Effects of dietary supplementation of multi-enzyme on growth performance, nutrient digestibility, small intestinal digestive enzyme activities, and large intestinal selected microbiota in weanling pigs G. G. Zhang, Z. B. Yang, Y. Wang, W. R. Yang and H. J. Zhou J ANIM SCI 2014, 92:2063-2069. doi: 10.2527/jas.2013-6672 originally published online March 18, 2014
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Effects of dietary supplementation of multi-enzyme on growth performance, nutrient digestibility, small intestinal digestive enzyme activities, and large intestinal selected microbiota in weanling pigs1 G. G. Zhang,* Z. B. Yang,* Y. Wang,† W. R. Yang,*2 and H. J. Zhou* *Animal Sciences and Technology, Shandong Agricultural University, Tai-an, Shandong, 271018, P. R. China; and †Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, Alberta T1J 4B1, Canada
ABSTRACT: Two experiments were conducted to assess the effects of dietary supplementation of an exogenous multi-enzyme (EME) preparation to 35to 65-d-old piglets on apparent total tract digestibility (ATTD), growth performance, digestive enzyme activities, and selected microbial populations in feces. In Exp.1, twenty eight 35-d-old piglets were randomly assigned to 7 dietary treatments (corn-soybean based diet supplemented with 0, 100, 150, 200, 250, 300, or 350 mg EME/kg) in a 14-d digestibility study. Piglets fed the diets supplemented with EME had greater ATTD of DM, CP, and GE (P = 0.001, 0.005, and 0.009, respectively) than those fed the diet without EME supplementation, and those ATTD values increased linearly and quadratically (P < 0.001) as the levels of supplemented EME increased. In Exp. 2, two hundred 35-d-old weanling piglets were randomly allocated to 20 pens. The pens were then randomly assigned to 5 dietary treatments (corn–soybean based diet supplemented with 0, 100, 150, 250, or 350 mg EME/kg) with 4 pens per treatment in a 30-d feeding experiment. Piglets has ad libitum access to diets and water, and they were weighed
at the beginning (35-d-old), middle (50-d-old), and end (65-d-old) of the experiment. Fecal samples were grabbed directly from the rectum and digesta samples from duodenum, jejunum, and ileum were taken at the end of the experiment for the analysis of selected bacteria populations and digestive-enzyme activities. The ADG and ADFI tended to be greater with the increasing levels of supplemented EME in both periods, whereas G:F was improved (P = 0.012 and 0.017) by EME in the period of 35 to 50 d of age and during the overall experimental period. Furthermore, inclusion of EME in diet increased the counts of Lactobacilli spp. and Bacillus subtilis spp., but reduced the populations of Salmonella spp. and Escherichia coli spp. in the feces. The EME supplementation also enhanced (P < 0.05) the activities of amylase, lipase, and protease in the small intestine. The growth performance—enhancing effects of EME appeared to be mediated by the age of the piglet and the dose of EME used. Supplementation of corn–soybean meal diets for 35- to 65-d-old piglets with EME has a potential to enhance gut health condition, increase nutrient digestion, and increase growth performance.
Key words: digestibility, enzyme activity, microbial populations, multi-enzyme, weanling pigs © 2014 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2014.92:2063–2069 doi:10.2527/jas2013-6672 INTRODUCTION Piglets are usually subjected to nutritional, environmental, immunological, and physiological stresses during the weaning process (Kim et al., 2011). Studies have shown that weaning stress can readily cause change in intestinal morphology, including a decrease 1This
study was funded by Shandong Modern Agricultural Technology and Industry System of China. 2Corresponding author: [email protected] Received May 6, 2013. Accepted February 24, 2014.
in villous height and an increase in crypt depth of small intestine (Leonard et al., 2011). This generally results in the loss of mature enterocytes-carrying brush border enzymes, such as aminopeptidases and various carbohydrases, and subsequently reduces the digestibility of nutrients, especially protein and carbohydrates (Kluess et al., 2010). Furthermore, the newly weaned piglets’ digestive tract and endogenous secretion systems are not fully developed (Leonard et al., 2011). All of these lead to insufficient digestive-enzyme activities and gastrointestinal dysbacteriosis (O’Doherty et al., 2010). Thus, it is vital to develop a feeding strategy to allevi-
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ate the weaning stresses of piglets and ensure the postweaned piglets a smooth transition in this risk period (Kim et al., 2011). One of the approaches studied is the supplementation of diets offered to newly-weaned piglets with appropriate exogenous multi-enzymes (EME; Smith et al., 2010; Willamil et al., 2012). However, the observed responses to the EME have been inconsistent (Omogbenigun et al., 2004). In addition, there is still controversy surrounding the changes in digestive tract enzyme activities and microbiota in response to the dietary supplementation of EME to pigs (Fan et al., 2009; Owusu-Asiedu et al., 2010; Smith et al., 2010). On the other hand, enzyme activities and microbial populations in the digestive tract are considered as vital factors that can influence gut health and nutrient digestibility (Yang et al., 2010). The objectives of this study were to assess the effects of EME supplementation of weanling piglet diets on nutrient digestibility, growth performance, digestive enzyme activities, and selected intestinal microbiota populations. MATERIALS AND METHODS All animals used in this study were cared for strictly following the animal care and use protocol that was approved by the Shandong Agricultural University Animal Nutrition Research Institute. Apparent Total Tract Digestibility (Exp. 1) Animal, Treatments, Diets, and Feeding Management. Twenty-eight 35-d-old piglets (weaned at 21 d and fed a commercial starter diet for a 14-d transitional period; PIC Hybrid, Jinan, China) with BW of 9.59 ± 0.36 kg (an equal number of barrows and gilts) were obtained from Shandong Agricultural University Research Farm (Shandong, China) and randomly allocated to 28 pens. The pens were plastic-covered expanded metal floors and located in a temperature-controlled room with good ventilation. The initial room temperature was set at 29.5°C and was gradually decreased by 1.5°C per week until the end of the experiment. At the start of the experiment, the 28 individually-fed piglets were randomly assigned to 7 dietary treatments (2 barrows and 2 gilts per treatment). The 7 dietary treatments were basal diet (Table 1) supplemented with 0 (control), 100, 150, 200, 250, 300, or 350 mg/kg of an EME preparation (Nopcozyme II; Diasham Resources Pte Ltd., Jurong, Singapore) in a completely randomized design. The product was a dry powder with starch as the carrier and contained amylase from Bacillus amyloliquefaciens (EC 3.2.1.1; 4,520), protease from Bacillus subtilis (EC 3.4.21; 8,660), and xylanase from Trichoderma (EC 3.2.1.8; 6,000). The same batch of the EME preparation was used for the entire experiment.
Table 1. Composition and energy and nutrient contents of the basal diet (Exp. 1 and 2) Item Ingredient, % Corn Wheat middling Whey powder Soybean oil Soybean meal Fish meal L-Lys×HCl DL-Met Thr Dicalcium phosphate Limestone Sodium chloride Vitamin-trace mineral premix1 Total Analyzed composition GE, MJ/kg DE, MJ/kg CP, % Ca, % Total P, % Lys, % Met, % Met + Cys, % Thr, %
Content 53.00 5.00 6.50 2.50 24.76 5.50 0.30 0.10 0.04 0.80 0.30 0.20 1.00 100.00 18.28 14.29 20.00 0.82 0.42 1.38 0.45 0.78 0.87
1Supplied per kilogram of complete diet: 11,025 IU vitamin A, 2203 IU vitamin D3, 80 IU vitamin E, 4.4 mg vitamin K3, 4.4 mg thiamine, 11 mg riboflavin, 35 mg d-pantothenic acid, 59.5 mg niacin, 330 mg choline, 0.9 mg folic acid, 0.5 mg biotin, 55 µg vitamin B12, 40 mg Mn as manganese sulfate, 130 mg Fe as ferrous sulfate, 130 mg Zn as zinc sulfate, 15 mg Cu as copper sulfate, 0.35 mg I as calcium iodide, and 0.3 mg Se as sodium selenite.
