Multiprotease improves amino acid release in vitro, energy, and nutrient

utilization in broilers fed diets varying in crude protein levels

Lina M. Pe~nuela-Sierra,*,y Valmiro L. Arag~ao-Neto,* Paula Lozano-Cruz,*,y

Juan Nicolas Mejia-Abaunza,*,y Muhammad Ali,* Joaquin Caba~ nas-Ojeda,* Yanlin Yang ,z
Maria Camila Alfaro-Wisaquillo,* Gustavo A. Quintana-Ospina ,*
Bindhu Lakshmibai Vasanthakumari ,x Alexandra Wealleans ,x Ye Lao,x and
Edgar O. Oviedo-Rondon *,1
*
Prestage Department of Poultry Science, North Carolina State University, Raleigh, NC, 27695, USA; yCollege of
Veterinary Medicine, Universyty of Tolima, Ibague, Tolima, 730006299, Colombia; zKemin (China) Technologies
Co., Zhuhai, 519040 , China; and xKemin Industries, Des Moines, Iowa 50317, USA

ABSTRACT Low crude protein (CP) diets can reduce
nitrogen (N) excretion and costs by increasing N utiliza-
tion efficiency. Exogenous proteases may further improve
protein digestibility in low CP diets. This study first eval-
uated in vitro the efficacy of a multiprotease on amino
acid (AA) release from feedstuffs and broiler feed. Later,
a broiler study evaluated the effect of feeding corn-soy-
bean meal diets containing 3 CP levels (17, 19, and 21%
CP) with supplementation on top of 0 or 2,400 U/kg mul-
tiprotease on chicken growth performance, total tract
CP, and ileal AA digestibilities, and energy utilization.
Ross 708 male chickens were placed in 42 cages and
assigned to 6 treatments resulting from a 3 £ 2 factorial
arrangement. Three isocaloric basal diets were formulated
to reduce CP, but all diets maintained digestible Lys:CP
in 5.47% and the same ideal protein profile. Data were
analyzed in a completely randomized design. On average,
the multiprotease increased (P < 0.05) in vitro free AA
release by 27.81% in most feedstuffs evaluated compared
to the control. For broiler feed, 1,200 U/g multiprotease
Key words: low crude protein, broiler, exogenous multiprotease, AA digestibility, AMEn
addition improved (P < 0.001) in vitro free AA release by
18.90%. This multiprotease showed interaction effects
(P < 0.05) on chicken FCR, energy, and CP digestibility.
As expected, BW at 24 d, BW gain, and FCR (8−24 d)
worsened (P < 0.001) as dietary CP reduced from 21 to
17%, and multiprotease addition did not improve (P >
0.05) these parameters. BW gain decreased by 12.9%
when N intake was reduced from 49.32 to 38.49 g/bird.
Multiprotease supplementation improved (P < 0.01)
AMEn by 71 kcal/kg, CP digestibility from 59.45 to
63.51%, ileal AA digestibility, and DM digestibility from
67.08 to 73.49%, but only in the 21% CP diet. No differ-
ences in ileal AA digestibility due to CP level (P > 0.05)
were detected, except for Cys digestibility (P < 0.01). In
conclusion, low CP diets reduced growth performance
and improved N utilization but negatively affected energy
utilization efficiency. Exogenous multiprotease supple-
mentation improved AME, AMEn, protein, ileal AA, and
DM digestibility in the 21% CP diet without significantly
affecting growth performance.
2024 Poultry Science 103:103546
https://doi.org/10.1016/j.psj.2024.103546

INTRODUCTION broiler production costs (Greenhalgh et al., 2020; Amer

Low crude protein (CP) diets can mitigate the envi-
ronmental impact of nitrogen (N) and ammonia emis-
sions, minimize soybean meal dependence, and reduce
Ó 2024 The Authors. Published by Elsevier Inc. on behalf of Poultry
Science Association Inc. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/
4.0/).
Mention of a trademark or proprietary product does not constitute a
guarantee or warranty of the product by North Carolina Agricultural
Research Service, nor does it imply approval to the exclusion of other
products that may be suitable.
1
Corresponding author: edgar_oviedo@ncsu.edu
et al., 2021). Corn-soybean meal diets remain one of the
most efficient feeds for poultry and are known to be
highly digestible. However, it is possible to further
improve the efficiency of nutrient utilization and digest-
ibility by observing details such as accurate feed formu-
lation close to broiler amino acid (AA) needs,
improving the balance between protein and starch
digestibility dynamics (Moss et al., 2018; Chrystal et al.,
2020; Liu et al., 2021), and adding exogenous enzymes
that increase AA digestibility (Pan et al., 2016; Borda-
Molina et al., 2019; Wealleans et al., 2023). Improve-
ments in apparent ileal AA digestibility in broilers are

