Scholarly article on topic 'Effect of replacing antibiotics using multi-enzyme preparations on production performance and antioxidant activity in piglets'

Effect of replacing antibiotics using multi-enzyme preparations on production performance and antioxidant activity in piglets Academic research paper on "Animal and dairy science"

CC BY-NC-ND
0
0
Share paper
Academic journal
Journal of Integrative Agriculture
OECD Field of science
Keywords
{antibiotics / "multi-enzyme preparations" / "growth performance" / digestibility / "antioxidant property"}

Abstract of research paper on Animal and dairy science, author of scientific article — Xin-yan HAN, Feng-ying YAN, Xin-zheng NIE, Wei XIA, Sha CHEN, et al.

Abstract The study was conducted to investigate the effects of replacing antibiotics using multi-enzyme preparations on growth performance, coefficient of total tract apparent digestibility, digestive enzyme activity, and antioxidant property in piglets. A total of 160 piglets ((21.35±0.22) kg) were randomly assigned to five dietary treatments: 1) basal diet supplemented with antibiotics (AC), 2) antibiotic diet supplemented with 0.5 g kg−1 multi-enzyme preparations (AC+0.5EP), 3) antibiotic diet supplemented with 1.5 g kg−1 multi-enzyme preparations (AC+1.5EP), 4) basal diet supplemented with a half dosage of antibiotics and 1.5 g kg−1 multi-enzyme preparations (AH+1.5EP), and 5) basal diet supplemented with 1.5 g kg−1 multi-enzyme preparations (BC+1.5EP). The results showed that AC+1.5EP significantly improved the feed efficiency, apparent digestibility of ether extract (EE) and crude ash (CA), lipase activity in pancreas and duodenum content, maltase and lactase activity in jejunum and ileum mucosa, glutathione peroxidase (GSH-Px) concentration in serum and liver, and decreased malondialdehyde (MDA) concentration in serum and liver compared with piglets receiving AC (P<0.05). Piglets receiving BC+1.5EP showed no significant difference in growth performance (P>0.05) but had lower MDA concentration than piglets receiving AC (P<0.05). The apparent digestibility of EE and crude fiber (CF), duodenal lipase activity, jejunum mucosa maltase, and ileum mucosa lactase activity of piglets receiving AH+1.5EP or BC+1.5EP were significantly improved compared with piglets receiving AC (P<0.05). These results indicated an additive growth promotion effect between antibiotics and multi-enzyme preparations on piglets, and the multi-enzyme preparations may be used as substitutes for antibiotics for improving piglet production performance and health status.

Academic research paper on topic "Effect of replacing antibiotics using multi-enzyme preparations on production performance and antioxidant activity in piglets"

jTa

□ Ei ".........

ELSEVIER

Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Effect of replacing antibiotics using multi-enzyme preparations on production performance and antioxidant activity in piglets ^

CrossMark

HAN Xin-yan, YAN Feng-ying, NIE Xin-zheng, XIA Wei, CHEN Sha, ZHANG Xiao-xu, QIAN Li-chun

Key Laboratory of Animal Nutrition and Feed Science in East China, Ministry of Agriculture/College of Animal Sciences, Zhejiang University, Hangzhou 310058, P.R.China

Abstract

The study was conducted to investigate the effects of replacing antibiotics using multi-enzyme preparations on growth performance, coefficient of total tract apparent digestibility, digestive enzyme activity, and antioxidant property in piglets. A total of 160 piglets ((21.35±0.22) kg) were randomly assigned to five dietary treatments: 1) basal diet supplemented with antibiotics (AC), 2) antibiotic diet supplemented with 0.5 g kg-1 multi-enzyme preparations (AC+0.5EP), 3) antibiotic diet supplemented with 1.5 g kg-1 multi-enzyme preparations (AC+1.5EP), 4) basal diet supplemented with a half dosage of antibiotics and 1.5 g kg-1 multi-enzyme preparations (AH+1.5EP), and 5) basal diet supplemented with 1.5 g kg-1 multienzyme preparations (BC+1.5EP). The results showed that AC+1.5EP significantly improved the feed efficiency, apparent digestibility of ether extract (EE) and crude ash (CA), lipase activity in pancreas and duodenum content, maltase and lactase activity in jejunum and ileum mucosa, glutathione peroxidase (GSH-Px) concentration in serum and liver, and decreased malondialdehyde (MDA) concentration in serum and liver compared with piglets receiving AC (P<0.05). Piglets receiving BC+1.5EP showed no significant difference in growth performance (P>0.05) but had lower MDA concentration than piglets receiving AC (P<0.05). The apparent digestibility of EE and crude fiber (CF), duodenal lipase activity, jejunum mucosa maltase, and ileum mucosa lactase activity of piglets receiving AH+1.5EP or BC+1.5EP were significantly improved compared with piglets receiving AC (P<0.05). These results indicated an additive growth promotion effect between antibiotics and multi-enzyme preparations on piglets, and the multi-enzyme preparations may be used as substitutes for antibiotics for improving piglet production performance and health status.

