Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers

Effects of protease and non-starch polysaccharide enzyme on performance, digestive function,... Three hundred one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets containing non-starch polysaccharide (NSP) enzyme and different levels of acid prote- ase from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth per- OPENACCESS formance, digestive function, activity of endogenous digestive enzymes in the pancreas and Citation: Yuan L, Wang M, Zhang X, Wang Z mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control (2017) Effects of protease and non-starch chickens, average daily feed intake (ADFI) and average daily gain (ADG) were significantly polysaccharide enzyme on performance, digestive function, activity and gene expression of enhanced by the addition of NSP enzyme in combination with protease supplementation at endogenous enzyme of broilers. PLoS ONE 12(3): 40 or 80 mg/kg (p<0.05). Feed-to-gain ratio (FGR) was significantly improved by supple- e0173941. https://doi.org/10.1371/journal. mentation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease pone.0173941 compared to the control diet (p<0.05). Apparent digestibility of crude protein (ADCP) was Editor: Gotthard Kunze, Leibniz-Institut fur significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or Pflanzengenetik und Kulturpflanzenforschung 80 mg/kg protease (p<0.05). Cholecystokinin (CCK) level in serum was reduced by 31.39% Gatersleben, GERMANY with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05), but the Received: October 31, 2016 CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. Accepted: March 1, 2017 After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 Published: March 21, 2017 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% Copyright:© 2017 Yuan et al. This is an open (p<0.05), respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme access article distributed under the terms of the combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and Creative Commons Attribution License, which 27.41%, respectively (p<0.05). However, supplementation with NSP enzyme and 80 or 160 permits unrestricted use, distribution, and reproduction in any medium, provided the original mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respec- author and source are credited. tively (p<0.05). The activities of pancreatic lipase and amylase were significantly higher in Data Availability Statement: All relevant data are treated animals than they were in the control group (p<0.05). Supplementation with NSP within the paper and its Supporting Information enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased pancreatic trypsin files. mRNA levels by 40%, 44% and 28%, respectively. Supplementation with NSP enzyme and Funding: The authors received no specific funding 160 mg/kg protease decreased pancreatic trypsin mRNA levels by 13%. Pancreatic lipase for this work. and amylase mRNA expression were significantly elevated in treated animals compared to Competing interests: The authors have declared the control group (p<0.05). These results suggest that the amount of NSP enzyme and acid that no competing interests exist. PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 1 / 13 Application of protease and NSP enzyme in broilers protease in the diet significantly affects digestive function, endogenous digestive-enzyme activity and mRNA expression in broilers. Introduction Soybean meal (SBM) is an important protein resource in poultry diets due to its high protein content and amino-acid balance. However, the nutritional value of SBM is reduced by the inclusion of non-starch polysaccharides (NSPs) and other anti-nutritional factors (ANFs) that cause poultry meal digestion to be incomplete[1, 2]. There are large variations in the nutrient contents of soybean meal. This difference is not only reflected in the composition of proteins and amino acids, but also in the levels of NSPs and other anti-nutritional factors[3]. Water-sol- uble NSPs are difficult to digest and decrease the amount of free water in the intestine, thus affecting the digestion and absorption of other nutrients[4]. The addition of enzymes can reduce the negative effects of NSPs and improve nutrient availability in poultry diets. The hydrolysis of NSPs can reduce the stickiness of pentosan, release nutrients from the cell wall, and break down starches into simple sugars, allowing nutrients as well as digestive enzymes to move more freely and improving growth perfor- mance, nutrient absorption and the efficiency of feed digestion. Previous trials have demon- strated that proteases and carbohydrate enzymes can improve the nutritional value of SBM[5± 8]. Enzymes added to poultry diets can improve weight gain, feed conversion, the viscosity of intestinal chyme, and the digestibility of dry matter [9±11]. The primary benefit of xylanase in feed is to the reduction in viscosity. Partial hydrolysis of NSP by xylanase decreases intestinal chyme viscosity[12] and degrades cell-wall polysaccha- rides to release nutrients[3, 13]. Gao et al. (2008) speculated that its growth-promoting effect may also be related to the oligosaccharides produced by endogenous enzymes or exogenous enzymes[14]. Due to the inadequate secretion of digestive enzymes by broilers, exogenous enzymes have been included in broiler diets for decades. The supplementation of corn-soybean-meal diets with exogenous enzymes can improve weight gain, feed conversion ratio and ileal digestibility of amino acids[10]. Supplementation improves the availability of calories, protein and other nutrients. Proteases added to soybean meal broiler diets can increase body weight gain, appar- ent nitrogen retention and apparent metabolizable energy, although the feed-conversion ratio remains unchanged[15]. Exogenous enzymes may also increase the secretion of endogenous substances in the gas- trointestinal tract of broilers[16±18]. However, previous works suggest that supplementation with exogenous digestive enzymes may hinder the development of metabolism in the digestive organs. Excessive levels of enzymes can affect levels of endogenous enzymes in the gastrointes- tinal tract, with negative effects on health[19]. Variations in the level of this effect depend on many factors, such as age, type of diet and enzyme dose[20±22]. We added NSP enzyme with different levels of proteases to the broiler diet to evaluate their effects on growth, physiological index, and the synthesis and secretion of endogenous enzymes. These findings provide evi- dence for the application of enzymes in poultry feed. Materials and methods The research was conducted in accordance with the Declaration of Helsinki and with the Guide for Care and Use of Laboratory Animals as adopted and promulgated by the United National Institutes of Health. All experimental protocols were approved by the Review PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 2 / 13 Application of protease and NSP enzyme in broilers Committee for the Use of Institutional Animal Care and Use Committee of Henan Agricul- tural University. Institutional Animal Care and Use Committee of Henan Agricultural Univer- sity approval Number (20161030). Chickens and diets Three hundred one-day-old male broiler chicks (Ross-308) were randomly allocated by body weight to five treatments, with six replicate pens of 10 broilers. The broilers were housed in electrically heated cages with 24 h light and provided with food and water for 42 days. The room temperature was maintained at 33 to 35ÊC during the first week and was gradually decreased to 24ÊC by the end of the third week. The birds were fed a corn-soybean basal diet formulated by the National Research Council (Table 1). Additionally, 150 mg/kg NSP enzyme and 40, 80 or 160 mg/kg acid protease were mixed into the corn-soybean diet to produce the experimental diets. NSP enzyme (containing 20 000 U/g xylanase, 2500 U/gβ-glucanase and 500 U/g cellulaseenzymatic activity) and acid protease (enzymatic activity 50 000 U/g) were obtained from the Shanghai Honest Biological Technology Co. Ltd. All diets were fed in mash form, The diets and water were supplied for ad libitum intake.\ Table 1. Diet compositions and nutrient levels. Item 1-21 d 22-42 d Ingredients, % Corn 51.31 54.60 Soybean meal 40.00 36.20 Soybean oil 4.60 5.60 Dicalcium phosphate 1.90 1.60 Limestone 1.30 1.20 NaCl 0.30 0.30 DL- Methionine 0.20 0.11 Choline chloride 0.20 0.20 Mineral premix 0.10 0.10 Maduramicin Ammonium 0.06 0.06 Vitamin premix 0.03 0.03 Total 100.00 100.00 Nutrient levels ME, kcal/kg 2984.72 3092.08 Crude protein 21.67 20.38 Met, % 0.55 0.44 Lys, % 1.10 1.02 Thr, % 0.91 0.84 Cys, % 0.37 0.35 Trp, % 0.31 0.28 Calcium, % 0.99 0.88 Total phosphorus, % 0.70 0.63 Non-phytate phosphorus, % 0.44 0.39 Provided per kg of diet: vitamin A, 15000 IU; vitamin D , 3900 IU; vitamin E, 30 IU; VK , 3 mg; VB , 2.4 mg; 3 3 1 VB 9 mg; B , 4.5 mg; B , 0.021 mg; Pantothenic acid, 30 mg; Niacin, 45 mg; Folic acid, 1.2 mg; Biotin, 0.18 2 6 12 mg. Provided per kg of diet: Cu(CuSO ·5H O), 8 mg; Zn(ZnSO ·7H O), 40 mg; Fe(FeSO ·7H O), 80 mg; Mn 4 2 4 2 4 2 (MnSO .5H O), 100mg; I (KI), 0.35 mg; Se(Na SeO ), 0.15 mg. 4 2 2 3 https://doi.org/10.1371/journal.pone.0173941.t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 3 / 13 Application of protease and NSP enzyme in broilers Sample collection The broilers were weighed after being starved for 12 h at 21 and 42 days of age, and feed con- sumption for each pen was recorded for the 1±21-day or 1±42-day phases. The average daily feed intake (ADFI), average daily gain (ADG), and feed-to-gain ratio (FGR) were recorded during the initial (1±21 days) and total (1±42 days) phases of the experiment. From 14-16 d and 35-37 d, fecal samples were collected in duplicate and then dried at 60ÊC. Feed and stool samples were used to determine the apparent digestibility of crude protein (ADCP). At 21 and 42 d of age, one chick per cage was selected and weighed. The blood sam- ples were taken from the wing vein, centrifuged at 2,500×g for 0.5 h at 4ÊC to separate the serum and then stored at -20ÊC until further analysis. The chickens were anesthetized and then killed by jugular bleeding. The pancreatic and intestinal contents were immediately fro- zen in liquid nitrogen and then stored at -70ÊC. Digestibility assays Fecal and feed samples were dried at 105ÊC for 24 h and filtered through a 0.5-mm sieve. All samples were analyzed in duplicate. CP was determined using the AOAC method (2000)[23]. Acid-insoluble ash (AIA) was determined using the method of Choct and Annison (1992)[3]. Nutrient digestibility was calculated as follows: Digestibility …%† ˆ 100 100…diet AIA % fecal AIA %†…fecal CP % diet CP %† Determination of cholecystokinin (CCK) in serum Serum CCK was analyzed by ELISA using commercial kits (Kit number EK-069-04; Phoenix- Biotech Co., LTD, Beijing, China). Determination of endogenous digestive enzyme activity in the pancreas -1 Approximately 1 g of pancreatic tissue was added to 9 ml distilled water to obtain a 100 mgg homogenate with Omni Prep Multi-Sample Homegenizer (Omni, USA), and then centrifuged at 1,500×g for 15 min. The supernatant was collected for the determination of digestive en- zyme activities. Trypsin, lipase and amylase activity were determined using different kits (A080, A054 and C016, Nanjing Jiancheng Bioengineering Institute). Trypsin activity was defined as the amount of enzyme in each milligram of protein that caused an increase in absor- bance of 0.003 per min at 253 nm wavelength, pH 8.0 and 37ÊC[24]. Lipase activity was deter- mined per the amount of enzyme that hydrolyzed olive oil to form 1 μmol product per minute [25]. Amylase activity was defined as the amount of enzyme that hydrolyzed 1.0 mg substrate per 3 min at pH 6.9 at 40ÊC[26]. The protein concentrations were measured using the Coo- massie Brilliant Blue method with bovine serum albumin as standard. Determination of the mRNA expression of pancreatic digestive-enzyme genes RNA extraction. We extracted total RNA from approximately 50 mg of pancreatic tissue using TRIzol reagent according to the manufacturer's instructions (Invitrogen, USA). The RNA concentration was determined by absorbance at 260 nm using a UV spectrophotometer (UV754N, Shenzhen, China). We evaluated RNA purity by measuring the OD260/OD280 ratio. The RNA samples typically had an OD260/OD280 ratio between 1.9 and 2.0. PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 4 / 13 Application of protease and NSP enzyme in broilers Table 2. Primer pairs for trypsin, amylase, lipase andβ-actin genes from broilers. Primers Sequence Length of fragment (bp) Trypsin P15’-AGCGAGCAGACCATTAGTTC-3’ 253 P25’-AGGAGAGTACAGGGGCATTC-3’ Amylase P35’-AAGTGGAATGGAGAGAAGATG-3’ 147 P45’-CCAGAAAGTAAGAATGGAAGC-3’ Lipase P55’-CCCTGCCCAACCTTATTT-3’ 176 P65’-GCATTTCCACTCCTCCGT-3’ β-actin P75’-ACCGCAAATGCTTCTAAAC-3’ 93 P85’-CCAATCTCGTCTTGTTTTATG-3’ https://doi.org/10.1371/journal.pone.0173941.t002 Primer synthesis. The primers were synthesized by the Shanghai Biological Technology Co. (China). The pancreatic trypsin, amylase and lipase primer pairs (P1 and P2, P3 and P4, and P5 and P6) were designed based on the trypsin, amylase and lipase coding sequences, respectively. Theβ-actin primer pair (P7 and P8) was designed based on the conservedβ-actin sequence. The trypsin, amylase, lipase andβ-actin cDNA primers were designed based on the GenBank sequences NM_205385, NM_001001473, DQ 334850 and NM_205518, respectively (Table 2). Fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR). We carried out a real-time PCR using a SYBR PrimeScript™ RT-PCR Kit (Takara Biotechnology, Dalian, China) according to the manufacturer's instructions. Total RNA was reverse-transcribed to obtain cDNA using oligo-dT and reverse transcriptase at 50ÊC for 30 min and then heat inactivation of the enzyme at 85ÊC for 5 min. The cDNA sam- ples were then mixed with SYBR dye. We carried out gene-specific and quantitative real- time PCR using a Bio-Rad iQ5 PCR machine (Bio-Rad, USA). The PCR program consisted of pre-denaturation at 95ÊC for 5 min, 35 cycles of denaturation at 95ÊC for 10 s, annealing at 55ÊC or 56ÊC (forβ-actin and amylase, respectively) for 15 s and extension at 72ÊC for 12 s. The PCR was then heated from 60 to 95ÊC to produce the melting curve. One negative control was included in all reactions. Digestive-enzyme mRNA. The quantity of digestive enzyme mRNA in each sample was normalized toβ-actin. The cDNA of the digestive enzymes was quantified using relative stan- dard-curve methods. Because the amplification efficiencies of the target and references genes were slightly different, the quantification of the gene copy number was obtained from different standard curves for the target and reference genes. The average value obtained for the control sample was defined as 1, and the experimental results are expressed as a percentage of those obtained for the control group. https://www.ncbi.nlm.nih.gov/pubmed/11846609?dopt=Abstract Statistical analysis The experimental data are expressed as means with standard errors. The data were analyzed using analysis of variance in SAS 8.0, and Duncan's multiple-range test was used to compare treatment means. Differences were considered statistically significant at P < 0.05. Results Performance The growth-performance results are presented in Table 3. For days 1-21, supplementary prote- ase and NSP enzyme had no effect on average daily feed intake (ADFI) and average daily gain (ADG) (p>0.05), although the feed conversion ratio (FCR) was significantly improved with PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 5 / 13 Application of protease and NSP enzyme in broilers Table 3. Effect of different levels of complex enzymes on body weight gain, feed intake, and feed conversion in broilers. Item Supplementary enzyme level (NSP enzyme + protease) (mg/kg) 1 2 3 4 5 (0+0) (150+0) (150+40) (150+80) (150+160) 1±21 days ADFI 44.31± 1.05 44.04± 2.08 44.29± 2.65 45.81± 1.97 46.59± 0.91 ADG 28.51± 0.75 28.73± 1.50 28.74± 1.96 29.79± 1.35 30.44± 0.64 a b ab ab b FCR 1.55± 0.01 1.53± 0.01 1.54± 0.02 1.54± 0.01 1.53± 0.01 1±42 days cd d b a bc ADFI 81.60± 1.42 80.95± 1.49 84.16± 1.06 86.26± 1.35 83.37± 2.48 b b a a b ADG 42.68± 0.70 43.03± 0.90 44.78± 0.72 45.55± 0.59 43.66± 1.32 a b b b a FCR 1.91± 0.01 1.88± 0.01 1.88± 0.01 1.89± 0.02 1.91± 0.01 Each value represents mean± SE of six replicates. In the same row, values with no superscript letter or the same superscript letter indicate no signi®cant difference (P> 0.05); those with different superscript letters indicate a signi®cant difference (P< 0.05). ADFI, average daily feed intake; ADG, average daily gain; FCR, feed conversion ratio. https://doi.org/10.1371/journal.pone.0173941.t003 supplementation with NSP enzyme or NSP enzyme combined with 160 mg/kg protease com- pared to the control (p<0.05). For days 1-42, ADFI and ADG were significantly enhanced by NSP enzyme combined with protease at 40 or 80 mg/kg (p<0.05), and supplementation with NSP enzyme combined with protease at 80 mg/kg yielded the highest ADG (6.79% increase compared to the control) compared to the control diet. FCR was significantly improved com- pared to the control by supplementation with NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05), but supplementation with NSP enzyme combined with 160 mg/kg protease had no effect on FCR (p>0.05). Digestibility The results for the apparent digestibility of crude protein are presented in Table 4. For days 14-16, apparent digestibility of crude protein (ADCP) was significantly enhanced by supplementation with NSP enzyme or NSP enzyme combined with 40 or 160 mg/kg protease (p<0.05), and sup- plementation with NSP enzyme combined with protease at 160 mg/kg yielded the highest ADCP (7.26% increase compared to the control). For days 35-37, compared to the control, ADCP was significantly enhanced by supplementation with NSP enzyme or NSP enzyme combined with protease at 40 or 80 mg/kg (p<0.05), and supplementation with NSP enzyme combined with 40 mg/kg protease yielded the highest ADCP (12.66% increase compared to the control). Serum CCK Results for the CCK level in the serum are shown in Table 4. At 21 days of age, compared to the control animals, the serum CCK in animals given NSP enzyme or NSP enzyme combined with protease supplementation at 40, 80 or 160 mg/kg (p>0.05). At 42 days of age, compared to the control animals, the CCK level in the serum of animals treated with NSP enzyme and protease at 160 mg/kg was reduced by 31.39% (p<0.05), but the CCK level in the serum of ani- mals given NSP enzyme alone was increased by 26.51%. Activity of pancreatic digestive enzymes The results for trypsin activity in the pancreas are presented in Table 5. After 21 days, com- pared to the control animals, animals given NSP enzyme or NSP enzyme combined with PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 6 / 13 Application of protease and NSP enzyme in broilers Table 4. Effect of different levels of complex enzymes on apparent digestibility of crude protein and serum CCK of broilers. Item Supplementary enzyme level (NSP enzyme + protease) (mg/kg) 1 2 3 4 5 (0+0) (150+0) (150+40) (150+80) (150+160) ADCP(%) c b b bc a 14±16 days 65.14±1.40 67.66±1.55 67.10±1.49 66.79±1.49 69.88±1.53 c ab a b c 35±37 days 54.90±1.06 60.43±1.63 61.86±2.10 59.40±1.32 55.23±1.08 CCK(pg/ml) a a a a a 21 days 98.22±41.40 162.28±14.41 134.38±36.80 130.39±79.57 127.93±48.62 a a a a b 42 days 248.43±43.34 314.30±11.82 299.81±102.02 283.72±58.47 170.46±61.57 Each value represents mean± SE of six replicates. In the same row, values with no superscript letter or the same superscript letter indicate no signi®cant difference (P> 0.05); those with different superscript letters indicate a signi®cant difference (P< 0.05). https://doi.org/10.1371/journal.pone.0173941.t004 protease at 40 or 80 mg/kg showed increased activity of pancreatic trypsin (74.13%, 70.66% and 42.59%) (p<0.05), respectively. However, trypsin activity in the pancreas of the animals -1 given NSP enzyme combined with 160 mg/kg protease group (245 Umg protein ) was similar -1 to that observed in the control group (252 Umg protein ). After 42 days, supplementation with NSP enzyme or NSP enzyme combined with 40 mg/kg protease significantly increased the activity of pancreatic trypsin (32.45% and 27.41%) (p<0.05), compared with the control group. However, supplementation with NSP enzyme combined with protease at 80 or 160 mg/ kg decreased the activity of pancreatic trypsin by 10.75% and 25.88% (p<0.05). Pancreatic lipase activity was significantly higher in treated groups than in the control group (p<0.05). Supplementation with NSP enzyme resulted in the highest levels of pancreatic lipase at 21 days of age (36.33% increase compared to the control group). After 42 days, supple- mentation with NSP enzyme combined with protease at 80 mg/kg had the highest pancreatic lipase (increased by 78.79% compared to the control group). After 21 days, compared to the control, supplementation with NSP enzyme or NSP enzyme combined with protease at 40 or 80 mg/kg increased the activity of pancreatic amylase by 15.09%, 13.78% and 18.64% (p<0.05), respectively. After 42 days, pancreatic amylase activity was significantly higher in treated animals than in the control group (p<0.05). Table 5. Effect of different levels of complex enzymes on digestive enzyme activity of broilers pancreas (U/mgprot). Item Supplementary enzyme level (NSP enzyme + protease) (mg/kg) 1 2 3 4 5 (0+0) (150+0) (150+40) (150+80) (150+160) Trypsin c a a b c 21 days 251.94±28.20 438.72±62.39 429.97±20.77 359.24±38.11 244.87±31.58 b a a c d 42 days 406.14±28.48 537.95±46.30 517.47±37.09 362.50±21.22 301.04±19.32 Lipase c a b b b 21 days 475.43±14.70 648.16±10.68 601.47±19.96 616.09±16.37 606.53±22.26 d b b a c 42 days 499.37±20.16 784.36±32.78 798.87±26.26 892.81±18.38 635.56±12.66 Amylase b a a a ab 21 days 117.69±6.39 135.45±18.09 133.91±1.68 139.63±3.09 129.59±18.93 e a a b b 42 days 178.83±3.75 205.08±1.45 201.26±2.07 193.04±1.72 195.63±6.17 Each value represents mean± SE of six replicates. In the same row, values with no superscript letter or the same superscript letter indicate no signi®cant difference (P> 0.05); those with different superscript letters indicate a signi®cant difference (P< 0.05). https://doi.org/10.1371/journal.pone.0173941.t005 PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 7 / 13 Application of protease and NSP enzyme in broilers Table 6. Effect of different levels of complex enzymes on Quantification of pancreatic digestive enzyme mRNA in broilers (relative ratio). Item Supplementary enzyme level (NSP enzyme + protease) (mg/kg) 1 2 3 4 5 (0+0) (150+0) (150+40) (150+80) (150+160) Trypsin c a a a b 21 days 1.00±0.09 1.53±0.10 1.55±0.10 1.51±0.08 1.35±0.06 c a a b d 42 days 1.00±0.12 1.40±0.06 1.44±0.08 1.28±0.03 0.87±0.11 Lipase b a a a a 21 days 1.00±0.07 1.27±0.07 1.24±0.02 1.26±0.01 1.23±0.03 d a ab ab bc 42 days 1.00±0.07 1.15±0.02 1.12±0.03 1.13±0.04 1.07±0.07 Amylase b a a a a 21 days 1.00±0.05 1.79±0.04 1.77±0.04 1.78±0.02 1.80±0.06 b a a a a 42 days 1.00±0.04 1.61±0.06 1.56±0.04 1.60±0.04 1.59±0.03 Each value represents mean± SE of six replicates. In the same row, values with no superscript letter or the same superscript letter indicate no signi®cant difference (P> 0.05); those with different superscript