1
2 ~
PENUELA-SIERRA ET AL.
reported to result in improvements in growth perfor-
mance and reduced N excretion and may improve over-
all gut health due to reduced putrefaction in the distal
intestine (Cowieson et al., 2017; Cardinal et al., 2019).
Reducing dietary CP levels affect AA digestibilities
and can cause AA imbalances (Liu et al., 2021). Overall
digestibility of AA in corn-based diets is reduced by
8.36% in the distal ileum when low CP diets are used
(Moss et al., 2018). Nevertheless, maize-based diets are
more conducive to CP reductions than wheat-based
diets (Chrystal et al., 2021). Low-CP corn-soybean meal
diets contain high quantities of maize starch that, at the
ileal level, may compete with AA for intestinal uptake
via Na+ -dependent and -independent transporters with
overlapping specificities (Moss et al., 2018; Chrystal et
al., 2020). Traditionally, non-bound AA has been added
to low-CP diets in the synthetic form to meet the
requirements of main limiting AA (Lys, Met, Thr, Val)
and other AA (Ile, Leu, Trp, Glu, Gly). However, these
synthetic AA sources are more digestible and increase
competition with starch.
Consequently, formulating low-CP diets with a better
AA balance and minimizing synthetic AA inclusion
could be proposed to improve the performance of
broilers fed low-CP diets. However, the best feed conver-
sion ratios (FCR) have been reported when dietary
starch:protein ratios are between 1.50 and 1.85. There-
fore, it is expected that FCR will irremediably worsen in
low-CP compared to a control diet with better starch:
protein ratios (Liu et al., 2021).
In order to estimate the optimum dietary levels of
AAs to use in low-CP diets where essential and nones-
sential AA will be reduced, the concept of digestible Lys
(dig. Lys) to true protein (TP) ratio can be used. Alho-
tan and Pesti (2016) estimated that dig. Lys require-
ments for BW gain and FCR can be between 4.92 §
0.51% and 5.58 § 0.70% of TP, respectively. To apply
the TP concept in feed formulation, Alhotan and Pesti
(2016) proposed obtaining each feedstuff’s N content
and determining TP using the prediction equation
TP = 0.873 x CP + 0.435 (R2 = 0.99). Since this concept
of TP is not widely applied, and optimums for each age
are still not completely elucidated, in this experiment,
the TP and dig. Lys: The TP ratio was calculated, but a
similar ratio was expressed as a dig. Lys: CP, as others
have used (Ng’ambi et al., 2009) and using one ideal AA
ratio. However, ideal AA ratios are probably different in
low-CP diets (Chrystal et al., 2020).
Exogenous protease enhances protein digestibility in
broiler nutrition when evaluating feedstuffs (Adebiyi
and Olukosi, 2015; Stefanello et al., 2016) or complete
diets (Angel et al., 2011; Cowieson and Roos, 2016). Pro-
teases can shift the site of protein digestion to more
proximal intestinal segments (Cowieson and Roos,
2016), which can benefit low CP diets. Exogenous pro-
teases have been reported to reduce the antinutritional
effects of various protein raw materials and to increase
intestinal resilience by reducing inflammatory responses,
improving tight junction and mucin integrity (Cowieson
and Roos, 2016; Cowieson et al., 2017), reducing
populations of intestinal Clostridium (Kamel et al.,
2015), and improving intestinal mucosa traits (Xu et al.,
2017). The simultaneous supplementation of multiple
proteases with different pH optima and substrate speci-
ficity may improve the efficacy and consistency of prote-
ase supplementation. It is important to test the
activities of these multiproteases under different pH con-
ditions and for diverse substrates to determine their effi-
cacy.
Enzymes, including mono-component protease, have
been proposed to improve the utilization of reduced CP
diets with considerable synthetic AA supplementation
(Vieira et al., 2016; Chrystal et al., 2020; Jabbar et al.,
2021). Broilers fed a protein-deficient diet (lower CP
and AA) supplemented with multiprotease were found
to improve growth performance with increased ileal AA
and CP digestibility (Cho et al. 2020).
The present study evaluated first the effects of a mul-
tiprotease supplementation in vitro across a range of
feed ingredients and commercial broiler feed to test its
efficacy in releasing free AA during in vitro digestibility.
The second objective was to evaluate in a broiler study
the in vivo efficacy of the multiprotease when applied to
common corn-soybean meal diets but varying in CP
level to observe variability in feed ingredient inclusion.
Grower broiler diets with 3 CP levels (17, 19, and 21%)
maintaining digestible Lys in 5.47% of CP, and the same
ideal protein profile was used for the study, and the
effects of protease supplementation on performance
energy utilization, CP, and AA ileal digestibility was
evaluated.
MATERIALS AND METHODS
The North Carolina State University Institutional
Animal Care and Use Committee approved all proce-
dures involving broiler chicken used in the present exper-
iment (Protocol No. 21-145).
In Vitro Substrate Degradation by a
Multiprotease
To test the efficacy of the multiprotease to release free
AA, an in vitro 2-step method with sequential incuba-
tions at acidic and neutral conditions was designed to
mimic the gastrointestinal conditions. In the acidic incu-
bation, each treatment was made up to 50 mL with
0.1 M phosphate buffer (pH 2.5) and 10% (w/v) of pep-
sin and incubated for 1 h at 37°C under 90 rpm of contin-
uous stirring. The samples were subsequently
neutralized by 50 mL of 0.1 M phosphate buffer (pH
12.0) to pH 6.8 and incubated at 37 °C for 2 h under
150 rpm of continuous stirring. 10.0 g of individual pro-
tein raw material was weighed into a 250 mL conical
flask for each treatment. Each type of protein raw mate-
rial was treated by blank control or an exogenous multi-
protease (KEMZYMETMProtease, Kemin Industries,
Des Moines, IA) supplemented at an equivalent dosage
to 1,200 U/kg feed. Furthermore, maize, sorghum, rice,
 MULTIPROTEASE IN LOW PROTEIN DIETS 3
Table 1. Feed ingredients used in the in vitro study. results were calculated based on the standard curve.
The level of improvement was calculated based on the
Material Commercial origin Crude protein, %
amount of free AA in the protease-supplemented group
Protein-sources vs. the control group.
Soybean meal 1 Guangdong province, 47.00
China
Soybean meal 2 Jiangxi province, China 43.36
Dehulled soybean Jiangxi province, China 46.00 In Vivo Evaluation of Multiprotease Efficacy
meal
Fermented soybean Jiangxi province, China 50.00 Chicken Husbandry A total of 294 Ross-708 d-old
meal
Cereals male chicks were placed in 42 Alternative Design
Corn Guangdong province, 8.24 (Siloam Springs, AR) Super Brooder battery cages
China (96.5 £ 78.7 £ 38.1 cm) in an environmentally con-
Sorghum Guangdong province, 9.36
China trolled room (3.50 £ 12 m). Each cage had one stainless
Rice Guangdong province, 10.40 steel linear feeder and 2 nipple drinkers with cups. Room
China temperature was recorded and adjusted daily to guaran-
Wheat Guangdong province, 13.90
China tee thermoneutral temperatures at each age. The room
Barley Guangdong province, 11.33 temperature decreased from 32 to 28°C the first wk, and
China was maintained at 27 and 23°C during the second and
Complete broiler feeds
Commercial feed 1 Guangdong province, 17.62 third wk, respectively. A 23 h light and one h dark cycle
China was used the first week, and 18 h light and 6 h darkness
Commercial feed 2 Guangdong province, 15.65 the following wk. Mortality was recorded daily. Feed
China
Commercial feed 3 Guangdong province, 17.03 and water were offered ad libitum to the chicks during
China the experimental period.

Treatments and Experimental Diets

wheat, and barley were supplemented with the multipro-
tease at a dose equivalent to 2,400 U/kg.
The nine types of protein and cereal raw materials and
3 complete diets used in this study, as summarized in
Table 1, were commercial products bought on the open
feed market. Pepsin from porcine stomach mucosa were
obtained from Sino pharm Group Co., Ltd., China. All
other chemicals were obtained from Guangzhou Chemi-
cal Reagent Co, China.
The Ninhydrin reaction has been widely used to analyze
AA, peptides, and proteins in biomedical studies (Fried-
man, 2004) and recently for chicken protein digestibility
in feedstuffs (Bryan et al., 2018; 2019; Bryan and Classen,
2020). The method was used to analyze the number of
amino groups, which was used to indicate the degree of
hydrolysis of protein materials. In this assay, 1 mL of
digestion supernatant was mixed with 1 mL of ninhydrin
reagent solution (1% solution of ninhydrin, Sigma, in eth-
ylene glycol monomethyl ether) and 1 mL of sodium ace-
tate-acetic acid buffer (0.2 M, pH 5.4, Guangshi, China).
The mixture was shaken to homogeneity by a mixture
(G560E, Scientific Industries, INC) and then boiled for
15 min in a 100°C water bath (HWS-24, Yiheng Instru-
ments, China). The solution was cooled to room tempera-
ture and diluted with 3 mL of 60% ethanol. The
absorbance against deionized water at 570 nm was mea-
sured by ultraviolet and visible spectrophotometer (2,100,
Unico). The absorbance value of the supernatant needed
to be within the range of 0.2 to 0.8. Otherwise, dilution
was needed till the absorbance reached the valid range.
A standard curve was plotted using different dilutions
of lysine (Sigma, 0.06−0.34 mmol/L) as the y-axis and
the corresponding absorbance as the x-axis. The assay
was performed in duplicate for each sample, and the
Six treatments resulting from a 3 £ 2 factorial
arrangement of 3 dietary CP levels (17, 19, and 21%)
and 2 levels of exogenous multiprotease (KEMZYMETM-
Protease, Kemin Industries, Des Moines, IA) supplemen-
tation (0 and 2,400 U/kg) were evaluated in a
completely randomized design with seven replicates per
treatment. The 2,400 U/kg equates to 300 g/t feed and
is in line with the use of this protease in other studies
(Chandrasekar et al., 2017; De Leon et al., 2021; Weal-
leans et al., 2023). The multiprotease includes a combi-
nation of acid (pepsin type protease), neutral (metallo
endo-peptidase), and alkaline (serine endopeptidase),
proteases, produced by Aspergillus niger, Bacillus subti-
lis, and Bacillus licheniformis, respectively (Lim et al.,
2014). All protease activities are measured in a stan-
dardized assay: one unit of protease activity is defined as
the amount of enzyme that solubilizes one microgram of
Azo-casein per minute at pH 7.0 and 37°C (Pan et al.,
2016).
Until 7 d of age, chicks received a standard corn-soy-
bean starter diet as crumbles, formulated to meet Ross
708 requirements (Aviagen, 2019). The experimental
grower diets were produced from 3 basal diets (Table 2)
with their respective CP level. The nutrient composition
for these experimental diets is presented in Table 2.
These 3 basal diets were isocaloric (3,100 kcal/kg ME)
formulated to reduce CP, but all diets maintained dig.
Lys in 5.47 % of CP and the same ideal protein profile.
This dig. Lys:CP ratio is estimated to be 6.08 § 0.04%
dig. Lys:TP ratio, which is within the optimum levels
suggested by Alhotan and Pesti (2016). The ideal pro-
tein profile chosen corresponds to Ross Broiler (Aviagen,
2019) nutrient recommendations, with dig. total sulfur
4 ~
PENUELA-SIERRA ET AL.

