Effect of Exogenous Protease, Mannanase, and Xylanase Supplementation in Corn and High Protein Corn DDGS Based Diets on Growth Performance, Intestinal Morphology and Nutrient Digestibility in Broiler Chickens

A study was conducted to evaluate the effects of exogenous enzymes supplementation in high protein (Hi-Pro) corn DDGS-based diets on broiler growth performance, intestinal health, and nutrient digestibility. A total of 200 one-day-old broiler chicks were randomly allocated to four dietary treatments with five replicates of 10 birds each. A basal diet (CON) was formulated (CP: 20%; ME: 2900 Kcal/kg) containing 15% of dietary crude protein share from Hi-Pro corn DDGS. Three experimental diets were developed using basal diets; diet one was supplemented with protease (CON-P), diet two with an enzyme mix of mannanase and xylanase (CON-MX) and diet three with a protease and enzyme mix of mannanase and xylanase. A digestibility assay was carried out using Celite (insoluble acid ash), from day 29 to day 35. On day 35, four birds from each replicate were picked randomly and killed by cervical dislocation to collect ileal digesta. In addition, duodenum, jejunum and ileum lengths were measured, and tissue samples were collected from midpoints of jejunum to note villus height and crypt depths. Feed intake, body weight gain and feed conversion ratio were not affected by dietary treatments during the entire study period (p>0.05). Likewise, no difference in carcass characteristics, soft organ weight, intestinal morphology, apparent amino acids digestibility and visible ileal digestible energy was observed by the dietary treatments (p>0.05).Supplementation of exogenous protease and enzyme mix (mannanase and xylanase) had no effect on growth performance, intestinal integrity and nutrient availability of broiler chickens fed HiPro DDGS-based diets.


INTRODUCTION
Distiller dried grains with soluble (DDGS) is a corn by-product that is produced by dry milling and fermentation process. The recent trend of biofuel production has provided nutritionists an opportunity to use DDGS as an alternative protein ingredient in poultry diets. Ethanol producers are continuously improving on fractioning technologies to enhance the production of ethanol and the quality of DDGS (Jung & Batal 2009). Mainly two fractioning techniques are employed by the ethanol industry, i.e., front end and back end fraction technology. Only endosperm fraction of corn is subjected to fermentation in front end fractionation technology that eliminates non-fermentable fraction (germ and bran) of corn and results in (High-Pro) corn DDGS (Robinson et al. 2008). High protein (Hi-Pro) corn DDGS is higher in crude protein (CP), amino acids and energy than conventional corn DDGS (Singh et al. 2005). The inclusion of corn DDGS in broiler diet is well documented, however, higher level of corn DDGS has been reported to reduce growth performance (Lumpkins et al. 2004;Wang et al. 2007;Choi et al. 2008;Youssef et al. 2008;Loar et al. 2010;Zhang et al. 2013).
The total content and availability of nutrients change after fermentation and drying process. During DDGS production, the concentration of non-phytate increased from 0.08% to 0.34% (NRC 1994). Matsui (2002) also observed that phytate phosphorus content reduced, when soybean meal (SBM) was subjected to fermentation. Lopez et al. (1983) also found similar results; fermentation reduced the phytate content of corn and increased non-phytate content. During ethanol production, process alteration in the structural components of corn grain takes place, and the availability of nutrients is also modified. The application of heat during the drying process has been reported to increase phosphorus bioavailability and reduce amino acids digestibility (Amezcua & Parsons 2007).
Exogenous protease supplementation in poultry diets as a mono-component or admixture of other enzymes has become quite common (Cowieson & Adeola 2005). The supplementation of exogenous protease not only improves the CP and amino acid digestibility (Cowieson & Roos 2014) but also tends to enhance ileal digestibility of energy (Kalmendal & Tauson 2012). Cowieson et al. (2017 a,b) also noted the valuable effect of exogenous protease on mucin secretion, intestinal integrity, and immunity in broiler studies.
Non-starch polysaccharides interfere with the mixing of digesta with endogenous enzymes by increasing the viscosity of digesta, thus acts as antinutritional factors (Slominski 2011). Gao et al. (2008) reported that the presence of arabinoxylan in the wheat-based diet leads to hypertrophy of the digestive organ. However, supplementation of xylanase in a wheat-based diet reduces the negative effect of arabinoxylan on the relative weight of digestive organs (duodenum, jejunum, and pancreas). In another study, it was pointing out that arabinoxylan not only reduces the digestibility of nutrients but also increases gastro-intestinal secretions (Angkanaporn et al. 1994). Supplementation of non-starch polysaccharides (NSPase) and proteases individually or in combination are reported to improve the nutrient utilization in poultry (Olukosi et al. 2010). Ludke et al. (2018) also reported that the supplementation of exogenous enzymes blend improved production in chicken. Campasino et al. (2015) found that the supplementation of NSPase in DDGS-based diets improved broiler performance, protein, and energy digestibility coefficient.
The effect of exogenous enzymes supplementation in Low-Pro corn DDGS based broiler diets is well explored. However, Hi-Pro corn DDGS being relatively new feedstuffs warrants the use of exogenous enzymes, more specifically mannanase, xylanase and proteases to further evaluate and improve its nutritive value for broiler chickens. Therefore, this study was planned to evaluate the effect of exogenous enzymes (mannanase, xylanase, and proteases) alone or in combination on broiler performance, apparent amino acid digestibility and apparent ileal digestible energy in Hi-Pro corn DDGS-based diet.

