Accepted Manuscript

Title: Effects of coated proteases on the performance, nutrient

retention, gut morphology and carcass traits of broilers fed
corn or sorghum based diets supplemented with soybean meal
Author: X. Xu H.L. Wang L. Pan X.K. Ma Q.Y. Tian Y.T. Xu
S.F. Long Z.H. Zhang X.S. Piao
PII:
DOI:
Reference:

S0377-8401(16)30874-4
http://dx.doi.org/doi:10.1016/j.anifeedsci.2016.10.015
ANIFEE 13653

To appear in:

Animal

Received date:
Revised date:
Accepted date:

19-5-2016
13-9-2016
21-10-2016

Feed

Science

and

Technology

Please cite this article as: Xu, X., Wang, H.L., Pan, L., Ma, X.K., Tian, Q.Y., Xu, Y.T.,
Long, S.F., Zhang, Z.H., Piao, X.S., Effects of coated proteases on the performance,
nutrient retention, gut morphology and carcass traits of broilers fed corn or sorghum
based diets supplemented with soybean meal.Animal Feed Science and Technology
http://dx.doi.org/10.1016/j.anifeedsci.2016.10.015
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.

Effects of coated proteases on the performance, nutrient retention, gut morphology and carcass traits of broilers fed corn or sorghum
based diets supplemented with soybean meal

X. Xua, H.L. Wanga, L. Pana, X.K. Maa, Q.Y. Tiana, Y.T. Xua, S.F. Longa, Z.H. Zhangb, X.S. Piaoa,*

State Key Laboratory of Animal Nutrition, Ministry of Agriculture Feed Industry Centre, China Agricultural University, Beijing 100193, China

Kemin Industries Company, Zhuhai 519040, China

Corresponding author. Tel.: +8615901013490, Fax: +861062733688, Email: piaoxsh@mafic.ac.cn.

Abbreviations: AA, amino acid; ADFI, average daily feed intake; ADG, average daily gain; AID, apparent ileal digestibility; AME, apparent
metabolisable energy; ATTD, apparent total tract digestibility; BW, body weight; CP, crude protein; DM, dry matter; GE, gross energy; ME,
metabolisable energy; SID, standardized ileal digestibility.

Highlights

Coated protease supplementation enhanced ADG during finisher phase and overall period.

Coated protease supplementation increased AME and the retention of DM, GE and nitrogen in starter phase.

Coated protease supplementation improved gut morphology in starter phase.

Coated protease supplementation improved carcass traits of broilers.

Sorghum-soybean meal diets had a similar effect on the performance of broilers to corn-soybean meal diets.

AB STRACT
The effects of proteases on the performance, nutrient retention, gut morphology and carcass traits of broilers fed corn or sorghum based diets
supplemented with soybean meal were studied in this experiment. A total of 256 male Arbor Acre broilers (one-day-old and weighing 43.8
1.31 g) were randomly allotted to 1 of 4 treatments in a 2 x 2 factorial arrangement that included diet type (corn vs. sorghum) and protease
supplementation (0 vs. 150 mg/kg). The feeding program consisted of a starter diet fed from d 1 to 21 and a finisher diet fed from d 22 to 42.
Supplementation of protease significantly increased the ADG of broilers during the finisher phase (P < 0.01) and the overall experiment (P <
0.05). Diet type had no effect on the performance of broilers during the starter phase, the finisher phase or the overall experiment (P > 0.05).
From d 19 to 21, the AME and DM, GE and nitrogen retention were all increased by supplementation with protease (P < 0.05). Moreover, the
birds fed sorghum-based diets showed a trend towards decreased AME and retention of GE (P = 0.05) and nitrogen (P = 0.08). From d 40 to 42,
the AME and the retention of DM, GE and nitrogen were all increased (P<0.05) by supplementation with protease. On d 21, the birds fed diets
supplemented with protease had significantly increased villus height and villus height:crypt depth ratio as well as decreased crypt depth in the
duodenum, jejunum and ileum (P < 0.01). On d 42, increased villus height, villus height:crypt depth ratio and decreased crypt depth were
observed in the jejunum of broilers fed diets containing protease (P < 0.05). Supplementing protease significantly increased breast muscle
weight, pH24h value and decreased breast muscle drip loss (P < 0.05). In conclusion, compound protease supplementation enhanced ADG of

broilers during finisher phase and overall period. The AME and DM, GE and nitrogen retention and gut morphology of broilers fed diets
supplemented protease were improved in the starter phase. Supplementing protease increased slaughter weight, breast muscle weight and pH24h
value while decreasing breast muscle drip loss. Furthermore, low tannin sorghum-based diets generated similar performance compared with
broilers fed corn-based diets.

