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Bacteriophage and probiotics both enhance the performance of growing pigs but bacteriophage are more effective
Accepted Manuscript Title: Bacteriophage and probiotics both enhance the performance of growing pigs but bacteriophage are more effective Author: K.H. Kim S.L. Ingale J.S. Kim S.H. Lee J.H. Lee I.K. Kwon B.J. Chae PII: DOI: Reference:
S0377-8401(14)00198-9 http://dx.doi.org/doi:10.1016/j.anifeedsci.2014.06.012 ANIFEE 13106
To appear in:
Animal
Received date: Revised date: Accepted date:
29-1-2014 20-6-2014 21-6-2014
Feed
Science
and
Technology
Please cite this article as: Kim, K.H., Ingale, S.L., Kim, J.S., Lee, S.H., Lee, J.H., Kwon, I.K., Chae, B.J.,Bacteriophage and probiotics both enhance the performance of growing pigs but bacteriophage are more effective, Animal Feed Science and Technology (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.06.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Bacteriophage and probiotics both enhance the performance of growing pigs but
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bacteriophage are more effective†
3 K.H. Kim a, 1, S.L. Ingale a, 1, J.S. Kim a, S.H. Lee a, J.H. Lee b, I.K. Kwon a, B.J. Chae a,*
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a
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of Korea b
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CTC Bio, Inc., Seoul, 138-858, Republic of Korea
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College of Animal Life Sciences, Kangwon National University, Chuncheon, 200-701, Republic
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Abbreviations: ADFI, average daily feed intake; ADG, average daily gain; apparent total tract
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digestibility; BW, body weight; CP, crude protein; DM, dry matter; G:F, gain:feed; GE, gross
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energy; NC, negative control; PC, positive control; TAB, total anaerobic bacteria;
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This study was supported by CTC Bio, Inc. and the Institute of Animal Resources at
Kangwon National University, Chuncheon, Republic of Korea.
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These authors contributed equally to this work.
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Corresponding author: Tel.:+82 33 250 8616; Fax: +82 33 244 4946
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E-mail address: [email protected] (B.J. Chae)
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ABSTRACT
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Two experiments were conducted to determine the effects of dietary supplementation with
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bacteriophage, probiotics and their combination on growth performance, apparent total tract
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digestibility (ATTD), fecal bacterial populations and serum immunoglobulins in growing pigs. In
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both experiments, 200 barrows (Landrace × Yorkshire × Duroc) were randomly allotted to 4
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treatments on the basis of BW. There were 5 replicate pens in each treatment with 10 pigs per
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pen. Experimental diets were fed in meal form for 35 d. In Exp. 1, dietary treatments included
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basal diet supplemented with 0 (control diet without any antimicrobials), 0.5, 1.0 and 1.5 g/kg
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commercial bacteriophage product. Bacteriophage product contained a cocktail of
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bacteriophages of Salmonella (S. typhimurium, S. enteritidis, S. cholerasuis and S. derby),
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Staphylococcus aureus, Escherichia coli and Clostridium perfringens types A and C. Dietary
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increasing levels of bacteriophage linearly improved (P<0.05) the ADG, ADFI and ATTD of
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DM. At d 35 of the experiment, pigs fed diets supplemented with increasing levels of
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bacteriophage had greater (linear, P<0.05) fecal TAB, Bifidobacterium spp. and Lactobacillus
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spp. and fewer (linear, P<0.05) fecal Clostridium spp. and coliforms. Dietary treatments had no
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effect (P>0.05) on serum immunoglobulin concentrations at d 35 of experiment. In Exp. 2,
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dietary treatments were basal diet without any antimicrobials (Control) and basal diets
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supplemented with 3.0 g/kg fermented probiotic product (P), 1.0 g/kg bacteriophage (B) and
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combination of 1.0 g/kg bacteriophage and 3.0 g/kg fermented probiotic product (BP). Probiotic
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products used herein contained Lactobacillus acidophilus, Bacillus subtilis and Saccharomyces
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cerevisiae. Pigs fed the B and BP diets had greater (P<0.05) ADG, ADFI, G:F and ATTD of
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DM, CP and GE than that of pigs fed the control and P diets. Pigs fed the P diet had greater
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(P<0.05) ADG, ADFI and ATTD of CP than that of pigs the fed control diet. At d 35, pigs fed
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the BP diet had greater (P<0.05) fecal TAB, Bifidobacterium spp. and Lactobacillus spp. and
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fewer (P<0.05) Clostridium spp. and coliforms than pigs fed the control diet. Also, pigs fed the P
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and B diets had greater (P<0.05) Lactobacillus spp and fewer (P<0.05) coliforms at d 35 than
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that of pigs fed the control diet. Dietary treatments had no effect (P>0.05) on serum
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immunoglobulin concentrations (d 35). The present results suggest that bacteriophages and
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probiotics both improve different aspects of grower pig’s performance but that bacteriophages
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are more effective than probiotics and would appear to offer an alternative to antibiotic type
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growth promoters.