letters indicate a signi®cant difference (P< 0.05). https://doi.org/10.1371/journal.pone.0173941.t006 Expression of pancreatic digestive-enzyme mRNA NSP enzyme or NSP enzyme combined with protease significantly affected trypsin, lipase and amylase mRNA expression in broilers (Table 6). After 21 days, trypsin mRNA expression in the pancreas had increased by 53%, 55%, 51% and 34% (P<0.05) in the animals given NSP enzyme and NSP enzyme combined with 40, 80 or 160 mg/kg protease, respectively. Com- pared to the control, after 42 days, animals given NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease showed increased pancreatic trypsin mRNA levels (increased by 40%, 44% and 28%, respectively). However, supplementation with NSP enzyme combined with 160 mg/kg significantly decreased (13% difference) pancreatic trypsin mRNA levels. The broilers pancreatic lipase and amylase mRNA expression were significantly higher than those of con- trol group (p<0.05). Discussion Amino acids are vital nutrients derived from food proteins (except for synthetic forms). Pro- tein degradation is largely determined by the secretion of the pancreas and stomach. Protein digestibility in broiler diets changes with dietary composition[27, 28]. Thus, incomplete diges- tion, including undigested proteins,can be ameliorated by the addition of exogenous proteases. Arabinoxylan is an important water-soluble non-starch polysaccharide (NSP) in feed that increases chyme viscosity, decreases the availability of nutrients, and alters the intestinal flora, thus affecting nutrient digestion and absorption[3, 29]. Previous studies have shown that the addition of xylanase to wheat-based diets significantly increases body weight gain (BWG) and improves the feed conversion rate (FCR) of broilers at 21 days of age[14, 30, 31] without affect- ing feed intake, indicating that improved nutrient utilization was responsible for the improved feed-conversion rate[32]. In this study, at 1-21 days and 1-42 days, the FCR was significantly improved compared to the control by supplementation with NSP enzyme alone, but there was no significant effect on ADG and ADFI. The effects of exogenous proteases added to poultry diets on growth performance are variable, possibly due to differences in tests and trial designs, especially in the negative control diets. Additionally, many studies have used complexes of enzymes rather than a single component enzyme, yielding more varied results[1, 10, 15, 33± 36] because the results cannot be attributed to one particular enzyme. The present study PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 8 / 13 Application of protease and NSP enzyme in broilers demonstrated that during days1-42, the addition of NSP enzyme combined with a protease at 40 or 80 mg/kg to a corn-soybean meal diet can increase ADFI and ADG. The results showed that addition of this enzyme could improve the FCR and ADG of broil- ers by increasing protein digestibility. At 21 days of age, CP was significantly enhanced by sup- plementation with NSP enzyme or NSP enzyme combined with 40 or 160 mg/kg protease (p<0.05). At 42 days of age, compared to the control, CP was significantly enhanced by supple- mentation with NSP enzyme or NSP enzyme combined with the protease at 40 or 80 mg/kg (p<0.05). Previous research results show that the addition of xylanase to wheat-based diets sig- nificantly reduces the viscosity of the digesta and improves the absorption of DM, CP and calo- ries in broilers, consistent with our results[31]. Corn-soybean-meal diets supplemented with compound enzyme (containing xylanase, protease and amylase) could improve crude protein digestibility by 15.6% while increasing body weight gain by 5.5% and FE by 4%[35]. The same enzyme used in corn-soybean-meal and wheat middlings-based diets increased crude-protein availability by 26.3% but had no effect on growth performance[37]. Therefore, our findings, along with those of Cowieson et al. [35] and Freitas et al. [38], suggest that multienzyme com- plexes can improve nutrient availability and protein digestibility. The intestinal tract has endocrine cells that secrete CCK, and 98% of the CCK in the intesti- nal tract is present in the mucosal layer, with the highest concentration in the duodenum. CCK has an important physiological function in the gastro intestinal tract, regulating pancre- atic exocrine secretion and promoting the synthesis of trypsin, chymotrypsin and amylase. CCK also inhibits gastric emptying, promotes bowel movements, and causes gallbladder contraction[39]. CCK A Receptor (CCK- R) is mainly distributed in the pancreatic gland, gallbladder, smooth muscle, vagus-nerve afferent fibers, brain etc.[40]. CCK- R in gastrointes- tinal tract is mainly mediated by secretion from the pancreas, contraction of the gallbladder and gastric smooth muscle and secretion from intestinal mucosal cells. It enhances sphincter muscle tension to delay gastric emptying[41] and reduces feed intake[42, 43]. Feed intake can be increased by reducing levels of CCK[44]. In this study, the level of CCK in serum was in- creased with NSP enzyme supplementation. However, with increased protease levels, CCK lev- els gradually decreased, and the ADFI increased significantly during days1-42. This research indicated that exogenous protease could increase the feed intake of broilers by decreasing serum levels of CCK. The amount of digestive juice synthesized or secreted by the pancreas, liver and intestinal mucosa and the increased enzymatic activity determine digestive function. Pancreatic juice includes a variety of enzymes involved in the degradation of nutrients, such as pancreatic amy- lase, trypsin, chymotrypsin, elastase, pancreatic lipase and colipase, all of which are directly related to digestive function. The strength of pancreatic enzyme activity is an excellent index by which to measure the digestive capacity[17]. In the present study, supplementation with NSP enzyme or NSP enzyme combined with protease significantly increased the activity of pancreatic lipase and amylase in broilers, but supplementation with NSP enzyme combined with protease at 80 or 160 mg/kg significantly decreased the activity of pancreatic trypsin. Almirall et al. reported that supplementaryβ-glucanase enzyme significantly increased the activity of lipase, amylase and trypsin in the intestinal contents of broilers on a barley-based diet[45]. Engberg et al. also found that supplementary xylanase significantly increased the activity of lipase and chymotrypsin in broilers[46]. A rat experiment showed that pancreatic secretion and enzyme activity increased significantly as active polysaccharide was added[47]. Mahagna et al. reported that supplementary amylase and protease significantly decreased the activity of amylase, chymotrypsin and trypsin in the intestinal contents of broilers on asor- ghum-soybean-meal diet[48]. Cowieson et al. added glucose solution containing seven types of monomeric enzymes to broiler diets and found that both phytase and protease significantly PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 9 / 13 Application of protease and NSP enzyme in broilers increased the excretion of endogenous nitrogen, amino acids, calories and dry matter, suggest- ing that exogenous proteases may participate in the feedback regulation of the pancreas[19]. Therefore, supplementation with exogenous digestive enzymes would decrease the activity of pancreatic digestive enzymes, and supplementation of viscous grain diets with NSP enzyme could reduce the inhibitory effect of NSP on digestive-enzyme activity. The synthesis of pancreatic digestive enzyme is controlled by a specific gene in acinar cells. The enzyme precursors are modified into zymogens, which are then transported to the top of the cell by the Golgi apparatus and packaged into vacuoles. In response to an external signal, the cell secretes the zymogen into the pancreatic duct[49]. In the present study, after 42 days, supplementation with NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease significantly increased levels of pancreatic trypsin, lipase and amylase mRNA, but supplemen- tation with NSP enzyme combined with 160 mg/kg of protease significantly decreased pancre- atic trypsin mRNA. Endogenous expression of digestive enzyme genesis regulated by dietary nutrient levels. A study on rats showed that when dietary protein content increased from 15% to 70%, pancreatic trypsin, chymotrypsin, and elastase expression increased 3.6, 3.9 and 1.8 times, respectively[50]. When the carbohydrate content increased from 11% to 75%, pancre- atic amylase mRNA increased 3.5-8 times[51]. It has been reported that when dietary fat content increased from 3% to 30%, lipase expression increased 2.2-3.9 times[52]. Thus, endog- enous expression of digestive-enzyme genes may be regulated through nutrition. Conclusions Supplementation with NSP enzyme and protease at 80 mg/kg yielded the best performance in ADG broilers.The addition of 150 mg/kg NSP enzyme improved FCR, promoted serum CCK secretion, increased the expression of pancreatic trypsin, amylase and lipase genes, and enhanced digestive enzyme activity in the pancreas. When the NSP enzyme was combined with 160 mg/kg protease, the growth of broilers was significantly improved during days 1-21 but decreased in the later stage. Broiler serum CCK, pancreatic trypsin activity and mRNA expression were decreased with the increase of exogenous protease, and the inhibitory effect was more obvious in the later stage. Supporting information S1 File. Effect of different levels of complex enzymes on body weight gain, feed intake, and feed conversion in broilers. (XLS) S2 File. Effect of different levels of complex enzymes on apparent digestibility of crude protein and serum CCK of broilers. (XLS) S3 File. Effect of different levels of complex enzymes on digestive enzyme activity of broil- ers pancreas (U/mgprot). (XLS) S4 File. Effect of different levels of complex enzymes on Quantification of pancreatic diges- tive enzyme mRNA in broilers (relative ratio). (XLS) Author Contributions Conceptualization: LY MW ZW. PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 10 / 13 Application of protease and NSP enzyme in broilers Data curation: LY MW. Formal analysis: LY XZ. Funding acquisition: LY ZW. Investigation: LY. Methodology: LY XZ. Project administration: LY MW ZW. Resources: LY ZW. Software: LY ZW. Supervision: LY MW XZ. Validation: LY MW. Visualization: LY. Writing ± original draft: LY MW XZ ZW. Writing ± review & editing: LY MW XZ ZW. References 1. Marsman GJ, Gruppen H, van der Poel AF, Kwakkel RP, Verstegen MW, Voragen AG. The effect of thermal processing and enzyme treatments of soybean meal on growth performance, ileal nutrient digestibilities, and chyme characteristics in broiler chicks. Poultry science. 1997; 76(6):864±72. Epub 1997/06/01. PMID: 9181620 2. Yin YL, Baidoo SK, Schulze H, Simmins PH. Effects of supplementing diets containing hulless barley varieties having different levels of non-starch polysaccharides with beta-glucanase and xylanase on the physiological status of the gastrointestinal tract and nutrient digestibility of weaned pigs. Livestock Pro- duction Science. 2001; 71(2±3):97±107. 3. Choct M, Annison G. Anti-nutritive effect of wheat pentosans in broiler chickens: roles of viscosity and gut microflora. British poultry science. 1992; 33(4):821±34. Epub 1992/09/01. https://doi.org/10.1080/ 00071669208417524 PMID: 1393677 4. Ward AT, Marquardt RR. The effect of saturation, chain length of pure triglycerides, and age of bird on the utilization of rye diets. Poultry science. 1983; 62(6):1054±62. Epub 1983/06/01. PMID: 6878135 5. Bedford MR. Mode of action of feed enzymes. J Appl Poult Res. 1993; 2(1):85±92. 