Amylase activity was determined using the method described by Somogyi (1960), with one amylase unit being defined as the amount of enzyme that hydrolyzes 10 mg of starch in 30 min at pH 6.5 and 37°C. Protease activity was analyzed according to the modified method of Hu et al. (2012) using casein as substrate. One protease unit is defined as the amount of enzyme that hydrolyzes casein to form 1 μmol product/min at pH 3.0 and 40°C. Xylanase activity was assayed using the method described by König et al. (2002), with 1 unit of xylanase being defined as the amount of enzyme that releases 1 μmol of reducing sugars/min at pH 4.8 and 50°C. Basal diets were formulated to meet nutrient requirements for weaned piglets recommended by the Feeding standard of swine in China (NY/T 65–2004; Ministry of Agriculture of China, 2004), and were fed as mash. All diets were prepared in a single batch. The EME was first mixed with a premix that was subsequently mixed with other ingredients and then stored in covered containers. The piglets had ad libitum access to feed and also water through nipple drinkers for the duration of the experiment.
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Multi-enzyme supplementation in weaned pig diet
Sample Collection, Preparation, and Analysis. The experiment was conducted for 14 d with the first 7 d as adaptation period and the remaining 7 d for sample collection. Feed offered and orts of each piglet were recorded daily for determination of feed intake and all feces were individually collected immediately after excretion during the 7-d period. Subsample of each diet was taken daily at feeding, dried at 55°C, and ground using a 1.0-mm screen for chemical analysis. The daily excreta of each piglet collected at each time was weighted, and 10% HCl was added at a ratio of 100 g of wet fecal sample to 10 mL 10% HCl, and subsequently stored in a sealed plastic bag at –20°C. At the end of the 7-d period, all bags containing daily feces of each piglet were thawed at room temperature and mixed thoroughly. An equal amount of daily fecal sample from the same piglet was pooled and a single subsample (10% of the total weight) was then oven dried at 55°C to constant weight and ground to pass a 1.0-mm screen for chemical analyses. The diet and fecal samples were analyzed for DM and CP according to the procedures described by Wang et al. (2011). Gross energy was determined using a Parradiabatic bomb calorimeter method as described by Zhang et al. (2012). The apparent total tract digestibility (ATTD) of DM, CP, and GE was calculated as following: ATTD (%) =
DMI × NCF − FW × NCf × 100 DMI × NCF
[1]
where DMI is in kg; NCF is the dietary content of DM, CP, or GE (%, DM basis); FW is the daily output of feces (kg); and NCf is the fecal content of DM, CP, or GE (%, DM basis). Growth Performance, Digestive Enzyme Activities, and Intestinal Microbiota (Exp. 2) Animal, Treatments, Diets, and Feeding Management. Two hundred 35-d-old piglets (weaned at 21-d of age and fed a commercial starter diet for a 14-d transition period; PIC Hybrid) with BW of 9.59 ± 0.36 kg (an equal number of barrows and gilts) were used in a 30-d feeding experiment. Piglets were randomly allocated into 20 pens (5 barrows and 5 gilts per pen), and pens were randomly assigned to 5 dietary treatment (basal diet supplemented with 0, 100, 150, 250, or 350 mg EME/kg) with 4 pens per treatment. The pens were plastic-covered expanded metal floors and located in a closed swine house with the same condition as described in Exp.1. The basal diet, its preparation, and method of EME supplementation were also the same as described for Exp.1. Piglets had ad libitum access to feed (mash form) and water during the entire experimental period. Determination of Feed Intake, Growth Rate, Feed Efficiency, and Diarrhea Index. Piglets were weighed
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on d 1 (35-d-old), 15 (50-d-old), and 30 (65-d-old) of the experiment to determine the ADG. Orts and spillages were collected and weighed daily to determine the ADFI. Feed efficiency was calculated as G:F. The piglets were visually observed daily in the morning for 3 to 4 h and incidence of diarrhea was recorded for each piglet based on the method described by Ball and Aherne (1982) and Song et al. (2012). Diarrhea score of each pig was assessed visually each day in the entire experimental period with a score from 1 to 5 (1 = normal feces, 2 = moist feces, 3 = mild diarrhea, 4 = severe diarrhea, and 5 = watery diarrhea). Diarrhea index was then calculated as the percentage of piglets with a diarrhea score of 3 and greater in the total piglets of each group. Determination of Selected Microbial Populations in Feces. Effects of EME supplementation on fecal populations of Escherichia coli spp., Salmonella spp., Lactobacillus spp., and Bacillus subtilis spp. were determined using the method described by Yang et al. (2010) and O’Doherty et al. (2010). Fresh fecal samples were collected by grabbing directly from rectums of 4 healthy piglets (1 piglet per pen) in each treatment (same sex for all treatment) on d 30 of the experiment. One gram of the sample was first added to 99 mL of sterile 0.1% peptone, homogenized for 1 min by blending, and then serially diluted in sterile 0.1% peptone. The dilutions were spread on Eosin Methylene Blue agar and Salmonella-Shigella agar, incubated aerobically at 37°C for 18 to 24 h to enumerate E. coli spp. and Salmonella spp., respectively. Lactobacillus spp. and B. subtilis spp. were isolated on de Man, Rogosa, and Sharp agar, and Luria-Bertani agar with overnight (18 to 24 h) incubation at 37°C in a 5% CO2 environment. Colonies of the bacterium in each plate were counted and the numbers of bacteria were calculated as colony-forming units per gram of fresh samples. Activities of Digestive Enzymes in Intestinal Digesta. Four healthy piglets from each treatment (1 per pen) were randomly selected in the morning of d 30 of the experiment. The piglets were euthanized 4 h after morning feeding by intracardiac injection of sodium pentobarbital (50 mg/kg BW; Omogbenigun et al., 2004; Fan et al., 2009). The abdominal cavity of the piglets was immediately opened and the small intestine was ligatured into duodenum, jejunum, and ileum according to the anatomical structure characteristic (i.e., the ligament linking duodenum and colon was taken as a symbol of distinguishing duodenum and jejunum, and the ligament jointing ileum and cecum was considered as a marker of parting jejunum and ileum). The digesta samples from each section were subsequently collected by massaging the tract from proximal and distal ends and stored immediately at –20°C. For the analysis, the digesta samples were thawed at room temperature and homogenized in 4 volumes of ice-cold 0.9% sodium chloride solution. The homog-
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Table 2. Effects of supplementing different levels of an exogenous multi-enzyme (EME) preparation to 35- to 49-dold piglets fed corn–soybean meal based diet on the apparent total tract digestibility of DM, CP, and GE (Exp. 1)1 Item DM CP GE 1n
0 84.45 84.35 84.06
100 85.45 85.30 85.20
EME, mg/kg 200 85.64 86.36 86.40
150 85.72 86.36 86.41
SEM 250 86.55 86.23 86.87
300 86.82 86.59 86.67
350 86.84 87.09 86.66
0.33 0.35 0.39
0 vs. others 0.001 0.005 0.009
P-value Linear < 0.001 < 0.001 < 0.001
Quadratic < 0.001 < 0.001 < 0.001
= 4.
enate was centrifuged at 13,800 × g for 20 min at 4°C and the supernatant was analyzed for amylase, protease, and lipase activities (Fan et al., 2009; Hu et al., 2012). Amylase and protease activity were determined using the method described in Exp. 1. Lipase (EC 3.l.l.3) activity was assayed using the method described by Tietz and Fiereck (1966), and one lipase unit is defined as the amount of enzyme that hydrolyzes 1 μmol of olive oil/ min. All determinations were performed in duplicates. Data Calculations and Statistical Analyses Data were statistically analyzed using the GLM procedure of SAS (Version 9.0; SAS Inst. Inc., Cary NC). The individual fed animal (Exp. 1) and the pen (Exp. 2) were used as the experimental unit. The effect of EME supplementation was determined by the preplanned contrast (0 vs. others) and linear and quadratic effects. Difference was declared significant when P < 0.05, and tendency was declared with P-values between 0.05 and 0.10.