Table 2. Ingredient and nutrient composition of the experimen- experimental crumbled diets were analyzed at Kemin
tal diets. Industries Labs.
Grower basal diets (8 - 24 d)
Items Starter 0-7d 21% 19% 17% Diet Manufacturing
Ingredients ————————- % ———————
Yellow dent-corn 51.70 55.77 61.73 68.67 Corn was ground in hammermill tip speed
Soybean meal solvent- 29.68 24.88 19.67 19.40 3,335 m/min and #24 screen and obtained a particle
extracted
Full-fat soybean meal 10.00 10.00 10.00 4.47
size of 927 mm of geometric mean diameter (dgw) and
Poultry fat 2.19 3.18 2.42 2.36 3.64 geometric standard deviation (Sgw). The soybean
DDGS 2.00 2.00 2.00 0.90 had 980 § 2.71 mm dgw, and full-fat soybean was ground
Limestone fine 1.32 1.00 1.01 1.01
Dicalcium phosphate 1.17 0.99 1.03 1.09
to obtain a similar particle size of 953 § 3.26 mm dgw.
DL-Methionine 0.37 0.33 0.28 0.24 Diets were mixed in a twin-shaft counterpoise ribbon
Sodium bicarbonate 0.34 0.26 0.27 0.28 mixer (Model TRDB126060, Hayes & Stolz, Fort
Salt 0.30 0.30 0.29 0.30
L-Lysine 0.24 0.22 0.24 0.24
Worth, TX) for 180 s. All diets were pelletized and
Trace mineral premix1 0.20 0.20 0.20 0.20 crumbled. Mixed mash feeds were conveyed to a single
Choline chloride 0.18 0.18 0.18 0.18 pass conditioner (model C18LL4/F6, California Pellet
L-Threonine 0.15 0.12 0.11 0.10
Vitamin premix2 0.10 0.10 0.10 0.10
Mill, Crawfordsville, IN) and conditioned at 85°C for
Coban 0.05 0.05 0.05 0.05 30 s. Diets were pelleted using a 30 HP CPM pellet mill
Phytase3 0.03 0.03 0.03 0.03 (model PM1112 2, California Pellet Mill, Crawfords-
Titanium dioxide4 0.00 0.40 0.40 0.40
Energy and nutrient content
ville, IN) equipped with a 4.4 £ 35.2 mm die with 548
Metabolizable energy, 3,000 3,100 3,100 3,100 cm2 working surface area at a production rate of
kcal/kg 16.34 kg/min (980 kg/h). The steam pressure was 207
Crude protein, % 23.00 21.00 19.00 17.00
Fat, % 7.03 8.05 7.40 6.50
kPa. The pellet mill die was warmed with feed before
Crude fiber, % 2.64 2.53 2.42 2.11 pelleting the experimental batches. After pelleting, pel-
Calcium, % 0.96 0.87 0.87 0.87 lets were cooled in a counterflow cooler (Model
Total phosphorus, % 0.58 0.52 0.51 0.50
Ash, % 5.56 5.22 4.98 4.74
VK09 £ 09KL, Geelen Counterflow USA, Inc, Orlando,
Available phosphorus, %4 0.48 0.43 0.43 0.43 FL) and crumbled.
Phytate phosphorus, % 0.22 0.22 0.21 0.20
Dig Lys:CP, % 5.57 5.47 5.47 5.47
Dig Lysine, % 1.28 1.15 1.04 0.93 Experimental Procedure
Dig. TSAA, % 0.95 0.87 0.79 0.70
Dig. Thr, % 0.86 0.77 0.70 0.62
Dig Trp, % 0.24 0.22 0.19 0.17
Group BW and feed intake were obtained at 7 and
Dig. Ile, % 0.88 0.80 0.71 0.63 24 d of age, and BW gain and FCR (adjusted for
Dig. Leu, % 1.83 1.73 1.63 1.53 mortality) were calculated. On d 23 and 24, excreta
Dig. Val, % 0.97 0.89 0.81 0.73
Dig. Arg, % 1.40 1.26 1.11 0.98
without feathers or down were collected in 2 shifts,
Dig. Lys:ME, % per Mcal 0.427 0.371 0.335 0.300 immediately mixed, pooled by cage, and stored in a
Dietary Electrolyte Bal- 300 268 246 225 freezer at -15°C. Ileal digesta contents were obtained
ance, mEq/kg
from all chicks at 25 d after euthanizing them by
1
Trace minerals provided per kg of premix: manganese (Mn SO4), 60 g; CO2 inhalation, and digesta samples were collected
zinc (ZnSO4), 60 g; iron (FeSO4), 40 g; copper (CuSO4), 5 g; iodine (Ca
(IO3)2),1.25 g. from the half-distal portion of the ileum. The ileum
2
Vitamins provided per kg of premix: vitamin A, 13,227,513 IU; vita- was defined as that portion of the small intestine
min D3, 3,968,253 IU; vitamin E, 66,137 IU; vitamin B12, 39.6 mg; ribofla- extending from the Meckel’s diverticulum to 40 mm
vin, 13,227 mg; niacin, 110,229 mg; d-pantothenic acid, 22,045 mg;
menadione, 3,968 mg; folic acid, 2,204 mg; vitamin B6, 7,936 mg; thia- proximal to the ileo-caecal junction. Ileal digesta con-
mine, 3,968 mg; biotin, 253.5 mg. tents were gently flushed with de-ionized water into
3
Phytase (1,000 FTU/kg) was expected to release at least 0.12% avail- plastic containers, pooled by cage, and immediately
able phosphorus and 0.06% Ca (Kemin Industries, Des Moines, Iowa
50317, USA). stored in a freezer at -15°C. The excreta samples
4
Undigestible marker Titanium dioxide (Venator, Hombitan were dried at 55°C in a forced-air oven, and ileal
AFDC101, CAS 13463-67-7, Duisburg, Germany). digesta samples were lyophilized using FreeZone 6
(NC State University Phytotron). Subsequently, sam-
ples were ground in a Retsch mill (Retsch USA
AA (TSAA), Thr, and Val maintained 76, 67, and 78%
Verder Scientific, Inc. Newtown, PA) to pass through
of dig. Lys, respectively. Synthetic Lys, Met, and Thr
a 0.5-mm screen in a grinder.
were used to obtain desired AA levels of balanced pro-
tein but not to keep similar AA concentrations across
the 3 CP diets. The Lys:energy ratios were 0.30, 0.34, Chemical Analysis and Parameter
and 0.37%/Mcal ME. All diets contained titanium Calculations
dioxide (TiO2) as an indigestible marker. Two batches
of each basal diet were used to add either sand or the Diets, excreta, and ileal samples were analyzed for dry
multiprotease on top at a rate of 2,400 units of prote- matter (DM), CP, and titanium content. Excreta sam-
ase. Phytase and protease activity in samples of ples were further analyzed for gross energy (GE) and
 MULTIPROTEASE IN LOW PROTEIN DIETS
nitrogen content. Feed and ileal digesta samples were
analyzed for AA content with HPLC (AOAC, 2006
method 982.30 Ea,b,c). Dry matter was determined
according to the method, and CP (N x 6.25) was deter-
mined by the combustion method (LECO, AOAC Inter-
national, 2006; method 990.03). The GE analysis was
determined using an oxygen bomb calorimeter (IKA
C5003; IKA Labortechnik), and AME was corrected to
zero N retention (AMEn) using a factor of 8.22 kcal/g
(Hill and Anderson, 1958). Titanium, CP, DM, and AA
concentrations were measured in triplicate for dietary
feeds and duplicates for fecal and ileal digesta samples.
Titanium was measured on a UV spectrophotometer fol-
lowing the method described by Myers et al. (2004).
Apparent ileal digestibility, total tract utilization, and
AMEn were calculated using the following
equations (Kong and Adeola, 2014):
Digestibility ð%Þ ¼ ½1  ðMi =Mo Þ  ðEo =Ei Þ  100
AMEn ðkcal=kgÞ
¼ GEi  ½GEo  ðMi =Mo Þ  8:22
fNi  ½No  Mi =Mo g
Where Mi represents the concentration of inert maker
titanium dioxide in the diet in g/kg DM; Mo repre-
sents the concentration of titanium dioxide in the
excreta and ileal digesta in g/kg DM output; Ei repre-
sents the concentration of DM, CP, energy, or AA in
the diet in mg/kg of DM; and Eo represents the con-
centration of DM, CP, AA and energy in the excreta
and ileal digesta, in mg/kg DM. The GEi is gross
energy (kcal/kg) in the diet; GEo is the GE (kcal/kg)
in the excreta; Ni represents nitrogen concentration in
the diet, and No represents nitrogen concentration in
the excreta in g/kg DM.
Additionally, the coefficients of apparent DM fecal
and ileal digestibility were calculated. The N intake was
calculated using N dietary content and feed intake.
5
Using calorie and nitrogen intake, the conversion ratios
to live BW were estimated.
Statistical Analysis
All data were evaluated in completely randomized
designs. The in vitro data was analyzed in a one-way
ANOVA to test the effect of multiprotease with mean
separation by student’s t or Tukey’s HSD test. In the
chicken experiment, a 3 £ 2 factorial arrangement of
treatments with 3 CP levels and 2 multiprotease supple-
mentation levels was evaluated. Each treatment had
seven replicates distributed equally in 42 cages. Data
was analyzed in 2-way ANOVA using JMP 16 (SAS
Institute. Inc., Cary, NC). Means were separated by the
LS means procedure using Tukey’s HSD at a significance
level of alpha 0.05. Correlation coefficients between for-
mulated and analyzed CP and AA content were calcu-
lated. Finally, energy, CP, and Lys intakes were
calculated, and correlation analysis with chicken growth
performance was conducted.
RESULTS AND DISCUSSION
In Vitro Protein Degradation by a
Multiprotease
As an initial investigative step, the ability of the mul-
tiprotease to release AA was tested in vitro in nine raw
feed materials and 3 broiler commercial feeds using a
methodology of CP digestibility developed and vali-
dated by Bryan et al. (2018, 2019) and positively corre-
lated with in vivo AA digestibility (Bryan and Classen,
2020). The results are shown in Table 3. This data was
intended to preliminarily explore the substrate-specific-
ity of the multiprotease as a foundational step before
animal trials. The addition of exogenous multiprotease
improved (P < 0.05) AA release on top of the action of
pepsin in all but one sample. The improvement ratio