6. Yin YL, Deng ZY, Huang HL, Li TJ, Zhong HY. The effect of arabinoxylanase and protease supplemen- tation on nutritional value of diets containing wheat bran or rice bran in growing pig. Journal of Animal & Feed Sciences. 2004; 13(3):445±61. 7. Li J, Li D, Yin YL, Piao XS, He JH, Chen GP, et al. Performance, nutrient digestibility and intestinal disaccharidase activity of weaner/grower pigs given diets containing extruded Chinese stored brown rice with exogenous enzyme supplements. Animal Science. 2005; 79(3):429±38. 8. Yin YL, Baidoo SK, Jin LZ, Liu YG, Schulze H, Simmins PH. The effect of different carbohydrase and protease supplementation on apparent (ileal and overall) digestibility of nutrients of five hulless barley varieties in young pigs. Livestock Production Science. 2001; 71(2±3):109±20. 9. Reddy VR, Qudratullah S. Utilisation of squilla meal (a novel animal protein source) by broilers. British poultry science. 1997; 38(3):263±9. Epub 1997/07/01. https://doi.org/10.1080/00071669708417984 PMID: 9280352 10. Zanella I, Sakomura NK, Silversides FG, Fiqueirdo A, Pack M. Effect of enzyme supplementation of broiler diets based on corn and soybeans. Poultry science. 1999; 78(4):561±8. Epub 1999/05/07. PMID: 10230910 11. Scheideler SE, Beck MM, Abudabos A, Wyatt C. Multiple-enzyme (Avizyme) supplementation of corn- soy based layer diets. J Applied Poult Res. 2005; 14(1):77±86. 12. Bedford MR, Classen HL. Reduction of intestinal viscosity through manipulation of dietary rye and pen- tosanase concentration is effected through changes in the carbohydrate composition of the intestinal PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 11 / 13 Application of protease and NSP enzyme in broilers aqueous phase and results in improved growth rate and food conversion efficiency of broiler chicks. The Journal of nutrition. 1992; 122(3):560±9. Epub 1992/03/01. PMID: 1542013 13. Meng X, Slominski BA, Nyachoti CM, Campbell LD, Guenter W. Degradation of cell wall polysaccha- rides by combinations of carbohydrase enzymes and their effect on nutrient utilization and broiler chicken performance. Poultry science. 2005; 84(1):37±47. Epub 2005/02/03. PMID: 15685940 14. Gao F, Jiang Y, Zhou GH, Han ZK. The effects of xylanase supplementation on performance, charac- teristics of the gastrointestinal tract, blood parameters and gut microflora in broilers fed on wheat-based diets. Animal Feed Science and Technology. 2008; 142(1-2):173±84. 15. Ghazi S, Rooke JA, Galbraith H, Bedford MR. The potential for the improvement of the nutritive value of soya-bean meal by different proteases in broiler chicks and broiler cockerels. British poultry science. 2002; 43(1):70±7. Epub 2002/05/11. https://doi.org/10.1080/00071660120109935 PMID: 12003341 16. Noy Y, Sklan D. Digestion and absorption in the young chick. Poultry science. 1995; 74(2):366±73. Epub 1995/02/01. PMID: 7724461 17. Krogdahl A, Sell JL. Influence of age on lipase, amylase, and protease activities in pancreatic tissue and intestinal contents of young turkeys. Poultry science. 1989; 68(11):1561±8. Epub 1989/11/01. PMID: 2481853 18. Nitsan Z, Ben-Avraham G, Zoref Z, Nir I. Growth and development of the digestive organs and some enzymes in broiler chicks after hatching. British poultry science. 1991; 32(3):515±23. Epub 1991/07/01. https://doi.org/10.1080/00071669108417376 PMID: 1716507 19. Cowieson AJ, Acamovic T, Bedford MR. Using the precision-feeding bioassay to determine the efficacy of exogenous enzymesÐA new perspective. Animal Feed Science and Technology. 2006; 129(1- 2):149±58. 20. Acamovic T. Commercial application of enzyme technology for poultry production. World's Poult Sci J. 2001; 57(3):225±42. 21. Bedford MR. Exogenous enzymes in monogastric nutrition Ð their current value and future benefits. Anim Feed Sci Technol. 2000; 86(1-2):1±13. Epub 2000/08/31. 22. Cowieson AJ. Factors that affect the nutritional value of maize for broilers. Animal Feed Science and Technology. 2005; 119(3-4):293±305. 23. AOAC. Official Methods of Analysis. 17th ed. Association of Official Analytical Chemists. 2000; Wash- ington, DC. 24. Iwamori M, Iwamori Y, Ito N. Sulfated lipids as inhibitors of pancreatic trypsin and chymotrypsin in epi- thelium of the mammalian digestive tract. Biochemical & Biophysical Research Communications. 1997; 237(2):262. 25. Verduin PA, Punt JMHM, Kreutzer HH. Studies on the determination of lipase activity. Clin Chim Acta. 1973; 46(1):11±9. PMID: 4732885 26. Somogyi M. Modification of two methods for the assay of amylase. Clinical Chemistry. 1960; 6:23±35. PMID: 13832788 27. Parsons CM, Castanon F, Han Y. Protein and amino acid quality of meat and bone meal. Poultry sci- ence. 1997; 76(2):361±8. Epub 1997/02/01. PMID: 9057220 28. Lemme A, Ravindran V, Bryden WL. Ileal digestibility of amino acids in feed ingredients for broilers. World Poultry Sci J. 2004; 60(4):423±38. 29. Choct M, Hughes RJ, Wang J, Bedford MR, Morgan AJ, Annison G. Increased small intestinal fermen- tation is partly responsible for the anti-nutritive activity of non-starch polysaccharides in chickens. British poultry science. 1996; 37(3):609±21. Epub 1996/07/01. https://doi.org/10.1080/00071669608417891 PMID: 8842468 30. Gao F, Jiang Y, Zhou GH, Han ZK. The effects of xylanase supplementation on growth, digestion, circu- lating hormone and metabolite levels, immunity and gut microflora in cockerels fed on wheat-based diets. British poultry science. 2007; 48(4):480±8. Epub 2007/08/19. https://doi.org/10.1080/ 00071660701477320 PMID: 17701501 31. Esmaeilipour O, Shivazad M, Moravej H, Aminzadeh S, Rezaian M, van Krimpen MM. Effects of xyla- nase and citric acid on the performance, nutrient retention, and characteristics of gastrointestinal tract of broilers fed low-phosphorus wheat-based diets. Poultry science. 2011; 90(9):1975±82. Epub 2011/ 08/17. https://doi.org/10.3382/ps.2010-01264 PMID: 21844263 32. Zhang L, Xu J, Lei L, Jiang Y, Gao F, Zhou GH. Effects of Xylanase Supplementation on Growth Perfor- mance, Nutrient Digestibility and Non-starch Polysaccharide Degradation in Different Sections of the Gastrointestinal Tract of Broilers Fed Wheat-based Diets. Asian-Australasian journal of animal sci- ences. 2014; 27(6):855±61. Epub 2014/07/23. PubMed Central PMCID: PMCPmc4093178. https://doi. org/10.5713/ajas.2014.14006 PMID: 25050024 PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 12 / 13 Application of protease and NSP enzyme in broilers 33. Pinheiro DF, Cruz VC, Sartori JR, Vicentini Paulino ML. Effect of early feed restriction and enzyme sup- plementation on digestive enzyme activities in broilers. Poultry science. 2004; 83(9):1544±50. Epub 2004/09/24. PMID: 15384906 34. Olukosi OA, Cowieson AJ, Adeola O. Age-related influence of a cocktail of xylanase, amylase, and pro- tease or phytase individually or in combination in broilers. Poultry science. 2007; 86(1):77±86. Epub 2006/12/21. PMID: 17179419 35. Cowieson AJ, Ravindran V. Effect of exogenous enzymes in maize-based diets varying in nutrient den- sity for young broilers: growth performance and digestibility of energy, minerals and amino acids. British poultry science. 2008; 49(1):37±44. Epub 2008/01/23. https://doi.org/10.1080/00071660701812989 PMID: 18210288 36. Walk CL, Cowieson AJ, Remus JC, Novak CL, McElroy AP. Effects of dietary enzymes on performance and intestinal goblet cell number of broilers exposed to a live coccidia oocyst vaccine. Poultry science. 2011; 90(1):91±8. Epub 2010/12/24. https://doi.org/10.3382/ps.2010-00760 PMID: 21177448 37. Gracia MI, La  zaro R, Latorre MA, Medel P, Aranõ Âbar MJ, Jime  nez-Moreno E, et al. Influence of enzyme supplementation of diets and cooking±flaking of maize on digestive traits and growth performance of broilers from 1 to 21 days of age. Animal Feed Science and Technology. 2009; 150(3-4):303±15. 38. Freitas DM, Vieira SL, Angel CR, Favero A, Maiorka A. Performance and nutrient utilization of broilers fed diets supplemented with a novel mono-component protease. The Journal of Applied Poultry Research. 2011; 20(3):322±34. 39. Rehfeld JF. Cholecystokinin. Clinics in gastroenterology. 1980; 9(3):593±607. Epub 1980/09/01. PMID: 40. Wang Y, Prpic V, Green GM, Reeve JR Jr., Liddle RA. Luminal CCK-releasing factor stimulates CCK release from human intestinal endocrine and STC-1 cells. American journal of physiology Gastrointesti- nal and liver physiology. 2002; 282(1):G16±22. Epub 2001/12/26. PMID: 11751153 41. Takiguchi S, Suzuki S, Sato Y, Kanai S, Miyasaka K, Jimi A, et al. Role of CCK-A receptor for pancreatic function in mice: a study in CCK-A receptor knockout mice. Pancreas. 2002; 24(3):276±83. Epub 2002/ 03/15. PMID: 11893936 42. Gibbs J, Young RC, Smith GP. Cholecystokinin decreases food intake in rats. Journal of comparative and physiological psychology. 1973; 84(3):488±95. Epub 1973/09/01. PMID: 4745816 43. Lukaszewski L, Praissman M. Effect of continuous infusions of CCK-8 on food intake and body and pan- creatic weights in rats. The American journal of physiology. 1988; 254(1 Pt 2):R17±22. Epub 1988/01/01. 44. Pekas JC. Effect of cholecystokinin immunization, enhanced food intake and growth of swine on lean yield and carcass composition. The Journal of nutrition. 1991; 121(4):563±7. Epub 1991/04/01. PMID: 45. Almirall M, Francesch M, Perez-Vendrell AM, Brufau J, Esteve-Garcia E. The differences in intestinal viscosity produced by barley and beta-glucanase alter digesta enzyme activities and ileal nutrient digestibilities more in broiler chicks than in cocks. The Journal of nutrition. 1995; 125(4):947±55. Epub 1995/04/01. PMID: 7536829 46. Engberg RM, Hedemann MS, Steenfeldt S, Jensen BB. Influence of whole wheat and xylanase on broiler performance and microbial composition and activity in the digestive tract. Poultry science. 2004; 83(6):925±38. Epub 2004/06/23. PMID: 15206619 47. Ikegami S, Tsuchihashi F, Harada H, Tsuchihashi N, Nishide E, Innami S. Effect of viscous indigestible polysaccharides on pancreatic-biliary secretion and digestive organs in rats. The Journal of nutrition. 1990; 120(4):353±60. Epub 1990/04/01. PMID: 2158535 48. Mahagna M, Nir I, Larbier M, Nitsan Z. Effect of age and exogenous amylase and protease on develop- ment of the digestive tract, pancreatic enzyme activities and digestibility of nutrients in young meat-type chicks. Reproduction, nutrition, development. 1995; 35(2):201±12. Epub 1995/01/01. PMID: 7537505 49. Konturek PC, Kania J, Kukharsky V, Ocker S, Hahn EG, Konturek SJ. Influence of gastrin on the expression of cyclooxygenase-2, hepatocyte growth factor and apoptosis-related proteins in gastric epi- thelial cells. Journal of physiology and pharmacology: an official journal of the Polish Physiological Soci- ety. 2003; 54(1):17±32. Epub 2003/04/04. 50. Giorgi D, Renaud W, Bernard JP, Dagorn JC. Regulation of proteolytic enzyme activities and mRNA concentrations in rat pancreas by food content. Biochemical and biophysical research communications. 1985; 127(3):937±42. Epub 1985/03/29. PMID: 3885943 51. Giorgi D, Bernard JP, Lapointe R, Dagorn JC. Regulation of amylase messenger RNA concentration in rat pancreas by food content. The EMBO journal. 1984; 3(7):1521±4. Epub 1984/07/01. PubMed Cen- tral PMCID: PMCPmc557553. PMID: 6204864 52. Wicker C, Scheele GA, Puigserver A. Pancreatic adaptation to dietary lipids is mediated by changes in lipase mRNA. Biochimie. 1988; 70(9):1277±83. Epub 1988/09/01. PMID: 2465787 PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 13 / 13 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png PLoS ONE Pubmed Central

Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers

PLoS ONE , Volume 12 (3) – Mar 21, 2017

Loading next page...