RESULTS Apparent Total Tract Digestibility of DM, CP, and GE In Exp. 1, piglets supplemented with EME had greater ATTD of DM, CP, and GE (P = 0.001, 0.005, and 0.009, respectively) than those not supplemented with EME, and there seemed to be no differences in ATTD of DM, CP, and GE among piglets fed the diets supplemented with EME (Table 2). Nevertheless, the ATTD of DM, CP, and GE increased linearly and quadratically (P < 0.001) as supplementation of EME increased. Animal Growth Performances and Diarrhea Index The effects of supplementing EME on ADFI, ADG, and G:F in Exp. 2 are presented in Table 3. Supplementation of EME to 35- to 50-d-old piglets tended (P = 0.065 and 0.088) to increase ADG and ADFI and increased (P = 0.012) G:F as compared to control. The G:F increased lin-
Table 3. Effects of supplementing different levels of an exogenous multi-enzyme (EME) preparation to 35- to 65-dold piglets fed corn–soybean meal based diet on the growth performance and diarrhea index (Exp. 2)1 Item 0 BW, kg 35 d of age 9.30 50 d of age 14.77 65 d of age 24.57 35 to 50 d of age ADG, g 364 ADFI, g 574 G:F 0.63 51 to 65 d of age ADG, g 653 ADFI, g 1,148 G:F 0.57 35 to 65 d of age ADG, g 509 ADFI, g 864 G:F 0.59 8.42 Diarrhea index, %2 1n
100
EME, mg/kg diet 150
250
350
SEM
0 vs. others
P-value Linear
Quadratic
9.85 15.71 25.58
9.23 15.21 25.27
9.94 16.1 26.24
9.77 16 26.44
0.65 0.85 1.10
0.050 0.052 0.113
0.065 0.027 0.104
0.092 0.064 0.277
391 575 0.68
399 574 0.69
410 591 0.69
415 582 0.71
18 22 0.03
0.065 0.088 0.012
0.092 0.271 < 0.001
0.248 0.095 < 0.001
658 1,149 0.57
671 1,170 0.57
677 1,174 0.58
696 1,181 0.59
25 46 0.04
0.068 0.089 0.075
0.028 0.069 < 0.001
0.391 0.062 < 0.001
524 847 0.62 5.31
535 851 0.63 4.91
543 872 0.62 4.09
556 859 0.65 3.25
19 30 0.03 0.85
0.058 0.091 0.017 0.013
< 0.001 0.426 < 0.001 < 0.001
0.002 0.373 < 0.001 < 0.001
= 4.
2Diarrhea
index was calculated as the percentage of diarrhea piglets with a score of 3 or greater in total piglets of each group. The diarrhea score is assessed according to the following criteria: 1 = normal feces, 2 = moist feces, 3 = mild diarrhea, 4 = severe diarrhea, and 5 = watery diarrhea. Downloaded from www.journalofanimalscience.org at Queen Mary, University of London on May 29, 2014
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Table 4. Effects of supplementing different levels of an exogenous multi-enzyme (EME) preparation to 35- to 65-dold piglets fed corn–soybean meal based diet on digestive enzymes activities (U/g digesta) in different sections of the small intestine (Exp. 2)1 Item Duodenum Amylase Lipase Protease Jejunum Amylase Lipase Protease Ileum Amylase Lipase Protease 1n
0 vs. others
P-value Linear
Quadratic
4 15 128
0.001 0.021 0.001
< 0.001 < 0.001 < 0.001
< 0.001 < 0.001 < 0.001
387 339 7,841
10 12 109
0.001 0.003 0.001
0.022 < 0.001 < 0.001
0.018 0.012 < 0.001
542 357 5,899
6 9 74
0.004 < 0.001 0.001
< 0.001 < 0.001 < 0.001
< 0.001 < 0.001 < 0.001
0
100
EME, mg/kg 150
250
350
300 406 5,943
348 628 6,477
352 638 6,607
371 1,197 6,856
378 1,212 7,087
320 308 6,646
370 319 7,172
375 327 7,386
377 330 7,569
294 224 5,015
336 323 5,471
339 326 5,587
452 347 5,615
SEM
= 4.
early and quadratically (P < 0.001) as supplementation of EME increased, even though there seems to be no difference in G:F among EME-supplemented groups. Supplementation of EME to 51- to 65-d-old piglets tended to increase ADG, ADFI, and G:F (P = 0.068, 0.089, and 0.075, respectively) compared with the control group, and G:F increased linearly and quadratically (P < 0.001) as EME supplementation increased. Over the entire experimental period (age of 35 to 65 d), ADG and ADFI tended (P = 0.058 and 0.091, respectively) to be increased, whereas G:F was increased (P = 0.017) by EME supplementation compared with the control group. Overall G:F increased linearly and quadratically (P < 0.001) as supplementation levels of EME increased. Supplementation of EME reduced (P = 0.013) the diarrhea index in piglets over the 30-d experimental period compared with the control group, and the diarrhea index decreased linearly and quadratically (P < 0.001) as EME supplemented increased. Activities of Digestive Enzymes in Small Intestine Digestive enzymes activities in the duodenum, jejunum, and ileum are presented in Table 4. Supplementation of EME enhanced activity of amylase, lipase, and protease in all sections of the small intestine (P < 0.021) compared
with the control group. All those enzymes activities in the duodenum, jejunum, and ileum increased linearly and quadratically (P < 0.05) with the increasing EME supplementation. However, the difference in enzymatic activities among different EME-supplemented groups varied depending on the enzymes and the sections of the small intestine. Selected Microbial Populations in Piglet Feces Populations of Lactobacilli spp., B. subtilis spp., Salmonella spp., and E.coli spp. in feces collected from the rectum at 65 d of age are presented in Table 5. All of them were affected by the treatments. Supplementation of EME increased fecal counts of Lactobacilli spp. and B. subtilis spp. (P = 0.007 and 0.004, respectively), but decreased that of Salmonella spp. and E. coli spp. (P = 0.002 and 0.015, respectively) compared with the control group. All of them showed linear and quadratic dose responses to the levels of EME supplementation. Piglets supplemented with EME had higher counts of Lactobacilli spp. and B. subtilis spp. (linear and quadratic, P < 0.001), but they had lower counts of Salmonella spp. and E. coli spp. (linear and quadratic, P < 0.002) as EME supplementation increased
Table 5. Effects of supplementing different levels of an exogenous multi-enzyme (EME) preparation to 35- to 65-d-old piglets fed corn–soybean meal based diet on the selected microbial populations in rectum feces (log cfu/g feces; Exp. 2)1 Item Lactobacilli spp. Bacillus Subtilis spp. Salmonella spp. Escherichia coli spp. 1n
0 8.99 7.59 7.11 7.12
100 9.16 7.81 6.91 6.99
EME, mg/kg diet 150 9.27 7.92 6.86 6.87
250 9.34 8.14 6.75 6.71
350 9.37 8.20 6.63 6.51
SEM 0.04 0.04 0.07 0.09
0 vs. others 0.007 0.004 0.002 0.015
P-value Linear < 0.001 < 0.001 < 0.001 < 0.001
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Quadratic < 0.001 < 0.001 0.002 < 0.001
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DISCUSSION An important finding of this study was that supplementation of EME improved feed efficiency of 35- to 65-d-old piglets fed corn–soybean based diet, with the effect being greater for the age of 35 to 50 d than for 51 to 60d old. The increased feed efficiency is probably due to the increased nutrient supply by EME because EME supplementation tended to increase both ADG and ADFI and increase ATTD of DM, CP, and GE. Effects of EME supplementation on energy and nutrient digestion and growth performance of pigs have been assessed previously and the results varied among studies. Positive effects of EME on nutrient digestibility and animal growth performance were observed in some studies (Li et al., 1996; Omogbenigun et al., 2004), but not in others (Thacker et al., 1988; Officer, 1995). The inconsistent results of animal responses to the EME supplementation can be explained by the differences in diet composition and variations of EME activities and age of animals used (Kim et al., 2011). Willamil et al. (2012) also reported that the effects of EME can be mediated by the type of cereal grain in the diet. This study used a corn–soybean based diet and the EME preparation contained protease activity in addition to fibrolytic activities that were the main activities in other studies. The results demonstrated that supplementation