Table 3. Release of free amino acids from plant-based protein sources and cereals, and complete commercial broiler feeds following sup-
plementation with 1,200 or 2,400 U/g of an exogenous multiprotease.

Free Amino Acid Content (mmol/L)

Control 1,200 U/g 2,400 U/g P-value
Protein-sources
Soybean meal 1 11.83 § 1.3 13.9 § 0.95 − NS
Soybean meal 2 11.47 § 0.03b 14.35 § 0.42a − 0.001
Dehulled soybean meal 15.33 § 0.63b 18.98 § 0.76a − 0.010
Fermented soybean meal 21.2 § 0.41b 24.07 § 0.25a − 0.001
Cereals
Corn 3.95 § 0.02c 4.56 § 0.01b 5.07 § 0.09a 0.001
Sorghum 3.26 § 0.18b 4.15 § 0.21a 4.94 § 0.23a 0.050
Rice 3.67 § 0.23b 5.88 § 0.20a 6.65 § 0.06a 0.001
Wheat 5.69 § 0.10c 7.96 § 0.19b 10.21 § 0.02a 0.001
Barley 4.08 § 0.13b 5.2 § 0.19a 6.06 § 0.20a 0.010
Complete broiler feeds
Commercial feed 1 7.21 § 0.09b 8.37 § 0.07a − 0.001
Commercial feed 2 9.23 § 0.17b 10.7 § 0.01a − 0.001
Commercial feed 3 7.21 § 0.12b 8.99 § 0.05a − 0.001
a-c
Means in rows followed by different superscript letters are statistically different (P < 0.05) by t-test or Tukey’s test. n = 2 per treatment and each
ingredient. NS = non-significant.
6 ~
PENUELA-SIERRA ET AL.

following supplementation with 1,200 U/g in the feed
ingredients ranged from 13.54 to 60.22 %, and in the
complete broiler feed, from 15.93 to 24.69 %, averaging
18.90%. Uplifts in free AA release from cereals were gen-
erally higher (P < 0.05) with supplementation of
2,400 U/g, with improvement ratios ranging from 28.35
to 87.20% compared to the control, averaging 57.81 %,
but not significantly different from 1,200 U/g in sor-
ghum, rice, and barley.
Across the nine feedstuffs tested, the average uplift in
free AA release was 27.81% compared to the control. No
significant uplifts were seen for one of the soybean meal
sources. This difference in efficacy may affect the
response to the multiprotease in vivo. This is in accor-
dance with the reported ileal digestibility values of such
alternate protein sources in broilers and provides an ave-
nue for future research into the correlation between in
vitro and in vivo efficacy of multiprotease.
Though the 2 assessed soybean meal samples had sim-
ilar basal digestibility in the presence of pepsin (11.83
and 11.47 mmol/L AA release), the relative uplift fol-
lowing multiprotease supplementation was different
between samples (17.5 and 25.1 %). Significant digest-
ibility improvement was observed in soybean meal 2
(P < 0.001) and dehulled soybean meal (P < 0.01) but
not in soybean meal 1 (P > 0.05). The presence of prote-
ase inhibitors in soybeans is well known, and the concen-
tration varies between soybean varieties, processing
conditions, and growing microclimate (Sakkas et al.,
2019; Yang et al., 2022). The difference in AA release as
a percentage of the control may be due to the undeter-
mined presence of such anti-nutritional factors. Aderi-
bigbe et al. (2021) suggested that the effect of protease
on AA release and bird performance was independent of
the levels of trypsin inhibitor present in the diet. How-
ever, Wedekind et al. (2020) suggested that exogenous
protease was more effective in diets containing high
trypsin inhibitor levels. Another possible reason could
be the differences in protein solubility between the 2 soy-
bean sources (Zhao et al., 2022). The uplift of AAs
observed in fermented soybean meal (P < 0.001) and
other types of soybean meal could be due to the destruc-
tion of macromolecular proteins, the deactivation of
anti-nutritional factors, or a combination of both factors
during the fermentation process (Bao et al., 2014).
In summary, the in vitro evaluation indicated that the
multiprotease applied in this test acts over various feed-
stuffs used in poultry diets and complete broiler feed.
However, the degree of efficacy may vary according to
the feed ingredient evaluated. The following step is to
test the efficacy of the multiprotease in vivo when
applied to chicken corn-soybean diets varying in nutri-
ent composition and feedstuff proportions. Modifying
the CP levels will cause the desired dietary variability.
The multiprotease will be added to chicken feed at
2,400 U/kg since corn free-AA release was increased
(P < 0.001) by 11.18 % using this level instead of
1,200 U/g (Table 3). The specific objectives were to test
the effects of the multiprotease on CP, AA, and energy
utilization by chickens fed these diets.
In Vivo Evaluation of a Multiprotease in
Broiler Diets Varying in CP Content
Growth Performance Results of laboratory analysis of
samples of experimental diets (Table 4) indicated good
agreement (P < 0.001) between formulated and ana-
lyzed values for CP (r = 0.991) and AA (r = 0.997). The
analyzed activity of phytase for all diets and protease for
supplemented diets is presented in Table 5. The values
observed in pelletized and crumbled diets matched the
expected activity of phytase supplemented in the basal
diets and the inclusion of protease according to the
treatment. No protease activity was detected in control
diets.

Table 4. Formulated and analyzed1 values for crude protein (CP) and amino acids (AA) of experimental diets per CP treatment.