 
/lp/pubmed-central/effects-of-protease-and-non-starch-polysaccharide-enzyme-on-IcM02U2Xq9

References (61)

Publisher
Pubmed Central
Copyright
© 2017 Yuan et al
eISSN
1932-6203
DOI
10.1371/journal.pone.0173941
Publisher site
See Article on Publisher Site

Abstract

Three hundred one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets containing non-starch polysaccharide (NSP) enzyme and different levels of acid prote- ase from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth per- OPENACCESS formance, digestive function, activity of endogenous digestive enzymes in the pancreas and Citation: Yuan L, Wang M, Zhang X, Wang Z mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control (2017) Effects of protease and non-starch chickens, average daily feed intake (ADFI) and average daily gain (ADG) were significantly polysaccharide enzyme on performance, digestive function, activity and gene expression of enhanced by the addition of NSP enzyme in combination with protease supplementation at endogenous enzyme of broilers. PLoS ONE 12(3): 40 or 80 mg/kg (p<0.05). Feed-to-gain ratio (FGR) was significantly improved by supple- e0173941. https://doi.org/10.1371/journal. mentation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease pone.0173941 compared to the control diet (p<0.05). Apparent digestibility of crude protein (ADCP) was Editor: Gotthard Kunze, Leibniz-Institut fur significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or Pflanzengenetik und Kulturpflanzenforschung 80 mg/kg protease (p<0.05). Cholecystokinin (CCK) level in serum was reduced by 31.39% Gatersleben, GERMANY with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05), but the Received: October 31, 2016 CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. Accepted: March 1, 2017 After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 Published: March 21, 2017 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% Copyright:© 2017 Yuan et al. This is an open (p<0.05), respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme access article distributed under the terms of the combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and Creative Commons Attribution License, which 27.41%, respectively (p<0.05). However, supplementation with NSP enzyme and 80 or 160 permits unrestricted use, distribution, and reproduction in any medium, provided the original mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respec- author and source are credited. tively (p<0.05). The activities of pancreatic lipase and amylase were significantly higher in Data Availability Statement: All relevant data are treated animals than they were in the control group (p<0.05). Supplementation with NSP within the paper and its Supporting Information enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased pancreatic trypsin files. mRNA levels by 40%, 44% and 28%, respectively. Supplementation with NSP enzyme and Funding: The authors received no specific funding 160 mg/kg protease decreased pancreatic trypsin mRNA levels by 13%. Pancreatic lipase for this work. and amylase mRNA expression were significantly elevated in treated animals compared to Competing interests: The authors have declared the control group (p<0.05). These results suggest that the amount of NSP enzyme and acid that no competing interests exist. PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 1 / 13 Application of protease and NSP enzyme in broilers protease in the diet significantly affects digestive function, endogenous digestive-enzyme activity and mRNA expression in broilers. Introduction Soybean meal (SBM) is an important protein resource in poultry diets due to its high protein content and amino-acid balance. However, the nutritional value of SBM is reduced by the inclusion of non-starch polysaccharides (NSPs) and other anti-nutritional factors (ANFs) that cause poultry meal digestion to be incomplete[1, 2]. There are large variations in the nutrient contents of soybean meal. This difference is not only reflected in the composition of proteins and amino acids, but also in the levels of NSPs and other anti-nutritional factors[3]. Water-sol- uble NSPs are difficult to digest and decrease the amount of free water in the intestine, thus affecting the digestion and absorption of other nutrients[4]. The addition of enzymes can reduce the negative effects of NSPs and improve nutrient availability in poultry diets. The hydrolysis of NSPs can reduce the stickiness of pentosan, release nutrients from the cell wall, and break down starches into simple sugars, allowing nutrients as well as digestive enzymes to move more freely and improving growth perfor- mance, nutrient absorption and the efficiency of feed digestion. Previous trials have demon- strated that proteases and carbohydrate enzymes can improve the nutritional value of SBM[5± 8]. Enzymes added to poultry diets can improve weight gain, feed conversion, the viscosity of intestinal chyme, and the digestibility of dry matter [9±11]. The primary benefit of xylanase in feed is to the reduction in viscosity. Partial hydrolysis of NSP by xylanase decreases intestinal chyme viscosity[12] and degrades cell-wall polysaccha- rides to release nutrients[3, 13]. Gao et al. (2008) speculated that its growth-promoting effect may also be related to the oligosaccharides produced by endogenous enzymes or exogenous enzymes[14]. Due to the inadequate secretion of digestive enzymes by broilers, exogenous enzymes have been included in broiler diets for decades. The supplementation of corn-soybean-meal diets with exogenous enzymes can improve weight gain, feed conversion ratio and ileal digestibility of amino acids[10]. Supplementation improves the availability of calories, protein and other nutrients. Proteases added to soybean meal broiler diets can increase body weight gain, appar- ent nitrogen retention and apparent metabolizable energy, although the feed-conversion ratio remains unchanged[15]. Exogenous enzymes may also increase the secretion of endogenous substances in the gas- trointestinal tract of broilers[16±18]. However, previous works suggest that supplementation with exogenous digestive enzymes may hinder the development of metabolism in the digestive organs. Excessive levels of enzymes can affect levels of endogenous enzymes in the gastrointes- tinal tract, with negative effects on health[19]. Variations in the level of this effect depend on many factors, such as age, type of diet and enzyme dose[20±22]. We added NSP enzyme with different levels of proteases to the broiler diet to evaluate their effects on growth, physiological index, and the synthesis and secretion of endogenous enzymes. These findings provide evi- dence for the application of enzymes in poultry feed. Materials and methods The research was conducted in accordance with the Declaration of Helsinki and with the Guide for Care and Use of Laboratory Animals as adopted and promulgated by the United National Institutes of Health. All experimental protocols were approved by the Review PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 2 / 13 Application of protease and NSP enzyme in broilers Committee for the Use of Institutional Animal Care and Use Committee of Henan Agricul- tural University. Institutional Animal Care and Use Committee of Henan Agricultural Univer- sity approval Number (20161030). Chickens and diets Three hundred one-day-old male broiler chicks (Ross-308) were randomly allocated by body weight to five treatments, with six replicate pens of 10 broilers. The broilers were housed in electrically heated cages with 24 h light and provided with food and water for 42 days. The room temperature was maintained at 33 to 35ÊC during the first week and was gradually decreased to 24ÊC by the end of the third week. The birds were fed a corn-soybean basal diet formulated by the National Research Council (Table 1). Additionally, 150 mg/kg NSP enzyme and 40, 80 or 160 mg/kg acid protease were mixed into the corn-soybean diet to produce the experimental diets. NSP enzyme (containing 20 000 U/g xylanase, 2500 U/gβ-glucanase and 500 U/g cellulaseenzymatic activity) and acid protease (enzymatic activity 50 000 U/g) were obtained from the Shanghai Honest Biological Technology Co. Ltd. All diets were fed in mash form, The diets and water were supplied for ad libitum intake.\ Table 1. Diet compositions and nutrient levels. Item 1-21 d 22-42 d Ingredients, % Corn 51.31 54.60 Soybean meal 40.00 36.20 Soybean oil 4.60 5.60 Dicalcium phosphate 1.90 1.60 Limestone 1.30 1.20 NaCl 0.30 0.30 DL- Methionine 0.20 0.11 Choline chloride 0.20 0.20 Mineral premix 0.10 0.10 Maduramicin Ammonium 0.06 0.06 Vitamin premix 0.03 0.03 Total 100.00 100.00 Nutrient levels ME, kcal/kg 2984.72 3092.08 Crude protein 21.67 20.38 Met, % 0.55 0.44 Lys, % 1.10 1.02 Thr, % 0.91 0.84 Cys, % 0.37 0.35 Trp, % 0.31 0.28 Calcium, % 0.99 0.88 Total phosphorus, % 0.70 0.63 Non-phytate phosphorus, % 0.44 0.39 Provided per kg of diet: vitamin A, 15000 IU; vitamin D , 3900 IU; vitamin E, 30 IU; VK , 3 mg; VB , 2.4 mg; 3 3 1 VB 9 mg; B , 4.5 mg; B , 0.021 mg; Pantothenic acid, 30 mg; Niacin, 45 mg; Folic acid, 1.2 mg; Biotin, 0.18 2 6 12 mg. Provided per kg of diet: Cu(CuSO ·5H O), 8 mg; Zn(ZnSO ·7H O), 40 mg; Fe(FeSO ·7H O), 80 mg; Mn 4 2 4 2 4 2 (MnSO .5H O), 100mg; I (KI), 0.35 mg; Se(Na SeO ), 0.15 mg. 4 2 2 3 https://doi.org/10.1371/journal.pone.0173941.t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 3 / 13 Application of protease and NSP enzyme in broilers Sample collection The broilers were weighed after being starved for 12 h at 21 and 42 days of age, and feed con- sumption for each pen was recorded for the 1±21-day or 1±42-day phases. The average daily feed intake (ADFI), average daily gain (ADG), and feed-to-gain ratio (FGR) were recorded during the initial (1±21 days) and total (1±42 days) phases of the experiment. From 14-16 d and 35-37 d, fecal samples were collected in duplicate and then dried at 60ÊC. Feed and stool samples were used to determine the apparent digestibility of crude protein (ADCP). At 21 and 42 d of age, one chick per cage was selected and weighed. The blood sam- ples were taken from the wing vein, centrifuged at 2,500×g for 0.5 h at 4ÊC to separate the serum and then stored at -20ÊC until further analysis. The chickens were anesthetized and then killed by jugular bleeding. The pancreatic and intestinal contents were immediately fro- zen in liquid nitrogen and then stored at -70ÊC. Digestibility assays Fecal and feed samples were dried at 105ÊC for 24 h and filtered through a 0.5-mm sieve. All samples were analyzed in duplicate. CP was determined using the AOAC method (2000)[23]. Acid-insoluble ash (AIA) was determined using the method of Choct and Annison (1992)[3]. Nutrient digestibility was calculated as follows: Digestibility …%† ˆ 100 100…diet AIA % fecal AIA %†…fecal CP % diet CP %† Determination of cholecystokinin (CCK) in serum Serum CCK was analyzed by ELISA using commercial kits (Kit number EK-069-04; Phoenix- Biotech Co., LTD, Beijing, China). Determination of endogenous digestive enzyme activity in the pancreas -1 Approximately 1 g of pancreatic tissue was added to 9 ml distilled water to obtain a 100 mgg homogenate with Omni Prep Multi-Sample Homegenizer (Omni, USA), and then centrifuged at 1,500×g for 15 min. The supernatant was collected for the determination of digestive en- zyme activities. Trypsin, lipase and amylase activity were determined using different kits (A080, A054 and C016, Nanjing Jiancheng Bioengineering Institute). Trypsin activity was defined as the amount of enzyme in each milligram of protein that caused an increase in absor- bance of 0.003 per min at 253 nm wavelength, pH 8.0 and 37ÊC[24]. Lipase activity