Keywords: antibiotics, multi-enzyme preparations, growth performance, digestibility, antioxidant property

1. Introduction

Received 1 April, 2016 Accepted 24 May, 2016

HAN Xin-yan, E-mail: xyhan@zju.edu.cn; Correspondence QIAN

Li-chun, Tel: +86-571-88982104, E-mail: lcqian@zju.edu.cn

© 2017, CAAS. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/) doi: 10.1016/S2095-3119(16)61425-9

Piglets have a limited ability to effectively utilize feed nutrients for their digestive tract and endogenous secretion system are not fully developed (Leonard et al. 2011). Weaning piglets commonly encounter low feed intake, body weight loss, and post-weaning diarrhea (Kim et al. 2012). Antibiotics are typically used as a cost-effective tool for improving feed efficiency and for preventing diseases in piglets (Allen et al. 2011). However, the widely used antibiotics have been scru-

tinized as contributors to the dissemination of antimicrobial resistance (Brewer et al. 2013), and consumers are becoming increasingly concerned about drug residues in meat products (Thacker 2013). In 2006, the use of antibiotics as growth promoters was forbidden in the European Union (EU), and there is a tendency all over the world to decrease antibiotics use as growth promoters. More attention was paid to exogenous enzyme preparations, which can complement the insufficiency of endogenous digestive enzymes and significantly improve the digestibility of some dietary components (Thacker 2013).

Traditionally, in pig diets, cereals and soybean meal were principal ingredient sources that supplied energy and protein (O'Doherty et al. 2001). However, studies indicated that non-starch polysaccharides (NSP) in corn and soybean meal negatively affected nutrient digestibility (Van Kempen et al. 2006; O'Shea et al. 2014). Thus, the inclusion of exogenous multi-enzymes in corn soybean meal-based diet contributed to improved nutrient value, particularly for monogastric animals with a limited digestive capacity (Li et al. 2010; Cozannet et al. 2012; Jo et al. 2012; Passos et al. 2015). Moreover, proteases combined with carbohydrates alleviated the negative effects of dietary fiber and significantly improved the digestion of legumes (Wenk 2000). Glucose oxidase catalyzes glucose to gluconic acid, which is consequently fermented to butyric acid via gastrointestinal microflora. This stimulates the growth of epithelial cells when glucose oxidase is fed to the animals (Tsukahara et al. 2002; Bankar et al. 2009).

Thus far, there were few reports about possible effects between multi-enzyme preparations and antibiotics on growth performance and health status of piglets. The aim of this study was to evaluate the possible effects between multi-enzyme preparations and antibiotics and to investigate the effect of replacing antibiotics with multi-enzyme preparations on growth performance, coefficient of total tract apparent digestibility (CTTAD), digestive enzyme activities, and antioxidant property in piglets.

2. Materials and methods

2.1. Multi-enzyme preparations

Multi-enzyme preparations were provided by the Feed Research Institute of the Chinese Academy of Agricultural Sciences and contained 2 000 U of acid protease, 150 U of fungal a-amylase, 3 500 U of xylanase, 350 U of p-man-nanase, 120 U of glucose oxidase, 120 U of acid cellulose, and 100 U of galactosidase activities per gram.

2.2. Animals and the experimental design

All animals that were used in this research were maintained according to the principles of the Animal Care and Use

Committee of Zhejiang University, China. In total, 160 crossbred piglets (Landrace*Yorkshire*Duroc) with a body weight (BW) of (21.35±0.22) kg were assigned to one of the following five dietary treatments, and each treatment was replicated four times with eight pigs per replication. These treatments were, as follows: 1) basal diet supplemented with 75 mg kg-1 auromycin and 20 mg kg-1 colistin (AC), 2) antibiotic diet supplemented with 0.5 g kg-1 multi-enzyme preparations (AC+0.5EP), 3) antibiotic diet supplemented with 1.5 g kg-1 multi-enzyme preparations (AC+1.5EP), 4) basal diet supplemented with 37.5 mg kg-1 auromycin and 10 mg kg-1 colistin, and 1.5 g kg-1 multi-enzyme preparations (AH+1.5EP), and 5) basal diet supplemented with 1.5 g kg-1 multi-enzyme preparations (BC+1.5EP). The basal diet was based on corn-soybean meal and was supplemented with minerals and vitamins to meet or exceed the requirement for piglets (NRC 1998). All diets were steam-pelleted at 80°C. The composition of the experimental diet is shown in Table 1.

The piglets were housed in an environmentally controlled nursery building. The pens were equipped with a feeder, a nipple drinker, and plastic-covered expanded metal floors. Water was available ad libitum. The piglets were fed a common diet for a 7-day adaptation period. The growth performance of piglets was evaluated in terms of the average daily gain (ADG), average daily feed intake (ADFI), and feed to gain (F/G).