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Key words: Bacteriophage, Fecal bacterial populations, Growing pigs, Immunoglobulins,
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Performance, Probiotics.
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1. Introduction
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In South Korea, use of antibiotics as growth promoters in animal feeds has been forbidden
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since 2011 (Global Agricultural Information Network, 2011). Therefore, the search continues for
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non-antibacterial growth promoters that are active in vivo, are fast acting, possess a broad
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spectrum in activity, do not induce bacterial resistance and subsequently promote growth
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performance of pigs. A number of research findings on the use of alternatives like probiotics,
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oligosaccharides, organic acids and antimicrobial peptides to replace antibiotics in feed have
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been documented with varying success (Choi et al., 2011a; Yan et al., 2011; Yoon et al., 2012,
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2013; Lee et al., 2014). In this context, bacteriophages are believed to be an ideal candidate, due
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to their natural antibacterial properties (Jamalludeen et al., 2009; Yan et al., 2012; Wang et al.,
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2013).
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Bacteriophages are obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery (McGrath et al., 2004). Bacteriophages are
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amongst the most abundant living entities on earth playing important roles in maintaining the
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natural abundance and distribution of microorganisms (Sulakvelidze, 2011) and have been used
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to both prevent and treat bacterial diseases in human and animals. Most of the previous studies
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on bacteriophages evaluated their therapeutic effects on disease challenged pigs (Barrow, 2001;
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Jamalludeen et al., 2009; Wall et al., 2010) and poultry (Huff et al., 2002; Toro et al., 2005;
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Atterbury et al., 2007). Previous studies with dietary supplementation of anti- Salmonella
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bacteriophage reported improved performance and reduced bacterial shedding in growing pigs
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(Gebru et al., 2010; Yan et al., 2012). Recently it has been reported that supplementation with
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bacteriophages to laying hens diets resulted in greater feed efficiency, egg production and
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improved excreta microbiota (Zhao et al., 2012; Wang et al., 2013). Previous studies in the
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authors laboratory reported that a multimicrobe probiotic products had potential to improve the
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performance and gut health and could be used as an alternative to antibiotics growth promoters
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in pigs and broilers (Choi et al., 2011b; Kim et al., 2012). The present study was designed to
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investigate the effects of dietary supplementation with bacteriophage, probiotics and
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combination of bacteriophage and probiotics on growth performance, ATTD of nutrients, fecal
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bacterial populations and serum immunoglobulins of growing pigs.
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2. Materials and methods
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The project underwent proper ethical standards and the experiments were approved by the
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Institutional Animal Care and Use Committee of Kangwon National University, Chuncheon,
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Republic of Korea. These experiments were conducted at the facility of Kangwon National
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University farm and the pigs (Landrace × Yorkshire × Duroc) were housed in partially slotted
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and concrete floor pens with a pen size of 2.80 m × 5.00 m. All pens were equipped with a self-
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feeder and nipple drinker to allow ad libitum access to feed and water.
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2.1. Bacteriophage
Bacteriophage product was obtained from a commercial feed company (CTC Bio, Inc., Seoul,
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Republic of Korea). In short, the product contained a cocktail of bacteriophages of Salmonella
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(S. typhimurium, S. enteritidis, S. cholerasuis and S. derby), Staphylococcus aureus, Escherichia
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coli (k88, k99 and f41) and Clostridium perfringens types A and C. The product contained 109
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plaque-forming units (pfu)/g bacteriophages.
