Authors Accepted Manuscript

Effects of dietary crude protein levels and

exogenous protease on performance, nutrient
digestibility, trypsin activity and intestinal
morphology in broilers
X.M. Ding, D.D. Li, Z.R. Li, J.P. Wang, Q.F.
Zeng, S.P. Bai, Z.W. Su, K.Y. Zhang
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DOI:
Reference:

S1871-1413(16)30199-8
http://dx.doi.org/10.1016/j.livsci.2016.09.002
LIVSCI3064

To appear in: Livestock Science

Received date: 29 January 2016
Revised date: 1 September 2016
Accepted date: 5 September 2016
Cite this article as: X.M. Ding, D.D. Li, Z.R. Li, J.P. Wang, Q.F. Zeng, S.P.
Bai, Z.W. Su and K.Y. Zhang, Effects of dietary crude protein levels and
exogenous protease on performance, nutrient digestibility, trypsin activity and
intestinal
morphology
in
broilers, Livestock
Science,
http://dx.doi.org/10.1016/j.livsci.2016.09.002
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Effects of dietary crude protein levels and exogenous protease on performance, nutrient
digestibility, trypsin activity and intestinal morphology in broilers
X. M. Ding, D. D. Li, Z. R. Li, J. P. Wang, Q. F. Zeng, S. P. Bai, Z. W. Su, K. Y. Zhang*
Institute of Animal Nutrition, Sichuan Agricultural University, China, Yaan, 625014
*

Corresponding author. Tel/fax: 86 835 2885630. zkeying@sicau.edu.cn

Abstract
The experiment was conducted to study the effect of exogenous protease in different crude
protein (CP) level diets on performance, nutrient utilization, trypsin activity and intestinal
morphology in broilers. A total of 1,080 1-d-old Arbor Acres (AA) male broilers were
allocated into a 3 3 factorial design that included 3 CP levels (CP1, CP starter 21%,
grower 19%; CP2, CP starter 20%, grower 18%; CP3, CP starter 19%, grower 17%) and 3
protease levels (0, 150, and 300 mg/kg, enzyme activity unit 1 105 U/g) for 42 d (starter,
d 1 to 21; grower, d 22 to 42). As CP was decreased on d 1 to 21 and from d 1 to 42, body
weight gain (BWG) and feed intake (FI) of birds were reduced (P 0.05), whereas the
ratio of feed to gain (F/G) was increased (P < 0.05). Protease supplementation significantly
improved the F/G of 1 to 21-d-old broilers (P < 0.05), regardless of dietary protein level.
The apparent CP digestibility of the CP3 diet was lower than that of the CP1 dietP<0.05),
and protease supplementation significantly improved CP digestibility (P<0.05). When CP
levels decreased, the activity of trypsin in the pancreas and duodenum content was
significantly decreased (P<0.05), and both the villus height and the villous height and the
crypt depth ratio (VCR) in the duodenum, jejunum, and ileum were significantly reduced
(P<0.05). Supplementation with 300 mg/kg of protease significantly increased the activity
of trypsin in the pancreas (P<0.05) and 21 d villus height and VCR in the duodenum,
jejunum (villus height), ileum (VCR). No interaction was observed among growth
performance, nutrient digestibility, trypsin activity and the intestinal morphology of the
1

broilers. These results suggest that supplementation with an exogenous protease enhanced
endogenous trypsin activity and intestinal morphology regardless of CP levels, which
resulted in improved CP digestibility during the starter period.

1. Introduction
Feed costs account for approximately 60-75% of broiler production costs and, in turn,
protein sources account for the majority of those feed costs. As a result of increasing feed
costs and the rapid development of the enzyme industry, the use of exogenous enzymes
(including phytase, the NSP enzyme, and enzyme blends) as a cost-effective means of
improving feed efficiency, performance and environmental quality is already relatively
commonplace.
In the past, research has mainly focused on improving the energy value of diets and
increasing the utilization of phytate phosphorus from plant based diets, with much less
attention being paid to crude protein (CP) digestion. The endogenous proteases synthesized
and released in the gastrointestinal tract (GIT) are generally considered to be sufficient for
optimizing feed protein utilization (Le Heurou-Luron et al., 1993; Nir et al., 1993).
However, poultry CP digestibility data indicate that valuable amounts of protein pass
through the GIT without being completely digested (Parsons et al., 1997; Wang and arsons,
1998; Lemme et al., 2004). This undigested protein represents a chance for the use of
supplemental exogenous proteases in broiler diets to improve protein digestibility.
Many studies have reported adding enzyme blends containing proteases to the poultry
diet (Morgan and Bedford, 1995; Cowieson and Ravindran, 2008; Rahman et al., 2009).
However, the interpretation of results from these protease studies is sometimes difficult
because of confounding effects resulting from the presence of several enzymatic activities
2