Keywords: Broilers, Corn, Nutrient retention, Performance, Protease, Sorghum

1. Introduction
Sorghum (Sorghum bicolor L. Moench) can be grown in semi-arid areas around the world (Gualtieri and Rapaccini, 1990) and is adapted
to low-quality soil. Sorghum has the potential to replace corn as an alternative poultry feed. However, sorghum may not always be comparable
to corn due to its relatively high tannin and kafirin contents which possess potent anti-nutritive properties for mono-gastric animals (Selle et al.,
2010; Liu et al., 2013; Khoddami et al., 2015).
Low-tannin sorghum has been shown to substitute for corn in swine and poultry feeds without affecting their performance (Garcia et al.,
2005; Bozutti, 2009). Conversely, high-tannin sorghum has been shown to adversely affect the performance of broilers (Pour-Reza and Edriss,
1997). Pan et al. (2016) reported that tannin is a key factor in the determination of the energy content in sorghum when fed to growing pigs.
Even in low-tannin sorghum, kafirin, the most abundant protein in sorghum, is the last to be digested. Kafirin has a concentration of
approximately 480 g/kg of total sorghum protein (Taylor et al., 1984). Kafirin has a lack of basic AA (Selle et al., 2003) and it can negatively
impact the digestibility of AA due to resistance to proteolysis (Selle et al., 2010). It also can impede starch digestion by physical and chemical
interactions (Duodu et al., 2003).
Some studies have indicated that exogenous protease improves broiler performance and nutrient digestibility (Barekatain et al., 2013; Liu
et al., 2013). New types of proteases containing acidic, neutral and alkaline proteases produced by Aspergillus niger, Bacillus subtilis and

Bacillus Licheniformis have recently been developed. These proteases can successively dissolve and work in acidic, neutral or alkaline
microenvironments in the digestive tract (Pan et al., 2016). These new proteases have been shown to improve AA digestibility of weanling and
growing pigs fed corn-soybean diets (Pan et al., 2016; Yu et al., 2016). However, there are few reports on the application of protease in broiler
diets containing sorghum. Therefore, the present study was conducted to test the effects of proteases on the performance, nutrient retention, gut
morphology and carcass traits of broilers fed corn or sorghum based diets.
2. Materials and methods
2.1. Experimental products
The corn and sorghum used in this study were obtained from China and Australia, respectively. The chemical composition of the corn or
sorghum is shown in Table 1. The new compound proteases were in granular form and were provided by the Kemin Industries Company
(Zhuhai, China).
The compound contains acidic, neutral and alkaline proteases produced by Aspergillus niger, Bacillus subtilis and Bacillus Licheniformis,
respectively. The percentage and activity of each protease are proprietary. The acidic protease is coated with a heat-resistant membrane, while
the neutral and alkaline proteases are both coated with heat-proof and acid-resistant layers which are not expected to be degraded by gastric acid
(Pan et al., 2016). Therefore, the compound proteases are expected to successively dissolve and work in an acidic, neutral or alkaline

microenvironment in the digestive tract.

2.2. Experimental birds
The experimental protocols used in these experiments were approved by the Institutional Animal Care and Use Committee of China
Agricultural University (Beijing, China). A total of 256, one-day-old male Arbor Acre broiler chickens (weighing 43.8 1.31 g), were
purchased from the Arbor Acres Poultry Breeding Company (Beijing, China). All birds were raised in wire-floored cages (90 x 60 x 40 cm3) in
an environmentally controlled room with continuous light and had ad libitum access to feed and water. The ambient temperature was
maintained at 34C at the start of experiment and was decreased as the birds progressed in age to ensure a final temperature of 24C at 35 d and
thereafter. The relative humidity was set at 45 to 55% and was kept within this range. All birds were inoculated with inactivated Infectious
Bursa Disease Vaccine on d 14 and 21 and Newcastle Disease Vaccine on d 7 and 28. The trial was conducted in 2 phases, consisting of a starter
phase from d 1 to 21 and a finisher phase from d 22 to 42.
2.3. Experimental design and diets
The broilers chickens were randomly allotted to 1 of 4 treatments in a 2 x 2 factorial arrangement that included protease supplementation
(0 vs. 150 mg/kg) and diet type (corn vs. sorghum). Eight cages per treatment were used with 8 birds per cage. All diets were fed in mash form
and were formulated to meet the nutritional requirements for broiler chickens recommended by the National Research Council (1994). The

composition of the experimental diets is listed in Table 2.

2.4. Broiler performance
All birds were weighed individually after their arrival from the hatchery. The birds were also weighed on d 21 and 42 after a 12 h fast.
Feed bags were weighed at the same time and these values were used to calculate average daily gain (ADG), average daily feed intake (ADFI)
and feed/gain (F/G).
2.5. Nutrient retention
On d 19 and 40, all feed and excreta were removed from the feeders and collection trays. A weighed amount of feed was then added to the
feeders in sufficient quantity that the feeders would not run out of feed during the 3 d collection period. The amount of feed consumed by the
birds in each pen during these 3 days was measured and all excreta from each pen was collected and then feathers and spilled feed were
removed and the excreta were weighed. A 500 gram sample of excreta was removed and dried at 60C for 72 h. Feed and dried excreta samples
were subsequently ground to pass through a 40-mesh screen and thoroughly mixed before analyses. The AME and rentention of DM, GE, as
well as nitrogen and were calculated with the following formulae:
DM retention = 100% * (DM intake DM in excreta)/DM intake
GE rentention = 100% * (GE intake GE in excreta)/GE intake