of these EME increased the digestive enzymes activities and improved digestion of DM, CP, and GE by piglets. This could contribute to the improved feed efficiency of EMEsupplemented piglets observed in this study. The results that efficacy of EME in improving feed efficiency was more pronounced for 35- to 50-d than 51- to 65-d-old piglets were in agreement with other reports (Omogbenigun et al., 2004; Parra et al., 2012). This is likely due to the fact that digestive function is not fully developed in the younger age and, therefore, supplementing EME at this growth stage would enable piglets to digest and utilize dietary energy and nutrients more efficiently. Omogbenigun et al. (2004) and Parra et al. (2012) also reported that the declined positive effects of the EME supplementation on growth performance of piglets as they get older was due to the improved digestive capacity of weanling piglets such as the maturation of endogenous enzyme-secreting system and the increase in gastrointestinal microbial populations. Ngoc et al. (2011) reported superior growth responses to EME in piglets weighing below 20 kg, but not in those weighing 20 to 40 kg. The BW of piglets at the ages of 35-, 50-, and 65-d-old in this study were 9.6, 15.6, and 25.6 kg, respectively, and, therefore, the efficacy of EME in improving feed efficiency was greater for the age of 35to 50-d than 51- to 65-d-old. Diarrhea caused by infectious disease is a serious problem in weanling piglets and usually leads to
an increased mortality (Wang et al., 2011). This study demonstrated that supplementing EME reduced diarrhea index. This is probably due to the effects of EME supplementation to increase Lactobacilli and B. subtilis and decrease E. coli and Salmonella populations, and also to increase enzyme activities in the digestive tract of piglets observed in this study and reported by others (O’Doherty et al., 2010; Smith et al., 2010). It has been showed that the species and populations of microorganisms in the digestive tract influenced the digestibility of nutrients and affected gut health conditions (Yang et al., 2010). Both Lactobacilli and B. subtilis are considered as beneficial intestinal bacteria, whereas E. coli and Salmonnella are considered major bacteria that often cause gut health problems such as diarrhea, especially for younger animals. Therefore, the increased Lactobacilli and B. subtilis and reduced E. coli and Salmonella populations by supplementation of EME may have contributed to the decreased diarrhea index observed in this study. In addition, the increased digestive enzymes activities by EME supplementation may have also contributed to the reduced diarrhea index through enhancing animals’ digestive function, as indicated by the increased energy and nutrient digestibility. Increased digestive enzyme activities by supplementation of varying EME preparations were also documented in other studies (e.g., Wen et al., 2012). The increased digestive enzyme activities observed in this study are probably combined results from ingested EME and the enhanced endogenous enzymes secretion caused by the increased amount of energy and nutrients available for digestion by the action of EME. On the contrary, Fan et al. (2009) found that dietary supplementation of xylanase and β-glucanase to weaned piglets decreased the activities of amylase and lipase of the duodenum digesta. The reason for the differences across experiments is probabily associated with differences in types and levels of EME used, the age of animal involved, and the nature of the diets used (Li et al., 2004; Fan et al., 2009; Mirzaie et al., 2012; Wen et al., 2012), and, clearly, further research is required to elucidate the more crucial factors involved. Although the results showed that the majority of the measured variables responded to EME supplementation in a dose-dependent manner, the nutrient digestibility and growth performance results seems to indicate minimal differences in the range of 100 to 350 mg/kg of EME supplementation. This indicates that EME supplementation levels between 150 and 250 would be more economically feasible than the greater dosage under the present experimental conditions. In conclusion, supplementation of diets for 35- to 65-d-old piglets with EME containing activities of amylase, protease, and xylanase, increased digestibility of DM, CP, and GE, improved feed efficiency, and tended
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Multi-enzyme supplementation in weaned pig diet
to increase the growth rate and feed intake. In addition, the EME supplementation had positive effects on amylase, lipase, and protease activity and microbiota in the digestive tract. It appeared that the efficacy of EME supplementation in enhancing growth performance was greater at the age of 35- to 50-d than 50- to 65-d-old, and that supplementation of the EME preparation at the levels between 150 and 250 mg/kg diet would seem to be economically feasible. LITERATURE CITED Ball, R. O., and F. X. Aherne. 1982. Effect of diet complexity and feed restriction on the incidence and severity of diarrhea in early-weaned pigs. Can. J. Anim. Sci. 62:907–913. Fan, C. L., X. Y. Han, Z. R. Xu, L. J. Wang, and L. R. Shi. 2009. Effects of β-glucanase and xylanase supplementation on gastrointestinal digestive enzyme activities of weaned piglets fed a barley-based diet. J. Anim. Physiol. Anim. Nutr. 93:271–276. Hu, C., J. Song, Z. You, Z. S. Luan, and W. Li. 2012. Zinc oxidemontmorillonite hybrid influences diarrhea, intestinal mucosal integrity, and digestive enzyme activity in weaned pigs. Biol. Trace Elem. Res. 12:1–7. Kim, J. C., C. F. Hansen, B. P. Mullan, and J. R. Pluske. 2011. Nutrition and pathology of weaner pigs: Nutritional strategies to support barrier function in the gastrointestinal tract. Anim. Feed Sci. Technol. 173:3–16. Kluess, J., U. Schoenhusen, W. B. Souffrant, P. H. Jones, and B. G. Miller. 2010. Impact of diet composition on ileal digestibility and small intestinal morphology in early-weaned pigs fitted with a T-cannula. Animal 4:586–594. König, J., R. Grasser, H. Pikor, and K. Vogel. 2002. Determination of xylanase, β-glucanase, and cellulase activity. Anal. Bioanal. Chem. 374:80–87. doi:10.1007/s00216-002-1379-7 Leonard, S. G., T. Sweeney, B. Bahar, B. P. Lynch, and J. V. O’Doherty. 2011. Effects of dietary seaweed extract supplementation in sows and post-weaned pigs on performance, intestinal morphology, intestinal microflora and immune status. Br. J. Nutr. 1:1–12. Li, S., W. C. Sauer, R. Mosenthin, and B. Kerr. 1996. Effect of β-glucanase supplementation of cereal-based diets for starter pigs on the apparent digestibilities of dry matter, crude protein and energy. Anim. Feed Sci. Technol. 59:223–231. Li, W. F., J. Feng, Z. R. Xu, and C. M. Yang. 2004. Effects of nonstarch polysaccharides enzymes on pancreatic and small intestinal digestive enzyme activities in piglet fed diets containing high amounts of barley. World J. Gastroenterol. 10:856–859. Ministry of Agriculture of China. 2004. Feeding standard of swine. NY/T 65–2004. Ministry of Agriculture, Beijing, China. Mirzaie, S., M. Zaghari, S. Aminzadeh, M. Shivazad, and G. G. Mateos. 2012. Effects of wheat inclusion and xylanase supplementation of the diet on productive performance, nutrient retention, and endogenous intestinal enzyme activity of laying hens. Poult. Sci. 91:413–425. doi:10.3382/ps.2011-01686. Ngoc, T.T.B., N.T. Len, B. Ogle, and J.E. Lindberg. 2011. Influence of particle size and multi-enzyme supplementation of fibrous diets on total tract digestibility and performance of weaning (8–20kg) and growing (20–40kg) pigs. Anim. Feed Sci. Technol. 169:86–95.