2.3. Sample collection and preparation

During the last week of the experiment, chromic oxide was

Table 1 Ingredients and composition of the experiment basal

diet (g kg-1 as feed)

Ingredient Content

Corn 625

Soybean meal 185

Extruded soybeans 100

Wheat middling 30

Limestone 10

Soybean oil 10

Salt 10

Premix1) 10 Composition (analyzed except for digestible energy)

Digestible energy (MJ kg-1)2) 14.02

Crude protein (g kg-1) 178

Crude fiber 31.2

Ether extract 53.6

Crude ash 39.9

11 Supplied per kilogram of diet: vitamin A (8 500 IU), vitamin D3 (1 000 IU), vitamin E (40 IU), thiamine (2.5 mg), riboflavin (7.0 mg), pantothenic acid (25 mg), pyridoxine (2.0 mg), vitamin B12 (0.08 mg), vitamin K3 (1.0 mg), niacin (30 mg), choline chloride (800 mg), biotin (0.08 mg), folic acid (0.6 mg), Fe (FeSO4, 130 mg), Zn (ZnSO4, 100 mg), Cu (CuSO4, 15 mg), Mn (MnSO4H2O, 14 mg), I (KI, 0.21 mg), Se (Na2SeO3, 0.2 mg).

2) Digestible energy was calculated from the data provided by the Feed Database of China (2012).

added as an indigestible marker at 5 g kg-1 diet. Feces were collected from each pen at 9:00 a.m. for a period of 3 consecutive days. Then, the collected feces were weighed, and 10% HCl was added at a ratio of 100 g of wet fecal sample to 10 mL of 10% HCl. The mixture was oven-dried at 65°C to constant weight. Then, it was ground to pass a 1.0-mm screen for the following chemical analyses.

At the end of the trial, eight piglets from each treatment (two per pen) were selected randomly and were slaughtered according to the welfare of animal slaughter regulations of China using an intracardiac injection of sodium pentobarbital (50 mg kg-1 BW). Blood samples were collected from the anterior vena cava, centrifuged at 3 000*g at 4°C for 15 min, and then, the serum was obtained. The serum samples were transferred to sterile tubes. Then, the samples were frozen in liquid nitrogen and stored at -80°C before determining biochemical indices. The pancreas and liver samples were excised, frozen in liquid nitrogen, and stored at -80°C until use. Jejunum and ileum mucosa were stripped with a flat knife from seromuscular layers at the middle of jejunum and ileum after a gentle wash with physiological saline. The mucosa samples containing samples collected from duodenum were quickly frozen in liquid nitrogen and immediately stored at -20°C.

2.4. CTTAD measurements of feed and feces

Fecal samples were oven-dried at 60°C for 4 d and were pooled from each pen along with diet samples. The samples were finely ground to pass through a 1-mm screen using a Cyclotec 1093 Sample Mill (FOSS North America, Eden Prairie, MN, USA) and a Thomas-Wiley Mill (Thomas Scientific Swedesboro, NJ, USA), respectively. The feed and feces samples were analyzed according to the procedures of AOAC (2000). Dry matter (DM), crude protein (CP) (N*6.25), ether extract (EE), crude ash (CA), and crude fiber (CF) were determined according to AOAC (2000). Chromic oxide was determined using the method of Schurch et al. (1950). The CTTAD of dietary components was calculated, as follows:

CTTAD (%)=[1-(DfxId)/(Dd*If)]x100 Where, Df is the nutrient concentration in feces (%, DM basis), Dd is the nutrient concentration in the diet (%, DM basis), Id is the chromic oxide concentration in feces (%, DM basis), If is the chromic oxide concentration in the diet (%, DM basis).

2.5. Biochemical analyses

The content samples were extracted with 1 mmol L-1 HCl (50 mg digesta in 1 mL 1 mmol L-1 HCl) for 1 h at 4°C followed by centrifugation (3 000*g), according to the method

of Jensen et al. (1998). The supernatants were collected for enzyme activity analyses. The tissue samples were homogenized using Ultra-Turrax (T8, IKA-Labortechnik, Staufen, Germany) in an ice-cold 0.9% NaCl with a ratio of 1:10 (w/v). The homogenate was centrifuged (3 000*g) at 4°C for 15 min, and then, the supernatant was used for the analysis. The total protein content of tissues was determined, as described by Lowry et al. (1951). Lipase (EC3.1.1.3) and trypsin (EC3.4.21.4) activities were determined according to the methodology described in the method of Erlanson-Albertsson et al. (1987). Amylase (EC3.2.1.1) was analyzed using starch (Sigma Chemical Co., St. Louis) as the substrate, according to the method of Howard and Yudkin (1963), and the rate of starch disappearance was measured. Sucrase (EC3.2.1.48), maltase (EC3.2.1.20), and lactase (EC3.2.1.23) activities were measured according to the method of Dahlqvist (1968). The MDA, superoxide dismutase (SOD), and glutathione per-oxidase (GSH-Px) were determined using the colorimetric method with a spectrophotometer (T6 new century, Beijing Purkinje General Instrument Co., Ltd., Beijing, China), according to the procedures of Placer et al. (1966), Panckenko et al. (1975), and Lawrence and Burk (1976), respectively. The enzyme activities of amylase, lipase, trypsin, sucrose, maltase, and lactase were expressed as the specific activity (U mg-1 pro) in tissues. The enzyme activities of SOD and GSH-Px were expressed as the specific activity (U mg-1 pro) in tissues and as U mL-1 in serum, respectively. The MDA concentration was expressed as nmol mg-1 pro in the liver sample and as nmol mL-1 in serum, respectively.