Treatment 21 19 17
Nutrient Formulated Analyzed Formulated Analyzed Formulated Analyzed
—————————————————————————- % —————————————————————————
CP 21.00 21.36 19.00 18.96 17.00 17.08
Total AA
Lys 1.26 1.38 1.14 1.24 1.01 1.10
TSAA 0.96 0.98 0.87 0.90 0.77 0.82
Thr 0.90 0.89 0.81 0.81 0.72 0.72
Trp 0.25 0.24 0.22 0.22 0.19 0.20
Ile 0.88 0.96 0.78 0.84 0.69 0.74
Leu 1.77 1.83 1.65 1.64 1.52 1.50
Val 0.97 1.08 0.88 0.88 0.78 0.81
Arg 1.38 1.36 1.22 1.18 1.07 1.04
Glu 3.65 3.78 3.30 3.31 2.96 2.95
Asp 2.08 2.13 1.83 1.82 1.60 1.60
Phe 1.02 1.08 0.92 0.95 0.82 0.85
Tyr 0.60 0.74 0.51 0.65 0.41 0.57
Ala 1.03 1.06 0.96 0.96 0.88 0.89
Gly 0.85 0.88 0.76 0.78 0.68 0.70
Ser 1.01 0.96 0.91 0.84 0.81 0.76
His 0.54 0.55 0.49 0.49 0.44 0.44
Pro 1.21 1.22 1.13 1.14 1.04 1.06
1
Analyzed values are the means of six samples, 3 replicates for each experimental diet.
 MULTIPROTEASE IN LOW PROTEIN DIETS 7
Table 5. Analyzed activity of enzymes in the pelletized and
crumbled experimental diets1

Diet Phytase (FTU/kg) Multiprotease (U/kg)2

Starter (0−7 d) 1,214
Grower basal diet (21%) 1,137
Grower basal diet (19%) 1,180
Grower basal diet (17%) 1,042
Grower basal diet (21%) 1,136
+ Protease
Grower basal diet (19%) 1,184
+ Protease
Grower basal diet (17%) 1,045
+ Protease
1
Values represent the average of 3 replicates samples per diet. Enzyme
recovery tests conducted by Kemin Industries (USA).
2
One unit of protease activity is defined as the amount of enzyme that
solubilizes one microgram of Azo-casein per minute at pH 7.0 and 37°C.
The results of the pre-experimental period up to 7 d of
age indicated the experiment started with a uniform
group of chickens (Table 6) for all treatments with an
average BW of 180 § 2 g, which is 23 g below the current
standard BW objective (203 g) for this genetic line
(Aviagen, 2022). No differences (P > 0.05) in BW
(Table 6), feed intake (132 § 2 g), and FCR (0.976 §
0.011 g:g) between groups of chickens assigned to the
different treatments, but fed a common starter diet were
observed up to 7 d of age.
Performance results from 8 to 24 d of age are pre-
sented in Table 6. Interaction effects (P < 0.05) were
observed on FCR (Figure 1). The main effects of CP
level were observed (P < 0.001) in BW at 24 d and BW
gain (8-24d). As expected, BW at 24 d (1,405, 1,338,
and 1,246 g), BW gain, and FCR (8−24 d) worsened as
dietary CP reduced from 21 to 17%, but multiprotease
supplementation did not improve (P > 0.05) these
parameters. The BW gain decreased by 12.9 % when N
intake reduced from 48.96 to 38.49 g/bird. Feed intake
was not affected (P > 0.05) by treatments.
These results partially agree with Yang et al. (2015),
who reported increments in feed intake and reduction in
growth and feed efficiency in Arbor Acres chickens when
CP and AAs were diluted 10, 20 and 30% (21.32 to
-
-
-
-
2,160
2,280
2,230
Figure 1. Effect of exogenous multiprotease (0 and 2,400 U/kg) on
feed conversion ratio of broilers at 24 d, fed grower diets (8 to 24 d)
with 3 levels of CP (17, 19, and 21%). a-c Means in bars with different
superscript letters are statistically different (P < 0.001) by Tukey’s
test.
15.40 % CP) in starter diets fed during similar dietary
phase (8−14 d). Several authors (Bregendahl et al.,
2002; Yadav and Sah, 2005; Angel et al., 2011; Freitas
et al., 2011) have reported that reduced-CP diets pro-
duced significant negative responses in growth perfor-
mance. Even with smaller CP reductions, several
authors reported worse chicken growth performance
when essential AA were not maintained at similar levels
using synthetic sources. Cowieson et al. (2017) reported
that Ross 308 broiler chickens fed diets containing 20.5%
CP and 1.08% dig. Lys had lower BW gain and higher
FCR than those fed diets with 21.0% CP and 1.13% dig.
Lys. Rehman et al. (2018) concluded that Hubbard chick-
ens receiving starter diets containing 19.3% CP were 21 g
heavier at d 14 of age than those receiving diets that con-
tained 18.8 % CP with 7% less dig. AA (1.00 vs. 0.93%
total lysine). Ndazigaruye et al. (2019) also observed 11 g
lighter Ross 308 male chickens at 21 d of age (962 vs. 973 g)
and worse FCR (1.364 vs. 1.399) by reducing CP from
20.58 or 19.72% CP (1.20 vs. 1.10% total Lys) in grower
diets fed after 8 d of age.

Table 6. Effect of exogenous multiprotease on growth performance of broilers at 24 d, fed grower diets (8 to 24 d) with 3 levels of CP (17,
19, and 21%).

CP level % Multiprotease U/kg BW 7 d BW 24 d BWG 8-24 d FI 8-24 d

———————————————−G—————————————————————
17 183 1,246c 1,067c 1,498
19 179 1,338b 1,159b 1,515
a a
21 180 1,405 1,225 1,508
SEM 1.7 11.6 11.4 16.9
0 181 1,324 1,145 1,498
2,400 179 1,334 1,155 1,516
SEM 1.4 9.4 9.3 13.8

CV, %1 3.49 3.25 3.70 4.20

Source of variation ————————————————−P-values———————————————————
CP level 0.342 <0.001 <0.001 0.772
Multiprotease 0.264 0.428 0.443 0.356
CP level*Multiprotease 0.792 0.104 0.159 0.107
a-c
Means in columns followed by different superscript letters are statistically different (P<0.05) by Tukey’s test. n = 7.
1
Coefficient of variation or normalized root-mean square deviation. Ratio of the standard deviation to the mean (%).
8 ~
PENUELA-SIERRA ET AL.

