi-
es the use of DIAAS-like calculations to evaluate
protein-based ingredients for use in pet foods and
complete diets because they not only consider AA
prole but also digestibility data and species-spe-
cic nutrient recommendations. Future studies
should evaluate the nutrient digestibility of these
ingredients as the main protein ingredients in com-
plete and balanced diets for dogs and cats.
Conict of interest statement. None declared.
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... The precision-fed rooster assay has proven to be an accurate model for companion animal nutrition based on its correlation to results from ileal-cannulated dogs Downloaded from https://academic.oup.com/tas/article-abstract/doi/10.1093/tas/txaa133/5874194 by guest on 21 July 2020 A c c e p t e d M a n u s c r i p t (Johnson et al., 1998). Although the use of DIAAS-like scores to evaluate ingredients used in pet foods is only just emerging (Oba et al., 2019;Do et al., 2020), it is the recommended method of evaluating protein quality according to the FAO Expert Consultation (FAO, 2013). ...
... These determined the DIAAS-like scores for raw chicken, retorted chicken, steamed chicken, and chicken meal. When using the AAFCO recommended values for adult dogs as the reference protein, methionine was the first-limiting amino acid for chicken meal while tryptophan was the first-limiting amino acid for the remaining chicken ingredients (Oba et al., 2019). Because methionine was the first-limiting amino acid in the black soldier fly larvae (Do et al., 2020) and chicken meal (Oba et al., 2020), these ingredients could not be used as complementary protein sources for the legume-based or yeast ingredients evaluated in the current study for adult dogs. ...
... These values are similar to the TMEn value reported byParsons et al. (1982) for dehulled soybean meal with a TMEn value of 2.95 kcal/g (DMB). Traditional protein sources used in the pet food industry, such as chicken-based ingredients, have been reported byOba et al. (2019). Chicken meal was reported to have a TMEn value of 3.72 kcal/g (DMB) while raw, steamed, and retorted chicken products had higher TMEn values, with an average of 5.59 kcal/g(Oba et al., 2019).The differences observed in theOba et al. (2019) study is reflective of how processing conditions and variation in the composition of animal-derived protein sources can influence energy content of ingredients, which is also observed, to a lesser extent, among the protein concentrates tested in the current study. ...
Article
Full-text available
Increased consumer interest in high-quality and novel protein sources has driven the demand for the inclusion of protein-rich ingredients in companion animal diets. Novel protein concentrates, with protein contents of at least 50%, have been used to satisfy these consumer demands. However, minimal information is available regarding the macronutrient composition and protein quality of these ingredients that is needed for proper formulation of pet foods. Therefore, the objectives of this study were to determine the macronutrient and amino acid compositions, standardized amino acid digestibility according to the precision-fed rooster assay, and determine protein quality using digestible indispensable amino acid scores (DIAAS-like) of pea protein (PP), potato protein (POP), faba bean protein (FBP), soy protein concentrate (SPC) and dried yeast (DY). Precision-fed rooster assays were conducted using cecectomized roosters to calculate standardized amino acid digestibility and true metabolizable energy corrected for nitrogen (TMEn). For all 5 protein concentrates, all essential amino acids were highly digestible (88.0-96.3%, dry matter basis [DMB]) with differences (P < 0.05) in only lysine, methionine, and tryptophan digestibilities. The TMEn values were highest for POP (4.22 kcal/g) and DY (3.61 kcal/g). The DIAAS-like values for adult dogs indicated that methionine was the first-limiting amino acid in all protein concentrates except POP, where the first-limiting amino acid was tryptophan. Using AAFCO recommended values for adult cats, DIAAS-like values for methionine were lowest (P < 0.05) for FBP at 81.5%, with all other amino acids for all protein concentrates over 100%. The NRC (2006) recommended allowances for adult cats indicated that DIAAS-like methionine values for PP (92.7%) and FBP (73.8%) were significantly lower (P < 0.05) with these being the first-limiting amino acids, with the remaining amino acids above 100% for the other protein concentrates. The protein quality and high essential amino acid digestibility of these protein concentrates indicate that they would be viable protein sources in canine and feline diets. However, additional complementary protein sources should be included to meet the requirements of all essential amino acids.