was deter- mined per the amount of enzyme that hydrolyzed olive oil to form 1 μmol product per minute [25]. Amylase activity was defined as the amount of enzyme that hydrolyzed 1.0 mg substrate per 3 min at pH 6.9 at 40ÊC[26]. The protein concentrations were measured using the Coo- massie Brilliant Blue method with bovine serum albumin as standard. Determination of the mRNA expression of pancreatic digestive-enzyme genes RNA extraction. We extracted total RNA from approximately 50 mg of pancreatic tissue using TRIzol reagent according to the manufacturer's instructions (Invitrogen, USA). The RNA concentration was determined by absorbance at 260 nm using a UV spectrophotometer (UV754N, Shenzhen, China). We evaluated RNA purity by measuring the OD260/OD280 ratio. The RNA samples typically had an OD260/OD280 ratio between 1.9 and 2.0. PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 4 / 13 Application of protease and NSP enzyme in broilers Table 2. Primer pairs for trypsin, amylase, lipase andβ-actin genes from broilers. Primers Sequence Length of fragment (bp) Trypsin P15’-AGCGAGCAGACCATTAGTTC-3’ 253 P25’-AGGAGAGTACAGGGGCATTC-3’ Amylase P35’-AAGTGGAATGGAGAGAAGATG-3’ 147 P45’-CCAGAAAGTAAGAATGGAAGC-3’ Lipase P55’-CCCTGCCCAACCTTATTT-3’ 176 P65’-GCATTTCCACTCCTCCGT-3’ β-actin P75’-ACCGCAAATGCTTCTAAAC-3’ 93 P85’-CCAATCTCGTCTTGTTTTATG-3’ https://doi.org/10.1371/journal.pone.0173941.t002 Primer synthesis. The primers were synthesized by the Shanghai Biological Technology Co. (China). The pancreatic trypsin, amylase and lipase primer pairs (P1 and P2, P3 and P4, and P5 and P6) were designed based on the trypsin, amylase and lipase coding sequences, respectively. Theβ-actin primer pair (P7 and P8) was designed based on the conservedβ-actin sequence. The trypsin, amylase, lipase andβ-actin cDNA primers were designed based on the GenBank sequences NM_205385, NM_001001473, DQ 334850 and NM_205518, respectively (Table 2). Fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR). We carried out a real-time PCR using a SYBR PrimeScript™ RT-PCR Kit (Takara Biotechnology, Dalian, China) according to the manufacturer's instructions. Total RNA was reverse-transcribed to obtain cDNA using oligo-dT and reverse transcriptase at 50ÊC for 30 min and then heat inactivation of the enzyme at 85ÊC for 5 min. The cDNA sam- ples were then mixed with SYBR dye. We carried out gene-specific and quantitative real- time PCR using a Bio-Rad iQ5 PCR machine (Bio-Rad, USA). The PCR program consisted of pre-denaturation at 95ÊC for 5 min, 35 cycles of denaturation at 95ÊC for 10 s, annealing at 55ÊC or 56ÊC (forβ-actin and amylase, respectively) for 15 s and extension at 72ÊC for 12 s. The PCR was then heated from 60 to 95ÊC to produce the melting curve. One negative control was included in all reactions. Digestive-enzyme mRNA. The quantity of digestive enzyme mRNA in each sample was normalized toβ-actin. The cDNA of the digestive enzymes was quantified using relative stan- dard-curve methods. Because the amplification efficiencies of the target and references genes were slightly different, the quantification of the gene copy number was obtained from different standard curves for the target and reference genes. The average value obtained for the control sample was defined as 1, and the experimental results are expressed as a percentage of those obtained for the control group. https://www.ncbi.nlm.nih.gov/pubmed/11846609?dopt=Abstract Statistical analysis The experimental data are expressed as means with standard errors. The data were analyzed using analysis of variance in SAS 8.0, and Duncan's multiple-range test was used to compare treatment means. Differences were considered statistically significant at P < 0.05. Results Performance The growth-performance results are presented in Table 3. For days 1-21, supplementary prote- ase and NSP enzyme had no effect on average daily feed intake (ADFI) and average daily gain (ADG) (p>0.05), although the feed conversion ratio (FCR) was significantly improved with PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 5 / 13 Application of protease and NSP enzyme in broilers Table 3. Effect of different levels of complex enzymes on body weight gain, feed intake, and feed conversion in broilers. Item Supplementary enzyme level (NSP enzyme + protease) (mg/kg) 1 2 3 4 5 (0+0) (150+0) (150+40) (150+80) (150+160) 1±21 days ADFI 44.31± 1.05 44.04± 2.08 44.29± 2.65 45.81± 1.97 46.59± 0.91 ADG 28.51± 0.75 28.73± 1.50 28.74± 1.96 29.79± 1.35 30.44± 0.64 a b ab ab b FCR 1.55± 0.01 1.53± 0.01 1.54± 0.02 1.54± 0.01 1.53± 0.01 1±42 days cd d b a bc ADFI 81.60± 1.42 80.95± 1.49 84.16± 1.06 86.26± 1.35 83.37± 2.48 b b a a b ADG 42.68± 0.70 43.03± 0.90 44.78± 0.72 45.55± 0.59 43.66± 1.32 a b b b a FCR 1.91± 0.01 1.88± 0.01 1.88± 0.01 1.89± 0.02 1.91± 0.01 Each value represents mean± SE of six replicates. In the same row, values with no superscript letter or the same superscript letter indicate no signi®cant difference (P> 0.05); those with different superscript letters indicate a signi®cant difference (P< 0.05). ADFI, average daily feed intake; ADG, average daily gain; FCR, feed conversion ratio. https://doi.org/10.1371/journal.pone.0173941.t003 supplementation with NSP enzyme or NSP enzyme combined with 160 mg/kg protease com- pared to the control (p<0.05). For days 1-42, ADFI and ADG were significantly enhanced by NSP enzyme combined with protease at 40 or 80 mg/kg (p<0.05), and supplementation with NSP enzyme combined with protease at 80 mg/kg yielded the highest ADG (6.79% increase compared to the control) compared to the control diet. FCR was significantly improved com- pared to the control by supplementation with NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05), but supplementation with NSP enzyme combined with 160 mg/kg protease had no effect on FCR (p>0.05). Digestibility The results for the apparent digestibility of crude protein are presented in Table 4. For days 14-16, apparent digestibility of crude protein (ADCP) was significantly enhanced by supplementation with NSP enzyme or NSP enzyme combined with 40 or 160 mg/kg protease (p<0.05), and sup- plementation with NSP enzyme combined with protease at 160 mg/kg yielded the highest ADCP (7.26% increase compared to the control). For days 35-37, compared to the control, ADCP was significantly enhanced by supplementation with NSP enzyme or NSP enzyme combined with protease at 40 or 80 mg/kg (p<0.05), and supplementation with NSP enzyme combined with 40 mg/kg protease yielded the highest ADCP (12.66% increase compared to the control). Serum CCK Results for the CCK level in the serum are shown in Table 4. At 21 days of age, compared to the control animals, the serum CCK in animals given NSP enzyme or NSP enzyme combined with protease supplementation at 40, 80 or 160 mg/kg (p>0.05). At 42 days of age, compared to the control animals, the CCK level in the serum of animals treated with NSP enzyme and protease at 160 mg/kg was reduced by 31.39% (p<0.05), but the CCK level in the serum of ani- mals given NSP enzyme alone was increased by 26.51%. Activity of pancreatic digestive enzymes The results for trypsin activity in the pancreas are presented in Table 5. After 21 days, com- pared to the control animals, animals given NSP enzyme or NSP enzyme combined with PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 6 / 13 Application of protease and NSP enzyme in broilers Table 4. Effect of different levels of complex enzymes on apparent digestibility of crude protein and serum CCK of broilers. Item Supplementary enzyme level (NSP enzyme + protease) (mg/kg) 1 2 3 4 5 (0+0) (150+0) (150+40) (150+80) (150+160) ADCP(%) c b b bc a 14±16 days 65.14±1.40 67.66±1.55 67.10±1.49 66.79±1.49 69.88±1.53 c ab a b c 35±37 days 54.90±1.06 60.43±1.63 61.86±2.10 59.40±1.32 55.23±1.08 CCK(pg/ml) a a a a a 21 days 98.22±41.40 162.28±14.41 134.38±36.80 130.39±79.57 127.93±48.62 a a a a b 42 days 248.43±43.34 314.30±11.82 299.81±102.02 283.72±58.47 170.46±61.57 Each value represents mean± SE of six replicates. In the same row, values with no superscript letter or the same superscript letter indicate no signi®cant difference (P> 0.05); those with different superscript letters indicate a signi®cant difference (P< 0.05). https://doi.org/10.1371/journal.pone.0173941.t004 protease at 40 or 80 mg/kg showed increased activity of pancreatic trypsin (74.13%, 70.66% and 42.59%) (p<0.05), respectively. However, trypsin activity in the pancreas of the animals -1 given NSP enzyme combined with 160 mg/kg protease group (245 Umg protein ) was similar -1 to that observed in the control group (252 Umg protein ). After 42 days, supplementation with NSP enzyme or NSP enzyme combined with 40 mg/kg protease significantly increased the activity of pancreatic trypsin (32.45% and 27.41%) (p<0.05), compared with the control group. However, supplementation with NSP enzyme combined with protease at 80 or 160 mg/ kg decreased the activity of pancreatic trypsin by 10.75% and 25.88% (p<0.05). Pancreatic lipase activity was significantly higher in treated groups than in the control group (p<0.05). Supplementation with NSP enzyme resulted in the highest levels of pancreatic lipase at 21 days of age (36.33% increase compared to the control group). After 42 days, supple- mentation with NSP enzyme combined with protease at 80 mg/kg had the highest pancreatic lipase (increased by 78.79% compared to the control group). After 21 days, compared to the control, supplementation with NSP enzyme or NSP enzyme combined with protease at 40 or 80 mg/kg increased the activity of pancreatic amylase by 15.09%, 13.78% and 18.64% (p<0.05), respectively. After 42 days, pancreatic amylase activity was significantly higher in treated animals than in the control group (p<0.05). Table 5. Effect of different levels of complex enzymes on digestive enzyme activity of broilers pancreas (U/mgprot). Item Supplementary enzyme level (NSP enzyme + protease) (mg/kg) 1 2 3 4 5 (0+0) (150+0) (150+40) (150+80) (150+160) Trypsin c a a b c 21 days 251.94±28.20 438.72±62.39 429.97±20.77 359.24±38.11 244.87±31.58 b a a c d 42 days 406.14±28.48 537.95±46.30 517.47±37.09 362.50±21.22 301.04±19.32 Lipase c a b b b 21 days 475.43±14.70 648.16±10.68 601.47±19.96 616.09±16.37 606.53±22.26 d b b a c 42 days 499.37±20.16 784.36±32.78 798.87±26.26 892.81±18.38 635.56±12.66 Amylase b a a a ab 21 days 117.69±6.39 135.45±18.09 133.91±1.68 139.63±3.09 129.59±18.93 e a a b b 42 days 178.83±3.75 205.08±1.45 201.26±2.07 193.04±1.72 195.63±6.17 Each value represents mean± SE of six replicates. In the same row, values with no superscript letter or the same superscript letter indicate no signi®cant difference (P> 0.05); those with different superscript letters indicate a signi®cant difference (P< 0.05). https://doi.org/10.1371/journal.pone.0173941.t005 PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 7 / 13 Application of protease and NSP enzyme in broilers Table 6. Effect of different levels of complex enzymes on Quantification of pancreatic digestive enzyme mRNA in broilers (relative ratio). Item Supplementary enzyme level (NSP enzyme + protease) (mg/kg) 1 2 3 4 5 (0+0) (150+0) (150+40) (150+80) (150+160) Trypsin c a a a b 21 days 1.00±0.09 1.53±0.10 1.55±0.10 1.51±0.08 1.35±0.06 c a a b d 42 days 1.00±0.12 1.40±0.06 1.44±0.08 1.28±0.03 0.87±0.11 Lipase b a a a a 21 days 1.00±0.07 1.27±0.07 1.24±0.02 1.26±0.01 1.23±0.03 d a ab ab bc 42 days 1.00±0.07 1.15±0.02 1.12±0.03 1.13±0.04 1.07±0.07 Amylase b a a a a 21 days 1.00±0.05 1.79±0.04 1.77±0.04 1.78±0.02 1.80±0.06 b a a a a 42 days 1.00±0.04 1.61±0.06 1.56±0.04 1.60±0.04 1.59±0.03 Each value represents mean± SE of six replicates. In the same row, values with no superscript letter or the