in a single treatment; studies using mono-component proteases are thus necessary. Most of
the single protease research has focused on performance and nutrient digestibility (Maiorka
et al., 2009; Favero et al., 2009; Fidelis et al., 2010; Angel et al., 2011; Freitas et al., 2011),
whereas reports on physiological responses are scarce. Some publications have shown
improvements in broiler performance and in nutrient utilization when proteases were added
to diets (Ghazi et al., 2003; Fru-Nji et al., 2011; Freitas et al., 2011; Angel et al., 2011);
however, other studies have reported inconsistent results (Simbaya et al., 1996; Marsman
et al., 1997; Naveed et al., 1998). Zanella et al. (1999) reported that enzyme
supplementation should allow for a reduction in CP formulation and that AA individuals
were not all improved by supplementation. Some studies have also evaluated practices that
involve a reduction in dietary protein levels because this not only reduces nitrogen
excretion into the environment (Aletor et al., 2000; Bregendahl et al., 2002) but also
reduces diet costs. Previous studies have explored the use of exogenous proteases in low
protein diets. Freitas et al. (2011) reported that protease supplementation had no effects on
performance, regardless of diet protein levels. However, the authors showed that the CP
digestibility and the determined AME were both greater when the protease was added to
high-protein diets (compared to low protein diets). There is no systematic information on
the effects of different protease supplementation levels in broiler diets. The objective of
this study was to evaluate the effects of crude protein levels and mono-component
proteases in broiler feeds on growth performance, apparent nutrient digestibility, trypsin
activity and intestinal morphology.
2. Materials and methods
2.1 Protease
The protease used in this study was produced by fermentation of Bacillus
licheniformis (Mianyang Habio Bio-tech Co. Ltd., Mianyang, China). One unit of this
3

protease enzyme activity is defined as the amount of enzyme that releases 1 g of

p-hydroxyphenylalanine from the Flynn positive amino acid and peptide per minute at pH
8.5 and 37C (Ren et al., 2011).
2.2 Experimental design and diet
A 3 3 factorial design that included 3 CP levels (CP1, CP starter 21%, grower 19%;
CP2, CP starter 20%, grower 18%; CP3, CP starter 19%, grower 17%) and 3 protease
levels (0, 150, and 300 mg/kg, enzyme activity unit of 1 105 U/g) was set up. A total of
1,080 1-d-old Arbor Acres (AA) male broilers, obtained from a commercial hatchery
(Sichuan Yuguan Agriculture Co., Ltd., Shehong, China), were randomly assigned to 54
pens (100200 cm) and maintained on a 24-h continuous light schedule. Each pen housed
20 broilers and was regarded as a statistical unit, resulting in 6 replicates for each of the 9
treatments, which were fed in the starter (d 1 to 21) and grower (d 22 to 42) period.
Temperature was gradually decreased from 33C at chick arrival to 23C on d 24, and
thereafter kept constant to trial termination on d 42. Feeds were presented ad libitum
during the entire trial. Water was provided in a drinker with 4 nipples per pen. The animal
experiment was conducted in accordance with the principles of the Animal Care and Use
Committee of the Sichuan Agricultural University (Wenjiang, China).
The CP1 diet (Table 1) was based on maize and soybean meal; composition and nutrient
levels were in line with the Agricultural Trade Standardization of China (NY/T33-2004).
The CP2 and CP3 diets were formulated with a reduction of 4.76% and 9.52% in CP and
amino acids (AA), respectively, compared to the CP1 diet. Other formulated nutrient and
energy targets remained similar across diets. Feeds were pelleted (~65C) using a small
granulator (Kefei, LQ-25 gantry type, Jiangsu, China).
2.3 Growth performance
Broilers were weighed, and feed consumption for each pen was measured on d 21 and
4