... Like ileal-cannulated animals, cecectomy allows for digestibility estimates to be made with minimal interference from the bacterial fermentation of proteins in the hindgut. Also, the cecectomized rooster assay is less expensive, time-consuming, and labor-intensive than other types of assays (Johnson et al., 1998;Faber et al., 2010;Kerr et al., 2014;Oba et al., 2019). Given these advantages, the cecectomized rooster assay is often a preferred model to evaluate the protein quality of novel ingredients for dogs and cats. ...
... Calculation of DIAAS-like values was followed according to Mathai et al. (2017) and Oba et al. (2019). The digestible indispensable AA reference ratios were calculated for each ingredient using the following equation (FAO, 2011): Digestible indispensable AA reference ratio = digestible indispensable AA content in 1 g protein of food (mg)/mg of the same dietary indispensable AA in 1 g of the reference protein. ...
... When evaluating protein quality of feed ingredients, AA and macronutrient composition and digestibility are necessary. Therefore, ileal-cannulated animals and the cecectomized rooster assay have been used to determine the quality of protein ingredients (Johnson et al., 1998;Faber et al., 2010;Kerr et al., 2014;Oba et al., 2019). Although the precision-fed cecectomized rooster assay includes a surgical procedure and does not allow the measure of AA metabolism, this assay is a good model to measure the nutrient and AA digestibility of pet food ingredients because ileal-cannulated dog and cat models have issues related to expense, animal welfare, and length of time (Engster et al., 1985;Kerr et al., 2014;Deng et al., 2016). ...
Article
Edible insects such as black soldier fly larvae (BSFL) are alternative protein sources for animal feeds due to their high-protein content and potential low environmental footprint. However, protein quality and AA content may vary across insect species and age. Our objective was to determine the effects of age on nutrient and AA digestibility of BSFL intended for use in pet foods using the precision-fed cecectomized rooster assay. All animal procedures were approved by the University of Illinois Institutional Animal Care and Use Committee prior to experimentation. Twenty-four cecectomized roosters (four roosters per substrate) were randomly assigned to test substrates [BSFL0 = day 0 (day of hatch); BSFL11 = day 11; BSFL14 = day 14; BSFL18 = day 18; BSFL23 = day 23; BSFL29 = day 29]. After 24 h of feed withdrawal, roosters were tube-fed 20 g of test substrates. 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 a completely randomized design and the GLM procedure of SAS 9.4. DM and OM digestibilities were not different among substrates, but acid-hydrolyzed fat digestibility tended to be greater (P < 0.10) for BSFL23 and BSFL29 than BSFL14 and BSFL18. Although all substrates had a high digestibility, BSFL0 and BSFL11 had the lowest (P < 0.05) digestibilities for most indispensable and dispensable AA. Digestible indispensable AA score (DIAAS)-like values were calculated to determine protein quality according to AAFCO nutrient profiles and NRC recommended allowances for dogs and cats. In general, BSFL18 had the highest, and BSFL11 had the lowest DIAAS-like values for most indispensable AA. Threonine, methionine, and tryptophan were often the first-limiting AA. Our results suggest that BSFL are a high-quality protein and AA source, but that age can affect the AA digestibility and protein quality of this alternative protein source.
... In this study, HG foods consistently had higher nutrient digestibilities than the fresh and extruded diets tested. A recent study calculated the nutrient digestibility of chicken-based ingredients differing by processing method (rendered, retorted, steamed, and raw) using the precision-fed cecectomized rooster assay (Oba et al., 2019), which has been demonstrated to accurately estimate canine ileal nutrient digestibility of ingredients or complete pet foods (Johnson et al., 1998). In Oba et al. (2019), DM and OM digestibilities of raw chicken (DM: 75.91% and OM: 80.51%), steamed chicken (DM: 76.46% and OM: 80.56%), and retorted chicken (DM: 73.49% and OM: 77.78%) were similar and much greater than that of chicken meal (DM: 60.05% and OM: 65.87%). ...