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2.2. Preparation of probiotic product
Lactobacillus acidophilus isolated from faces of weaned pigs, Bacillus subtilis isolated from
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natto (fermented soybeans), and Saccharomyces cerevisiae isolated from koji (malted wheat)
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were maintained in the laboratory as stock culture. A culture broth (CB) medium containing 60.0
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ml corn steep liquor, 40.0 ml molasses, 3.0 g/l yeast extract, 5.0 g/l KH2PO4 and 2.5 g/l K2HPO4
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in distilled water was prepared and autoclaved before being used.
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Two litres of autoclaved CB were inoculated with 2.0 mL of culture of each microbe
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separately and subjected to fermentation for 48 h. L. acidophilus and B. subtilis were incubated
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at 37 oC at pH 7.0, whereas S. cerevisiae was incubated at 32 oC at pH 4.0. The microbes grown
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on CB were directly sprayed on corn-soybean meal (1:1) followed by drying at 40 oC for 72 h.
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The microbes grown on CB were used as starter and pasteurized corn: soybean meal (1:1) was
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used as the substrate for carrying out fermentation as described previously by Shim et al. (2010).
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Then the substrates (13.0 kg) were inoculated with 2.0 litres of starter and fermented for 7 d at 32
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72 h and mixed to obtain the fermented probiotic product. The counts of L. acidophilus, B.
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subtilis and S. cerevisiae in fermented probiotic product were 4.0 × 108, 4.8 × 109 and 1.0 × 104
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cfu/g respectively.
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2.3. Animals, housing and treatments
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In Exp. 1, 200 barrows (average initial BW: 50.9 ± 0.530 kg) were randomly allotted to 4 treatments on the basis of initial BW. There were 5 replicate pens in each treatment with 10 pigs
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per pen. Dietary treatments consisted of a basal diet supplemented with 0 (control diet without
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any antimicrobial), 0.5, 1.0 and 1.5 g/kg commercial bacteriophage product (109 pfu/g). In Exp.
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2, 200 barrows (average initial BW: 50.5 ± 0.928 kg) were randomly allotted to 4 treatments on
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the basis of initial BW. There were 5 replicate pens in each treatment with 10 pigs per pen.
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Dietary treatments consisted of a basal diet without any antimicrobial (Control), basal diet
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supplemented with 1.0 g/kg bacteriophage (B; 109 pfu/g), basal diet supplemented with 3.0 g/kg
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fermented probiotic product (P) and basal diet supplemented with combination of 1.0 g/kg
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bacteriophage and 3.0 g/kg fermented probiotic product (BP). Fermented probiotic products used
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herein contained Lactobacillus acidophilus, Bacillus subtilis and Saccharomyces cerevisiae.
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Corn was replaced with bacteriophage (Exp. 1 and 2), probiotics and combination of
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bacteriophage and fermented probiotic product (Exp. 2) on an equal percentage basis. In both
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experiments, all diets met or exceeded the nutrient requirements as suggested by NRC (1998)
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and fed in meal form for 35 d (Tables 1 and 2).
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2.4. Experimental procedures, measurements, and analyses Pigs were weighed individually, and feed consumption was calculated at the end of each experiment to calculate ADG, ADFI and G:F. To evaluate the effects of dietary treatments on the
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ATTD of energy and nutrients, 2.50 g/kg chromic oxide (an inert indigestible indicator) was
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included in each diet from d 28 to 35 of each experiment. Fecal grab samples were collected
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from the floor of each pen during last 4 days of each experiment to determine the ATTD of DM,
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GE and CP. The fecal samples were pooled within pen and dried in a forced air oven at 60°C for
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72 h, and ground in a Wiley mill (Thomas Model 4 Wiley Mill, Thomas Scientific, Swedesboro,
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NJ) using a 1-mm screen and used for chemical analysis.
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At d 35 of both experiments, fresh fecal samples were collected from 2 pigs in each pen and used for measuring fecal bacterial populations. The samples collected for bacterial population
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analysis were immediately placed on ice until analyses were conducted later on the
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corresponding day. On d 35 of each experiment, a 10.0-mL blood sample was collected by
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jugular vein puncture from 2 randomly selected pigs in each pen using a disposable vacutainer
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tube without anticoagulant (Becton Dickinson, Franklin, NJ). After centrifugation (3,000 × g for
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15 min at 4°C), serum samples were separated and stored at -20°C and later analyzed for
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concentrations serum immunoglobulins (IgG, IgA and IgM).