same superscript letter indicate no signi®cant difference (P> 0.05); those with different superscript letters indicate a signi®cant difference (P< 0.05). https://doi.org/10.1371/journal.pone.0173941.t006 Expression of pancreatic digestive-enzyme mRNA NSP enzyme or NSP enzyme combined with protease significantly affected trypsin, lipase and amylase mRNA expression in broilers (Table 6). After 21 days, trypsin mRNA expression in the pancreas had increased by 53%, 55%, 51% and 34% (P<0.05) in the animals given NSP enzyme and NSP enzyme combined with 40, 80 or 160 mg/kg protease, respectively. Com- pared to the control, after 42 days, animals given NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease showed increased pancreatic trypsin mRNA levels (increased by 40%, 44% and 28%, respectively). However, supplementation with NSP enzyme combined with 160 mg/kg significantly decreased (13% difference) pancreatic trypsin mRNA levels. The broilers pancreatic lipase and amylase mRNA expression were significantly higher than those of con- trol group (p<0.05). Discussion Amino acids are vital nutrients derived from food proteins (except for synthetic forms). Pro- tein degradation is largely determined by the secretion of the pancreas and stomach. Protein digestibility in broiler diets changes with dietary composition[27, 28]. Thus, incomplete diges- tion, including undigested proteins,can be ameliorated by the addition of exogenous proteases. Arabinoxylan is an important water-soluble non-starch polysaccharide (NSP) in feed that increases chyme viscosity, decreases the availability of nutrients, and alters the intestinal flora, thus affecting nutrient digestion and absorption[3, 29]. Previous studies have shown that the addition of xylanase to wheat-based diets significantly increases body weight gain (BWG) and improves the feed conversion rate (FCR) of broilers at 21 days of age[14, 30, 31] without affect- ing feed intake, indicating that improved nutrient utilization was responsible for the improved feed-conversion rate[32]. In this study, at 1-21 days and 1-42 days, the FCR was significantly improved compared to the control by supplementation with NSP enzyme alone, but there was no significant effect on ADG and ADFI. The effects of exogenous proteases added to poultry diets on growth performance are variable, possibly due to differences in tests and trial designs, especially in the negative control diets. Additionally, many studies have used complexes of enzymes rather than a single component enzyme, yielding more varied results[1, 10, 15, 33± 36] because the results cannot be attributed to one particular enzyme. The present study PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 8 / 13 Application of protease and NSP enzyme in broilers demonstrated that during days1-42, the addition of NSP enzyme combined with a protease at 40 or 80 mg/kg to a corn-soybean meal diet can increase ADFI and ADG. The results showed that addition of this enzyme could improve the FCR and ADG of broil- ers by increasing protein digestibility. At 21 days of age, CP was significantly enhanced by sup- plementation with NSP enzyme or NSP enzyme combined with 40 or 160 mg/kg protease (p<0.05). At 42 days of age, compared to the control, CP was significantly enhanced by supple- mentation with NSP enzyme or NSP enzyme combined with the protease at 40 or 80 mg/kg (p<0.05). Previous research results show that the addition of xylanase to wheat-based diets sig- nificantly reduces the viscosity of the digesta and improves the absorption of DM, CP and calo- ries in broilers, consistent with our results[31]. Corn-soybean-meal diets supplemented with compound enzyme (containing xylanase, protease and amylase) could improve crude protein digestibility by 15.6% while increasing body weight gain by 5.5% and FE by 4%[35]. The same enzyme used in corn-soybean-meal and wheat middlings-based diets increased crude-protein availability by 26.3% but had no effect on growth performance[37]. Therefore, our findings, along with those of Cowieson et al. [35] and Freitas et al. [38], suggest that multienzyme com- plexes can improve nutrient availability and protein digestibility. The intestinal tract has endocrine cells that secrete CCK, and 98% of the CCK in the intesti- nal tract is present in the mucosal layer, with the highest concentration in the duodenum. CCK has an important physiological function in the gastro intestinal tract, regulating pancre- atic exocrine secretion and promoting the synthesis of trypsin, chymotrypsin and amylase. CCK also inhibits gastric emptying, promotes bowel movements, and causes gallbladder contraction[39]. CCK A Receptor (CCK- R) is mainly distributed in the pancreatic gland, gallbladder, smooth muscle, vagus-nerve afferent fibers, brain etc.[40]. CCK- R in gastrointes- tinal tract is mainly mediated by secretion from the pancreas, contraction of the gallbladder and gastric smooth muscle and secretion from intestinal mucosal cells. It enhances sphincter muscle tension to delay gastric emptying[41] and reduces feed intake[42, 43]. Feed intake can be increased by reducing levels of CCK[44]. In this study, the level of CCK in serum was in- creased with NSP enzyme supplementation. However, with increased protease levels, CCK lev- els gradually decreased, and the ADFI increased significantly during days1-42. This research indicated that exogenous protease could increase the feed intake of broilers by decreasing serum levels of CCK. The amount of digestive juice synthesized or secreted by the pancreas, liver and intestinal mucosa and the increased enzymatic activity determine digestive function. Pancreatic juice includes a variety of enzymes involved in the degradation of nutrients, such as pancreatic amy- lase, trypsin, chymotrypsin, elastase, pancreatic lipase and colipase, all of which are directly related to digestive function. The strength of pancreatic enzyme activity is an excellent index by which to measure the digestive capacity[17]. In the present study, supplementation with NSP enzyme or NSP enzyme combined with protease significantly increased the activity of pancreatic lipase and amylase in broilers, but supplementation with NSP enzyme combined with protease at 80 or 160 mg/kg significantly decreased the activity of pancreatic trypsin. Almirall et al. reported that supplementaryβ-glucanase enzyme significantly increased the activity of lipase, amylase and trypsin in the intestinal contents of broilers on a barley-based diet[45]. Engberg et al. also found that supplementary xylanase significantly increased the activity of lipase and chymotrypsin in broilers[46]. A rat experiment showed that pancreatic secretion and enzyme activity increased significantly as active polysaccharide was added[47]. Mahagna et al. reported that supplementary amylase and protease significantly decreased the activity of amylase, chymotrypsin and trypsin in the intestinal contents of broilers on asor- ghum-soybean-meal diet[48]. Cowieson et al. added glucose solution containing seven types of monomeric enzymes to broiler diets and found that both phytase and protease significantly PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 9 / 13 Application of protease and NSP enzyme in broilers increased the excretion of endogenous nitrogen, amino acids, calories and dry matter, suggest- ing that exogenous proteases may participate in the feedback regulation of the pancreas[19]. Therefore, supplementation with exogenous digestive enzymes would decrease the activity of pancreatic digestive enzymes, and supplementation of viscous grain diets with NSP enzyme could reduce the inhibitory effect of NSP on digestive-enzyme activity. The synthesis of pancreatic digestive enzyme is controlled by a specific gene in acinar cells. The enzyme precursors are modified into zymogens, which are then transported to the top of the cell by the Golgi apparatus and packaged into vacuoles. In response to an external signal, the cell secretes the zymogen into the pancreatic duct[49]. In the present study, after 42 days, supplementation with NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease significantly increased levels of pancreatic trypsin, lipase and amylase mRNA, but supplemen- tation with NSP enzyme combined with 160 mg/kg of protease significantly decreased pancre- atic trypsin mRNA. Endogenous expression of digestive enzyme genesis regulated by dietary nutrient levels. A study on rats showed that when dietary protein content increased from 15% to 70%, pancreatic trypsin, chymotrypsin, and elastase expression increased 3.6, 3.9 and 1.8 times, respectively[50]. When the carbohydrate content increased from 11% to 75%, pancre- atic amylase mRNA increased 3.5-8 times[51]. It has been reported that when dietary fat content increased from 3% to 30%, lipase expression increased 2.2-3.9 times[52]. Thus, endog- enous expression of digestive-enzyme genes may be regulated through nutrition. Conclusions Supplementation with NSP enzyme and protease at 80 mg/kg yielded the best performance in ADG broilers.The addition of 150 mg/kg NSP enzyme improved FCR, promoted serum CCK secretion, increased the expression of pancreatic trypsin, amylase and lipase genes, and enhanced digestive enzyme activity in the pancreas. When the NSP enzyme was combined with 160 mg/kg protease, the growth of broilers was significantly improved during days 1-21 but decreased in the later stage. Broiler serum CCK, pancreatic trypsin activity and mRNA expression were decreased with the increase of exogenous protease, and the inhibitory effect was more obvious in the later stage. Supporting information S1 File. Effect of different levels of complex enzymes on body weight gain, feed intake, and feed conversion in broilers. (XLS) S2 File. Effect of different levels of complex enzymes on apparent digestibility of crude protein and serum CCK of broilers. (XLS) S3 File. Effect of different levels of complex enzymes on digestive enzyme activity of broil- ers pancreas (U/mgprot). (XLS) S4 File. Effect of different levels of complex enzymes on Quantification of pancreatic diges- tive enzyme mRNA in broilers (relative ratio). (XLS) Author Contributions Conceptualization: LY MW ZW. PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 10 / 13 Application of protease and NSP enzyme in broilers Data curation: LY MW. Formal analysis: LY XZ. Funding acquisition: LY ZW. Investigation: LY. Methodology: LY XZ. Project administration: LY MW ZW. Resources: LY ZW. Software: LY ZW. Supervision: LY MW XZ. Validation: LY MW. Visualization: LY. Writing ± original draft: LY MW XZ ZW. Writing ± review & editing: LY MW XZ ZW. References 1. Marsman GJ, Gruppen H, van der Poel AF, Kwakkel RP, Verstegen MW, Voragen AG. The effect of thermal processing and enzyme treatments of soybean meal on growth performance, ileal nutrient digestibilities, and chyme characteristics in broiler chicks. Poultry science. 1997; 76(6):864±72. Epub 1997/06/01. PMID: 9181620 2. Yin YL, Baidoo SK, Schulze H, Simmins PH. Effects of supplementing diets containing hulless barley varieties having different levels of non-starch polysaccharides with beta-glucanase and xylanase on the physiological status of the gastrointestinal tract and nutrient digestibility of weaned pigs. Livestock Pro- duction Science. 2001; 71(2±3):97±107. 3. Choct M, Annison G. Anti-nutritive effect of wheat pentosans in broiler chickens: roles of viscosity and gut microflora. British poultry science. 1992; 33(4):821±34. Epub 1992/09/01. https://doi.org/10.1080/ 00071669208417524 PMID: 1393677 4. Ward AT, Marquardt RR. The effect of saturation, chain length of pure triglycerides, and age of bird on the utilization of rye diets. Poultry science. 