AME = GEdiet * GE retention

Nitrogen retention = 100% * (Nitrogen intake Nitrogen in excreta)/Nitrogen intake
2.6. Chemical analyses
The corn, sorghum, diets, and faecal samples were analyzed for Kjeldahl N (Thiex et al., 2002) and ether extract (Thiex et al., 2003). Dry
matter (DM) (AOAC Method 930.15), starch (AOAC Method 948.02), ash (AOAC Method 942.05), calcium (AOAC Method 927.02) and
phosphorus (AOAC Method 965.17) in corn, sorghum, diets and faecal samples were determined using the methods of the Association of
Official Analytical Chemists (AOAC, 2000). Neutral detergent fibre (NDF) and acid detergent fibre (ADF) concentrations in corn, sorghum and
diets samples were determined using the methods of Van Soest et al. (1991). GE in corn, sorghum, diets and faecal samples was determined
with a bomb calorimeter (Parr Instruments, Moline, IL). The tannins in corn and sorghum were determined according to the methods described
by Price et al. (1978).
Before analysis for AA, the corn and sorghum samples were hydrolyzed with 6 N HCl for 24 h at 110C (AOAC Method 999.13) and
analyzed for AA using an Amino Acid Analyzer (Hitachi L-8900, Tokyo, Japan). Methionine and cystine were determined as methionine
sulfone and cysteic acid after cold performic acid oxidation overnight and hydrolyzed with 7.5 N HCl for 24 h at 110C (AOAC Method 994.12)
using an Amino Acid Analyzer (Hitachi L-8800, Tokyo, Japan). Tryptophan was determined after LiOH hydrolysis for 22 h at 110C (AOAC

Method 998.15) using High Performance Liquid Chromatography (Agilent 1200 Series, Santa Clara, CA). All analyses were conducted in
duplicate.
2.7. Determination of intestinal morphology
One bird per cage was selected according to the average weight of the birds in the pen and euthanized on d 21 and 42. Immediately after
euthanasia, two-cm tissue samples of the small intestine were obtained from the upper (start point of organ), middle (center point of organ) and
lower (end point of organ) parts of the duodenum (from the gizzard outlet to the end of the pancreatic loop), jejunum (from the pancreatic loop
to Meckels diverticulum) and ileum (from Meckels diverticulum to the ileo-caeco-colic junction), according to the method of Giannenas et al.
(2010). The intestinal samples were flushed with 0.9% salt solution, fixed with 10% formaldehyde-phosphate buffer and kept at 4C for
microscopic assessment of intestinal morphology.
Villus height and crypt depth were measured according to Li et al. (1990) with some modifications. In brief, fixed intestinal samples were
prepared using conventional paraffin embedding techniques. Samples were sectioned at a thickness of 5 m and then placed on a glass slide and
stained with hematoxylin and eosin. Villus height and crypt depth were measured under 10 times magnification using an Olympus CK 40
Microscope (Olympus Optical Company, Shenzhen, China). Villus height was measured from the tip (lamina propria) of the villus to the base
(villus-crypt junction) while the crypt depth was measured from the villus-crypt junction to the distal limit of the crypt. Three sections from

each part (upper, middle and lower) of the duodenum, jejunum and ileum (2 villi/sections/segment/bird) were measured. Eighteen villi were
counted from 9 different sections in each segment (duodenum, jejunum and ileum) per bird, and their average was expressed as the mean villus
height and crypt depth for each bird. Finally, the villus height and crypt depth from 8 birds were averaged to obtain the mean villus height and
crypt depth for each treatment group.
2.8. Determination of carcass traits
The birds that were euthanized on d 42 were also used to determine carcass traits. The breast and thigh muscle located on the left side
were removed and weighed. The percentage of the breast and thigh muscle weight relative to slaughter weight was determined. Meat colour,
including lightness (L*), redness (a*), and yellowness (b*) values, was measured from 3 orientations (middle, medial, and lateral) using a
Chromameter (CR-410, Konica Minota, Tokyo, Japan). The pH values at 45 min and 24 h postmortem were also measured at 3 locations using
a glass penetration pH electrode (pH-star, Matthaus, Germany). Drip loss for 24 was measured using the plastic bag method as described
previously (Straadt et al., 2007).
2.9. Statistical analysis
All data were analyzed as a 2 x 2 factorial design using the GLM procedure of SAS (SAS Institute Inc., Cary, NC). The cage was the
experimental unit for performance and rentention and individual bird was the experimental unit for other parameters. The factors in the model

included diet type, protease supplementation and their interaction. Least squares means were derived for all treatments and were compared
using the PDIFF (Adjusted Tukey) and STDERR options of SAS. Results are expressed as least squares means and SEM. The results were
considered significant at P < 0.05 and a trend at P < 0.10.
3. Results
3.1 Broiler performance
There were no significant interactions between diet type and supplementation of proteases for broiler performance (P > 0.05, Table 3). The
type of diet had no significant effect on the performance of the broilers during the starter phase, finisher phase or the overall experiment (P >
0.05). Supplementation with protease significantly improved the ADG of birds in the finisher phase (P < 0.01) and overall (P < 0.05). The
mortality of the broilers was not affected by type of diet, protease supplementation or their interaction (P > 0.05).
3.2 Nutrient retention
No interactions between diet type and protease supplementation were observed for nutrient retention (P > 0.05, Table 4). From d 19 to 21,
the AME and retention of DM, GE and nitrogen of birds were all increased with supplementation of protease in the diets (P < 0.05). Moreover,
the birds fed sorghum-based diets demonstrated a trend for decreased AME, retention of GE (P = 0.05) and nitrogen (P = 0.08) compared with
those fed corn-based diets. From d 22 to 42, the AME and retention of DM, GE and nitrogen of birds were all increased with supplementation