42 during the 42-day experiment. The cumulative weight gain (BWG), feed intake (FI),
and F:G (F/G) ratio were calculated.
2.4 Apparent nutrient and energy digestibilit
A total excreta collection method was used to determine the apparent nutrient and gross
energy digestibility. At day 43, one bird per replicate was selected based on the average
body weight and housed individually in cages. Feed and water were provided ad libitum.
The digestibility trial consisted of a two-day adaptation period followed by a four-day
collection period. Excreta was collected every 3 hours and stored at 20C. At the end of
the collection period, feed was collected and weighed. The excreta samples were
freeze-dried. The dried samples were ground to pass through a 0.5 mm sieve and stored in
airtight plastic containers at -4C until chemical analysis. The feed and excreta samples
were analyzed in duplicate using AOAC methods (2000) (dry matter, AOAC method
930.15; CP, AOAC method 990.03). The feed and excreta gross energies were determined
using an adiabatic bomb calorimetric method according to manufacturers instructions
(Parr Instrument Company, IL, USA).
2.5 Digestive enzyme activity assay
On days 22 and 43, one bird per replicate with the BW close to the replicate mean after a
12-h fast was selected and killed by cervical dislocation. These birds were then dissected to
remove the pancreas and the intestinal (duodenum) contents. The pancreas and duodenal
contents were immediately frozen in liquid nitrogen and stored at -20. Trypsin activity in
the pancreas and duodenal contents was measured following the detection kit instructions
obtained from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
2.6 Intestinal morphology
Duodenal, jejunal and ileal samples of approximately 2 cm in length were stored in a 4%
methanol solution for histological analyses. These samples were stained with haematoxylin
5

and eosin using standard paraffin embedding procedures. A total of five intact,
well-oriented cryptvillus units were selected in triplicate from each broiler intestine.
Villous height and crypt depth were measured using an image processing and analysis
system (Optimus software version 6.5; Media Cybergenetics, North Reading, MA).
2.7 Statistical Analyses
Data were subjected to analysis of variance using the general linear model procedures
implemented in SAS (1990) using a 33 factorial arrangement with the pen being the
experimental unit. The model included protein, protease and their interaction as the main
effects. Mean values were compared using Duncans multiple range test when significant
difference existed. In all analyses, significance was declared at P<0.05.
3. Results
3.1 Growth performance
Broilers fed CP2 and CP3 diets had lower (P<0.05) BWG and FI and higher F/G
(P<0.05) compared to broilers that were fed the CP1 diet from d 1 to 21. The BWG of
broilers from the CP3 treatment was lower than that of broilers from the CP2 treatment
during the 42-d experimental period (Table 2). The supplementation with exogenous
protease (150 and 300 mg/kg) had no significant effects (P>0.05) on growth performance
during the 42-d experimental period.
3.2 Apparent nutrient and energy digestibility
There were no significant differences in dry matter (DM) and gross energy digestibility
among treatments (Table 3). For broilers that were fed low-protein diets (CP2 and CP3),
CP digestibility was decreased (P<0.008) and the addition of protease at the highest level
improved CP digestibility at 42 d.
3.3 Trypsin activity in the pancreas and duodenum contents
Trypsin activity was significantly decreased (P<0.001) in the pancreas and the
6