MATERIALS AND METHODS
All procedures followed in the conduct of the experiment were approved by the Graduate Studies and Research Board, University of Agriculture, Faisalabad, Pakistan.

Animal Husbandry and Experimental Procedure
All the ingredients except corn DDGS used in experimental diets were provided by a commercial feed mill, Sadiq Feed Mill (Pvt. Ltd.) Rawalpindi, Pakistan. Hi-Pro corn DDGS was imported from the USA, and its nutrient profile is shown in Table 1. A basal diet (CP: 20% and ME: 2900 Kcal/ Kg) was formulated, 15% dietary protein contributed by Hi-Pro corn DDGS. Three experimental treatments were given to the birds. Experimental treatments were: Basal diet supplemented with protease (Cibenza DP 100 at 250 g/ ton of feed); Basel diet supplemented with an enzyme mix of mannanase and xylanase (CON-MX; Winzyme MX-100 g/ ton of feed) and Basel diet supplemented with a protease and enzyme mix of mannanase and xylanase (CON-MXP; Winzyme MX 100g/ ton of feed + Cibenza DP 100 at 250g/ton of feed). Diets were formulated using software WinFeed 2.8 (WinFeed Ltd., Cambridge, UK). Two hundred 1-day-old broiler chicks (Ross 308) were purchased from a local commercial hatchery, Sadiq Brother Poultry Hatcheries (Pvt. Ltd.) Rawalpindi. These chicks were randomly allotted to one of four dietary treatments. Each dietary treatment had five replicates with ten birds in each replicate. The birds were reared on the concrete floor, covered with a 2-inch layer of rice husk as bedding material. The feed was offered ad libitum while the supply of fresh and clean water was made available round the clock. The room temperature was maintained at 33°C during the first week of the trial with a reduction of 3°C every week. All the birds were reared under identical managemental conditions throughout the experiment.

Effect of Exogenous Protease, Mannanase, and Xylanase Supplementation in Corn and High Protein Corn DDGS Based Diets on Growth Performance, Intestinal Morphology and Nutrient Digestibility in Broiler Chickens
eRBCA-2019-1088

Exogenous enzymes
The NSPase (Winzyme MX ® @100 g/mt) used in this study was provided by Suntaq International Limited, China. Winzyme MX ® was a combination of endo1, 4 D xylanase and β-mannanase made by advanced deep ventilation fermentation method and contained 5000 IU/g β-mannanase and 15000 IU/g Xylanase. One unit of β-mannanase was added to the amount of enzyme, which liberates 1 μmol of reducing sugar (mannose) from 3 mg/ml of mannan solution in 1 min, at 37°C and pH5.5 and one unit of Xylanase is defined as that amount of enzyme required to liberate 1 μmol reducing sugar from xylan solution per minute under the condition of xylan solution concentration 5 mg/ml, at 37°C and pH value 5.50, The exogenous enzyme protease (Cibenza DP 100 @250g/MT) used in this experiment was provided by Novus International Inc. One gram of Cibenza DP 100 contained 600000 U/g. Cibenza DP 100 produced by Bacillus licheniformis.