of protease in the diets (P < 0.05). However, there was no significant difference in nutrient retention of the birds fed the two different type diets
(P > 0.05).
3.3 Gut morphology
The data displayed in Table 5 shows the gut morphology of the birds on d 21 and 42. On d 21, the birds fed diets supplemented with
protease had significantly increased villus height and villus height:crypt depth ratio while they had decreased crypt depth in the duodenum,
jejunum and ileum compared with birds fed diets containing no protease (P < 0.01). However, there were no significant differences in gut
morphology between birds fed the two diet types (P > 0.05) and no interaction between diet type and protease supplementation was obtained
(P > 0.05). On d 42, increased villus height, villus height:crypt depth ratio and decreased crypt depth was observed in the jejunum of broilers
fed diets containing protease (P < 0.05). However, the gut morphology in the duodenum and ileum of birds did not differ (P > 0.05).
3.4 Carcass traits
The effects of protease on the carcass traits of broilers fed corn or sorghum-based diets are shown in Table 6. No interactions were
observed between diet type and protease supplementation and diet type had no significant effect on the carcass traits of broilers (P > 0.05).
Supplementing protease significantly increased slaughter weight, breast muscle weight and pH24h value while decreasing breast muscle drip loss
(P < 0.05). However, there were no significant effects on the thigh and breast muscle yield of broilers fed diets containing protease (P > 0.05).

4. Discussion
4.1 The effect of corn or sorghum in broiler diets
The effects of replacing corn with low-tannin sorghum (Sorghum bicolor L. Moench) on broiler performance, nutrient retention, gut
morphology and carcass traits were evaluated in this experiment. No significant differences in the above parameters were found indicating that
all of the dietary corn can be replaced by low-tannin sorghum grain without negative effects during the entire growth phase. Similar to our
observations, Pour-Reza and Edriss (1997) reported that tannins did not significantly affect broiler performance when the dietary tannin
concentration did not exceed 2.6 g/kg. The sorghum used in our study contained a low concentration of tannins (0.5 g/kg) and therefore few
negative effects were caused by feeding sorghum. Torres et al. (2013) also found that low-tannin sorghum could replace corn in broiler diets
without any significant effect on performance and carcass traits which is consistent with our results.
The birds fed sorghum-soybean diets showed a trend towards decreased AME, retention of GE and nitrogen compared with the birds fed
corn-soybean diets from d 19 to 21. This may have been caused by the kafirin protein fraction because of its inherent hydrophobicity and
disulphide cross-linking especially in the - and -kafirin periphery of protein-bodies (Liu et al., 2013). Another potential reason may be the
relatively high level of phytate and low phytase activity in sorghum. Sorghum contains about 2.92 g/kg total phosphorus (P) and 82.7% of total
P is phytate bond P. Moreover, natural phytase activity in sorghum is extremely low compared to wheat and barley (Selle et al., 2003). During

the starter phase, the tissues of young broilers develop quite rapidly and fewer endogenous enzymes are secreted (Kaczmarek et al., 2014),
which may lead to the trend for reduced GE and nitrogen retention. As the birds aged, the negative trend disappeared during the finisher phase
and the overall experiment.
The effects of sorghum-soybean diets on the gut morphology and carcass traits of broilers have seldom been reported. Similar to broiler
performance, the gut morphology and carcass traits showed no significant difference indicating that replacement of corn by sorghum in diets
had no negative effects on the broilers.
4.2 The effect of protease supplementation
It has been confirmed in many studies that protease supplementation in corn-, wheat-, or maize-soybean diets can improve the performance
and nutrient digestibility in mono-gastric animals (Cafe et al., 2002; Cowieson and Adeola, 2005; Zanella et al., 1999). Similarly, our results
showed that supplementation of protease improved the performance of broilers fed sorghum- or corn-soybean diets. The improved performance
may have been caused by the increased nutrient retention of birds fed diets containing exogenous protease.
Exogenous proteases have been used in poultry diets as an effective additive to improve nutrient retention by hydrolyzing protein to free
AA and peptides which are available for absorption in the small intestine (Liu et al., 2013; Adebiyi and Olukosi, 2015). Using the same
compound proteases as those used in the present experiment, Yu et al. (2016) and Pan et al. (2016) found that dietary supplementation with

protease increased the coefficients of AID and SID of CP and most AA in weaned and growing pigs, respectively. An increased ATTD of DM,
GE and CP of pigs fed diets containing proteases were also obtained in the study of Pan et al. (2016). These results were all in agreement with
our study. Angel et al. (2011) reported a 6% improvement in CP digestibility of a low protein diet (maize-soybean diet) which is in agreement
with this study. In the present study, the diets were formulated with the same total Lys, Met and Thr level, however, the SID Lys level in corn
(8.2 g/kg) or sorghum (7.7 g/kg) diets were different. Yu et al. (2016) reported the retention rate of coated proteases in the stomach was over
90% while the release rate in the small intestine was about 85%, whereas uncoated proteases had a release rate less than 35% indicating that
coating proteases reduced the activity loss in the stomach which likely increased nutrient retention.
For young chicks, a longer villus increases the absorptive surface of the intestine, while a shorter crypt depth indicates lower tissue
turnover as well as a lower demand for tissue development (Khodambashi et al., 2012). The longer villus and shorter crypt depths of broilers
fed diets containing proteases were consistent with the increased DM, GE and CP utilization observed indicating more nutrients were digested
and absorbed in the intestine.
In present study, the breast muscle weight of broilers fed diets supplemented with proteases was significantly increased compared with
broilers fed diets containing no proteases, which is in agreement with the improved ADG of broilers fed diets containing proteases. The meat
quality (pH24h, drip loss) of breast and thigh muscle was different which may have been caused by the different fibre types in the two muscles