Nevertheless, the present study evaluated a more sig-
nificant CP reduction from 21 to 17% CP and dig. Lys
from 1.15% to 0.93. In the current study, chickens fed
21% and 19% CP grower diets were heavier than the
current standard for Ross-708 male broilers (Aviagen,
2022), while chickens fed the 17% CP diets had similar
BW (1,246 g) to this standard (1,250 g) at d 24. The die-
tary balance (5.47 % dig. Lys:CP) and AA profile used
in this experiment supported adequate growth. Opti-
mum dig. Lys:CP ratios have been previously evaluated
(Ng’ambi et al., 2009), and results indicated that in each
feeding phase, one ratio could optimize growth rate,
FCR, and breast meat yield; however, the ratios can be
different for each parameter and dig. Lys:TP ratio could
be a better parameter to formulate these low-CP diets
(Alhotan and Pesti, 2016). However, FCR worsened sig-
nificantly as CP decreased from 21 % CP (1.230 g:g) to
19% (1.273) and 17% CP (1.355 g:g). The interaction
effect (P > 0.001) indicated FCR response was depen-
dent on multiprotease supplementation; however, no sig-
nificant improvement due to multiprotease was detected
at any CP level (Figure 1). Higher starch content in low-
CP diets could partially explain the adverse effects of
increasing FCR (Chrystal et al., 2020; Liu et al., 2021).
Positive effects of serine proteases on growth perfor-
mance parameters have been reported (Cowieson et al.,
2017; Rehman et al., 2018; Park et al., 2020; Saleh et al.,
2020; Jabbar et al., 2021), while others did not show a
response over the same chicken growing period from 7 to
24 d of age (Ding et al., 2016, Law et al., 2018; Cardinal
et al., 2019). Proteases have been tested to improve the
performance of chickens fed low-CP diets (Ding et al.,
2016; Cho et al., 2020; Jabbar et al., 2021). Ding et al.
(2016) reported that a reduced CP diet (starter diets 21,
20, and 19% CP) negatively affected performance. The
supplementation with an exogenous protease produced
by fermentation using Bacillus licheniformis at 150 and
300 mg/kg had no significant effects on growth perfor-
mance. Those results are similar to the data obtained in
the present study. In contrast, Mohammadigheisar and
Kim (2018) showed significant improvements in BW
gain due to the addition of a serine protease, also
expressed in Bacillus licheniformis to low CP diets
(19.4% CP in starter and 17.5% CP in grower diets) and
a positive trend was observed in improving FCR. In a
recent study by Cho et al. (2020), formulating diets with
the same concept used in the current study and using
the same multiprotease observed similar responses in
growth performance with a reduction in dietary CP
(21.5−18.5% CP). However, multiprotease supplemen-
tation could restore the production parameters.
It is evident that the dietary reduction of CP affects
growth performance. However, the response to protease
supplementation has been inconsistent across different
studies, presumably due to the differences in feed ingre-
dients or the research methodologies used. In the present
study, it should be emphasized that all diets maintained
dig. Lys:CP at 5.47 % and the same ideal protein profile
and only synthetic Lys, Met, and Thr were used to
obtain the desired AA levels of balanced protein but not
to keep similar essential AA concentrations across the
three CP diets.
Energy and Total Tract Digestibility
Energy utilization and total tract digestibility results
are presented in Table 7. Interaction effects (P < 0.05)
were observed for AME, AMEn, CP, and total and
apparent ileal DM digestibility. Chickens fed diets with
17 and 19 % CP had better AME and AMEn than chick-
ens fed 21% CP diets. The highest total tract CP digest-
ibility was observed in chickens fed diets with 17 % CP
and the worst in chickens fed a diet with 21% CP with-
out the multiprotease. Multiprotease supplementation
improved (P < 0.01) AMEn by 71 kcal/kg, CP digest-
ibility from 59.45 to 63.51 %, and ileal DM digestibility
from 67.08 to 73.49%, but only in diets containing 21 %
CP. Ileal DM and total tract CP digestibility only
improved with multiprotease supplementation in 21%
CP diets. However, the total tract digestibility of DM
decreased (P < 0.05) as dietary CP increased, and the

Table 7. Effect of exogenous multiprotease (2,400 U/kg) on apparent metabolizable energy (AME), nitrogen-corrected AME (AMEn),
total tract apparent digestibility of CP and dry matter, and the apparent ileal dry matter digestibility of broilers at 24 d fed diets with 3
levels of crude protein (17, 19, and 21%).