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O’Doherty, J. V., S. Dillon, S. Figat, J. J. Callan, and T. Sweeney. 2010. The effects of lactose inclusion and seaweed extract derived from Laminaria spp. on performance, digestibility of diet components and microbial populations in newly weaned pigs. Anim. Feed Sci. Technol. 157:173–180. Officer, D. I. 1995. Effect of multi-enzyme supplements on the growth performance of piglets during the pre-and post-weaning periods. Anim. Feed Sci. Technol. 56:55–65. Omogbenigun, F. O., C. M. Nyachoti, and B. A. Slominski. 2004. Dietary supplementation with multienzyme preparations improves nutrient utilization and growth performance in weaned pigs. J. Anim. Sci. 82:1053–1061. Owusu-Asiedu, A., P. H. Simmins, J. Brufau, R. Lizardo, and A. Péron. 2010. Effect of xylanase and β-glucanase on growth performance and nutrient digestibility in piglets fed wheat-barleybased diets. Livest. Sci. 134:76–78. Parra, J., J. Agudelo, L. Ortiz, M. C. Ramírez, B. Rodríguez, and A. López Herrera. 2012. Lipopolysaccharide (LPS) from E. coli has detrimental effects on the intestinal morphology of weaned pigs. Revista Colombiana de Ciencias Pecuarias 24:598–608. Smith, A. G., P. Reilly, T. Sweeney, K. M. Pierce, D. A. Gahan, J. J. Callan, and J. V. O’Doherty. 2010. The effect of cereal type and exogenous enzyme supplementation on intestinal microbiota and nutrient digestibility in finisher pigs. Livest. Sci. 133:148–150. Somogyi, M. 1960. Modifications of two methods for the assay of amylase. Clin. Chem. 6:23–35 Song, M., Y. Liu, J. A. Soares, T. M. Che, O. Osuna, C. W. Maddox, and J. E. Pettigrew. 2012. Dietary clays alleviate diarrhea of weaned pigs. J. Anim. Sci. 90:345–360. Thacker, P. A., G. L. Campbell, and J. GrootWassink. 1988. The effect of beta-glucanase supplementation on the performance of pigs fed hulless barley. Nutr. Rep. Int. 38:91–99. Tietz, N. W., and E. A. Fiereck. 1966. A specific method for serum lipase determination. Clin. Chim. Acta 13:352–358. Wang, D., X. S. Piao, Z. K. Zeng, T. Lu, Q. Zhang, P. F. Li, L. F. Xue, and S. W. Kim. 2011. Effects of keratinase on performance, nutrient utilization, intestinal morphology, intestinal ecology and inflammatory response of weaned piglets fed diets with different levels of protein. Asian-Aust. J. Anim. Sci. 24:1718–1728. Wen, C., L. C. Wang, Y. M. Zhou, Z. Y. Jiang, and T. Wang. 2012. Effect of enzyme preparation on egg production, nutrient retention, digestive enzyme activities and pancreatic enzyme messenger RNA expression of late-phase laying hens. Anim. Feed Sci. Technol. 172:180–186. Willamil, J., I. Badiola, E. Devillard, P. A. Geraert, and D. Torrallardona. 2012. Wheat-barley-rye- or corn-fed growing pigs respond differently to dietary supplementation with a carbohydrase complex. J. Anim. Sci. 90:824–832. Yang, Y., E. Kiarie, B. A. Slominski, A. Brûlé-Babel, and C. M. Nyachoti. 2010. Amino acid and fiber digestibility, intestinal bacterial profile, and enzyme activity in growing pigs fed dried distillers grains with solubles-based diets. J. Anim. Sci. 88:3304–3312. Zhang, G. G., Z. B. Yang, Q. Q. Zhang, W. R. Yang, and S. Z. Jiang. 2012. A multienzyme preparation enhances the utilization of nutrients and energy from pure corn and wheat diets in broilers. J. Appl. Poult. Res. 21:216–225.
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References
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Effects of dietary supplementation of multi-enzyme on growth performance, nutrient digestibility, small intestinal digestive enzyme activities, and large intestinal selected microbiota in weanling pigs1 G. G. Zhang,* Z. B. Yang,* Y. Wang,† W. R. Yang,*2 and H. J. Zhou* *Animal Sciences and Technology, Shandong Agricultural University, Tai-an, Shandong, 271018, P. R. China; and †Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, Alberta T1J 4B1, Canada
ABSTRACT: Two experiments were conducted to assess the effects of dietary supplementation of an exogenous multi-enzyme (EME) preparation to 35to 65-d-old piglets on apparent total tract digestibility (ATTD), growth performance, digestive enzyme activities, and selected microbial populations in feces. In Exp.1, twenty eight 35-d-old piglets were randomly assigned to 7 dietary treatments (corn-soybean based diet supplemented with 0, 100, 150, 200, 250, 300, or 350 mg EME/kg) in a 14-d digestibility study. Piglets fed the diets supplemented with EME had greater ATTD of DM, CP, and GE (P = 0.001, 0.005, and 0.009, respectively) than those fed the diet without EME supplementation, and those ATTD values increased linearly and quadratically (P < 0.001) as the levels of supplemented EME increased. In Exp. 2, two hundred 35-d-old weanling piglets were randomly allocated to 20 pens. The pens were then randomly assigned to 5 dietary treatments (corn–soybean based diet supplemented with 0, 100, 150, 250, or 350 mg EME/kg) with 4 pens per treatment in a 30-d feeding experiment. Piglets has ad libitum access to diets and water, and they were weighed
at the beginning (35-d-old), middle (50-d-old), and end (65-d-old) of the experiment. Fecal samples were grabbed directly from the rectum and digesta samples from duodenum, jejunum, and ileum were taken at the end of the experiment for the analysis of selected bacteria populations and digestive-enzyme activities. The ADG and ADFI tended to be greater with the increasing levels of supplemented EME in both periods, whereas G:F was improved (P = 0.012 and 0.017) by EME in the period of 35 to 50 d of age and during the overall experimental period. Furthermore, inclusion of EME in diet increased the counts of Lactobacilli spp. and Bacillus subtilis spp., but reduced the populations of Salmonella spp. and Escherichia coli spp. in the feces. The EME supplementation also enhanced (P < 0.05) the activities of amylase, lipase, and protease in the small intestine. The growth performance—enhancing effects of EME appeared to be mediated by the age of the piglet and the dose of EME used. Supplementation of corn–soybean meal diets for 35- to 65-d-old piglets with EME has a potential to enhance gut health condition, increase nutrient digestion, and increase growth performance.
Key words: digestibility, enzyme activity, microbial populations, multi-enzyme, weanling pigs © 2014 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2014.92:2063–2069 doi:10.2527/jas2013-6672 INTRODUCTION Piglets are usually subjected to nutritional, environmental, immunological, and physiological stresses during the weaning process (Kim et al., 2011). Studies have shown that weaning stress can readily cause change in intestinal morphology, including a decrease 1This
study was funded by Shandong Modern Agricultural Technology and Industry System of China. 2Corresponding author: [email protected] Received May 6, 2013. Accepted February 24, 2014.
in villous height and an increase in crypt depth of small intestine (Leonard et al., 2011). This generally results in the loss of mature enterocytes-carrying brush border enzymes, such as aminopeptidases and various carbohydrases, and subsequently reduces the digestibility of nutrients, especially protein and carbohydrates (Kluess et al., 2010). Furthermore, the newly weaned piglets’ digestive tract and endogenous secretion systems are not fully developed (Leonard et al., 2011). All of these lead to insufficient digestive-enzyme activities and gastrointestinal dysbacteriosis (O’Doherty et al., 2010). Thus, it is vital to develop a feeding strategy to allevi-
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ate the weaning stresses of piglets and ensure the postweaned piglets a smooth transition in this risk period (Kim et al., 2011). One of the approaches studied is the supplementation of diets offered to newly-weaned piglets with appropriate exogenous multi-enzymes (EME; Smith et al., 2010; Willamil et al., 2012). However, the observed responses to the EME have been inconsistent (Omogbenigun et al., 2004). In addition, there is still controversy surrounding the changes in digestive tract enzyme activities and microbiota in response to the dietary supplementation of EME to pigs (Fan et al., 2009; Owusu-Asiedu et al., 2010; Smith et al., 2010). On the other hand, enzyme activities and microbial populations in the digestive tract are considered as vital factors that can influence gut health and nutrient digestibility (Yang et al., 2010). The objectives of this study were to assess the effects of EME supplementation of weanling piglet diets on nutrient digestibility, growth performance, digestive enzyme activities, and selected intestinal microbiota populations. MATERIALS AND METHODS All animals used in this study were cared for strictly following the animal care and use protocol that was approved by the Shandong Agricultural University Animal Nutrition Research Institute. Apparent Total Tract Digestibility (Exp. 1) Animal, Treatments, Diets, and Feeding Management. Twenty-eight 35-d-old piglets (weaned at 21 d and fed a commercial starter diet for a 14-d transitional period; PIC Hybrid, Jinan, China) with BW of 9.59 ± 0.36 kg (an equal number of barrows and gilts) were obtained from Shandong Agricultural University Research Farm (Shandong, China) and randomly allocated to 28 pens. The pens were plastic-covered expanded metal floors and located in a temperature-controlled room with good ventilation. The initial room temperature was set at 29.5°C and was gradually decreased by 1.5°C per week until the end of the experiment. At the start of the experiment, the 28 individually-fed piglets were randomly assigned to 7 dietary treatments (2 barrows and 2 gilts per treatment). The 7 dietary treatments were basal diet (Table 1) supplemented with 0 (control), 100, 150, 200, 250, 300, or 350 mg/kg of an EME preparation (Nopcozyme II; Diasham Resources Pte Ltd., Jurong, Singapore) in a completely randomized design. The product was a dry powder with starch as the carrier and contained amylase from Bacillus amyloliquefaciens (EC 3.2.1.1; 4,520), protease from Bacillus subtilis (EC 3.4.21; 8,660), and xylanase from Trichoderma (EC 3.2.1.8; 6,000). The same batch of the EME preparation was used for the entire experiment.