(Zhang et al., 2009). The thigh muscle consists of type I, type IIa, and type IIb fibres which produce energy relying primarily on aerobic
glycolysis of type I fibres. However, the breast muscles mainly consist of type IIb fibres which produce energy primarily by anaerobic
glycolysis of glycogen (Wang et al., 2015). Solomon et al. (1998) suggested that pork containing a higher proportion of type IIb fibres tended to
be soft and exudative-like due to its anaerobic nature, higher glycogen concentration, and lower pH24h. In the current study, supplementation
with proteases significantly increased the pH24h and decreased the drip loss which may have been caused by changing the proportion of the
muscle fibre types. However, the muscle fibre type was not determined in this trial and this should be studied further in future research.
5. Conclusions
Our results indicate that compound protease supplementation enhanced ADG of broilers during finisher phase and overall period. The
AME, DM, GE and nitrogen retention and gut morphology of broilers fed diets supplemented protease were improved in the starter phase.
Supplementing protease increased slaughter weight, breast muscle weight and pH24h value while decreasing breast muscle drip loss.
Furthermore, low tannin sorghum-based diets generated similar performance compared with broilers fed corn-based diets.
Acknowledgments
This study was completed at China Agriculture University Animal Experiment Base (Fengning, China). The authors thank Jianjun Zang for
his input to this study. This study was made possible by a grant from Kemin Industries (Zhuhai, China), the National Natural Science

Foundation (No. 31372316) and the 111 Project (B16044).

This work has no conflict of interest.

References
Adebiyi, A.O., Olukosi, O.A., 2015. Apparent and standardized ileal amino acid digestibility of wheat distillers dried grains with solubles with
or without exogenous protease in broilers and turkeys. Br. Poult. Sci. 56, 239-246.
Angel, C., Saylor, W., Vieira, S., Ward, N., 2011. Effects of a monocomponent protease on performance and protein utilization in 7-to
22-day-old broiler chickens. Poult. Sci. 90, 2281-2286.
Association of Official Analytical Chemists, 2000. Official Methods of Analysis. 17th ed. Association of Official Analytical Chemists.
Arlington, VA.
Barekatain, M.R., Antipatis, C., Choct, M., Iji, P.A., 2013. Interaction between protease and xylanase in broiler chicken diets containing
sorghum distillers dried grains with solubles. Anim. Feed Sci. Technol. 182, 71-81.
Bozutti, S.R.A., 2009. Avaliacao de ingredientes alternativos na alimentacao de frangos de corte com a adicao de enzimas. MSci Diss.
Faculdade de Zootecnia e Engenharia de Alimentos/Universidade de Sao Paulo, Pirassununga, Sao Paulo, Brazil.
Cafe, M.B., Borges, C.A., Fritts, C.A., Waldroup, P.W., 2002. Avizyme improves performance of broilers fed corn-soybean, meal-based diets. J.
Appl. Poult. Res. 11, 29-33.

Cowieson, A.J., Adeola, O., 2005. Carbohydrases, proteases, and phytase have an additive beneficial effect in nutritionally marginal diets for
broiler chicks. Poult. Sci. 84, 1860-1867.
Duodu, K.G., Taylor, J.R.N., Belton, P.S., Hamaker, B.R., 2003. Factors affecting sorghum protein digestibility. J. Cereal Sci. 38, 117-131.
Garcia, R.G., Mendes, A.A., Costa, C., Paz, I.C.L.A., Takahashi, S.E., Pelicia, K.P., Komiyama, C.M., Quinteiro, R.R., 2005. Desempenho e
qualidade de carne de frangos de corte alimentados com diferentes niveis de sorgo em substituicao ao milho. Arq. Bras. Med. Vet. Zootec.
57, 634-643.
Giannenas, D.Y., Tsalie, E., Chronis, E.F., Doukas, D., Kyriazakis, I., 2010. Influence of dietary mushroom Afaricus bisporus on intestinal
morphology and microflora composition in broiler chickens. Res. Vet. Sci. 89, 78-84.
Gualtieri, M., Rapaccini, S., 1990. Sorghum grain in poultry feeding. Worlds Poult. Sci. J. 46, 246-253.
Kaczmarek, S.A., Rogiewicz, A., Mogielnicka, M., Rutkowski, A., Jones, R.O., Slominski, B.A., 2014. The effect of protease, amylase, and
non-starch polysaccharide-degrading enzyme supplementation on nutrient utilization and growth performance of broiler chickens fed
corn-soybean meal-based diets. Poult. Sci. 93, 1745-1753.
Khodambashi Emami, N., Samie, A., Rahmani, H.R., Ruiz-Feriab, C.A., 2012. The effect of peppermint essential oil and frutooligosaccharides,
as alternatives to virginiamycin, on growth performance, digestibility, gut morphology and immune response of male broilers. Anim. Feed

Sci. Technol. 175, 57-64.