duodenum as the dietary protein levels increased for the 21- and 42-day periods (Table 4).
On d 42, trypsin activity was significantly increased in the pancreas (P<0.05). No
interaction was observed between protein levels, exogenous protease and trypsin activity.
3.4 Intestinal morphology
As shown in Table 5 and Table 6, as dietary CP levels decreased, villus height and VCR
in the duodenum, jejunum, and ileum were significantly reduced (P<0.05), whereas crypt
depth remained unaffected (P>0.05). Supplementation with a protease significantly
increased villus height and VCR in the duodenum, villus height in the jejunum, and VCR
in the ileum on d 21. There was no evidence that intestinal morphology was affected on d
42. There was no significant interaction between protein levels and added protease
affecting broiler intestinal morphology.
4. Discussion
4.1 Growth performance
In the present study, CP2 and CP3 treatments lowered dietary CP and amino acid levels
4.76% and 9.52%, respectively, compared to the CP1 treatment. As CP levels decreased,
the BWG and FI decreased while the F/G ratio significantly increased. Compared with the
CP1 diet, CP2 and CP3 treatments: 1) lowered BWG 3.8% and 7.4% (respectively), 2)
lowered FI 1.5% and 2.3% (respectively), 3) increased F/G 2.1% and 5.7% (respectively)
at d 1 to 21, 4) lowered BWG 0.8% and 4.4% (respectively) at days 1 to 42, and 5)
increased F/G 1.6% and 2.7% (respectively) at d 1 to 42. Therefore, the CP3 diet resulted
in a worse performance than the CP2 diet. These results are in agreement with previous
findings (Bregendahl et al., 2002; Yadav and Sah, 2005; Angel et al., 2011; Freitas et al.,
2011)that reported that supplementing a reduced-protein diet produced significant negative
responses in growth performance. In our study, the protease supplementation had no
effects on BWG or FI, though F/G was slightly improved (1.4%) between 1 to 21 d of age.
7

These results are mostly consistent with previous reports for turkeys (Vieira et al., 2013)
and broilers (Yadav and Sah, 2005; Fru-Nji et al., 2011; Angel et al., 2011; Freitas et al.,
2011). However, Ghazi et al. (2003) reported that the use of a mono-component protease
resulted in an increase of BWG and FI, while F/G was either negatively affected or not
affected at all, depending on the protease concentration used.
4.2 Apparent nutrient digestibility
The current study indicates that a low protein diet significantly (P<0.008) decreased CP
digestibility: a decrease of 5.2% was observed in the CP3 diet. This result is consistent
with a previous study by Yadav and Sah (2005), who reported that a one percent reduction
in dietary CP (but not in ether extract, crude fiber, or nitrogen free extract) significantly
decreased CP digestibility in broilers. In addition, Freitas et al. (2011) observed that
reduced protein levels significantly decreased protein digestibility. In contrast, Angel et al.
(2011) reported that a low protein diet had no effects (P>0.05) on CP digestibility in
broilers. Vieira et al. (2013) found no significant differences in apparent CP digestibility
when comparing normal protein and low protein diets. In these studies, growth
performance effects resulting from protease addition occurred in parallel with increases in
protein digestibility. Angel et al. (2011) indicated that CP digestibility improved as a result
of protease-supplemented diets when compared with PC or low protein diets, though they
did not observe differences among various concentrations of protease supplementation
(100, 200, 400, and 800 mg/kg). Freitas et al. (2011) reported that an improvement of 1.8%
or 1.0% in CP digestibility was observed when the protease was added to the high protein
or the low protein diet, respectively. In our study, supplementation with protease at the
highest level (300 mg/kg) increased the CP digestibility by 4.2% regardless of the CP
levels.
4.3 Trypsin activity
8

In the case of endogenous proteases, peptide bond specificity directly affects the rate
of protein hydrolysis, the quantity of peptides and the number of AA released and made
available for absorption (Moran, 1982). Exogenous proteases may augment endogenous
peptidases