Table 1. Composition and energy and nutrient contents of the basal diet (Exp. 1 and 2) Item Ingredient, % Corn Wheat middling Whey powder Soybean oil Soybean meal Fish meal L-Lys×HCl DL-Met Thr Dicalcium phosphate Limestone Sodium chloride Vitamin-trace mineral premix1 Total Analyzed composition GE, MJ/kg DE, MJ/kg CP, % Ca, % Total P, % Lys, % Met, % Met + Cys, % Thr, %
Content 53.00 5.00 6.50 2.50 24.76 5.50 0.30 0.10 0.04 0.80 0.30 0.20 1.00 100.00 18.28 14.29 20.00 0.82 0.42 1.38 0.45 0.78 0.87
1Supplied per kilogram of complete diet: 11,025 IU vitamin A, 2203 IU vitamin D3, 80 IU vitamin E, 4.4 mg vitamin K3, 4.4 mg thiamine, 11 mg riboflavin, 35 mg d-pantothenic acid, 59.5 mg niacin, 330 mg choline, 0.9 mg folic acid, 0.5 mg biotin, 55 µg vitamin B12, 40 mg Mn as manganese sulfate, 130 mg Fe as ferrous sulfate, 130 mg Zn as zinc sulfate, 15 mg Cu as copper sulfate, 0.35 mg I as calcium iodide, and 0.3 mg Se as sodium selenite.
Amylase activity was determined using the method described by Somogyi (1960), with one amylase unit being defined as the amount of enzyme that hydrolyzes 10 mg of starch in 30 min at pH 6.5 and 37°C. Protease activity was analyzed according to the modified method of Hu et al. (2012) using casein as substrate. One protease unit is defined as the amount of enzyme that hydrolyzes casein to form 1 μmol product/min at pH 3.0 and 40°C. Xylanase activity was assayed using the method described by König et al. (2002), with 1 unit of xylanase being defined as the amount of enzyme that releases 1 μmol of reducing sugars/min at pH 4.8 and 50°C. Basal diets were formulated to meet nutrient requirements for weaned piglets recommended by the Feeding standard of swine in China (NY/T 65–2004; Ministry of Agriculture of China, 2004), and were fed as mash. All diets were prepared in a single batch. The EME was first mixed with a premix that was subsequently mixed with other ingredients and then stored in covered containers. The piglets had ad libitum access to feed and also water through nipple drinkers for the duration of the experiment.
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Multi-enzyme supplementation in weaned pig diet
Sample Collection, Preparation, and Analysis. The experiment was conducted for 14 d with the first 7 d as adaptation period and the remaining 7 d for sample collection. Feed offered and orts of each piglet were recorded daily for determination of feed intake and all feces were individually collected immediately after excretion during the 7-d period. Subsample of each diet was taken daily at feeding, dried at 55°C, and ground using a 1.0-mm screen for chemical analysis. The daily excreta of each piglet collected at each time was weighted, and 10% HCl was added at a ratio of 100 g of wet fecal sample to 10 mL 10% HCl, and subsequently stored in a sealed plastic bag at –20°C. At the end of the 7-d period, all bags containing daily feces of each piglet were thawed at room temperature and mixed thoroughly. An equal amount of daily fecal sample from the same piglet was pooled and a single subsample (10% of the total weight) was then oven dried at 55°C to constant weight and ground to pass a 1.0-mm screen for chemical analyses. The diet and fecal samples were analyzed for DM and CP according to the procedures described by Wang et al. (2011). Gross energy was determined using a Parradiabatic bomb calorimeter method as described by Zhang et al. (2012). The apparent total tract digestibility (ATTD) of DM, CP, and GE was calculated as following: ATTD (%) =
DMI × NCF − FW × NCf × 100 DMI × NCF
[1]
where DMI is in kg; NCF is the dietary content of DM, CP, or GE (%, DM basis); FW is the daily output of feces (kg); and NCf is the fecal content of DM, CP, or GE (%, DM basis). Growth Performance, Digestive Enzyme Activities, and Intestinal Microbiota (Exp. 2) Animal, Treatments, Diets, and Feeding Management. Two hundred 35-d-old piglets (weaned at 21-d of age and fed a commercial starter diet for a 14-d transition period; PIC Hybrid) with BW of 9.59 ± 0.36 kg (an equal number of barrows and gilts) were used in a 30-d feeding experiment. Piglets were randomly allocated into 20 pens (5 barrows and 5 gilts per pen), and pens were randomly assigned to 5 dietary treatment (basal diet supplemented with 0, 100, 150, 250, or 350 mg EME/kg) with 4 pens per treatment. The pens were plastic-covered expanded metal floors and located in a closed swine house with the same condition as described in Exp.1. The basal diet, its preparation, and method of EME supplementation were also the same as described for Exp.1. Piglets had ad libitum access to feed (mash form) and water during the entire experimental period. Determination of Feed Intake, Growth Rate, Feed Efficiency, and Diarrhea Index. Piglets were weighed
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on d 1 (35-d-old), 15 (50-d-old), and 30 (65-d-old) of the experiment to determine the ADG. Orts and spillages were collected and weighed daily to determine the ADFI. Feed efficiency was calculated as G:F. The piglets were visually observed daily in the morning for 3 to 4 h and incidence of diarrhea was recorded for each piglet based on the method described by Ball and Aherne (1982) and Song et al. (2012). Diarrhea score of each pig was assessed visually each day in the entire experimental period with a score from 1 to 5 (1 = normal feces, 2 = moist feces, 3 = mild diarrhea, 4 = severe diarrhea, and 5 = watery diarrhea). Diarrhea index was then calculated as the percentage of piglets with a diarrhea score of 3 and greater in the total piglets of each group. Determination of Selected Microbial Populations in Feces. Effects of EME supplementation on fecal populations of Escherichia coli spp., Salmonella spp., Lactobacillus spp., and Bacillus subtilis spp. were determined using the method described by Yang et al. (2010) and O’Doherty et al. (2010). Fresh fecal samples were collected by grabbing directly from rectums of 4 healthy piglets (1 piglet per pen) in each treatment (same sex for all treatment) on d 30 of the experiment. One gram of the sample was first added to 99 mL of sterile 0.1% peptone, homogenized for 1 min by blending, and then serially diluted in sterile 0.1% peptone. The dilutions were spread on Eosin Methylene Blue agar and Salmonella-Shigella agar, incubated aerobically at 37°C for 18 to 24 h to enumerate E. coli spp. and Salmonella spp., respectively. Lactobacillus spp. and B. subtilis spp. were isolated on de Man, Rogosa, and Sharp agar, and Luria-Bertani agar with overnight (18 to 24 h) incubation at 37°C in a 5% CO2 environment. Colonies of the bacterium in each plate were counted and the numbers of bacteria were calculated as colony-forming units per gram of fresh samples. Activities of Digestive Enzymes in Intestinal Digesta. Four healthy piglets from each treatment (1 per pen) were randomly selected in the morning of d 30 of the experiment. The piglets were euthanized 4 h after morning feeding by intracardiac injection of sodium pentobarbital (50 mg/kg BW; Omogbenigun et al., 2004; Fan et al., 2009). The abdominal cavity of the piglets was immediately opened and the small intestine was ligatured into duodenum, jejunum, and ileum according to the anatomical structure characteristic (i.e., the ligament linking duodenum and colon was taken as a symbol of distinguishing duodenum and jejunum, and the ligament jointing ileum and cecum was considered as a marker of parting jejunum and ileum). The digesta samples from each section were subsequently collected by massaging the tract from proximal and distal ends and stored immediately at –20°C. For the analysis, the digesta samples were thawed at room temperature and homogenized in 4 volumes of ice-cold 0.9% sodium chloride solution. The homog-
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Table 2. Effects of supplementing different levels of an exogenous multi-enzyme (EME) preparation to 35- to 49-dold piglets fed corn–soybean meal based diet on the apparent total tract digestibility of DM, CP, and GE (Exp. 1)1 Item DM CP GE 1n
0 84.45 84.35 84.06
100 85.45 85.30 85.20
EME, mg/kg 200 85.64 86.36 86.40
150 85.72 86.36 86.41
SEM 250 86.55 86.23 86.87
300 86.82 86.59 86.67
350 86.84 87.09 86.66
0.33 0.35 0.39
0 vs. others 0.001 0.005 0.009
P-value Linear < 0.001 < 0.001 < 0.001
Quadratic < 0.001 < 0.001 < 0.001
= 4.
enate was centrifuged at 13,800 × g for 20 min at 4°C and the supernatant was analyzed for amylase, protease, and lipase activities (Fan et al., 2009; Hu et al., 2012). Amylase and protease activity were determined using the method described in Exp. 1. Lipase (EC 3.l.l.3) activity was assayed using the method described by Tietz and Fiereck (1966), and one lipase unit is defined as the amount of enzyme that hydrolyzes 1 μmol of olive oil/ min. All determinations were performed in duplicates. Data Calculations and Statistical Analyses Data were statistically analyzed using the GLM procedure of SAS (Version 9.0; SAS Inst. Inc., Cary NC). The individual fed animal (Exp. 1) and the pen (Exp. 2) were used as the experimental unit. The effect of EME supplementation was determined by the preplanned contrast (0 vs. others) and linear and quadratic effects. Difference was declared significant when P < 0.05, and tendency was declared with P-values between 0.05 and 0.10.