Khoddami, A., Truong, H.H., Liu, S.Y., Roberts, T.H., Selle, P.H., 2015. Concentrations of specific phenolic compounds in six red sorghums
influence nutrient utilization in broiler chickens. Anim. Feed Sci. Technol. 210, 190-199.
Li, D.F., Thaler, R.C., Nelssen, J.L., Harmon, D.L., Allee, G.L., Weeden, T.L., 1990. Effect of fat sources and combinations on starter pig
performance, nutrient digestibility and intestinal morphology. J. Anim. Sci. 68, 3694-3704.
Liu, S.Y., Selle, P.H., Court, S.G., Cowieson, A.J., 2013. Protease supplementation of sorghum-based broiler diets enhances amino acid
digestibility coefficients in four small intestinal sites and accelerates their rates of digestion. Anim. Feed Sci. Technol. 183, 175-183.
NRC. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC.
Pan, L., Li, P., Ma, X.K., Xu, Y.T., Tian, Q.Y., Liu, L., Li, D.F., Piao, X.S., 2016. Tannin is a key factor in the determination and prediction of
energy content in sorghum grains fed to growing pigs. J. Anim. Sci. (In press)
Pour-Reza, J., Edriss, M.A., 1997. Effects of dietary sorghum of different tannin concentration and tallow supplementation on the performance
of broiler chicks. Br. Poult. Sci. 38, 512-517.
Price, M.L., Steve, V.S., Butler, L.C., 1978. A critical evaluation of the vanillin reaction as an assay for tannin in sorghum grain. J. Agric. Food
Chem. 26, 1214-1218.

SAS, 2001. Statistical Analysis System, Version 8.2. SAS Inc. Cary, NC, USA.
Selle, P.H., Cadogan, D.J., Li, X., Bryden, W.L., 2010. Implication of sorghum in broiler chicken nutrition. Anim. Feed Sci. Technol. 156,
57-74.
Selle, P.H., Walker, A.R., Bryden, W.L., 2003. Total and phytate-phosphorus contents and phytase activity of Australian-sourced feed
ingredients for pigs and poultry. Aust. J. Exp. Agric. 43, 475-479.
Solomon, M.B., Van Laack, R.L.J.M., Eastridge, J.S., 1998. Biophysical basis of pale, soft, exudative (PSE) pork and poultry muscle: A review.
J. Muscle Foods. 9, 1-12.
Straadt, I.K., Rasmussen, M., Andersen, H.J., Bertram, H.C., 2007. Aging-induced changes in microstructure and water distribution in fresh and
cooked pork in relation to water-holding capacity and cooking loss A combined confocal laser scanning microscopy (CLSM) and
low-field nuclear magnetic resonance relaxation study. Meat Sci. 75, 687-695.
Taylor, J.R.N., Schussler, L., Van der Walt, W.H., 1984. Fractionation of proteins from low-tannin sorghum grain. J. Agric. Food Chem. 32,
149-154.
Thiex, N.J., Manson, H., Anderson, S., Persson, J.A., 2002. Determination of crude protein in animal feed, forage, grain, and oilseeds by using
block digestion with copper catalyst and steam distillation into boric acid: Collaborative study. J. AOAC Int. 85, 309-317.

Thiex, N.J., Anderson, S., Gildemeister, B., 2003. Crude fat, diethyl ester extraction, in feed, cereal grain, and forage
(Randall/Soxtec/Submersion Method): Collaborative study. J. AOAC Int. 86, 888-898.
Torres, K.A.A., Pizauro Jr., J.M., Soares, C.P., Silva, T.G.A., Nogueira, W.C.L., Campos, D.M.B., Furlan, R.L., Macari, M., 2013. Effects of
corn replacement by sorghum in broiler diets on performance and intestinal mucosa integrity. Poult. Sci. 92, 1564-1571.
Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber and non-starch polysaccharides in relation to animal nutrition. J.
Dairy. Sci. 74, 3568-3597.
Wang, X.F., Zhu, X.D., Li, Y.J., Liu, Y., Li, J.L., Gao, F., Zhou, G.H., Zhang, L., 2015. Effect of dietary creatine monohydrate supplementation
on muscle lipid peroxidation and antioxidant capacity of transported broilers in summer. Poult. Sci. 94, 2797-2804.
Yu, G.X., Chen, D.W., Yu, B., He, J., Zheng, P., Mao, X.B., Huang, Z.Q., Luo, J.Q., Zhang, Z.H., Yu, J., 2016. Coated protease increases ileal
digestibility of protein and amino acids in weaned piglets. Anim. Feed Sci. Technol. 214, 142-147.
Zanella, I., Sakomura, N.K., Silversides, F.G., Fiqueirdo, A., Pack, M., 1999. Effect of enzyme supplementation of broiler diets based on corn
and soybeans. Poult. Sci. 78, 561-568.
Zhang, L., Yue, H.Y., Zhang, H.J., Xu, L., Wu, S.G., Yan, H.J., Gong, Y.S., Qi, G.H., 2009. Transport stress in broilers: I. Blood metabolism,
glycolytic potential, and meat quality. Poult. Sci. 88, 2033-2041.

Table 1
Analyzed nutrient content of the corn or sorghum used in this study (g/kg of dry matter).1
Item
Corn
Dry matter
884.2
Crude protein
89.9
Calcium
0.24
Total phosphorus
2.6
Ash
13.2
Ether extract
40.4
Neutral detergent fibre
125.4
Acid detergent fibre
30.5
Starch
676.0
Tannin
0.2
Essential amino acids
Arginine
3.5
Histidine
2.6
Isoleucine
2.8
Leucine
11.8
Lysine
2.6
Methionine
2.0
Phenylalanine
8.4
Threonine
3.3
Tryptophan
0.7
Valine
4.5
Non-essential amino acids

Sorghum
876.3
90.1
0.23
3.0
15.2
20.7
119.8
34.9
770.0
0.5
3.8
2.3
4.0
12.5
2.4
1.8
7.7
3.4
0.8
5.2

Alanine
Aspartic acid
Cystine
Glutamic acid
Glycine
Proline
Serine
Tyrosine
1

All analyses were conducted in duplicate.