by

increasing

protein

digestibility

and

hydrolyzing

proteinaceous

anti-nutritional factors such as lectins, trypsin inhibitors and antigenic proteins (Ghazi et al.,
2002; Douglas et al., 2000). In the present study, decreasing the protein levels significantly
decreased trypsin activity in the pancreas (P<0.001) and in the duodenum contents
(P<0.001). An improvement of 16% in trypsin activity in the pancreas was observed when
protease was added to the diet at the dose of 300 mg/kg. However, limited comparisons
can be made due to the lack of mono-component protease studies in broilers. Yu et al.
(2002) reported that the intestinal protease activity did not change when broilers were fed
plant proteases on d 21. These seemingly inconsistent results may be because the quantity,
type of protease and species were different.
4.4 Intestinal morphology
The intestinal morphology (reflected by the villus height, crypt depth and the VCR of
the duodenum, jejunum and ileum) is one of the major indicators of gut health in an animal.
Less than optimal quality and concentration of proteins can adversely impact intestinal
development and function (Wijtten et al., 2010; Gilbert et al., 2008). In the present study,
the CP2 and CP3 diets reduced both the villus height and the VCR. We found that
supplementation with protease significantly increased villus height and VCR in the
duodenum, villus height in the jejunum, and VCR in the ileum. This is consistent with
results described by Yuan et al. (2008), who found that the addition of an enzyme complex
containing a protease increased villus height and VCR in the intestine. However, no
findings could be drawn due to the lack of mono-component protease studies on broilers.
More studies are needed to evaluate repeatability and to shed light on the exact mechanism
9

that mediates these protease effects. Furthermore, it is important to note that the extent of
the effect on d 21 was greater than that on d 42, which was probably because intestine
development occurs mainly in the early stages and further implies that young broilers
might be more sensitive to proteases.
5. Conclusions
In conclusion, a reduced-protein diet negatively affected the performance, crude protein
digestibility, trypsin activity in the pancreas and duodenum contents, and intestinal
morphology in broilers. In addition, supplementation with an exogenous protease (300
mg/kg) improved crude protein digestibility, trypsin activity in the pancreas, and
duodenum and jejunum morphology at d 21.
Conflicts of interest
There were no conflicts of interest. No conflict of interest exits in the submission of this
manuscript, and manuscript is approved by all authors for publication. I would like to
declare on behalf of my co-authors that the work described was original research that has
not been published previously, and not under consideration for publication elsewhere, in
whole or in part. All the authors listed have approved the manuscript that is enclosed.

Acknowledgments
The present study was supported by the science and technology support program of the
Sichuan province (no. 2014NZ0044) and by the Chinese Ministry of Sciences and
Technology (no. 2014DFA32860). L.Z. Wang received the funding.

10

Table 1. Ingredients and nutrient composition of the basal diets (as fed-basis).
Item

Starter (d 1 to 21)
CP1a

Ingredient,%
Maize
57.93
Soybean meal(CP 43%)
35.16
Soybean oil
2.77
CaCO3
1.16
Dicalcium phosphate (Ca 21.8%,
1.84
AP 16.8%)
DL-Methionine(98.5%)
0.21
L-Lys HCl(78%)
0.1
L-Threonine(99%)
0.04
NaCl
0.4
Vitamins Premixb
0.03
Choline chloride(50%)
0.16
Minerals Premixc
0.2
Total
100
calculated composition, % unless noted
AME, kcal/kg
2950
Crude Protein
21.0
Ca
1.0
Available P
0.45
Lysine
1.15
Methionine
0.50
Threonine
0.81
Analytical compositiond, % unless noted
Crude Protein
21.10
Lysine
1.17
Methionine
0.52
Threonine
0.82

Grower (d 22 to 42)