2.6. Statistical analysis

Statistical analysis was performed using a one-way ANOVA procedure from the SPSS 19.0 statistical software package. When a significant P-value for the treatment mean values was observed in the analysis of variance, the treatment mean values were compared using Duncan's multiple range tests. The variance was declared significant when P<0.05, and the tendency was declared with P-values between 0.05 and 0.10.

3. Results

3.1. Growth performance

The growth performance of piglets is presented in Table 2. The incremental inclusion of multi-enzyme preparations in the antibiotic diet improved the growth performance of piglets. Piglets receiving AC+0.5EP had a higher ADG and feed efficiency than those receiving AC, but it was not significantly higher. Piglets that were fed AC+1.5EP had

increased ADG by 9.20% (P=0.054), and the feed efficiency was improved by 4.79% (P<0.05) compared with piglets receiving AC. However, the ADFI was not affected by multi-enzyme supplementation (P>0.05). Piglets receiving AH+1.5EP or BC+1.5EP had no difference in ADG and feed efficiency compared with piglets receiving AC. The AC+1.5EP group achieved the best growth performance among the five treatments.

3.2. Coefficient of total tract apparent digestibility

The CTTAD of dietary component is presented in Table 3. The incremental inclusion of multi-enzyme preparations in antibiotic diet improved the CTTAD of EE (P<0.05). Moreover, piglets receiving AC+1.5EP had a higher CTTAD of CA compared with piglets receiving AC (P<0.05). However, the CTTAD of CP, CF, and DM was not affected by multi-enzyme preparations (P>0.05). AH+1.5EP or BC+1.5EP increased the CTTAD of EE, CF and decreased CA (P<0.05), but made no differences in CP and DM compared with AC (P>0.05).

3.3. Digestive enzyme activity

Digestive enzyme activities of piglets are presented in Table 4. The incremental inclusion of multi-enzyme preparations in antibiotic diet improved the duodenum lipase activity of piglets compared with AC (P<0.05). Piglets receiving AC+0.5EP tended to have increased duodenum amylase (P=0.075) and ileum mucosa lactase activities (P=0.054)

compared with piglets receiving AC. AC+1.5EP increased pancreas lipase and trypsin activities, duodenum amylase activity, maltase and lactase activities both in jejunum and ileum mucosa compared with AC (P<0.05). Piglets receiving AH+1.5EP or BC+1.5EP had a higher duodenum lipase activity, jejunum mucosa maltase and ileum mucosa lactase activities than those receiving AC (P<0.05).

3.4. Antioxidant activity

The antioxidant property of piglets is presented in Table 5. AC+0.5EP significantly decreased MDA concentration in serum and increased GSH-Px in liver compared with AC (P<0.05). Piglets that were fed AC+1.5EP had a lower MDA concentration and a higher GSH-Px concentration both in liver and in serum compared with those receiving AC (P<0.05). Piglets receiving AH+1.5EP showed a lower MDA concentration and a higher GSH-Px concentration both in liver and serum compared with AC (P<0.05). The BC+1.5EP treatment decreased the MDA concentration in serum and in liver and increased the GSH-Px concentration in liver compared with the AC group (P<0.05).

4. Discussion

As the public presses for antibiotic-free diets, there has been an increasing interest in the use of exogenous enzyme to improve the digestibility and growth performance of piglets. The basic principle of exogenous enzyme application is to

Table 2 Effect of multi-enzyme preparations and antibiotics on growth performance

Item1) Antibiotic Antibiotic 1.5 g kg- 1 enzymes SEM2) P-value

control 0.5 g kg-1 enzymes 1.5 g kg-1 enzymes 50% antibiotic 0% antibiotic

IW (kg) 21.35 21.41 21.41 21.34 21.39 0.221 1.000

FW (kg) 39.63 40.64 41.37 39.22 40.89 0.328 0.195

ADG (g) 522.19 ab 549.43 ab 570.24 b 510.76 a 557.14 ab 8.335 0.106

ADFI (g) 1 127.34 1 152.08 1 169.93 1 112.17 1 158.92 15.976 0.831

F/G 2.16 bc 2.10 abc 2.05 a 2.18 bc 2.08 ab 0.018 0.108

1) IW, initial weight; FW, final weight; ADG, average daily gain; ADFI, average daily feed intake; F/G, feed to gain.

2) SEM, standard error of mean. The same as below.

Values are presented as the means (n=4). The mean values within rows with different letters differ significantly (P<0.05). The same as below.

Table 3 Effect of multi-enzyme preparations and antibiotics on total tract apparent digestibility

Item1) Antibiotic Antibiotic 1.5 g kg- 1 enzymes SEM P-value

control 0.5 g kg-1 enzymes 1.5 g kg-1 enzymes 50% antibiotic 0% antibiotic

EE 0.45 a 0.54 b 0.58 c 0.50 b 0.60 c 0.062 0.003

CP 0.67 0.68 0.69 0.67 0.69 0.007 0.845

CA 0.25 c 0.26 c 0.30 d 0.18 a 0.21 b 0.017 0.000

CF 0.61 a 0.61 a 0.63 a 0.65 b 0.67 c 0.009 0.001

DM 0.78 0.78 0.77 0.77 0.79 0.005 0.346

1) EE, ether extract; CP, crude protein; CA, crude ash; CF, crude fiber; DM, dry matter. n=4.