Total tract Ileal

Multiprotease, Crude protein Dry matter Dry matter
CP level, % U/kg AME AMEn digestibility digestibility digestibility
—————- Kcal/kg ————— —————————- % ——————−———
17 0 3,417a 3,389a 65.02a 72.32a 72.93ab
a ab
19 3,388 3,351 60.77bc 69.97ab 73.80ab
21 3,286b 3,234c 59.45b 66.89c 67.08c
17 2,400 3,366ab 3,337ab 61.83abc 70.64ab 72.78b
19 3,366ab 3,328ab 61.56abc 70.32ab 75.14a
21 3,351ab 3,305b 63.51a 68.96bc 73.49ab
SEM 29 29 0.90 0.61 0.46
CV, %1 2.17 2.15 3.81 2.30 1.58
Source of variation ————————————————— P-values ——————————————————
CP level 0.009 0.001 0.465 <0.001 <0.001
Multiprotease 0.907 0.945 0.047 0.629 <0.001
CP level*Multiprotease 0.048 0.038 0.002 0.0172 <0.001
a-c
Means in columns followed by different superscript letters are statistically different (P <0.05) by Tukey’s test. n = 7.
1
Coefficient of variation or normalized root-mean square deviation. Ratio of the standard deviation to the mean (%).
 MULTIPROTEASE IN LOW PROTEIN DIETS
Table 8. Effect of exogenous multiprotease on nitrogen intake, calorie, and nitrogen conversion of broilers at 24 d, fed grower diets (8 to
24 d) with 3 crude protein levels (17, 19, and 21%).
Multiprotease,
CP level, % U/kg Nitrogen intake
− g/bird −
17 38.49c
19 43.79b
21 49.32a
SEM 0.50
0 43.64
2,400 44.10
SEM 0.41
CV, %1 4.23
Source of variation ———————————————− P-values ————————————————
CP level 0.001
Multiprotease 0.437
CP level*Multiprotease 0.196
a-c
Means in columns followed by different superscript letters are statistically different (P < 0.05) by Tukey’s test. n = 7.
1
Coefficient of variation or normalized root-mean square deviation. Ratio of the standard deviation to the mean (%)
addition of multiprotease did not produce any signifi-
cant effect. However, calorie conversion worsened from
3.66 to 4.25 kcal/g BW gain as the CP level decreased
while N conversion improved from 40.0 to 36.1 g/kg
BWG (Table 8). These results indicated better N utiliza-
tion but decreased energy utilization with reduced die-
tary CP content. Research has shown better N
utilization with dietary CP reduction both without and
with performance depression. Belloir et al. (2017)
reported increased N retention efficiency with reducing
CP content (3.5 points/CP percentage point) while
observing a reduced performance. Reduction in CP lev-
els (20.5−17.4% CP) increased the N-utilization from 62
% to 73 %, with no performance drop in the study by
Ulrich et al. (2018).
Some studies have reported increased N retention and
improvements in growth performance or carcass traits
by supplementing exogenous proteases in broiler diets
(Xu et al., 2017; Rehman et al., 2018; Jabbar et al.,
2021), differing from the present study. Xu et al. (2017)
used the same multiprotease evaluated in the present
experiment in corn and sorghum-soybean diets with
corn gluten meal (21.8 and 21.3% CP). Rehman et al.
(2018) compared keratinase and serine proteases in diets
(19.3 and 18.8% CP) containing maize, rice polishing,
soybean, rapeseed, sunflower, cottonseed, and fish
meals. Jabbar et al. (2021) evaluated a neutral protease
in diets with a reduction in CP (17, 19, and 21% CP)
containing corn, soybean, canola, sunflower, gluten, and
bone meals, and poultry by-products, balanced with sev-
eral synthetic AA (Lys, Met, Val, Thr, Ile, Arg). Pro-
teases can have more beneficial effects in more complex
plant-based diets.
Nevertheless, Yu et al. (2007) reported that the sup-
plementation of a cocktail of a serine protease in combi-
nation with carbohydrases in low-CP corn-soybean diets
(20.39 and 18.93% CP in starter and grower diets)
resulted in lower dietary-protein waste, N excretion, and
emissions into the environment without a reduction in
the performance of broiler chickens. In the same way,
9
Calorie conversion Nitrogen conversion
− cal/g BWG − − g/kg BWG −
4.25a 36.09c
3.99b 37.77b
3.66c 40.11a
0.03 0.22
3.96 38.00
3.97 37.97
0.02 0.18
2.77 2.12
0.001 0.001
0.651 0.908
0.969 0.409
Freitas et al. (2011) observed improvements in FCR,
fat, and CP digestibilities using low-CP corn-soybean
meal-based diets (21.5 % CP in starter and 19.0% CP in
grower diets) with meat and bone meal (»6%) supple-
mented with increased levels of protease (7,500 to
120,000 PROT units/kg) in 42-day-old broilers. Like-
wise, Angel et al. (2011) reported that CP digestibility
improved due to serine protease supplementation com-
pared to control (22.5% CP) or low-CP (20.25% CP)
diets. However, they did not observe differences among
various concentrations of protease supplementation
(7,500, 15,000, 30,000, and 60,000 PROT units/kg) in
22-day-old chickens. Similarly, Fru-Nji et al. (2011) con-
cluded that exogenous serine protease (15,000 PROT/kg
feed) enhanced the protein and energy digestibility of
broilers fed corn-soybean meal diets (21.1 and 20.0 %
CP in starter and 20.0 and 19 % CP in grower diets). In
more recent work, Kamel et al. (2015) indicated that the
same serine protease on top (15,000 PROT/g) of corn-
soybean meal diets with corn gluten meal (21.5 % CP in
starter diets) improved growth performance, carcass
dressing percentage, CP digestibility in broiler chickens
and reduced ileal clostridia species counts. Proteases
may also affect endogenous enzyme activity. Ding et al.
(2016) reported an improvement of 16 % in trypsin
activity in the pancreas when a serine protease was
added to the diet (21 % CP in starter and 19 % in grower
diets) at the dose of 300 mg/kg, which could help
improve protein digestibility.
Cho et al. (2020), who worked with the same multi-
protease used in the present study, demonstrated that
supplementation of multiprotease to 21.5 to 18.5% CP
diets improved the ileal digestibility of DM and CP of
chickens on d 21 and 35 compared to those fed diets
without a multiprotease. In another broiler study con-
ducted by De Leon et al. (2021), the same multiprotease
improved the growth performance of low-CP (0.5%
reduction) and AA (2% reduction) diets when supple-
mented at 300g/ton (2,400 U/kg). De Leon et al. (2021)
observed improvements in DM, CP, and energy ileal
10 ~
PENUELA-SIERRA ET AL.
digestibility coefficient. On the other hand, Saleh et al.
(2020) studied the effect of serine-protease, and they
found that the digestibility of DM and CP was enhanced
by adding both 200 and 300 mg/kg of protease com-
pared to the control without supplementation in starter
diets containing 21 % CP.
Likewise, Jabbar et al. (2021) concluded that a neu-
tral protease expressed in Bacillus subtilis (30,000
PROT units/kg feed) improved BW gain and FCR in
diets with 17 and 19% CP during a trial period of 15 to
28 d. Protease supplementation only improved N reten-
tion (62.71 vs. 64.00 %) and reduced abdominal fat in
17% CP diets. However, no significant protease improve-
ments in growth performance or N retention, or changes
in abdominal fat were observed during that study in
chickens fed a 21 % CP diet. In the study described by
Jabbar et al. (2021), all diets maintain similar Lys (1.15
%) and TSAA (0.90%) levels by adding variable levels of
synthetic AA (L-Lys HCl, DL-Met, Val, Thr, Iso, Arg),
and did not contain phytase, or it was not reported.
This neutral protease improved the response of this low-
CP but had no significant benefits in the 21% CP diet.
This is opposite to the effects observed with the multi-
protease evaluated in the experiment reported here,
where phytase was added to all diets.
Several exogenous enzymes are frequently utilized in
poultry production. Velasquez-De Lucio et al. (2021)
mentioned that the enzyme most commonly used in
poultry diets worldwide is phytase, followed by xylanase
and amylase. All diets used in the present experiment
contained phytase, which has been suggested to improve
the utilization of low-CP diets (Chrystal et al., 2020).
Protease is currently less well adopted in commercial
poultry diets, and the results of studies conducted with
mono-component proteases are frequently inconsistent.
In this context, the effects of exogenous protease on
broilers are unclear due to differences in the types of pro-
teases tested and dissimilar methodology, differences in
the composition of the negative control diet, and the
addition of other enzymes like phytase and carbohy-
drases.
Inconsistency in the effect of exogenous proteases on
growth performance or AA digestibility in poultry diets
has been attributed to the inherent digestibility of AA
in the diets (Cowieson and Roos, 2016), which vary in
low-CP diets (Liu et al., 2021). The neutral proteases
supplemented in most of the experiments in the litera-
ture are categorized as serine proteases, the same family
of proteases as trypsin and chymotrypsin, and have
activity over a broad range of substrates (Odetallah et
al., 2005). Additionally, Freitas et al. (2011) suggested
that proteases supplemented in combination with other
enzymes might contribute to the conflicting reports sur-
rounding protease supplementation in poultry diets. In
the present study, all diets contained at least
1,000 FTU/kg phytase. The interactive effects between
enzymes could be partially responsible for some of the
results observed herein, as Erdaw et al. (2017) had dis-
cussed, but differentiating these effects was not the
scope of this project.
Amino Acid Ileal Digestibility
Interaction effects (P < 0.05) were observed for almost
all ileal AA digestibility values except for Cys and Trp
(Table 9). The multiprotease improved the ileal digest-
ibility of all AA in diets with 21% CP, except for Cys
and Trp. The multiprotease improved the digestibility
of Cys and Trp (P < 0.05) independently of the CP level.
No differences in ileal AA digestibility due to dietary CP
level (P > 0.05) were detected, except for Cys digestibil-
ity (P < 0.01), which was better in 19 % diets than in 21
% CP, while 17% was intermediate. A similar balance of
AA across all diets may cause similar ileal AA digestibil-
ity. The multiprotease had positive effects when AA lev-
els exceeded chicken requirements in 21% CP diets.
These results differ from those reported by Rada et al.
(2016). They concluded that mono-component serine
protease did not significantly affect apparent ileal AA
digestibility or protease activity in jejunum when feed-
ing diets containing 19.9 and 20.7% CP and different
inclusion levels of rapeseed meal. On the other hand,
Walk et al. (2019) observed improved AA digestibility
with the supplementation of serine and acid proteases in
diets containing 19.00 or 21.50% CP but with no
improvement in broiler performance parameters, similar
to the present study.
Several authors have reported results similar to the
present experiment in AA digestibility. For example,
Angel et al. (2011) observed that exogenous protease
supplementation improved the apparent digestibility
(0.010.08 %) of Arg, Thr, Ile, Asp, Lys, His, Ser, and
Cys in broiler chickens fed control (22.5% CP) or low-
CP (20.25% CP) diets. Other authors have reported sig-
nificant improvements in feed ingredients’ apparent ileal
AA digestibility (Adebiyi and Olukosi, 2015; Stefanello
et al., 2016). Park et al. (2020), researching the effects of
protease (0.03, 0.06, and 0.09%) on growth performance
and nutrient digestibility, concluded that the digestibil-
ity of a variety of AA, like Lys, Met, Cys, Thr, Ile, Leu,
His, and Try was enhanced significantly as exogenous
protease supplementation increased in broiler starter
(21% CP) and grower (19%) diets.
Plant proteins like soybean meal are rich in anti-
nutritional factors, particularly protease inhibitors
that can suppress the activity of the proteolytic
enzymes trypsin and chymotrypsin (Erdaw et al.,
2017). A reduction in the activity of these digestive
enzymes may decrease the protein and AA digestibility
of plant-based diets. Therefore, including protease in
livestock feed could effectively attenuate the consider-
able adverse effects of unbalanced AA and anti-nutri-
tional factors contained in feed materials that
primarily consist of plant proteins (Erdaw et al., 2017;
Park et al., 2020; Jabbar et al., 2021). In the present
experiment, the protease supplementation improved
energy utilization and digestibility in the 21% CP diet.
This diet had up to 34.88% soybean products (solvent-
extracted and full-fat) and more plant protein sub-
strate than the other diets. In this diet type, multipro-
teases will probably have more pronounced effects.
 MULTIPROTEASE IN LOW PROTEIN DIETS 11
Table 9. Effect of exogenous multiprotease on apparent ileal amino acid digestibility coefficients at 24 d of age for grower broiler diets with 3 crude protein levels (17, 19, and 21%), fed from

90.25ab 89.01ab 85.90ab 83.44ab 85.79ab 84.84ab 79.32ab 85.30ab

86.30ab 84.40ab 84.76ab 90.64ab 89.30ab 86.37ab 83.76ab 86.16ab 83.96ab 79.80ab 85.49ab

85.60ab 83.34b 84.15ab 89.75b 88.51b 85.37ab 82.34b 85.30ab 83.95ab 78.15b 84.67ab
90.99ab 89.53ab 87.11ab 84.07ab 86.74ab 85.07ab 80.36ab 85.88ab
CdCP

0.508
0.088
0.021
The in vivo improvements in ileal AA digestibility

83.81b 82.64b 82.01b 89.73b 88.16b 85.01b 82.53b 84.88b 82.78b 78.06b 84.07b

87.06a
———————————————————————————————- % ————————————————————————————————————

0.45

0.37

0.64
1.98
84.99
85.69
85.57

84.96
85.87

————————————————————————————— P-values ——————————————————————————————————

observed with multiprotease supplementation are con-
sistent with the in vitro evaluation results (Table 3).