... A recent study calculated the nutrient digestibility of chicken-based ingredients differing by processing method (rendered, retorted, steamed, and raw) using the precision-fed cecectomized rooster assay (Oba et al., 2019), which has been demonstrated to accurately estimate canine ileal nutrient digestibility of ingredients or complete pet foods (Johnson et al., 1998). In Oba et al. (2019), DM and OM digestibilities of raw chicken (DM: 75.91% and OM: 80.51%), steamed chicken (DM: 76.46% and OM: 80.56%), and retorted chicken (DM: 73.49% and OM: 77.78%) were similar and much greater than that of chicken meal (DM: 60.05% and OM: 65.87%). In a separate study, Oba et al. (2020) used the cecectomized rooster assay to test six HG dog foods, including the beef-and chicken-based HG foods tested in the current study. ...
... Additionally, the number of DIAAS-reference A c c e p t e d M a n u s c r i p t lowest DIAAS-like values for arginine and leucine were over 100%. Oba et al. (2019) determined threonine is the first-limiting amino acid in all the chicken ingredients when compared to AAFCO recommended values (91.5%) and NRC recommended allowances (98.8%) for adult cats at maintenance. ...
... chicken, steamed chicken, and chicken meal. When using AAFCO recommended values for adult dogs as the reference protein, methionine was the first-limiting amino acid for chicken meal while tryptophan was first-limiting for the other chicken ingredients(Oba et al., 2019). ...
Article
Full-text available
Traditionally, protein by-products from oil seeds and cereal grains have been used in pet foods as sustainable, inexpensive, and protein-rich ingredients. However, the on-going demonization of soy- and corn-based ingredients continue to hinder their use in pet food and treat formulations. Ideally, the further demonstration of their protein quality and nutrient composition may encourage their favorable return as acceptable ingredients in pet foods and treats. Therefore, the objectives of this study were to determine the macronutrient composition, indispensable amino acid profile, standardized amino acid digestibility, true metabolizable energy content corrected for nitrogen (TMEn), and digestible indispensable amino acid scores (DIAAS-like) of soy flakes (SF), peanut flour (PF), soybean meal (SBM), and corn gluten meal (CGM). Standardized amino acid digestibility was assessed using the precision-fed cecectomized rooster assay. All test ingredients demonstrated a profile of highly digestible indispensable amino acids except for lysine in PF, which was lowest (P < 0.05) at 45.5%. The SBM and CGM had the highest (P > 0.05) digestibilities of indispensable amino acids. A DIAAS-like values was calculated for each ingredient using either AAFCO (2020) recommended values or NRC (2006) recommended allowances as the reference protein pattern. For adult dogs compared to AAFCO recommended values, the first-limiting amino acid was lysine for PF and CGM but it was methionine for SF and SBM. For adult cats compared to AAFCO recommended values, the first-limiting amino acid was lysine for PF and CGM but it was threonine for SF. There was no first-limiting amino acid in SBM for cats as DIAAS-like values were over 100% for all indispensable amino acids. The TMEn values were highest (P < 0.05) for PF and CGM (4.58 and 4.31 kcal/g [dry matter basis], respectively). The protein quality of these plant-based protein by-products reflect their value as nutritional ingredients for canine and feline diets. However, the prior processing of these by-products must be considered before exposing them to additional processing methods, such as extrusion. Additionally, the inclusion of complementary proteins or supplemental amino acids will be needed to meet all indispensable amino acid requirements for a nutritionally complete and balanced pet food.