RESULTS Apparent Total Tract Digestibility of DM, CP, and GE In Exp. 1, piglets supplemented with EME had greater ATTD of DM, CP, and GE (P = 0.001, 0.005, and 0.009, respectively) than those not supplemented with EME, and there seemed to be no differences in ATTD of DM, CP, and GE among piglets fed the diets supplemented with EME (Table 2). Nevertheless, the ATTD of DM, CP, and GE increased linearly and quadratically (P < 0.001) as supplementation of EME increased. Animal Growth Performances and Diarrhea Index The effects of supplementing EME on ADFI, ADG, and G:F in Exp. 2 are presented in Table 3. Supplementation of EME to 35- to 50-d-old piglets tended (P = 0.065 and 0.088) to increase ADG and ADFI and increased (P = 0.012) G:F as compared to control. The G:F increased lin-
Table 3. Effects of supplementing different levels of an exogenous multi-enzyme (EME) preparation to 35- to 65-dold piglets fed corn–soybean meal based diet on the growth performance and diarrhea index (Exp. 2)1 Item 0 BW, kg 35 d of age 9.30 50 d of age 14.77 65 d of age 24.57 35 to 50 d of age ADG, g 364 ADFI, g 574 G:F 0.63 51 to 65 d of age ADG, g 653 ADFI, g 1,148 G:F 0.57 35 to 65 d of age ADG, g 509 ADFI, g 864 G:F 0.59 8.42 Diarrhea index, %2 1n
100
EME, mg/kg diet 150
250
350
SEM
0 vs. others
P-value Linear
Quadratic
9.85 15.71 25.58
9.23 15.21 25.27
9.94 16.1 26.24
9.77 16 26.44
0.65 0.85 1.10
0.050 0.052 0.113
0.065 0.027 0.104
0.092 0.064 0.277
391 575 0.68
399 574 0.69
410 591 0.69
415 582 0.71
18 22 0.03
0.065 0.088 0.012
0.092 0.271 < 0.001
0.248 0.095 < 0.001
658 1,149 0.57
671 1,170 0.57
677 1,174 0.58
696 1,181 0.59
25 46 0.04
0.068 0.089 0.075
0.028 0.069 < 0.001
0.391 0.062 < 0.001
524 847 0.62 5.31
535 851 0.63 4.91
543 872 0.62 4.09
556 859 0.65 3.25
19 30 0.03 0.85
0.058 0.091 0.017 0.013
< 0.001 0.426 < 0.001 < 0.001
0.002 0.373 < 0.001 < 0.001
= 4.
2Diarrhea
index was calculated as the percentage of diarrhea piglets with a score of 3 or greater in total piglets of each group. The diarrhea score is assessed according to the following criteria: 1 = normal feces, 2 = moist feces, 3 = mild diarrhea, 4 = severe diarrhea, and 5 = watery diarrhea. Downloaded from www.journalofanimalscience.org at Queen Mary, University of London on May 29, 2014
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Table 4. Effects of supplementing different levels of an exogenous multi-enzyme (EME) preparation to 35- to 65-dold piglets fed corn–soybean meal based diet on digestive enzymes activities (U/g digesta) in different sections of the small intestine (Exp. 2)1 Item Duodenum Amylase Lipase Protease Jejunum Amylase Lipase Protease Ileum Amylase Lipase Protease 1n
0 vs. others
P-value Linear
Quadratic
4 15 128
0.001 0.021 0.001
< 0.001 < 0.001 < 0.001
< 0.001 < 0.001 < 0.001
387 339 7,841
10 12 109
0.001 0.003 0.001
0.022 < 0.001 < 0.001
0.018 0.012 < 0.001
542 357 5,899
6 9 74
0.004 < 0.001 0.001
< 0.001 < 0.001 < 0.001
< 0.001 < 0.001 < 0.001
0
100
EME, mg/kg 150
250
350
300 406 5,943
348 628 6,477
352 638 6,607
371 1,197 6,856
378 1,212 7,087
320 308 6,646
370 319 7,172
375 327 7,386
377 330 7,569
294 224 5,015
336 323 5,471
339 326 5,587
452 347 5,615
SEM
= 4.
early and quadratically (P < 0.001) as supplementation of EME increased, even though there seems to be no difference in G:F among EME-supplemented groups. Supplementation of EME to 51- to 65-d-old piglets tended to increase ADG, ADFI, and G:F (P = 0.068, 0.089, and 0.075, respectively) compared with the control group, and G:F increased linearly and quadratically (P < 0.001) as EME supplementation increased. Over the entire experimental period (age of 35 to 65 d), ADG and ADFI tended (P = 0.058 and 0.091, respectively) to be increased, whereas G:F was increased (P = 0.017) by EME supplementation compared with the control group. Overall G:F increased linearly and quadratically (P < 0.001) as supplementation levels of EME increased. Supplementation of EME reduced (P = 0.013) the diarrhea index in piglets over the 30-d experimental period compared with the control group, and the diarrhea index decreased linearly and quadratically (P < 0.001) as EME supplemented increased. Activities of Digestive Enzymes in Small Intestine Digestive enzymes activities in the duodenum, jejunum, and ileum are presented in Table 4. Supplementation of EME enhanced activity of amylase, lipase, and protease in all sections of the small intestine (P < 0.021) compared
with the control group. All those enzymes activities in the duodenum, jejunum, and ileum increased linearly and quadratically (P < 0.05) with the increasing EME supplementation. However, the difference in enzymatic activities among different EME-supplemented groups varied depending on the enzymes and the sections of the small intestine. Selected Microbial Populations in Piglet Feces Populations of Lactobacilli spp., B. subtilis spp., Salmonella spp., and E.coli spp. in feces collected from the rectum at 65 d of age are presented in Table 5. All of them were affected by the treatments. Supplementation of EME increased fecal counts of Lactobacilli spp. and B. subtilis spp. (P = 0.007 and 0.004, respectively), but decreased that of Salmonella spp. and E. coli spp. (P = 0.002 and 0.015, respectively) compared with the control group. All of them showed linear and quadratic dose responses to the levels of EME supplementation. Piglets supplemented with EME had higher counts of Lactobacilli spp. and B. subtilis spp. (linear and quadratic, P < 0.001), but they had lower counts of Salmonella spp. and E. coli spp. (linear and quadratic, P < 0.002) as EME supplementation increased
Table 5. Effects of supplementing different levels of an exogenous multi-enzyme (EME) preparation to 35- to 65-d-old piglets fed corn–soybean meal based diet on the selected microbial populations in rectum feces (log cfu/g feces; Exp. 2)1 Item Lactobacilli spp. Bacillus Subtilis spp. Salmonella spp. Escherichia coli spp. 1n
0 8.99 7.59 7.11 7.12
100 9.16 7.81 6.91 6.99
EME, mg/kg diet 150 9.27 7.92 6.86 6.87
250 9.34 8.14 6.75 6.71
350 9.37 8.20 6.63 6.51
SEM 0.04 0.04 0.07 0.09
0 vs. others 0.007 0.004 0.002 0.015
P-value Linear < 0.001 < 0.001 < 0.001 < 0.001
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Quadratic < 0.001 < 0.001 0.002 < 0.001
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DISCUSSION An important finding of this study was that supplementation of EME improved feed efficiency of 35- to 65-d-old piglets fed corn–soybean based diet, with the effect being greater for the age of 35 to 50 d than for 51 to 60d old. The increased feed efficiency is probably due to the increased nutrient supply by EME because EME supplementation tended to increase both ADG and ADFI and increase ATTD of DM, CP, and GE. Effects of EME supplementation on energy and nutrient digestion and growth performance of pigs have been assessed previously and the results varied among studies. Positive effects of EME on nutrient digestibility and animal growth performance were observed in some studies (Li et al., 1996; Omogbenigun et al., 2004), but not in others (Thacker et al., 1988; Officer, 1995). The inconsistent results of animal responses to the EME supplementation can be explained by the differences in diet composition and variations of EME activities and age of animals used (Kim et al., 2011). Willamil et al. (2012) also reported that the effects of EME can be mediated by the type of cereal grain in the diet. This study used a corn–soybean based diet and the EME preparation contained protease activity in addition to fibrolytic activities that were the main activities in other studies. The results demonstrated that supplementation of these EME increased the digestive enzymes activities and