Table 4 Effect of multi-enzyme preparations and antibiotics on digestive enzyme activity

Antibiotic

Antibiotic

control 0.5 g kg-1 enzymes 1.5 g kg-1 enzymes

1.5 g kg-1 enzymes

50% antibiotic

0% antibiotic

SEM P-value

Pancreas Amylase Lipase Trypsin Duodenum Amylase Lipase Trypsin Jejunum mucosa Maltase Sucrase Lactase Ileum mucosa Maltase Sucrase Lactase

279.71 186.80 a 944.02 a

27.42 a 94.81 a 117.78

203.29 a 107.67

53.51 a

155.77 ab

58.52 6.55 a

282.93 195.62 a 973.73 a

29.93 ab 104.13 b 121.13

212.34 ab 106.83 55.07 a

164.67 b 59.56 6.93 ab

283.26 217.70 b 1111.08 b

30.27 b 110.48 c 123.99

228.70 bc 108.70 59.32 b

190.77 c 60.32 7.20 b

279.58 202.52 ab 976.99 a

29.26 ab 102.90 b 119.41

246.66 cd 104.62 55.07 a

145.26 a 59.53 7.26 b

273.48 207.45 ab 1000.60 a

28.07 ab 103.05 b 120.07

256.43 d 105.05 55.52 a

166.33 b 57.76 7.24 b

4.32 3.54 14.21

0.44 1.12

5.05 0.82 0.64

3.76 0.71 0.073

0.963 0.054 0.000

0.201 0.000 0.437

0.000 0.499 0.046

0.000 0.835 0.003

Table 5 Effect of multi-enzyme preparations and antibiotics on antioxidant activity

Item1) Antibiotic Antibiotic 1.5 g kg-1 enzymes SEM P-value

control 0.5 g kg-1 enzymes 1.5 g kg-1 enzymes 50% antibiotic 0% antibiotic

MDA 10.62 b 9.21 a 8.47 a 8.55 a 8.60 a 0.20 0.000

SOD 95.88 98.61 104.28 103.26 104.71 1.47 0.227

GSH-Px 572.13 a 581.60 ab 625.56 c 614.60 bc 609.92 abc 6.43 0.026

MDA 3.32 b 3.04 b 2.18 a 2.23 a 2.30 a 0.10 0.000

SOD 103.03 104.63 102.75 101.72 100.15 1.35 0.894

GSH-Px 134.59 a 155.20 b 168.79 b 163.19 b 154.55 b 3.36 0.008

1) MDA, malondialdehyde; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase. n=8.

improve the nutritive value of feedstuffs by: 1) breaking down anti-nutritional factors that may be present in many plant-based feed ingredients, 2) increasing availability of starch, proteins, and minerals that may be encapsulated within fiber-rich cell walls or bound up in a chemical form that the animal is unable to digest, and 3) supplementing enzymes that may be present in insufficient amounts in animal gastrointestinal tract (Owusu-Asiedu et al. 2010; Adeola and Cowieson 2011; Thacker 2013).

The corn-soybean meal-based diet is commonly used in pig production, with corn containing 10%, and soybean meal containing 22.7% non-starch polysaccharides (NSP) such as a-galactosides and p-galactomannan (CVB 1998). These NSPs are not digested by pigs because they lack enzymes that targeting a-1,6-galactosyl bonds and p-1,4-mannosyl bonds (Veum and Odle 2001). It was reported that piglets that were fed corn-soybean meal-based diet supplemented with an enzyme mixture containing a-amylase, p-mannan-ase, and protease significantly improved ADG and feed

efficiency (Jo et al. 2012). Similarly, pigs that were fed a soybean protein-based diet supplemented with protease increased the ADG, ADFI, and feed efficiency (Zuo et al. 2015). In the present study, the ADG, ADFI, and feed efficiency of piglets that were fed corn-soybean meal-based diets supplemented with a multi-enzyme preparation (BC+1.5EP) showed no significant difference compared with those receiving antibiotics treatment. This suggested positive effects of enzyme mixture on the pig growth performance. This beneficial effect may be due to synergistic effects of different types of enzymes, particularly a-galacto-sidase and p-mannanase. Moreover, piglets that were fed a diet supplemented with both antibiotics and an enzyme mixture (AC+1.5EP) had efficiency increase by 4.79% (P<0.05) compared with piglets receiving and antibiotics treatment. This additive growth promotion effect caused by antibiotics and enzyme supplementation was reported by Wenk (2000), who observed that the addition of both carbohydrases and antibiotics increased the piglet weight

gain by approximately 25%. Besides, Birzer et al. (1992) and Vranjes and Wenk (1995) showed an additive effect of two supplements in their studies.