... In this study, HG foods consistently had higher nutrient digestibilities than the fresh and extruded diets tested. A recent study calculated the nutrient digestibility of chicken-based ingredients differing by processing method (rendered, retorted, steamed, and raw) using the precision-fed cecectomized rooster assay (Oba et al., 2019), which has been demonstrated to accurately estimate canine ileal nutrient digestibility of ingredients or complete pet foods (Johnson et al., 1998). In Oba et al. (2019), DM and OM digestibilities of raw chicken (DM: 75.91% and OM: 80.51%), steamed chicken (DM: 76.46% and OM: 80.56%), and retorted chicken (DM: 73.49% and OM: 77.78%) were similar and much greater than that of chicken meal (DM: 60.05% and OM: 65.87%). ...
... A recent study calculated the nutrient digestibility of chicken-based ingredients differing by processing method (rendered, retorted, steamed, and raw) using the precision-fed cecectomized rooster assay (Oba et al., 2019), which has been demonstrated to accurately estimate canine ileal nutrient digestibility of ingredients or complete pet foods (Johnson et al., 1998). In Oba et al. (2019), DM and OM digestibilities of raw chicken (DM: 75.91% and OM: 80.51%), steamed chicken (DM: 76.46% and OM: 80.56%), and retorted chicken (DM: 73.49% and OM: 77.78%) were similar and much greater than that of chicken meal (DM: 60.05% and OM: 65.87%). In a separate study, Oba et al. (2020) used the cecectomized rooster assay to test six HG dog foods, including the beef-and chicken-based HG foods tested in the current study. ...
Article
Full-text available
Human-grade (HG) pet foods are commercially available, but they have not been well studied. Our objective was to determine the apparent total tract digestibility (ATTD) of HG pet foods and evaluate their effects on fecal characteristics, microbiota, and metabolites, serum metabolites, and hematology of dogs. Twelve dogs (mean age=5.5±1.0; BW=11.6±1.6 kg) were used in a replicated 4×4 Latin square design (n=12/treatment). The diets included: 1) Chicken and Brown Rice Recipe (extruded; Blue Buffalo); 2) Roasted Meals Tender Chicken Recipe (fresh; Freshpet); 3) Beef & Russet Potato Recipe (HG beef; JustFoodForDogs); and 4) Chicken & White Rice Recipe (HG chicken; JustFoodForDogs). Each period consisted of 28 d, with a 6-d diet transition phase, 16 d of consuming 100% of the diet, a 5-d phase for fecal collection, and 1 d for blood collection. All data were analyzed using the Mixed Models procedure of SAS 9.4. Dogs fed the extruded diet required a higher (P<0.05) daily food intake (dry matter basis; DMB) to maintain BW. The ATTD of dry matter (DM), organic matter (OM), energy, and acid-hydrolyzed fat (AHF) were greater (P<0.05) in dogs fed the HG diets than those fed the fresh diet, and greater (P<0.05) in dogs fed the fresh diet than those fed the extruded diet. Crude protein ATTD was lower (P<0.05) for dogs fed the extruded diet than those fed all other diets. Dogs fed the extruded diet had greater (P<0.05) fecal output (as-is; DMB) than dogs fed fresh (1.5-1.7 times greater) or HG foods (2.0-2.9 times greater). There were no differences in fecal pH, scores, and metabolites, but microbiota were affected by diet. Dogs fed HG beef had higher (P<0.05) relative abundance of Bacteroidetes and lower (P<0.05) relative abundance of Firmicutes than dogs fed the fresh or HG chicken diets. The Actinobacteria, Fusobacteria, Proteobacteria, and Spirochaetes phyla were unchanged (P>0.05), but diet modified the relative abundance of nearly 20 bacterial genera. Similar to previous reports, these data demonstrate that the fecal microbiota of dogs fed HG or fresh diets is markedly different than those consuming extruded diets, likely due to ingredient, nutrient, and processing differences. Serum metabolites and hematology was not greatly impacted by diet. In conclusion, the HG pet foods tested resulted in significantly reduced fecal output, were highly digestible, maintained fecal characteristics, serum chemistry, and hematology, and modified the fecal microbiota of dogs.