CP2a

CP3a

CP1

CP2

CP3

61.13
32.36
2.34
1.19

64.28
29.64
1.91
1.2

62.39
30.2
3.72
1.13

65.63
27.42
3.27
1.13

68.93
24.57
2.81
1.13

1.84

1.84

1.6

1.6

1.6

0.19
0.12
0.04
0.4
0.03
0.16
0.2
100

0.18
0.12
0.04
0.4
0.03
0.16
0.2
100

0.13
0.07
0.03
0.4
0.03
0.1
0.2
100

0.12
0.08
0.02
0.4
0.03
0.1
0.2
100

0.11
0.1
0.02
0.4
0.03
0.1
0.2
100

2950
20.0
1.0
0.45
1.10
0.47
0.77

2950
19.0
1.0
0.45
1.04
0.45
0.73

3050
19.0
0.9
0.40
1.01
0.40
0.73

3050
18.0
0.9
0.40
0.95
0.38
0.68

3050
17.0
0.9
0.40
0.90
0.36
0.64

20.07
1.09
0.45
0.76

19.31
1.04
0.43
0.75

19.12
1.03
0.38
0.75

18.07
0.99
0.37
0.70

17.08
0.86
0.35
0.63

a
CP1: crude protein (CP) levels, starter 21%, grower 19%; CP2: CP levels, starter 20%, grower 18%; CP3: CP levels,
starter 19%, grower 17%.
b
Supplied per kilogram of diet: vitamin A, 10000 IU; vitamin D3, 3000 IU; vitamin E 7.5 IU; vitamin K2, 1.5 mg;
vitamin B1, 0.6 mg; vitamin B2, 4.8 mg; vitamin B6, 1.8 mg; vitamin B12, 0.009 mg; pantothenic acid, 7.5 mg; folic acid,
0.15 mg/kg; niacin, 10.5 mg; biotin, 0.135 mg.
c
Supplied per kilogram of diet: Fe (FeSO4H2O), 100 mg; Cu (CuSO45H2O), 8 mg; Mn (MnSO4H2O), 120 mg; Zn
(ZnSO4H2O), 100 mg/kg; I(KI), 0.7 mg; Se (Na2SeO3), 0.35 mg.
d
Analyzed data correspond to duplicate analysis per sample.

11

Table 2. Effect of dietary crude protein levels and exogenous protease on broiler
performance.
Experimental Groups

Average body weight gain

(g)
d 1 to
d 22 to
d 1 to
21
42
42
866.8
1553.8
2420.6

Average feed intake (g)

d 1 to
d 22 to
d 1 to
21
42
42
1220.1
3250.5
4436.5

d 1 to
21
1.41

F/G
d 22 to
42
2.09

d 1 to
42
1.83

150

881.7

1533.2

2414.9

1231.0

3294.0

4493.1

1.40

2.15

1.86

300

877.9

1629.4

2507.3

1223.8

3311.1

4501.2

1.39

2.03

1.80

829.5

1632.2

2461.7

1201.3

3416.6

4550.6

1.45

2.09

1.85

150

846.4

1564.3

2410.6

1207.2

3341.1

4508.6

1.43

2.14

1.87

300

849.0

1559.8

2408.8

1211.6

3281.7

4453.3

1.43

2.10

1.85

818.3

1521.0

2339.3

1214.8

1.48

2.18

1.92

150

814.7

1518.8

2333.5

1208.1

3314.7
3243.2

4490.3
4418.2

1.48

2.14

1.89

800.4

1543.5

2343.9

1169.2

3181.7

4308.9

1.46

2.06

1.84

9.2

33.7

35.2
2447.6

13.6
1225.0

86.5

81.8

0.01

0.04

0.02

875.5c

1572

3285

4477

1.40a

2.09

1.83a

2427.0

1206.7
3347

4504

1.43b

2.11

1.86ab

3247

4406

1.48c

2.13

1.88b

4493

2.12

1.87

ab

2.14

1.88

2.07

1.83

0.586

0.035

Crude
protein

Protea
se

CP1

CP2

CP3

300
SEM
Crude
proteinb

CP1
CP2
CP3

Protease

0
150
300

Crude
protein

841.6b
811.1a
838
848
842
P<0.00
1

1585
1528
1569
1539

ab

2338.9

1197.4

2407
2386

1212
1215

3327
3293

4473

1578

2420

1202

3258

4421

0.102

0.001

0.050

0.369

0.324

1.45
1.44

1.43
P<0.00
1

Protease
0.459
0.343
0.503
0.433
0.622
0.547
0.043
0.109
0.057
Crude protein
Protease
0.343
0.254
0.365
0.259
0.788
0.673
0.712
0.538
0.511
a
CP1: crude protein (CP) levels, starter 21%, grower 19%; CP2: CP levels, starter 20%, grower 18%; CP3:
CP levels, starter 19%, grower 17%.
b
In the same column, values with different superscripts indicate significant differences (P<0.05).