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2.5. Chemical and microbial analyses Experimental diets and excreta samples were analyzed in triplicate for DM (Method 930.15)
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and CP (Method 990.03) using AOAC (2007) methods. Gross energy of diets and feces were
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measured by a bomb calorimeter (Model 1261, Parr Instrument Co., Moline, IL), and chromium
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concentration was determined with an automated spectrophotometer (Jasco V-650; Jasco Corp.,
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Tokyo, Japan) according to the procedure of Fenton and Fenton (1979). Serum IgG, IgA and
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IgM were determined using radial immune-diffusion kit (Tripple J farm, Bellingham, WA,
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USA). The microbiological assay of fecal samples was carried out by culturing in different media as
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suggested by Choi et al. (2011a). For the determination of total anaerobic bacteria (Tryptic soy
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agar), Lactobacillus spp. (using MRS agar + 0.200 g/l NaN3 + 0.500 g/l L-cystine hydrochloride
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monohydrate), Bifidobacterium spp. (MRS-NPNL: MRS agar + nalidixic acid, paromomycin +
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neomycin sulphate + lithium chloride), Clostridium spp. (TSC agar) and coliforms (violet red
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bile agar) were used. The microbiological assay of potential probiotic products was also carried
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out by culturing technique. Lactobacillus acidophilus was enumerated using MRS agar + 0.200
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g/l NaN3 + 0.500 g/l L-cystine hydrochloride monohydrate, B. subtilis by using plate count agar,
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S. cerevisiae by potato dextrose agar. The anaerobic conditions during the assay of total
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anaerobic bacteria and Clostridium spp. were created by using gas pak anaerobic system (BBL,
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No. 260678, Difco, Detroit, MI). The tryptic soy agar (No. 236950), MRS agar (No. 288130),
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violet red bile agar (No. 216695), plate count agar (No. 247940), and potato dextrose agar (No.
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213400) used were purchased from Difco Laboratories (Detroit, MI), and TSC agar (CM0589)
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was purchased from Oxoid (Hampshire, UK). The bacterial concentrations were transformed
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(log) before statistical analysis.
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2.6. Statistical Analysis In Exp. 1, statistical analysis was conducted using the GLM procedure of SAS (SAS Institute
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Inc., Cary, NC) as a randomized complete block design. Orthogonal polynomials were used to
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evaluate linear and quadratic effects of dietary bacteriophage supplementation (0, 0.5, 1.0 and
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1.5 g/kg). In Exp. 2, statistical analysis was conducted using the one way ANOVA procedure
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(SAS Institute Inc., Cary, NC), and when significant differences were identified among treatment
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means, they were separated using Tukey’s Honestly Significant Difference test. In both
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experiments, the pen was used as the experimental unit for all analysis of all the parameters.
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Probability values of ≤0.05 were considered as significant in both experiments.
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3. Results
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3.1. Growth performance
In experiment 1, ADG and ADFI increased linearly (P<0.05) with increasing dietary
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bacteriophage supplementation but there was no treatment effect on F: G (Table 3). In
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experiment 2, pigs fed the B and BP diets exhibited similar performance and had significantly
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better ADG, ADFI and F: G than those on the other two treatments. Pigs on the P treatment also
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grew faster and ate more than their control counterparts (Table 4).
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3.2. Apparent total tract digestibility
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In experiment 1, ATTD of DM increased linearly (P<0.05) with increasing dietary
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bacteriophage supplementation but there was no treatment effect on ATTD of CP and GE (Table
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3). In experiment 2, pigs fed the B and BP diets exhibited similar ATTD of nutrients and had
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significantly greater (P<0.05) ATTD of DM, CP and GE than those on the other two treatments.
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Pigs fed the control and P diets exhibited similar ATTD of all nutrients.