... Chicken-based ingredients are commonly used in pet food products. They are highly palatable, relatively inexpensive and provide an excellent source of protein, although individual products vary greatly in nutrient composition and processing conditions that may affect their protein quality and digestibility [3]. Dog caregivers are interested in OTC foods with limited protein sources because of their easy availability and relatively lower cost compared to veterinary therapeutic diets (VDT). ...
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Background These days the number of potential food allergens is very large, but chicken is one of the most common allergens in dogs. Elimination diet is one of the clinical tools for the diagnosis of allergies and allergy tests are not very reliable. The restriction diet is most commonly carried out by feeding pet foods, relying on the ingredients on the label to select an elimination diet not containing previously eaten foods. Unfortunately, mislabeling of pet food is quite common. The purpose of this study was to determine the absence or presence of chicken DNA using both qualitative and quantitative polymerase chain reaction (PCR) analysis methods in dry and wet maintenance complete pet foods for adult dogs. Results were used to verify the declared composition on the labels. Results Eleven out of fifteen (73%) dog foods were produced as declared by the manufacturer, two of which showed the presence of chicken protein as stated on the label. The remaining nine foods contained amounts of chicken DNA below 1%, consistent with declarations that no chicken was added in the composition. Four of tested dog foods (27%) were not produced consistently with the declaration on the packaging. Two dog foods (one dry and one wet) did not contain the claimed chicken protein. In two foods the addition of chicken DNA was detected at the level of over 2% and almost 6%, respectively. Conclusions In this study, we focused on one of the most commonly undeclared animal species on the label—chicken protein—and performed DNA analyzes to investigate possible contamination and mislabeling. The results showed some inaccuracies. However, most of them are trace amounts below 1%, which proves compliance with the label. Our results showed that undeclared animal species can be as common as missing an animal protein declared on the label. The conducted research indicates that both dry and wet analyzed foods should not be recommended as a diagnostic tool in elimination tests, because it may result in false negative results. Over-the-counter maintenance foods for dogs should not be recommended for the diagnosis and treatment of food hypersensitivity.
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New protein ingredients are used to support pet food market growth and the development of new products while maintaining animal dietary needs. However, novel protein sources (e.g., spray-dried chicken, and (or) rice, pea, and potato protein concentrates) have limited data available regarding their protein quality. The objective of this study was to evaluate protein ingredients used in the pet food industry by laboratory analysis and a chick growth assay as a model. Following analysis for proximate and amino acid composition, chicks (six birds per pen with four pens per treatment) were fed experimental diets for 10 d. Diets contained 10% crude protein from each of the experimental protein sources (spray-dried egg-SDEG; spray-dried egg white-SDEW, spray-dried inedible whole egg-SDIE, chicken by-product meal-CBPM, chicken meal-CKML, low-temperature fluid bed air-dried chicken-LTCK, low-temperature and pressure fluid bed dried chicken-LTPC, spray-dried chicken-SDCK, whey protein concentrate-WPCT, corn gluten meal-CGML, corn protein concentrate-CPCT, potato protein isolate-PPIS, rice protein concentrate-RPCT, pea protein isolate-PEPI, soy protein isolate-SPIS, and soybean meal-SBML) along with an N-free diet (negative control). Chicks fed SDEG, SDIE, and LTPC had the highest protein efficiency ratio (PER; P < 0.0001; 5.18, 5.37, and 5.33, respectively), LTCK and SDCK were intermediate (4.54 and 4.79), and the CBPM and CKML were the lowest among the poultry proteins for EAA:NEAA, PER, and Lys availability. Among the vegetable proteins, PPIS and SBML had the highest PER values (3.60 and 3.48, P < 0.0001). In general, the chick PER method ranked the quality of animal protein sources higher than vegetable proteins, and these results were consistent with the EAA:NEAA ratio and Lys availability.