Data collection
Weekly data on body weight and feed intake were recorded to calculate feed conversion ratio (FCR). On day 35, two birds per replicate were randomly picked and killed to record carcass %, relative breast, thigh and soft organ weight (liver, heart, and gizzard).

Digestibility assay and intestinal sampling
On day 29, a digestibility assay was conducted to measure the nutrient digestibility of experimental diets. Celite ® (AIA) was used as an external digestibility marker. The celite ® was mixed at 1% in all the experimental diets. These diets were fed from day 29 to 35 of the trial. On day 35, two birds from each replicate were randomly selected and killed by cervical dislocation for determining duodenum, jejunum and ileum lengths. The jejunum samples were collected by following Wang et al. (2015). Tissue samples (1.5 cm) obtained from the midpoint of jejunum were preserved in 10% buffer formalin phosphate solution until further analysis. Villi was photographed under a light microscope (Fasina et al. 2010).
Ileal digesta samples were collected from the ileum of 4 birds per replicate to determine the nutrient digestibility and apparent ileal digestible energy (AIDE) of experimental diets by the method described by Scott and Boldaji (1997). For this purpose, the intestinal tract was excised, and the contents of the tract from Meckel's diverticulum to 40 mm cranial to ileal-cecal junction were flushed into 200 ml plastic cups and shifted immediately to an ice container. A few drops of formalin were also added to digesta samples to stop any bacterial activity. The digest samples within the pen were pooled and dried in hot air oven at 65ºC till constant weight was achieved. Dried samples were ground to pass through 0.5 mm sieve and were stored at -10 o C till further analyses.

Chemical analysis
All diets and ileal digesta samples were analyzed for total nitrogen , AIA and gross energy (GE). Total N was analyzed by using micro Kjeldahl's apparatus (AOAC, 2000) as described in recent studies (Rahman et al. 2019). The AIA contents of the experimental diets and digested samples were determined following Vogtmann et al. (1975) and GE determined by Bomb calorimeter (Parr Instrument Co., Moline, IL). The total amino acids (AAs) contents of the experimental diets and ileal digesta were analyzed by using amino acid analyzer (Biochrom 30 Plus, Biochrom Ltd. Cambridge UK). All samples were oxidized with hydrogen peroxide + formic acid + phenol solution and sodium disulfide was used to decompose the excess oxidation reagent. After oxidation the samples were hydrolyze using 6M HCl for 24 hours. The pH of the hydrolyzed sample was adjusted to 2.20 and filtered; the amino acid profile was determined.

Effect of Exogenous Protease, Mannanase, and Xylanase Supplementation in Corn and High Protein Corn DDGS Based Diets on Growth Performance, Intestinal Morphology and Nutrient Digestibility in Broiler Chickens
eRBCA-2019-1088

Calculation and statistical analysis
The apparent digestibility coefficient of the nutrient was calculated by Ravindran et al. (1999).
N= Nutrient and AIA= Acid insoluble ash The AIDE of the diets was calculated by the following formula: Data were subjected to one-way ANOVA using the GLM procedure at a probability level of p<0.05 to determine the statistical significance and means were analyzed by Tukey test (SAS, 2000).