12

Table 3. Effects of dietary crude protein levels and exogenous protease on apparent dry
matter, crude protein, and gross energy digestibility in 42-d-old broilers (%).
Experimental Groupsa

Crude protein
CP1

CP2

CP3

Protease
0
150
300
0
150
300
0
150
300

SEM
Crude proteinb
Proteaseb

P-value
Crude protein
Protease
Crude protein Protease

CP1
CP2
CP3
0
150
300

Dry matter

Crude protein

Gross energy

77.4
76.7
77.2
76.1
77.3
77.5
77.8
74.5
75.5
0.90
77.1
76.5
75.9
77.1
76.2
76.7

63.7
65.3
66.3
62.5
64.4
64.4
60.0
61.7
63.4
1.38
65.1b
63.7ab
61.7a
62.1a
63.8ab
64.7b

78.4
77.6
78.7
77.3
78.8
79.3
78.9
75.3
77.1
0.95
78.3
78.4
77.0
78.2
77.2
78.3

0.171
0.381
0.088

0.008
0.048
0.970

0.150
0.246
0.084

CP1: crude protein (CP) levels, starter 21%, grower 19%; CP2: CP levels, starter 20%, grower 18%; CP3: CP
levels, starter 19%, grower 17%.
b
In the same column, values with different superscripts indicate significant differences (P<0.05).

13

Table 4. Effects of dietary crude protein levels and exogenous protease on trypsin activity
in pancreas and duodenum of broilers (103U/mg prot)
Experimental Groups a

Crude
protein
CP1

Protease

CP2

SEM
Crude
proteinb

Protease

42 d

5.78

Duodenum
contents
7.78

150

6.14

300
0
150

CP3

21 d

5.04

Duodenum
contents
7.82

8.16

4.91

7.69

6.38
4.75

8.68
7.32

5.86
4.61

7.87
6.31

Pancreas

Pancreas

5.76

8.67

3.79

5.95

300

5.94

7.81

5.20

6.52

0
150
300

3.69
3.64
3.63
0.51

5.23
5.01
5.81
0.52

2.70
3.56
3.54
0.59

4.20
4.42
4.70
0.60

CP1

6.08b

8.17b

5.23c

7.79c

CP2

5.58b
3.65a
4.74

8.01b
5.38a
6.64

4.53b
3.29a
4.00a

6.25b
4.46a
6.06

5.09

7.07

4.13ab

6.03

5.01

7.16

4.64

6.10

<0.001
0.179

<0.001
0.180

<0.001
0.026

<0.001
0.591

0.569

0.319

0.235

0.954

CP3
0
150
300

Crude protein
Protease
Crude protein Protease
a

CP1: crude protein (CP) levels, starter 21%, grower 19%; CP2: CP levels, starter 20%, grower 18%; CP3: CP
levels, starter 19%, grower 17%.
b
In the same column, values with different superscripts indicate significant differences (P<0.05). Same letter or no
superscripts indicate non-significant differences (P>0.05)
c
Means represent 6 birds per treatment, 1 bird per pen.

14

Table 5. Effect of dietary crude protein levels and exogenous protease on intestinal
morphology of 21-d-old broilers
Experimental
Groups a
Crude
Protease
protein
CP1