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3.3. Fecal bacterial populations
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In experiment 1, at d 35, pigs fed diets supplemented with increasing levels of bacteriophage
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had greater (linear, P<0.05; Table 5) fecal TAB, Bifidobacterium spp. and Lactobacillus spp. and
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fewer (linear, P<0.05) fecal Clostridium spp. and coliforms. In experiment 2, pigs fed the BP
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diets had greater (P<0.05; Table 6) fecal TAB, Bifidobacterium spp. and Lactobacillus spp. and
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fewer (P<0.05) Clostridium spp. and coliforms than that of pigs fed the control diet. Pigs fed the
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P and B diets had greater (P<0.05) Lactobacillus spp. and fewer (P<0.05) coliforms than that of
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pigs fed the control diet. Moreover, pigs fed the BP diets had greater (P<0.05) Lactobacillus spp.
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and fewer (P<0.05) coliforms than that of pigs fed the B diet.
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At d 35 of experiment 1 and 2, serum IgG, IgA and IgM levels averaged 6.53, 0.417 and
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0.956 respectively. They were similar in both experiments and not significantly affected by the
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treatments investigated
4. Discussion
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Altering and enhancing normal gut bacterial populations by targeting intestinal pathogens
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through nonantibiotic approaches can improve the gut health, immunity and performance of pigs
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and poultry (Choi et al., 2011 b; Sen et al., 2012; Jo et al., 2012; Kim et al., 2012; Yoon et al.,
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2012). Among various nonantibiotic alternatives, bacteriophages have received attention due to
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their natural antimicrobial properties (Gebru et al., 2010; Wang et al., 2013).
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Results obtained in the current study indicated that dietary supplementation with probiotics
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and bacteriophages but particularly with bacteriophages has the potential to improve the
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performance of growing pigs. Our results are in agreement with Gebru et al. (2010), who
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observed improvement in overall ADG and G:F of Salmonella challenged pigs fed diets
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supplemented with 3× 109 pfu/kg diet Salmonella typhimurium bacteriophage. A recent study in
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author’s laboratory also reported that dietary supplementation with a bacteriophage cocktail
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improved the growth performance of weanling pigs (Kim et al., 2013). In contrast Yan et al.
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(2012) reported that dietary supplementation with anti-Salmonella bacteriophage had no effect
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on ADG and G:F of growing pigs. The differences between experiments are likely associated
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with differences in the level and type of bacteriophage investigated, health status within herds,
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farm hygiene, diets composition, feed forms and interactions with other dietary feed additives.
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The results of our second study confirmed the growth promoting potential of the bacteriophage
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product and showed there were no synergistic effects of combining the probiotics with the
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bacteriophages. This suggests there was no additional benefit of addition of probiotic product to
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bacteriophage diets of growing pigs. In contrast Kim et al. (2013) working with weanling pigs
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reported that a combination of probiotics and bacteriophages supported better growth
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performance than bacteriophage alone.
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In the preset study, pigs fed diets supplemented with a bacteriophage cocktail, probiotics and
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both had greater ATTD of DM (Exp. 1 and 2), GE and CP (Exp. 2). In agreement with present
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results, Yan et al. (2012) reported improved ATTD of DM, nitrogen and energy of growing pigs
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fed diets supplemented with 0.5 g/kg diet anti-Salmonella bacteriophage. The results of
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Experiment 2 are in agreement with Kim et al. (2013), who observed greater ATTD of DM, CP
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and GE in weaning pigs fed diet supplemented with combination of bacteriophage and
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probiotics. The improved growth rate elicited in both experiments and in feed efficiency in
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experiment 2 by bacteriophage supplementation was likely associated with the concomitant
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changes in nutrient digestibility and in the balance of gut microbiota. The probiotic supplement
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elicited small changes in the gut microbiota, had no effect on nutrient digestibility and
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marginally improved rate compared to the control.
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The reduced populations of Clostridium spp. and coliform and increased Bifidobacterium and Lactobacillus found in pigs fed the diet supplemented with bacteriophage (Exp. 1) and a
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combination of bacteriophage and probiotics (Exp. 2) observed in the present study are in
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agreement with the results of other studies involving growing pigs, broilers and laying hens fed
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diets supplemented with anti-Salmonella bacteriophage (Yan et al., 2012; Zhao et al., 2012;
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Wang et al., 2013). Gebru et al. (2010) observed reduced bacterial shedding score in Salmonella
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typhimurium challenged pigs fed diets supplemented with bacteriophage. It should be noted
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however that the changes in bacterial populations reported here for pigs fed diets supplemented
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with bacteriophage and/or probiotics were not large and unlikely to contribute entirely to the
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improvement in growth performance elicited by the technologies and in particular by the
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bacteriophage product. Further research is required to better understand the mechanisms behind
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the quite significant improvement in performance associated with supplementing the diets with
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bacteriophages.