improved digestion of DM, CP, and GE by piglets. This could contribute to the improved feed efficiency of EMEsupplemented piglets observed in this study. The results that efficacy of EME in improving feed efficiency was more pronounced for 35- to 50-d than 51- to 65-d-old piglets were in agreement with other reports (Omogbenigun et al., 2004; Parra et al., 2012). This is likely due to the fact that digestive function is not fully developed in the younger age and, therefore, supplementing EME at this growth stage would enable piglets to digest and utilize dietary energy and nutrients more efficiently. Omogbenigun et al. (2004) and Parra et al. (2012) also reported that the declined positive effects of the EME supplementation on growth performance of piglets as they get older was due to the improved digestive capacity of weanling piglets such as the maturation of endogenous enzyme-secreting system and the increase in gastrointestinal microbial populations. Ngoc et al. (2011) reported superior growth responses to EME in piglets weighing below 20 kg, but not in those weighing 20 to 40 kg. The BW of piglets at the ages of 35-, 50-, and 65-d-old in this study were 9.6, 15.6, and 25.6 kg, respectively, and, therefore, the efficacy of EME in improving feed efficiency was greater for the age of 35to 50-d than 51- to 65-d-old. Diarrhea caused by infectious disease is a serious problem in weanling piglets and usually leads to
an increased mortality (Wang et al., 2011). This study demonstrated that supplementing EME reduced diarrhea index. This is probably due to the effects of EME supplementation to increase Lactobacilli and B. subtilis and decrease E. coli and Salmonella populations, and also to increase enzyme activities in the digestive tract of piglets observed in this study and reported by others (O’Doherty et al., 2010; Smith et al., 2010). It has been showed that the species and populations of microorganisms in the digestive tract influenced the digestibility of nutrients and affected gut health conditions (Yang et al., 2010). Both Lactobacilli and B. subtilis are considered as beneficial intestinal bacteria, whereas E. coli and Salmonnella are considered major bacteria that often cause gut health problems such as diarrhea, especially for younger animals. Therefore, the increased Lactobacilli and B. subtilis and reduced E. coli and Salmonella populations by supplementation of EME may have contributed to the decreased diarrhea index observed in this study. In addition, the increased digestive enzymes activities by EME supplementation may have also contributed to the reduced diarrhea index through enhancing animals’ digestive function, as indicated by the increased energy and nutrient digestibility. Increased digestive enzyme activities by supplementation of varying EME preparations were also documented in other studies (e.g., Wen et al., 2012). The increased digestive enzyme activities observed in this study are probably combined results from ingested EME and the enhanced endogenous enzymes secretion caused by the increased amount of energy and nutrients available for digestion by the action of EME. On the contrary, Fan et al. (2009) found that dietary supplementation of xylanase and β-glucanase to weaned piglets decreased the activities of amylase and lipase of the duodenum digesta. The reason for the differences across experiments is probabily associated with differences in types and levels of EME used, the age of animal involved, and the nature of the diets used (Li et al., 2004; Fan et al., 2009; Mirzaie et al., 2012; Wen et al., 2012), and, clearly, further research is required to elucidate the more crucial factors involved. Although the results showed that the majority of the measured variables responded to EME supplementation in a dose-dependent manner, the nutrient digestibility and growth performance results seems to indicate minimal differences in the range of 100 to 350 mg/kg of EME supplementation. This indicates that EME supplementation levels between 150 and 250 would be more economically feasible than the greater dosage under the present experimental conditions. In conclusion, supplementation of diets for 35- to 65-d-old piglets with EME containing activities of amylase, protease, and xylanase, increased digestibility of DM, CP, and GE, improved feed efficiency, and tended
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Multi-enzyme supplementation in weaned pig diet
to increase the growth rate and feed intake. In addition, the EME supplementation had positive effects on amylase, lipase, and protease activity and microbiota in the digestive tract. It appeared that the efficacy of EME supplementation in enhancing growth performance was greater at the age of 35- to 50-d than 50- to 65-d-old, and that supplementation of the EME preparation at the levels between 150 and 250 mg/kg diet would seem to be economically feasible. LITERATURE CITED Ball, R. O., and F. X. Aherne. 1982. Effect of diet complexity and feed restriction on the incidence and severity of diarrhea in early-weaned pigs. Can. J. Anim. Sci. 62:907–913. Fan, C. L., X. Y. Han, Z. R. Xu, L. J. Wang, and L. R. Shi. 2009. Effects of β-glucanase and xylanase supplementation on gastrointestinal digestive enzyme activities of weaned piglets fed a barley-based diet. J. Anim. Physiol. Anim. Nutr. 93:271–276. Hu, C., J. Song, Z. You, Z. S. Luan, and W. Li. 2012. Zinc oxidemontmorillonite hybrid influences diarrhea, intestinal mucosal integrity, and digestive enzyme activity in weaned pigs. Biol. Trace Elem. Res. 12:1–7. Kim, J. C., C. F. Hansen, B. P. Mullan, and J. R. Pluske. 2011. Nutrition and pathology of weaner pigs: Nutritional strategies to support barrier function in the gastrointestinal tract. Anim. Feed Sci. Technol. 173:3–16. Kluess, J., U. Schoenhusen, W. B. Souffrant, P. H. Jones, and B. G. Miller. 2010. Impact of diet composition on ileal digestibility and small intestinal morphology in early-weaned pigs fitted with a T-cannula. Animal 4:586–594. König, J., R. Grasser, H. Pikor, and K. Vogel. 2002. Determination of xylanase, β-glucanase, and cellulase activity. Anal. Bioanal. Chem. 374:80–87. doi:10.1007/s00216-002-1379-7 Leonard, S. G., T. Sweeney, B. Bahar, B. P. Lynch, and J. V. O’Doherty. 2011. Effects of dietary seaweed extract supplementation in sows and post-weaned pigs on performance, intestinal morphology, intestinal microflora and immune status. Br. J. Nutr. 1:1–12. Li, S., W. C. Sauer, R. Mosenthin, and B. Kerr. 1996. Effect of β-glucanase supplementation of cereal-based diets for starter pigs on the apparent digestibilities of dry matter, crude protein and energy. Anim. Feed Sci. Technol. 59:223–231. Li, W. F., J. Feng, Z. R. Xu, and C. M. Yang. 2004. Effects of nonstarch polysaccharides enzymes on pancreatic and small intestinal digestive enzyme activities in piglet fed diets containing high amounts of barley. World J. Gastroenterol. 10:856–859. Ministry of Agriculture of China. 2004. Feeding standard of swine. NY/T 65–2004. Ministry of Agriculture, Beijing, China. Mirzaie, S., M. Zaghari, S. Aminzadeh, M. Shivazad, and G. G. Mateos. 2012. Effects of wheat inclusion and xylanase supplementation of the diet on productive performance, nutrient retention, and endogenous intestinal enzyme activity of laying hens. Poult. Sci. 91:413–425. doi:10.3382/ps.2011-01686. Ngoc, T.T.B., N.T. Len, B. Ogle, and J.E. Lindberg. 2011. Influence of particle size and multi-enzyme supplementation of fibrous diets on total tract digestibility and performance of weaning (8–20kg) and growing (20–40kg) pigs. Anim. Feed Sci. Technol. 169:86–95.
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References
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