In the present study, both the addition of multi-enzyme preparations to the antibiotic diet (AC+0.5EP and AC+1.5EP) and the replacement of antibiotics with multi-enzyme preparations (AH+1.5EP and BC+1.5EP) positively influenced fat utilization. It is well known that bile salts play a crucial role in fat digestion, emulsification, and absorption (Maldona-do-Valderrama et al. 2011) and that bacterial flora is vitally important for enterohepatic circulation (Stamp and Jenkins 2008). Thus, the reduction of microorganisms in the digestive tract due to antibiotics leads to the deficiency of bile salts and, consequently, to low fat utilization (Vranjes and Wenk 1995). Thus, the removal of antibiotics (AH+1.5EP and BC+1.5EP) increased fat retention. On the other hand, the supplementation of animal diet with enzymes can decrease digesta viscosity and increase the utilization of nutrients, which influences the conditions and composition of intestinal microflora (Zhang et al. 2014; Passos et al. 2015). Additionally, the use of cell wall degrading enzymes (i.e., xylanase and cellulose) was reported to improve fat utilization, often to a greater extent than any other nutrient (Bedford and Schulze 1998). Further, the depressive effect of antibiotics on fat utilization was overcome by the multi-enzyme preparation when both supplements were added to the diet (AH+1.5EP and BC+1.5EP). Therefore, the improvement in CTTAD of EE was mainly ascribed to the multi-enzyme preparation, which led to the digesta viscosity reduction and to better diffusion of nutrients, which influenced gastrointestinal microflora. In addition, cellulolytic bacteria in the gastrointestinal tract help degrade fiber components (Varel and Yen 1997), and antibiotic supplementation depresses fiber degradability by reducing the microbial flora in the animal gastrointestinal tract (Vranjes and Wenk 1995). In the present study, all of the abovementioned observations supported the identified significant improvement in CTTAD of CF when antibiotics were replaced with multi-enzyme preparations (AH+1.5EP and BC+1.5EP). The piglets that received 1.5 g kg-1 multi-enzyme preparation (BC+1.5EP) showed the highest fiber degradability among the five treatments. This was caused not only by the removal of antibiotics but also by the addition of carbohydrases, which were employed to enhance digestion of carbohydrates including resistant starch or dietary fibers (Johnson et al. 1993).

The digestion of dietary nutrients is closely related to the digestive enzyme activity (Qian et al. 2015). Previous work reported that the digestive function of piglets was stimulated by enzyme supplementation (O'Doherty et al. 2010; Wen et al. 2012). The incremental amount of nutrients available for digestion due to the action of multi-enzyme supplementation in turn increased the excretion of endogenous enzymes

and led to the improvement of digestive enzyme activities (Bedford and Schulze 1998; Wen et al. 2012; Zhang et al. 2014). Therefore, the improvement of lipase activities in duodenum was ascribed to the multi-enzyme supplementation (AC+0.5EP and AC+1.5EP) and to the increased concentration of bile salts, which was caused by the removal of antibiotic (AH+1.5EP and BC+1.5EP), because bile salts stimulated the lipase activity (Albro et al. 1985; Bauer et al. 2005). Additionally, glucose oxidase that was added to diet catalyzed the oxidation of p-D-glucose to gluconic acid by utilizing molecular oxygen as an electron acceptor with a simultaneous production of hydrogen peroxide (Bankar et al. 2009). Gluconic acid that was produced by the action of glucose oxidase was mainly fermented to butyric acid, which is the main energy source of epithelial cells of intestine (Biagi et al. 2006). Therefore, feeding of glucose oxidase could indirectly stimulate the epithelial cells growth, digestive enzyme secretion, and enhanced the intestinal immune function of piglets. Therefore, enzyme supplementation will be more beneficial when using diets based on low quality ingredients or on poorly digestible ingredients as well as for young piglets that do not have a proper enzyme system or a gut microbial population (Omogbenigun et al. 2004; Parra et al. 2012; Zhang et al. 2014). Moreover, it was reported that proteases combined with carbohydrases increased digestion processes especially of legumes such as soybeans (Pugh and Charlton 1995). Therefore, the enhanced digestive enzyme activity and digestive function were mainly ascribed to the proper combination of enzyme mixture and of feed components.

The addition of multi-enzyme preparations to the antibiotic diet (AC+0.5EP and AC+1.5EP) increased GSH-Px and decreased MDA in piglets, which was similar to when antibiotics were replaced with multi-enzyme preparations (AH+1.5EP and BC+1.5EP). This suggested that multi-enzyme preparations could improve the antioxidant property and health status of piglets. In addition, multi-enzyme preparations can be used as healthy and free of any toxic substitutions of antibiotics. However, to date, there have been few studies investigating the influence of enzyme mixture on the antioxidant property and on the mechanism by which multi-enzyme preparations improve the antioxidant property in piglets. Therefore, this requires further research.

5. Conclusion

The supplementation of both multi-enzyme preparations and antibiotics had an additive effect on the pig growth performance. Specifically, the feed efficiency and related digestive enzyme activities significantly improved. By replacing antibiotics with multi-enzyme preparations, the feed efficiency was improved, and the fat and fiber utili-

zation was increased, and improved the related digestive enzyme activities and antioxidant properties. These results suggested that the multi-enzyme preparation was worthy of further investigation as a potential candidate for improving the growth performance and health status of piglets.

Acknowledgements

This study was financially supported by the National 863 Program of China (2013AA102803D).