6.2
5.5
2.8
14.9
3.4
7.3
4.0
2.1

8.3
6.7
2.5
19.4
3.4
7.7
4.4
3.5

Table 2
Ingredient composition of diets based on corn or sorghum used in the experiment (g/kg as fed)
d 0 to 21
Ingredient
Corn
Sorghum
Corn
600.0
Sorghum
600.0
Soybean meal
271.8
255.6
Corn gluten meal
61.5
75.0
Soybean oil
14.4
19.6
Dicalcium phosphate
17.0
19.8
Limestone
14.0
12.4
Salt
3.0
3.0
L-lysine HCl
2.0
2.8
DL-methionine
1.4
1.4
L-threonine
0.4
0.4
L-tryptophan
4.5
1
Vitamin-mineral premix
10.0
10.0
2
Chemical composition (g/kg DM)
ME, MJ/kg
12.55
12.55
Crude protein
218.3
213.2
Calcium
12.1
11.6
Total phosphorus
6.9
7.5
ATTD phosphorus
4.5
4.5
Lysine
10.8
11.2

d 22 to 42
Corn
600.0

0.8
0.2
4.4
10.0

Sorghum
644.4
234.2
53.4
24.0
15.0
12.5
3.0
2.5
0.5
0.5
10.0

12.76
195.6
9.7
5.9
3.5
10.3

12.76
197.1
9.9
6.5
3.5
10.1

319.1
36.6
11.5
14.3
3.0
-

SID lysine
Methionine
SID methionine
Threonine
SID threonine

8.2
5.4
3.4
8.4
6.6

7.7
5.5
3.4
8.2
6.6

8.8
3.6
2.8
7.5
6.2

8.2
3.6
2.8
7.4
6.2

Premix supplied per kg diet: vitamin A, 11,000 IU; vitamin D, 3,025 IU; vitamin E, 22 mg; vitamin K3, 2.2mg; thiamine, 1.65 mg; riboflavin, 6.6 mg; pyridoxine,
3.3 mg; cobalamin, 17.6 g; nicotinic acid, 22mg; pantothenic acid, 13.2 mg; folic acid, 0.33 mg; biotin, 88g; choline chloride, 500 mg; iron, 48 mg; zinc,
96.6 mg; manganese, 101.76 mg; copper, 10 mg; selenium, 0.05 mg; iodine, 0.96 mg; cobalt, 0.3 mg.
2
Analyzed values except for ME, ATTD P, SID Lysine, SID Methionine, and SID Threonine.
3
The diet was supplemented with 0.15 g/kg of compound proteases (Kemin Industries Company, Zhuhai, China). The compound protease, with a protease activity of
8000 units/g measured under the conditions of 37C and pH = 6, was in granular form, and contained acidic, neutral and alkaline proteases produced by
Aspergillus niger, Bacillus subtilis and Bacillus Licheniformis, respectively.

Table 3
Effects of compound proteases on the performance of broilers fed corn- or sorghum-soybean diets1
Corn-soybean
Sorghum-soybean
Diet
Protease
Item
+ Protease
+ Protease
Corn
Sorghum
+
d 1 to 21
ADG, g
ADFI, g
F/G
d 22 to 42
ADG, g
ADFI, g
F/G
d 1 to 42
ADG, g
ADFI, g
F/G
Mortality,
%
1

N=8

P-values
SEM

Diet

Protease

Diet*
protease

30.87
46.69
1.52

31.30
44.35
1.42

30.59
46.30
1.52

31.01
45.99
1.49

31.09
45.52
1.47

30.80
46.15
1.51

30.73
46.50
1.52

31.16
45.17
1.46

0.85
1.38
0.04

0.73
0.65
0.46

0.62
0.34
0.14

0.99
0.47
0.42

78.01
145.1
1.86

81.72
150.2
1.84

73.31
134.1
1.83

79.88
142.9
1.79

79.87
147.7
1.85

76.60
138.5
1.81

75.66
139.6
1.85

80.80
146.6
1.82

1.81
5.64
0.06

0.08
0.12
0.50

< 0.01
0.23
0.58

0.43
0.75
0.91

54.44
95.89
1.76

56.36
97.10
1.72

51.95
90.21
1.74

55.45
94.42
1.70

55.40
96.50
1.74

53.70
92.32
1.72

53.20
93.05
1.75

55.91
95.76
1.71

1.12
2.98
0.04

0.14
0.17
0.60

0.02
0.37
0.37

0.49
0.62
0.91

3.13

1.56

1.56

4.69

2.35

3.13

2.35

3.13

1.32

0.76

0.76

0.63

Table 4
Effects of compound proteases on AME (MJ/kg) and the retention of DM, GE and nitrogen (%) of broilers fed corn- or sorghum-soybean diets 1
Corn-soybean
Sorghum-soybean
Diet
Protease
P-value
Item
SEM
+ Protease
+ Protease
Corn Sorghum
+
Diet Protease Diet*protease
d 19 to 21
DM
GE
Nitrogen
retention
AME
d 40 to 42
DM
GE
Nitrogen
retention
AME
1