1983; 62(6):1054±62. Epub 1983/06/01. PMID: 6878135 5. Bedford MR. Mode of action of feed enzymes. J Appl Poult Res. 1993; 2(1):85±92. 6. Yin YL, Deng ZY, Huang HL, Li TJ, Zhong HY. The effect of arabinoxylanase and protease supplemen- tation on nutritional value of diets containing wheat bran or rice bran in growing pig. Journal of Animal & Feed Sciences. 2004; 13(3):445±61. 7. Li J, Li D, Yin YL, Piao XS, He JH, Chen GP, et al. Performance, nutrient digestibility and intestinal disaccharidase activity of weaner/grower pigs given diets containing extruded Chinese stored brown rice with exogenous enzyme supplements. Animal Science. 2005; 79(3):429±38. 8. Yin YL, Baidoo SK, Jin LZ, Liu YG, Schulze H, Simmins PH. The effect of different carbohydrase and protease supplementation on apparent (ileal and overall) digestibility of nutrients of five hulless barley varieties in young pigs. Livestock Production Science. 2001; 71(2±3):109±20. 9. Reddy VR, Qudratullah S. Utilisation of squilla meal (a novel animal protein source) by broilers. British poultry science. 1997; 38(3):263±9. Epub 1997/07/01. https://doi.org/10.1080/00071669708417984 PMID: 9280352 10. Zanella I, Sakomura NK, Silversides FG, Fiqueirdo A, Pack M. Effect of enzyme supplementation of broiler diets based on corn and soybeans. Poultry science. 1999; 78(4):561±8. Epub 1999/05/07. PMID: 10230910 11. Scheideler SE, Beck MM, Abudabos A, Wyatt C. Multiple-enzyme (Avizyme) supplementation of corn- soy based layer diets. J Applied Poult Res. 2005; 14(1):77±86. 12. Bedford MR, Classen HL. Reduction of intestinal viscosity through manipulation of dietary rye and pen- tosanase concentration is effected through changes in the carbohydrate composition of the intestinal PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 11 / 13 Application of protease and NSP enzyme in broilers aqueous phase and results in improved growth rate and food conversion efficiency of broiler chicks. The Journal of nutrition. 1992; 122(3):560±9. Epub 1992/03/01. PMID: 1542013 13. Meng X, Slominski BA, Nyachoti CM, Campbell LD, Guenter W. Degradation of cell wall polysaccha- rides by combinations of carbohydrase enzymes and their effect on nutrient utilization and broiler chicken performance. Poultry science. 2005; 84(1):37±47. Epub 2005/02/03. PMID: 15685940 14. Gao F, Jiang Y, Zhou GH, Han ZK. The effects of xylanase supplementation on performance, charac- teristics of the gastrointestinal tract, blood parameters and gut microflora in broilers fed on wheat-based diets. Animal Feed Science and Technology. 2008; 142(1-2):173±84. 15. Ghazi S, Rooke JA, Galbraith H, Bedford MR. The potential for the improvement of the nutritive value of soya-bean meal by different proteases in broiler chicks and broiler cockerels. British poultry science. 2002; 43(1):70±7. Epub 2002/05/11. https://doi.org/10.1080/00071660120109935 PMID: 12003341 16. Noy Y, Sklan D. Digestion and absorption in the young chick. Poultry science. 1995; 74(2):366±73. Epub 1995/02/01. PMID: 7724461 17. Krogdahl A, Sell JL. Influence of age on lipase, amylase, and protease activities in pancreatic tissue and intestinal contents of young turkeys. Poultry science. 1989; 68(11):1561±8. Epub 1989/11/01. PMID: 2481853 18. Nitsan Z, Ben-Avraham G, Zoref Z, Nir I. Growth and development of the digestive organs and some enzymes in broiler chicks after hatching. British poultry science. 1991; 32(3):515±23. Epub 1991/07/01. https://doi.org/10.1080/00071669108417376 PMID: 1716507 19. Cowieson AJ, Acamovic T, Bedford MR. Using the precision-feeding bioassay to determine the efficacy of exogenous enzymesÐA new perspective. Animal Feed Science and Technology. 2006; 129(1- 2):149±58. 20. Acamovic T. Commercial application of enzyme technology for poultry production. World's Poult Sci J. 2001; 57(3):225±42. 21. Bedford MR. Exogenous enzymes in monogastric nutrition Ð their current value and future benefits. Anim Feed Sci Technol. 2000; 86(1-2):1±13. Epub 2000/08/31. 22. Cowieson AJ. Factors that affect the nutritional value of maize for broilers. Animal Feed Science and Technology. 2005; 119(3-4):293±305. 23. AOAC. Official Methods of Analysis. 17th ed. Association of Official Analytical Chemists. 2000; Wash- ington, DC. 24. Iwamori M, Iwamori Y, Ito N. Sulfated lipids as inhibitors of pancreatic trypsin and chymotrypsin in epi- thelium of the mammalian digestive tract. Biochemical & Biophysical Research Communications. 1997; 237(2):262. 25. Verduin PA, Punt JMHM, Kreutzer HH. Studies on the determination of lipase activity. Clin Chim Acta. 1973; 46(1):11±9. PMID: 4732885 26. Somogyi M. Modification of two methods for the assay of amylase. Clinical Chemistry. 1960; 6:23±35. PMID: 13832788 27. Parsons CM, Castanon F, Han Y. Protein and amino acid quality of meat and bone meal. Poultry sci- ence. 1997; 76(2):361±8. Epub 1997/02/01. PMID: 9057220 28. Lemme A, Ravindran V, Bryden WL. Ileal digestibility of amino acids in feed ingredients for broilers. World Poultry Sci J. 2004; 60(4):423±38. 29. Choct M, Hughes RJ, Wang J, Bedford MR, Morgan AJ, Annison G. Increased small intestinal fermen- tation is partly responsible for the anti-nutritive activity of non-starch polysaccharides in chickens. British poultry science. 1996; 37(3):609±21. Epub 1996/07/01. https://doi.org/10.1080/00071669608417891 PMID: 8842468 30. Gao F, Jiang Y, Zhou GH, Han ZK. The effects of xylanase supplementation on growth, digestion, circu- lating hormone and metabolite levels, immunity and gut microflora in cockerels fed on wheat-based diets. British poultry science. 2007; 48(4):480±8. Epub 2007/08/19. https://doi.org/10.1080/ 00071660701477320 PMID: 17701501 31. Esmaeilipour O, Shivazad M, Moravej H, Aminzadeh S, Rezaian M, van Krimpen MM. Effects of xyla- nase and citric acid on the performance, nutrient retention, and characteristics of gastrointestinal tract of broilers fed low-phosphorus wheat-based diets. Poultry science. 2011; 90(9):1975±82. Epub 2011/ 08/17. https://doi.org/10.3382/ps.2010-01264 PMID: 21844263 32. Zhang L, Xu J, Lei L, Jiang Y, Gao F, Zhou GH. Effects of Xylanase Supplementation on Growth Perfor- mance, Nutrient Digestibility and Non-starch Polysaccharide Degradation in Different Sections of the Gastrointestinal Tract of Broilers Fed Wheat-based Diets. Asian-Australasian journal of animal sci- ences. 2014; 27(6):855±61. Epub 2014/07/23. PubMed Central PMCID: PMCPmc4093178. https://doi. org/10.5713/ajas.2014.14006 PMID: 25050024 PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 12 / 13 Application of protease and NSP enzyme in broilers 33. Pinheiro DF, Cruz VC, Sartori JR, Vicentini Paulino ML. Effect of early feed restriction and enzyme sup- plementation on digestive enzyme activities in broilers. Poultry science. 2004; 83(9):1544±50. Epub 2004/09/24. PMID: 15384906 34. Olukosi OA, Cowieson AJ, Adeola O. Age-related influence of a cocktail of xylanase, amylase, and pro- tease or phytase individually or in combination in broilers. Poultry science. 2007; 86(1):77±86. Epub 2006/12/21. PMID: 17179419 35. Cowieson AJ, Ravindran V. Effect of exogenous enzymes in maize-based diets varying in nutrient den- sity for young broilers: growth performance and digestibility of energy, minerals and amino acids. British poultry science. 2008; 49(1):37±44. Epub 2008/01/23. https://doi.org/10.1080/00071660701812989 PMID: 18210288 36. Walk CL, Cowieson AJ, Remus JC, Novak CL, McElroy AP. Effects of dietary enzymes on performance and intestinal goblet cell number of broilers exposed to a live coccidia oocyst vaccine. Poultry science. 2011; 90(1):91±8. Epub 2010/12/24. https://doi.org/10.3382/ps.2010-00760 PMID: 21177448 37. Gracia MI, La  zaro R, Latorre MA, Medel P, Aranõ Âbar MJ, Jime  nez-Moreno E, et al. Influence of enzyme supplementation of diets and cooking±flaking of maize on digestive traits and growth performance of broilers from 1 to 21 days of age. Animal Feed Science and Technology. 2009; 150(3-4):303±15. 38. Freitas DM, Vieira SL, Angel CR, Favero A, Maiorka A. Performance and nutrient utilization of broilers fed diets supplemented with a novel mono-component protease. The Journal of Applied Poultry Research. 2011; 20(3):322±34. 39. Rehfeld JF. Cholecystokinin. Clinics in gastroenterology. 1980; 9(3):593±607. Epub 1980/09/01. PMID: 40. Wang Y, Prpic V, Green GM, Reeve JR Jr., Liddle RA. Luminal CCK-releasing factor stimulates CCK release from human intestinal endocrine and STC-1 cells. American journal of physiology Gastrointesti- nal and liver physiology. 2002; 282(1):G16±22. Epub 2001/12/26. PMID: 11751153 41. Takiguchi S, Suzuki S, Sato Y, Kanai S, Miyasaka K, Jimi A, et al. Role of CCK-A receptor for pancreatic function in mice: a study in CCK-A receptor knockout mice. Pancreas. 2002; 24(3):276±83. Epub 2002/ 03/15. PMID: 11893936 42. Gibbs J, Young RC, Smith GP. Cholecystokinin decreases food intake in rats. Journal of comparative and physiological psychology. 1973; 84(3):488±95. Epub 1973/09/01. PMID: 4745816 43. Lukaszewski L, Praissman M. Effect of continuous infusions of CCK-8 on food intake and body and pan- creatic weights in rats. The American journal of physiology. 1988; 254(1 Pt 2):R17±22. Epub 1988/01/01. 44. Pekas JC. Effect of cholecystokinin immunization, enhanced food intake and growth of swine on lean yield and carcass composition. The Journal of nutrition. 1991; 121(4):563±7. Epub 1991/04/01. PMID: 45. Almirall M, Francesch M, Perez-Vendrell AM, Brufau J, Esteve-Garcia E. The differences in intestinal viscosity produced by barley and beta-glucanase alter digesta enzyme activities and ileal nutrient digestibilities more in broiler chicks than in cocks. The Journal of nutrition. 1995; 125(4):947±55. Epub 1995/04/01. PMID: 7536829 46. Engberg RM, Hedemann MS, Steenfeldt S, Jensen BB. Influence of whole wheat and xylanase on broiler performance and microbial composition and activity in the digestive tract. Poultry science. 2004; 83(6):925±38. Epub 2004/06/23. PMID: 15206619 47. Ikegami S, Tsuchihashi F, Harada H, Tsuchihashi N, Nishide E, Innami S. Effect of viscous indigestible polysaccharides on pancreatic-biliary secretion and digestive organs in rats. The Journal of nutrition. 1990; 120(4):353±60. Epub 1990/04/01. PMID: 2158535 48. Mahagna M, Nir I, Larbier M, Nitsan Z. Effect of age and exogenous amylase and protease on develop- ment of the digestive tract, pancreatic enzyme activities and digestibility of nutrients in young meat-type chicks. Reproduction, nutrition, development. 1995; 35(2):201±12. Epub 1995/01/01. PMID: 7537505 49. Konturek PC, Kania J, Kukharsky V, Ocker S, Hahn EG, Konturek SJ. Influence of gastrin on the expression of cyclooxygenase-2, hepatocyte growth factor and apoptosis-related proteins in gastric epi- thelial cells. Journal of physiology and pharmacology: an official journal of the Polish Physiological Soci- ety. 2003; 54(1):17±32. Epub 2003/04/04. 50. Giorgi D, Renaud W, Bernard JP, Dagorn JC. Regulation of proteolytic enzyme activities and mRNA concentrations in rat pancreas by food content. Biochemical and biophysical research communications. 1985; 127(3):937±42. Epub 1985/03/29. PMID: 3885943 51. Giorgi D, Bernard JP, Lapointe R, Dagorn JC. Regulation of amylase messenger RNA concentration in rat pancreas by food content. The EMBO journal. 1984; 3(7):1521±4. Epub 1984/07/01. PubMed Cen- tral PMCID: PMCPmc557553. PMID: 6204864 52. Wicker C, Scheele GA, Puigserver A. Pancreatic adaptation to dietary lipids is mediated by changes in lipase mRNA. Biochimie. 1988; 70(9):1277±83. Epub 1988/09/01. PMID: 2465787 PLOS ONE | https://doi.org/10.1371/journal.pone.0173941 March 21, 2017 13 / 13

Journal

PLoS ONEPubmed Central

Published: Mar 21, 2017

There are no references for this article.