References

Adeola O, Cowieson A J. 2011. Board-invited review: Opportunities and challenges in using exogenous enzymes to improve nonruminant animal production. Journal of Animal Science, 89, 3189-3218. Albro P W, Hall R D, Corbett J T, Schroeder J. 1985. Activation of nonspecific lipase (EC 3.1. 1.-) by bile salts. Biochimica et Biophysica Acta (Lipids and Lipid Metabolism), 835, 477-490.

Allen H K, Looft T, Bayles D O, Humphrey S, Levine U Y, Alt D, Stanton T B. 2011. Antibiotics in feed induce prophages in swine fecal microbiomes. mBio, 2, e00260-e002711. AOAC International (Association of Official Analytical Chemists). 2000. In: Horwitz W, ed., Official Methods of Analyses. 17th ed. AOAC, Gaithersburg, MD. Bankar S B, Bule M V, Singhal R S, Ananthanarayan L. 2009. Glucose oxidase - An overview. Biotechnology Advances, 27, 489-501. Bauer E, Jakob S, Mosenthin R. 2005. Principles of physiology of lipid digestion. Asian-Australasian Journal of Animal Sciences, 18, 282-295. Bedford M R, Schulze H. 1998. Exogenous enzymes for pigs

and poultry. Nutrition Research Reviews, 11, 91-114. Biagi G, Piva A, Moschini M, Vezzali E, Roth F X. 2006. Effect of gluconic acid on piglet growth performance, intestinal microflora, and intestinal wall morphology. Journal of Animal Science, 84, 370-378. Birzer D, Kronseder G, Stadler E, Gropp J. 1992. The performance of broiler administered an enzyme preparation breaking down carbohydrates (Roxazyme G(R)) in varying diet formulation. In: The Proceedings of the 3rd Symposium. 26-27 Sep., 1991. Stadtroda, Jena, Germany. (in German) Brewer M T, Xiong N, Anderson K L, Carlson S A. 2013. Effects of subtherapeutic concentrations of antimicrobials on gene acquisition events in Yersinia, Proteus, Shigella, and Salmonella recipient organisms in isolated ligated intestinal loops of swine. American Journal of Veterinary Research, 74, 1078-1083. Cozannet P, Preynat A, Noblet J. 2012. Digestible energy values of feed ingredients with or without addition of enzymes complex in growing pigs. Journal of Animal Science, 90, 209-211.

CVB (Centraal Veevoeder Bureau). 1998. Centraal veevoeder

bureau. In: Veevoedertalel (Feeding Value of Feed ingredients). Centraal Veevoeder Bureau, Runderweg 6, Lelystad, the Netherlands.

Dahlqvist A. 1968. Assay of intestinal disaccharidases. Analytical Biochemistry, 22, 99-107.

Erlanson-Albertsson C, Larsson A, Duan R. 1987. Secretion of pancreatic lipase and colipase from rat pancreas. Pancreas, 2, 531-535.

Chinese Feed Database. 2012. Table of Feed Composition and Nutritive Value in China. 23rd ed. China Feed, Beijing, China. (in Chinese)

Howard F, Yudkin J. 1963. Effect of dietary change upon the amylase and trypsin activities of the rat pancreas. British Journal of Nutrition, 17, 281-294.

Jensen M S, Bach Knudsen K E, Inborr J, Jakobsen K. 1998. Effect of p-glucanase supplementation on pancreatic enzyme activity and nutrient digestibility in piglets fed diets based on hulled and hulless barley varieties. Animal Feed Science and Technology, 72, 329-345.

Jo J K, Ingale S L, Kim J S, Kim Y W, Kim K H, Lohakare J D, Lee J H, Chae B J. 2012. Effects of exogenous enzyme supplementation to corn-and soybean meal-based or complex diets on growth performance, nutrient digestibility, and blood metabolites in growing pigs. Journal of Animal Science, 90, 3041-3048.

Johnson R, Williams P, Campbell R. 1993. Use of enzymes in pig production. Enzymes in Animal Nutrition, 1, 49-60.

Van Kempen T A T G, Van Heugten E, Moeser A J, Muley N S, Sewalt V J H. 2006. Selecting soybean meal characteristics preferred for swine nutrition. Journal of Animal Science, 84, 1387-1395.

Kim J C, Hansen C F, Mullan B P, Pluske J R. 2012. Nutrition and pathology of weaner pigs: Nutritional strategies to support barrier function in the gastrointestinal tract. Animal Feed Science and Technology, 173, 3-16.

Lawrence R A, Burk R F. 1976. Glutathione peroxidase activity in selenium-deficient rat liver. Biochemical and Biophysical Research Communications, 71 , 952-958.

Leonard S G, Sweeney T, Bahar B, Lynch B P, O'Doherty J V. 2011. Effects of dietary seaweed extract supplementation in sows and post-weaned pigs on performance, intestinal morphology, intestinal microflora and immune status. British Journal of Nutrition, 106, 688-699.

Li Y, Fang Z, Dai J, Partridge G, Ru Y, Peng, J. 2010. Corn extrusion and enzyme addition improves digestibility of corn/ soy based diets by pigs: In vitro and in vivo studies. Animal Feed Science and Technology, 158, 146-154.

Lowry O H, Rosebrough N J, Farr A L, Randall R J. 1951. Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193, 265-275.