0.160
0.084
0.007
82.09a
Although the in vivo study was not designed to evaluate

0.56

0.46

0.79
Gly

2.62
78.79
80.09
80.08

83.86b 79.06
85.01a 80.21
the correlation between in vitro and in vivo efficacy of
protease, our results indicated the possibility of deriving

0.979
0.041
0.014
86.01a
0.47

0.39

0.67
2.09
84.40
84.51
84.39
Ser

a correlation between the 2. Previous attempts to

develop a correlation between in vitro and in vivo prote-
ase efficacy were unsuccessful (Zheng et al., 2023).

0.338
0.078
0.046
87.64a
Phe

0.45

0.37

0.64
1.96
85.55
86.45
86.27

85.62
86.56

Despite the improvement in in vitro crude protein

digestibility (IVCPD), the addition of 4 proteases alone
and in combination significantly reduced the apparent

0.209
0.188
0.018
85.70a
Asp

0.51

0.42

0.73
2.30
82.89
83.92
84.12

83.24
84.04

ileal digestibility of CP in broilers at 31 d and the appar-

ent ileal digestibility of specific AA (Asp, Thr, Ser, Glu,

0.228
0.065
0.045
87.80a
Gly, Ala, Cys, Met, and Phe). This necessitates more
0.46

0.37

0.64
1.97
85.64
86.75
86.41

85.77
86.77
His

judiciously designed in vitro and in vivo experiments to

0.331 develop such correlation between the in vitro and in
0.078
0.016
90.65a
Glu

0.35

0.29

0.50
1.47
88.76
89.42
89.51

88.86
89.57

vivo effect of multiprotease.

0.134
0.078
0.021
91.89a
Arg

Energy and Nutrient Intake

0.32

0.26

0.46
1.32
90.00
90.81
90.89

88.04b 85.46b 83.67b 84.00b 90.23

90.88

The energy utilization, CP, and AA digestibility val-

0.318
0.036
0.004
85.10a

86.01ab 83.96ab 84.96a

85.28ab 85.24a
a

ues varied across treatments of CP levels. Dietary treat-

0.47

0.39

0.67
Ala

2.07
84.56
85.00
84.11

85.91

ments did not affect (P > 0.05) feed intake (Table 6).
Still, considering the variability in feed intake, energy
0.189
0.020
0.010
84.98a

Means in columns followed by different superscript letters are statistically different (P < 0.05) by Tukey’s test. n = 7.
0.47

0.38

0.66
2.05
83.66
84.85
84.63

86.32
Ile

utilization, and digestibility values per diet, it is possible

Coefficient of variation or normalized root-mean square deviation. Ratio of the standard deviation to the mean (%).

to calculate the total energy and nutrient intake or

retention (Table 10) during the experimental period.
0.374
0.025
0.003
89.21a 86.48a

86.47a
a
Leu

0.41

0.34

0.58
1.76
85.81
86.39
85.73

87.36

These results explain the chicken growth performance

results better.
No effects (P > 0.05) of CP level or protease supple-
0.111
0.010
0.334
0.38

0.31

0.53
89.10
88.78
87.99

80.97ab 83.02ab 88.93

81.16ab 83.40ab 88.19
78.92b 81.15b 87.00
80.02ab 82.25ab 89.29
82.00ab 83.63ab 89.37
88.98

1.57
Trp

mentation were observed in total energy intake, CP, and

Lys intake across treatments (Table 10). However, more
0.480
0.061
0.007
a
0.52

0.43

0.74
Val

82.64
83.52
83.34

82.60
83.70

85.20

2.32

protein and AA consumption was related to better BW

gain and FCR. Lysine and CP intake had a highly posi-
tive (P < 0.001) correlation with BW gain (r = 0.92,
0.464
0.073
0.022
a
Thr

0.62

0.50

0.87
75.68ab 80.49
81.58
80.96

92.96b 74.93b 80.35

81.67

82.99

2.86

0.85) and a negative correlation with FCR (r = -0.82;

-0.77), respectively. Energy consumption (AME and
AMEn) was only moderately correlated (r = 0.50) (P <
0.005
0.042
0.106
b
76.79a

76.21a
Cys

0.61

0.50

0.86
73.90

88.34ab 93.51ab 75.80

88.12ab 93.31ab 76.21
86.93b 92.00b 71.93
87.29b 93.28ab 75.52
77.36
75.54

2.86

0.001) with BW gain but not correlated (P > 0.05) with

other performance parameters.
0.415
0.027
0.023
93.61a

88.45ab 93.75a
a
Met

0.26

0.22

0.37
93.38
93.53
92.97

93.80

1.06

CONCLUSIONS
0.579
0.121
a
0.37

0.30

0.52
Lys

87.78
88.29
88.41

87.82
88.48

89.67

1.52

In line with the in vitro results, the multiprotease sup-

plementation improved the chicken AA and CP total
Multiprotease U/kg

tract digestibilities in 21% CP diets. The AMEn was

mainly affected by dietary CP levels, and multiprotease
0.004

supplementation improved the AMEn by 71kcal/kg.

SEM
0
2,400

2,400

Broiler performance was influenced primarily by dietary

CP levels, and adding multiprotease did not improve
growth performance at any CP level. Dietary CP level
CP level*Multi-protease

or multiprotease supplementation did not affect total

8 to 24 d of age. a, 1

energy, CP, and lysine intake. The similarity in energy,

Source of variation

CP, and AA intake partially explains the lack of differ-

Multi-protease

ences in BW gain and FCR between treatments with or

CP level %

without multiprotease supplementation. However,

CP level
CV, %1

higher CP and AA intake were correlated with better

a-c
SEM

SEM

1
17
19
21

17
19
21
17
19
21

performance. Further evaluation of the effect of

12 ~
PENUELA-SIERRA ET AL.
Table 10. Effect of exogenous multiprotease on total intake of Apparent Metabolizable Energy (AME) and Apparent Metabolizable Energy corrected by nitrogen (AMEn), crude protein multiprotease in poultry diets with lower CP levels and
different feedstuffs is recommended to better understand
protease application to broiler diets.

0.977
0.978
0.546
0.70
14.43
15.23
14.70
14.97
14.33
15.01

12.49
Lys

————————————————————- P-values ———————————————————————————

ACKNOWLEDGMENTS
——− g/broiler ——-

This work was supported by Kemin Industries, Des

Moines, Iowa, USA. The funding sponsor, Kemin Indus-
tries, provided financial support to conduct this
research, and Kemin Technologies Co. conducted part
of laboratory analyses related to enzyme activities in
vitro.
0.966
0.838
0.779
7.88

13.02
157.86
163.08
160.64
160.72
155.40
161.48
CP

DISCLOSURES
Yanlin Yang works for Kemin Technologies Co, Zhu-
Energy and nutrient intake

hai, China. Bindhu Lakshmibai Vasanthakumari, Ye

Lao and Alexandra Wealleans work for Kemin Indus-
tries, Des Moines, Iowa, USA. Kemin Industries was the
sponsor of this project. However, the in vivo experiment
and almost all laboratory analyses were conducted by
NC State personnel without intervention from Kemin
Industries or Kemin Technologies.
0.745
0.817
0.217
4.84
AMEn
(CP), and Lysine of broilers at 24 d, fed grower diets (8 to 24 d) with 3 crude protein levels (17, 19 and 21%).1

82
4,468
4,576
4,498
4,611
4,430
4,454

Coefficient of variation or normalized root-mean square deviation. Ratio of the standard deviation to the mean (%).

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