N=8

78.23 81.61
81.38 84.33

78.03
80.83

79.33
81.69

79.92 78.68
82.86 81.26

78.13 80.47
81.11 83.01

0.76 0.13
0.73 0.05

< 0.01
0.02

0.19
0.18

72.95 76.12

71.95

72.89

74.54 72.42

72.45 74.51

1.10 0.08

0.04

0.33

13.75 14.25

13.70

13.85

14.00 13.77

13.73 14.05

0.09 0.05

0.01

0.29

79.19 80.25
81.52 82.72

78.21
80.69

80.42
82.98

79.72 79.32
82.12 81.84

78.70 80.34
81.11 82.85

0.68 0.57
0.66 0.68

0.03
0.02

0.42
0.43

70.49 74.30

68.64

72.45

72.40 70.55

69.57 73.38

1.33 0.19

0.01

0.99

13.44 13.64

13.35

13.73

13.54 13.54

13.40 13.69

0.09 0.94

0.02

0.58

Table 5
Effects of compound proteases on gut morphology of broilers fed corn- or sorghum-soybean diets1
Corn-soybean
Sorghum-soybean
Diet
Protease
Item
+ Protease
+ Protease
Corn Sorghum
+
d 21
Duodenum (m)
Villus height 1625
Crypt depth
216
Villus: Crypt
8
ratio
Jejunum (m)
Villus height
935
Crypt depth
185
Villus: Crypt
5
ratio
Ileum (m)
Villus height
801
Crypt depth
162
Villus: Crypt
5
ratio
d 42
Duodenum (m)
Villus height 1963
Crypt depth
316
Villus: Crypt
6
ratio

P-value
SEM

1756
201

1614
211

1728
206

1691
209

1671
209

1620
214

985
163

942
172

976
161

960
174

959
167

939
179

886
143

825
158

873
141

843.5
153

1987
312

1952
336

Diet Protease Diet*protease

1742 13.26 0.46

204 3.58 0.68

< 0.01
< 0.01

0.65
0.68

0.13 0.58

< 0.01

0.53

981
162

8.35 0.77
4.58 0.68

< 0.01
< 0.01

0.22
0.49

0.18 0.59

< 0.01

0.89

849
150

813
160

880
142

7.25 0.59
3.68 0.62

< 0.01
< 0.01

0.86
0.92

0.18 0.75

< 0.01

0.69

1965
325

1975
314

1959
331

1958
326

1976 24.21 0.42

319 6.28 0.59

0.84
0.74

0.68
0.55

0.71

0.92

0.17 0.69

Jejunum (m)
Villus height 1384
Crypt depth
186
Villus: Crypt
7
ratio
Ileum (m)
Villus height 1269
Crypt depth
189
Villus: Crypt
7
ratio
1

N=8

1405
172

1368
184

1423
173

1395
179

1396
179

1376
185

1414
173

8.68 0.25
3.01 0.69

0.02
0.04

0.86
0.65

0.28 0.36

0.02

0.56

1265
188

1285
186

1256
184

1267
189

1271
185

1277
188

1261
186

8.36 0.87
2.69 0.66

0.92
0.76

0.85
0.21

0.26 0.78

0.84

0.63

Table 6
Effects of compound proteases on carcass traits of broilers fed corn- or sorghum-soybean diets1
Corn-soybean
Sorghum-soybean
Diet
Protease
Item
+ Protease
+ Protease
Corn Sorghum
+
Slaughter weight, g
Breast muscle
Breast weight, g
Breast muscle
yield, %
pH40min
pH24h
Lightness
Redness
Yellowness
Drip loss, %
Thigh muscle
Thigh w eight, g
Thigh muscle yield, %
pH40min
pH24h
Lightness
Redness
Yellowness
Drip loss, %
1
N=8

P-value
SEM

Diet Protease Diet*protease

2264

2342

2225

2351

2303

2288

2245

2347

27.4

0.91

<0.01

0.76

194

208

199

212

201

206

197

210

3.58

0.14

0.02

0.62

8.57

8.88

8.94

9.02

8.73

8.99

8.78

8.95

0.29

0.64

0.48

0.63

6.38
5.38
51.9
5.61
10.5
1.98

6.39
5.56
51.2
5.47
9.64
1.66

6.49
5.15
49.3
5.06
11.5
1.95

6.29
5.47
47.4
6.09
9.78
1.65

6.39
5.47
51.6
5.54
10.1
1.82

6.39
5.31
48.4
5.58
10.6
1.80

6.44
5.27
50.6
5.34
11.0
1.97

6.34
5.52
49.3
5.78
9.71
1.66

0.12
0.08
2.86
0.56
1.05
0.13

0.58
0.26
0.54
0.62
0.49
0.86

0.49
< 0.01
0.75
0.71
0.15
< 0.01

0.58
0.62
0.67
0.89
0.88
0.90

220
9.72
6.38
6.02
52.5
5.56
9.51
1.58

224
9.56
6.41
6.06
49.8
6.03
9.51
1.62

223
10.0
6.46
5.96
50.5
5.92
10.6
1.62

234
9.95
6.42
6.22
48.9
6.14
10.6
1.55

222
9.64
6.40
6.04
51.2
5.80
9.51
1.60

229
9.99
6.44
6.09
49.7
6.03
10.6
1.59

222
229
9.86 9.76
6.42 6.42
5.99 6.14
51.5 49.4
5.74 6.09
10.06 10.06
1.60 1.59

3.86
0.32
0.10
0.12
2.58
0.78
1.01
0.16

0.69
0.52
0.72
0.38
0.66
0.87
0.25
0.47

0.42
0.78
0.52
0.41
0.14
0.19
0.89
0.62

0.64
0.72
0.47
0.84
0.63
0.76
0.75
0.35