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Meat protein digestibility can be impaired because of indigestible protein aggregates that form during cooking. When the aggregates are subsequently fermented by microbiota, they can generate potentially harmful compounds for the colonic mucosa. This study aimed to evaluate the quantity of bovine meat protein escaping digestion in the human small intestine and the metabolic fate of exogenous nitrogen, depending on cooking processes. Sixteen volunteers (5 women and 11 men; aged 28 ± 8 y) were equipped with a double lumen intestinal tube positioned at the ileal level. They received a test meal exclusively composed of 120 g of intrinsically (15)N-labeled bovine meat, cooked either at 55°C for 5 min (n = 8) or at 90°C for 30 min (n = 8). Ileal effluents and blood and urine samples were collected over an 8-h period after the meal ingestion, and (15)N enrichments were measured to assess the digestibility of meat proteins and the transfer of dietary nitrogen into the metabolic pools. Proteins tended to be less digestible for the meat cooked at 90°C for 30 min than at 55°C for 5 min (90.1% ± 2.1% vs. 94.1% ± 0.7% of ingested N; P = 0.08). However, the particle number and size in ileal digesta did not differ between groups. The appearance of variable amounts of intact fibers was observed by microscopy. The kinetics of (15)N appearance in plasma proteins, amino acids, and urea were similar between groups. The amount of exogenous nitrogen lost through deamination did not differ between groups (21.2% ± 0.8% of ingested N). Cooking bovine meat at a high temperature for a long time can moderately decrease protein digestibility compared with cooking at a lower temperature for a short time and does not affect postprandial exogenous protein metabolism in young adults. The study was registered at www.clinicaltrials.gov as NCT01685307. © 2015 American Society for Nutrition.
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Commercial, dehulled, solvent-extracted soybean meal (DSBM) was overprocessed by autoclaving at 121 C and 105 kPa for 0, 20, 40, or 60 min. Analyzed concentrations (acid hydrolysis followed by ion-exchange chromatography) of lysine and cystine, and to a lesser extent arginine, in DSBM were reduced by increased autoclaving time. Autoclaving time had little or no effect on levels of other amino acids. Performance of chicks fed 40-min autoclaved DSBM as the sole source of dietary protein was inferior to that of chicks fed unautoclaved DSBM. True digestibilities of several amino acids in DSBM decreased (P<.05) as autoclaving time increased, with the largest decreases being observed for lysine, cystine, histidine, and aspartic acid. The effect of autoclaving on lysine was due both to reduced concentration of analyzed lysine and reduced digestibility of the lysine remaining after autoclaving. True digestibilities of most amino acids determined in conventional cockerels were higher (P<.05) than those determined in cecectomized cockerels. Bioavailabilities determined by chick growth assay of lysine in unautoclaved DSBM and DSBM autoclaved for 40 min were 82 and 70%, respectively. Autoclaving had no significant effect on TMEn of DSBM.
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Background: Cooking processes affect the physical, chemical, and structural properties of meat proteins. Cooking may also affect the protein quality of meat, as indicated by the true ileal digestibility of individual amino acids, the content of each truly digestible amino acid, and the digestible indispensable amino acid score (DIAAS). Objective: The study aimed to determine the effect of the cooking process (raw, not cooked; boiled; grilled; pan-fried; roasted) of beef on true (standardized) ileal amino acid digestibility, true ileal digestible amino acid content, and DIAAS. Methods: Beef topside steak was subjected to one of the following conditions: raw, boiled, grilled, pan-fried, or roasted, followed by mincing. The growing pig was used as an animal model for the adult human. Diets containing the raw or cooked meats (10% crude protein content) were fed to growing pigs (n = 6 per diet; mean ± SEM bodyweight, 23.6 ± 0.48 kg) and samples of terminal ileal digesta were collected under anesthesia. True ileal amino acid digestibility of the beef was determined and DIAAS values were calculated. Results: There were only minor differences in true ileal amino acid digestibility across cooking conditions with all amino acids having true ileal amino acid digestibility in the range of 90-100%. In general, boiled meat had the highest true ileal digestible amino acid content (total of 724 g/kg dry matter), and roasted meat the lowest (total of 641 g/kg dry matter; P < 0.001). The DIAAS was greater (P < 0.001) for the raw, boiled, and pan-fried meat treatments (97-99%) than for roasted meat (91%) or grilled meat (80%). The high DIAAS (range 80-99%) across cooking conditions confirms that bovine meat is a high-quality protein source. Conclusion: Cooking conditions affect the true ileal digestible amino acid content and DIAAS of beef, as determined with the use of the pig model.