Growth performance
Data on feed intake (FI), body weight gain (BWG) and feed conversion ratio (FCR) are present in Table  3. The supplementation of NSPase (combination of mannanase and xylanase) and protease alone or in combination did not improve (p>0.05) the FI, BWG and FCR during starter (day 1-21), finisher (day 22-35) and overall phase (day 1-35). However, a tendency of improvement in BWG was noted by the supplementation of exogenous enzymes (protease and NSPase) during the starter phase (day 1-21) over control. This effect was not observed during the finisher phase. The combination of enzymes (mannanase, xylanase, and protease) did not show any effect over control and or when supplemented alone (Table 3).
Carcass yield, breast and thigh yield and relative organs weight (gizzard, liver, and heart) are presented in Table 4. The supplementation of exogenous enzymes did not affect (p>0.05) these parameters.

Intestinal health
There was no difference (p>0.05) in the small intestine's length (duodenum, jejunum, ileum, and total length) with the supplementation of exogenous enzymes compared to the control. Duodenum length tended to increase numerically (p=0.78) in those birds fed the control diet compared to the diets supplemented with exogenous enzymes. Villus height, crypt depth, and crypt width were also not improved (p>0.05) by the supplementation of exogenous enzymes alone or in combination.

Nutrient digestibility
No difference was observed in protein, and apparent amino acids digestibility (p>0.05) in response to protease and NSPase compared to the control. Protein digestibility tended to numerically increase by 1.49 and 1.68%, respectively in the diets supplemented with protease and combination of protease and NSPase

Effect of Exogenous Protease, Mannanase, and Xylanase Supplementation in Corn and High Protein Corn DDGS Based Diets on Growth Performance, Intestinal Morphology and Nutrient Digestibility in Broiler Chickens
eRBCA-2019-1088 compared to the control. A similar trend was observed on apparent amino acids digestibility and AIDE by the supplementation of exogenous enzymes in corn and Hi-Pro DDGS based diets.

DISCUSSION
It has been reported that starch content of corn during dry milling process is utilized for ethanol production and non-starch polysaccharide content of the remaining proportion increased 2 to 3 folds over intact grain (Spiehs et al. 2002). Kim et al. (2008) analyzed the polymeric sugars content of DDGS and reported that corn DDGS contained a high content of glucan, xylan, and arabinan. In another study Pedersen et al. (2014) reported that NSP content of corn DDGS ranged 250-337 g/kg on DM basis and the distribution of sugar constituent was in the order of xylose >