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In the current study, supplementation of growing pigs diet with bacteriophage, probiotics or
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their combination had no effects on serum immunoglobulins. These findings are consistent with
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results of Kim et al. (2013), who reported no effects of supplementation of bacteriophage,
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probiotics or their combination to weanling pig’s diet. Previous studies with dietary
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supplementation with probiotics (Bacillus or Lactobacillus spp. alone or in combination)
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reported enhanced immune response of the piglets (European Food Safety Authority, 2010; Lee
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et al., 2014).
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5. Conclusions The present results suggest that bacteriophages and probiotics both improve different aspects of grower pig’s performance but that bacteriophages are more effective than probiotics and
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would appear to offer an alternative to antibiotic type growth promoters.
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Kim, J.S., Ingale, S.L., Kim, Y.W., Kim, K.H., Sen, S., Ryu, M.H., Lohakare, J.D., Kwon, I.K.,
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Chae, B.J., 2012. Effect of supplementation of multi-microbe probiotic product on growth
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performance, apparent digestibility, cecal microbiota and small intestinal morphology of
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Proceedings of the 2013 Annual Congress of KSAST, Jeju, South Korea.
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Lee, S.H., Ingale, S.L., Kim, J.S., Kim, K H., Lokhande, A., Kim, E.K., Kwon, I.K., Kim, Y.H., Chae, B.J., 2014. Effects of dietary supplementation with Bacillus subtilis LS 1-2
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fermentation biomass on growth performance, nutrient digestibility, cecal microflora and
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intestinal morphology of weanling pig. Anim. Feed Sci. Technol. 188, 102-110.
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Chem. Phys. Microbiol. 1, 163-189.
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McGrath, S., Fitzgerald, G.F., van Sinderen, D., 2004. Starter cultures: Bacteriophage. Cheese
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Ryu, M.H., Kwon, I.K., Chae, B.J., 2012. Effect of supplementation of Bacillus subtilis LS
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1-2 to broiler diet on growth performance, nutrient retention, caecal microbiology and small
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Shim, Y.H., Shinde, P.L., Choi, J.Y., Kim, J.S., Seo, D.K., Park, J.I., Chae, B.J., Kwon, I.K.,
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2010. Evaluation of multimicrobial probiotics produced by submerged liquid and solid
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substrate fermentation methods in broilers. Asian-Aust. J. Anim. Sci. 23, 521–529.
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Sulakvelidze, A., 2011. Bacteriophage: A new journal for the most ubiquitous organisms on
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Earth. Bacteriophage, 1:1-2; PMID: 21687529; http://dx.doi.org/10.4161/bact.1.1.15030
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Toro, H., Price, S.B., McKee, A.S., Hoerr, J.F., Krehling, J., Perdue, M., Bauermeister, L., 2005.
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Use of bacteriophages in combination with competitive exclusion to reduce Salmonella from
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infected chickens. Avian Dis. 49, 118-124. Wall, S.K., Zhang, J., Rostagno, M.H., Ebner, P.D., 2010. Phage therapy to reduce pre-
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processing Salmonella infections in market weight swine. Appl. Environ. Microbiol. 76, 48–
344
53.
Wang, J.P., Yan, L., Lee, J.H., Kim, I.H., 2013. Evaluation of bacteriophage supplementation on
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growth performance, blood characteristics, relative organ weight, breast muscle
347
characteristics and excreta microbial shedding in broilers. Asian-Aust. J. Anim. Sci. 26, 573-
348
578.
Yan, L., Meng, Q.W., Kim, I.H., 2011. The effects of an herb extract mixture on growth
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performance, nutrient digestibility, blood characteristics and fecal noxious gas content in
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growing pigs. Livest. Sci. 141, 143-147.