CP2

CP3

Villus
height
m

Crypt
depth
m

1163.4

183.3

150

1248.1

300

Proteaseb

Jejunum
Villus
height
m

Crypt
depth
m

6.4

769.4

136.7

186.5

6.7

786.9

1200.8

178.2

6.8

VCR

Ileum
Villus
height
m

Crypt
depth
m

VCR

5.7

480.4

122.4

4.0

132.3

6.1

492.4

130.4

3.9

840.2

148.1

5.7

488.2

137.5

3.6

VCR

1131.1

186.5

6.1

653.6

135.2

4.9

434.1

137.9

3.2

150

1206.4

181.8

6.7

676.5

144.7

4.7

456.0

118.3

3.9

300

1224.0

182.7

6.7

779.7

147.0

5.4

438.8

127.9

3.5

928.4

179.2

5.2

589.8

133.4

4.5

406.2

127.7

3.2

150

1020.2

177.9

5.8

609.3

148.1

4.2

445.2

119.0

3.8

300

998.2
39.7

179.8
5.4

5.6
0.3

592.8
26.3

133.4
6.8

4.5
0.3

438.0
19.5

129.2
5.9

3.4
0.2

CP1

1204.1b

182.6

6.6b

798.8c

139.0

5.9c

487.0b

130.1

3.8

183.7

6.5

703.3

142.3

5.0

442.9

128.0

3.5

5.5

597.3

138.3

4.4

429.8

125.3

3.5

671.0

135.1

5.0

440.2

129.4

3.4a

SEM
Crude
proteinb

duodenum

CP2

1187.2

CP3

982.3

179.0

1074.3

183.0

5.9

150

1158.2b

182.1

6.4b

691.0a

147.0

5.0

464.5

122.6

3.8b

300

1141.0b

180.2

6.4b

737.6b

137.6

5.2

455.0

131.5

3.5a

<0.001

0.536

<0.001

<0.001

0.743

<0.001

0.002

0.608

0.084

P-value
Crude protein
Protease

0.031
0.815
0.046
0.01
0.332
0.651
0.315
0.16
0.045
Crude protein
0.936
0.861
0.979
0.149
0.263
0.433
0.938
0.163
0.309
Protease
a
CP1: crude protein (CP) levels, starter 21%, grower 19%; CP2: CP levels, starter 20%, grower 18%; CP3: CP levels,
starter 19%, grower 17%.
b
In the same column, values with different superscripts indicate significant differences (P<0.05). Same letter or no
superscripts indicate non-significant differences (P>0.05).
c
Means represent 6 birds per treatment, 1 bird per pen.
d
VCR: villous height: crypt depth ratio.

15

Table 6. Effects of dietary crude protein levels and exogenous protease on intestinal
morphology of 42-d-old broilers.
Experimental Groups

duodenum

Crude
protein

Jejunum

Ileum

Protease

Villus
height
m

Crypt
depth
m

VCR

Villus
height
m

Crypt
depth
m

VCR

Villus
height
m

Crypt
depth
m

VCR

1405.2

220.6

6.4

1008.6

173.7

5.8

797.9

162.1

4.9

150

1575.1

225.8

7.0

1032.0

164.8

6.4

833.0

155.3

5.4

300

1571.1

219.5

7.2

1097.8

162.0

6.9

796.8

155.1

5.2

1433.6

207.4

7.0

982.2

165.4

6.0

795.7

159.2

5.1

150

1361.8

229.5

6.0

1062.3

165.2

6.5

800.2

157.4

5.2

300

1388.0

224.2

6.2

1034.4

171.1

6.1

811.2

152.7

5.4

1171.3

204.2

5.8

972.7

172.0

5.7

770.0

145.3

5.3

150

1241.1

203.1

6.2

939.5

175.7

5.4

752.1

145.9

5.2

300

1193.7
45.2

222.2
8.3

5.4
0.3

905.2
29.8

167.1
7.5

5.5
0.3

805.3
30.5

147.1
6.8

5.5
0.3

CP1

1517.2c

221.9

6.9b

1046.3b

166.9

6.4b

809.2

157.5b

5.2

220.4

1026.3

167.2

802.4

5.2

939.1

5.3

CP1

CP2

CP3

SEM
Crude
proteinb

CP2

1394.4

6.4

CP3

1202.1

209.8

5.8

1336.7

210.8

6.4

150

1392.7

219.5

300

1384.3

Crude protein
Protease
Crude protein Protease

Protease

6.2

156.4

171.6

5.5

775.8

146.1

987.8

170.4

5.8

787.9

155.5

5.1

6.4

1011.3

168.6

6.1

795.1

152.9

5.2

222.0

6.3

1012.5

166.7

6.1

804.4

151.6

5.4

<0.001

0.165

0.001

<0.001

0.696

0.006

0.373

0.009

0.676

0.274

0.235

0.869

0.524

0.839

0.474

0.801

0.773

0.542

0.077

0.36

0.056

0.059

0.733

0.238

0.704

0.952

0.823

P-value

CP1: crude protein (CP) levels, starter 21%, grower 19%; CP2: CP levels, starter 20%, grower 18%; CP3: CP levels,
starter 19%, grower 17%.
b
In the same column, values with different superscripts indicate significant differences (P<0.05). Same letter or no
superscripts indicate non-significant differences (P>0.05).
c
Means represent 6 birds per treatment, 1 bird per pen.
d
VCR: villous height: crypt depth ratio.

16

17