Effect of Exogenous Protease, Mannanase, and Xylanase Supplementation in Corn and High Protein Corn DDGS Based Diets on Growth Performance, Intestinal Morphology and Nutrient Digestibility in Broiler Chickens
eRBCA-2019-1088 arabinose > NCP-glucose > mannose > galactose. It is also well documented that during drying process amino acids digestibility were compromised due to heat treatments (Amezcua and Parsons 2007;Bandegan et al. 2009;Olukosi et al. 2010). Previous studies that evaluated the supplemental effect of exogenous enzymes reported inconsistent findings (Choct et al. 1999;Cowieson et al. 2006;Gao et al. 2007). In the present study, supplementation of protease, NSPase and the combination of protease and NSPase did not improve weight gain and feed conversion ratio_ENREF_24_ENREF_48. The reason for the lack of improvement due to exogenous enzymes supplementation on growth performance in the present study could be because Hi-Pro DDGS has less content of non-starch polysaccharide and less exposure to heat. Another reason for the lack of improvement might be due to the negative effect of exogenous protease supplementation on the secretion of the endogenous enzyme as reported by Kaczmarek et al. (2014). Olukosi et al. (2007Olukosi et al. ( , 2010 reported similar results. Olukosi et al. (2007Olukosi et al. ( , 2010 reported that the blend of xylanase, amylase and protease had no effect on growth performance and feed efficiency. Campasino et al. (2015) reported that the supplementation of NSPase (xylanase, glucanase, and galactosidase) in corn DDGSbased diet did not improve weight gain and FCR in the control diet. However, the enzyme supplementation in low energy diet (132 Kcal/kg less energy than control) improved weight gain. Zou et al. (2013) reported that the enzyme supplementation (mannanase, xylanase, and glucanase) in broiler diet did not improve weight gain. But FCR was improved. This improvement in FCR was attributed to the increased pancreatic secretions and activities of digestive enzymes.
Cowieson & Ravindran (2008) reported that the supplementation of enzymes (xylanase, protease, and amylase) in corn -SBM-based diet improved weight gain and feed efficiency, but feed intake remained unaffected. In another study, Cowieson et al. (2006) reported that the supplementation of xylanase, amylase, and protease improved the weight gain but did not improve the FCR. O'Neil et al. (2012) reported that the supplementation of xylanase in corn-SBM based diets cause reduction of digesta viscosity and convert arabinoxylans to Xylooligomers which may act as prebiotic for beneficial bacteria. A similar finding was also observed by Ferreira et al. (2016), and they reported that the supplementation of mannanase alone in corn-SBM based diet improve weight gain and FCR. In the present study, the supplementation of exogenous enzymes did not improve growth performance.
Carcass and soft organ weights were not affected by different dietary treatments. Mahmood et al. (2018) also reported that the supplementation of protease enzyme did not improve carcass yield, breast meat yield, thigh meat yield, gizzard, liver, and heart. Similar findings were also reported by various scientists (Gao et al. 2007;Hajati 2010;Opoku et al. 2015;Dalólio et al. 2016).
The small intestine is a major component of the digestive tract for digestion and nutrient absorption. Pluripotent columnar cells of the small intestine can differentiate into digestive, absorptive, or mucinproducing roles (Moog 1950;Cheng and Leblond 1974). Intestinal integrity (villus height, crypt depth, and goblet cell) are influenced by the dietary manipulation especially the supplementation of enzymes (Baurhoo et al. 2007;Salim et al. 2013). Mehri et al. (2010) reported that supplementation of 0.09% βmannanase in corn-SBM basal diet improved villus height. Zou et al. (2013), also reported improved intestinal integrity by the supplementation of xylanase, glucanase, and mannanase in broilers. In the current study, no improvements in intestinal integrity were observed by the supplementation of exogenous enzymes (mix of mannanase, xylanase, and protease). Duodenum, jejunum and ileum weight and length remained the same by the enzyme (proteases and a mix of mannanase and xylanase) when supplemented alone or in combination. Opoku et al. (2015) also found that protease and mannase alone in 30% wheat DDGS based broiler diet did not affect duodenum, jejunum and ileum weight and length. In the present study, the supplementation of protease and mix of mannanase and xylanase did not improve the apparent ileal digestibility of CP and amino acids. Opoku et al. (2015) found similar findings when they supplemented mannanase in 30% wheat DDGS based diet; however, with the supplementation of protease nitrogen digestibility was improved. Improvement in nitrogen digestibility by protease supplementation in Opoku et al. (2015) study might be due to high inclusion level of DDGS that provided more undigested nitrogen fraction for the action of protease compared to the present study. Campasino et al. (2015), also reported that the enzyme supplementation did not affect protein digestibility up to 10% inclusion level of corn DDGS, however, the improvement was observed at15% DDGS inclusion. Olukosi et al. (2015), reported that the supplementation of protease alone or the combination with xylanase and amylase improved nitrogen digestibility.
Apparent ileal digestible energy did not vary in response to protease and the mix of mannanase and xylanase. Similar results were reported by Olukosi et al. (2010). Olukosi et al. (2010) supplemented the blend of enzymes (Xylanase, amylase, and protease) and observed no improvement in AIDE in corn-SBM meal and corn DDGS based diet. However, Campasino et al.(2015) reported contradictory results and found that the supplementation of NSPase improved ME and digestible energy in corn DDGS based broiler diets. The improvement by enzyme supplementation was more pronounced when higher levels of corn DDGS were used. (Olukosi et al. (2015))reported an improvement in metabolizable energy with NSPase and protease. They used Low Pro corn DDGS which has high fiber content and might have led to more pronounced improvement.
In conclusion, the supplementation of NSPase and protease in Hi-Pro corn DDGS based diet did not improve the growth performance, intestinal morphology and nutrient digestibility in broiler chicken.