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Yan, L., Hong, S., Kim, I.H., 2012. Effects of bacteriophage supplementation on the growth
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performance, nutrient digestibility, blood characteristics, and fecal microbial shedding in
354
growing pigs. Asian-Aust. J. Anim. Sci. 25, 1451-1456.
355
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Yoon, J.H., Ingale, S.L., Kim, J.S., Kim, K.H., Lee, S.H., Park, Y.K., Kwon, I.K., Chae, B.J.,
356
2012. Effects of dietary supplementation of antimicrobial peptide-A3 on growth
357
performance, nutrient digestibility, intestinal and fecal microflora and intestinal morphology
358
in weanling pigs. Anim. Feed Sci. Technol. 177, 98-107.
359
Yoon, J.H., Ingale, S.L., Kim, J.S., Kim, K.H., Lohakare, J., Park, Y.K., Park, J.C., Kwon, I.K.,
360
Chae, B.J., 2013. Effects of dietary supplementation of antimicrobial peptide-P5 on growth
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performance, apparent total tract digestibility, intestinal microflora and intestinal morphology
362
of weanling pigs. J. Sci. Food Agri. 93, 587-592.
363
Zhao, P.Y., Kim, I.H., 2012. Effects of bacteriophage supplementation on egg performance, egg quality, excreta microflora, and moisture content in laying hens. Asian-Aust. J. Anim. Sci.
365
25, 1015-1020.
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17 Page 17 of 24
366 Table 1 Ingredient and chemical composition of experimental diets (as-fed basis; Exp. 1) 0.5
1.0
462.6
462.1
461.6
461.1
Wheat
110
110
110
110
Soybean meal (450 g/kg)
340
340
340
340
Animal fat
45.0
45.0
45.0
45.0
Molasses
10.0
10.0
10.0
10.0
Choline chloride (500 g/kg)
0.200
0.200
0.200
0.200
9.00
9.00
9.00
9.00
3.20
3.20
3.20
6.00
6.00
6.00
8.00
8.00
8.00
L-Lysine
(230 g/kg) (880 g/kg)
3.20 6.00
Dicalcium phosphate
8.00
Salt
3.00
3.00
3.00
3.00
Vitamin premix
1.50
1.50
1.50
1.50
Mineral premix2
1.50
1.50
1.50
1.50
-
0.500
1.00
1.50
13.8
13.8
13.8
13.8
Crude protein, g/kg
200
200
200
200
Lysine, g/kg
10.0
10.0
10.0
10.0
Methionine + Cysteine, g/kg
5.90
5.90
5.90
5.90
Calcium, g/kg
7.00
7.00
7.00
7.00
Available phosphorus, g/kg
3.00
3.00
3.00
3.00
M
Limestone
d
DL-Methionine
Bacteriophage Chemical composition
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ME, MJ/kg
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1
an
Corn
us
Ingredient, g/kg
1.5
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0
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Bacteriophage supplementation (g/kg)
Supplied per kilogram of diet: 16,000 IU vitamin A, 3,000 IU vitamin D3, 40 IU vitamin E, 5.0 mg vitamin K3, 5.0 mg vitamin B1, 20 mg vitamin B2, 4 mg vitamin B6, 0.08 mg vitamin B12, 40 mg pantothenic acid, 75 mg niacin, 0.15 mg biotin, 0.65 mg folic acid. 2 Supplied per kilogram of diet: 45 mg Fe, 0.25 mg Co, 50 mg Cu, 15 mg Mn, 25 mg Zn, 0.35 mg I, 0.13 mg Se. 367 368 369
18 Page 18 of 24
Table 2 Ingredient and chemical composition of experimental diets (as-fed basis; Exp. 2) Treatments1
Control
P
B
BP
462.6
459.6
461.6
458.6
Wheat
110
110
110
110
Soybean meal (450 g/kg)
340
340
340
340
Animal fat
45.0
45.0
45.0
45.0
Molasses
10.0
10.0
10.0
10.0
Choline chloride (500 g/kg)
0.200
0.200
0.200
0.200
9.00
9.00
9.00
9.00
3.20
3.20
3.20
6.00
6.00
6.00
8.00
8.00
8.00
(230 g/kg)
DL-Methionine
(880 g/kg)
3.20