Maldonado-Valderrama J, Wilde P, Macierzanka A, Mackie A. 2011. The role of bile salts in digestion. Advances in Colloid and Interface Science, 165, 36-46.

NRC (National Research Council). 1998. Nutrient Requirements of Swine. 10th ed. National Academy Press, USA.

O'Doherty J V, Dillon S, Figat S, Callan J J, Sweeney T. 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. Animal Feed Science and Technology, 157, 173-180.

O'Doherty J V, Murphy D, McGlynn S G. 2001. The effects of expander processing and by-product inclusion levels on performance of grower-finisher pigs. Animal Science, 73, 479-487.

Omogbenigun F O, Nyachoti C M, Slominski B A. 2004. Dietary supplementation with multienzyme preparations improves nutrient utilization and growth performance in weaned pigs. Journal of Animal Science, 82, 1053-1061.

O'Shea C J, Mc Alpine P O, Solan P, Curran T, Varley P F, Walsh A M, Doherty J V O. 2014. The effect of protease and xylanase enzymes on growth performance, nutrient digestibility, and manure odour in grower-finisher pigs. Animal Feed Science and Technology, 189, 88-97.

Owusu-Asiedu A, Simmins P H, Brufau J, Lizardo R, Peron A. 2010. Effect of xylanase and p-glucanase on growth performance and nutrient digestibility in piglets fed wheat-barley-based diets. Livestock Science, 134, 76-78.

Panchenko L F, Brusov O S, Gerasimov A M, Loktaeva T D. 1975. Intramitochondrial localization and release of rat liver superoxide dismutase. FEBS Letters, 55, 84-87.

Parra S J, Agudelo T J, Ortiz L, Ramírez M C, Rodríguez B, López Herrera A. 2012. Lipopolysaccharide (LPS) from E. coli has detrimental effects on the intestinal morphology of weaned pigs. Revista Colombiana de Ciencias Pecuarias, 24, 585-597.

Passos A A, Park I, Ferket P, von Heimendahl E, Kim S W. 2015. Effect of dietary supplementation of xylanase on apparent ileal digestibility of nutrients, viscosity of digesta, and intestinal morphology of growing pigs fed corn and soybean meal based diet. Animal Nutrition, 1, 36-40.

Placer Z A, Cushman L L, Johnson B C. 1966. Estimation of product of lipid peroxidation (malonyl dialdehyde) in biochemical systems. Analytical Biochemistry, 16, 359-364.

Pugh R, Charlton P. 1995. Enzyme applications for plant proteins: Time to look beyond cereals. In: Biotechnology in the FeedIndutry (Alltech's Annual Symposium). Nottingham University Press, Loughborough. pp. 393-396.

Qian L, Yue X, Hu L, Ma Y, Han X. 2015. Changes in diarrhea, nutrients apparent digestibility, digestive enzyme activities of weaned piglets in response to chitosan-zinc chelate. Animal Science Journal, 87, 564-569.

Schurch A F, Lloyd L E, Crampton E W. 1950. The use of chromic oxide as an index for determining the digestibility of a diet two figures. The Journal of Nutrition, 41, 629-636.

Stamp D, Jenkins G. 2008. An Overview of Bile-Acid Synthesis, Chemistry and Function. Bile Acids Toxicology and Bioactivity. Royal Society of Chemistry, London, United Kingdom. pp. 1-13.

Thacker P A. 2013. Alternatives to antibiotics as growth promoters for use in swine production: A review. Journal of Animal Science and Biotechnology, 4, 35.

Tsukahara T, Koyama H, Okada M, Ushida K. 2002. Stimulation of butyrate production by gluconic acid in batch culture of pig cecal digesta and identification of butyrate-producing bacteria. The Journal of Nutrition, 132, 2229-2234.

Varel V H, Yen J T. 1997. Microbial perspective on fiber utilization by swine. Journal of Animal Science, 75, 2715-2722.

Veum T L, Odle J. 2001. Feeding neonatal pigs. In: Lewis A J, Southern L L, eds., Swine Nutrition. CRC Press, New York. pp. 671-690.

Vranjes M V, Wenk C. 1995 Influence of dietary enzyme complex on the performance of broilers fed on diets with and without antibiotic supplementation. British Poultry Science, 36, 265-275.

Wen C, Wang L C, Zhou Y M, Jiang Z Y, Wang T. 2012. Effect of enzyme preparation on egg production, nutrient retention, digestive enzyme activities and pancreatic enzyme messenger RNA expression of late-phase laying hens. Animal Feed Science and Technology, 172, 180-186.

Wenk C. 2000. Recent advances in animal feed additives such as metabolic modifiers, antimicrobial agents, probiotics, enzymes and highly available minerals. Asian Australasian Journal of Animal Sciences, 13, 86-95.

Zhang G G, Yang Z B, Wang Y, Yang W R, Zhou H J. 2014. 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. Journal of Animal Science, 92, 2063-2069.

Zuo J, Ling B, Long L, Li T, Lahaye L, Yang C, Feng D. 2015. Effect of dietary supplementation with protease on growth performance, nutrient digestibility, intestinal morphology, digestive enzymes and gene expression of weaned piglets. Animal Nutrition, 1, 276-282.

(Managing editor ZHANG Juan)