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An experiment was conducted to compare values for digestible indispensable amino acid scores (DIAAS) for four animal proteins and four plant proteins with values calculated as recommended for protein digestibility-corrected amino acid scores (PDCAAS), but determined in pigs instead of in rats. Values for standardised total tract digestibility (STTD) of crude protein (CP) and standardised ileal digestibility (SID) of amino acids (AA) were calculated for whey protein isolate (WPI), whey protein concentrate (WPC), milk protein concentrate (MPC), skimmed milk powder (SMP), pea protein concentrate (PPC), soya protein isolate (SPI), soya flour and whole-grain wheat. The PDCAAS-like values were calculated using the STTD of CP to estimate AA digestibility and values for DIAAS were calculated from values for SID of AA. Results indicated that values for SID of most indispensable AA in WPI, WPC and MPC were greater ( P <0·05) than for SMP, PPC, SPI, soya flour and wheat. With the exception of arginine and tryptophan, the SID of all indispensable AA in SPI was greater ( P <0·05) than in soya flour, and with the exception of threonine, the SID of all indispensable AA in wheat was less ( P <0·05) than in all other ingredients. If the same scoring pattern for children between 6 and 36 months was used to calculate PDCAAS-like values and DIAAS, PDCAAS-like values were greater ( P <0·05) than DIAAS values for SMP, PPC, SPI, soya flour and wheat indicating that PDCAAS-like values estimated in pigs may overestimate the quality of these proteins.
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A wide variety of animal protein-based ingredients is commonly used in the pet food products. The raw ingredients and processing procedures used may greatly affect protein quality. Testing the quality of alternative protein sources is necessary and contributes to the sustainability of pet foods. The objective of this study was to test the chemical composition of 8 protein sources intended for use in dog and cat foods (calamari meal, pork peptone, alligator meal, lamb meal, venison meal, chicken meal, and 2 duck meals), and evaluate their true nutrient digestibility and nitrogen-corrected true ME (TMEn) using the precision-fed cecectomized rooster assay. Calamari meal and pork peptone had lower ash (4.4 and 3.6% of DM, respectively) but greater CP (88.1 and 80.5% of DM, respectively) and either greater or similar GE (5.6 and 5.3 kcal/g of DM, respectively) compared with alligator, lamb, venison, chicken, and duck meals (11.8 to 24.5% ash, 58.7 to 65.9% CP, and 4.6 to 5.3 kcal GE/g). Acid-hydrolyzed fat (AHF) was lower in calamari meal (8.7% of DM) compared with the other proteins tested (15.5-22.1% of DM). True nutrient digestibility was variable among the protein sources (52 to 79% of DM, 60 to 83% of OM, 78 to 92% of AHF, and 70 to 89% of GE) with pork peptone having the highest DM, AHF, and GE digestibility and calamari meal having the highest OM digestibility. True indispensable AA digestibility was highest for calamari meal, with all AA having a digestibility greater than 90%. Except for histidine, all indispensable AA had a digestibility over 85% for pork peptone. In contrast, true indispensable AA digestibility was lowest for lamb meal, with histidine having digestibility less than 70% and the other entire indispensable AA having digestibility between 72 and 88%. The TMEn of calamari meal (4.82 kcal/g DM and 86.9% of GE) was greater ( < 0.05) than the other protein sources. The lamb meal had the lowest TMEn value (3.12 kcal/g DM and 66.9% of GE), with others being intermediate (3.46-4.09 kcal/g DM and 71.2-77.9% of GE). This study demonstrates the considerable variability that exists not only in the chemical composition but also in the true nutrient digestibility among protein sources intended for use in dog and cat foods and justifies further in vivo testing of novel protein sources.