3.00
3.00
3.00
3.00
2
1.50
1.50
1.50
1.50
3
1.50
1.50
1.50
1.50
-
3.00
-
3.00
-
-
1.00
1.00
ME, MJ/kg
13.8
13.8
13.8
13.8
Crude protein, g/kg
200
200
200
200
Lysine, g/kg
10.0
10.0
10.0
10.0
Methionine + Cysteine, g/kg
5.90
5.90
5.90
5.90
Calcium, g/kg
7.00
7.00
7.00
7.00
Available phosphorus, g/kg
3.00
3.00
3.00
3.00
6.00
Dicalcium phosphate
8.00
Salt
M
Limestone
Vitamin premix
d
Mineral premix Bacteriophage
Ac ce p
Chemical composition
te
Probiotics
1
cr
an
L-Lysine
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Corn
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Ingredient, %
Dietary treatments were basal diet without any antimicrobial (Control), basal diet supplemented with 1.0 g/kg bacteriophage cocktail (B), basal diet supplemented with 3.0 g/kg probiotic (P) and basal diet supplemented with combination of 1.0 g/kg bacteriophage and 3.0 g/kg probiotics (BP) 2 Supplied per kilogram of diet: 16,000 IU vitamin A, 3,000 IU vitamin D3, 40 IU vitamin E, 5.0 mg vitamin K3, 5.0 mg vitamin B1, 20 mg vitamin B2, 4 mg vitamin B6, 0.08 mg vitamin B12, 40 mg pantothenic acid, 75 mg niacin, 0.15 mg biotin, 0.65 mg folic acid. 3 Supplied per kilogram of diet: 45 mg Fe, 0.25 mg Co, 50 mg Cu, 15 mg Mn, 25 mg Zn, 0.35 mg I, 0.13 mg Se. 370 19 Page 19 of 24
370 Table 3 Effects of dietary supplementation with a bacteriophage cocktail on the performance and apparent total tract digestibility of nutrients in pigs offered feed ad libitum for 35 days commencing at 50 kg (Exp. 1) x 1.0
1.5
SEM
737
764
815
822
Average daily feed intake, g
2,079
2,129
2,240
2,222
Feed:Gain
2.82
2.79
2.75
19. 5
0.001
0.592
us
Quadratic
61.2
0.043
0.544
0.103
0.318
0.941
0.852
0.012
0.984
2.71
Dry matter
0.841
0.846
0.025
Gross energy
0.874
0.875
0.875
0.879
0.048
0.458
0.754
Crude protein
0.831
0.832
0.838
0.845
0.046
0.088
0.371
0.847
d
M
Apparent total tract digestibility of nutrients
te
Data represents mean based on 5 replicate pens per treatment
Ac ce p
371 372
Linear
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Average daily gain, g
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0.5
Growth performance
x
P-values
0
cr
Bacteriophage supplementation (g/kg)
20 Page 20 of 24
372 Table 4. Effects of supplementing diet with a probiotics (P), a bacteriophage cocktail (B) and both (BP) on the performance and apparent total tract digestibility of nutrients in pigs offered feed ad libitum for 35 days commencing at 50 kg (Exp. 2). x Control
B
P
PB
a
Average daily feed intake, g
2,032
Feed:Gain
2.77
c
a
a
b
0.835
Gross energy
0.871
Crude protein
0.822
a
0.839
M
Dry matter
b
826
b
2,089
a
2.72
an
2.63
a
768
2,121
Apparent total tract digestibility of nutrients
b
a
0.874
b
d
x
b
809
us
c
733
cr
Growth performance Average daily gain, g
a
0.829
SEM
ip t
Item y
b
0.836
b
0.871
b
0.825
a
2,137
b
2.59
a
0.840
a
0.876
a
0.832
17.8 46.9 0.052
0.057 0.046 0.053
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373
te
Data represents mean based on 5 replicate pens per treatment Dietary treatments were basal diet without any antimicrobial (Control), basal diet supplemented with 1.0 g/kg bacteriophage cocktail (B), basal diet supplemented with 3.0 g/kg probiotic (P) and basal diet supplemented with combination of 1.0 g/kg bacteriophage and 3.0 g/kg probiotics (BP) abc Means with different superscripts in the same row are different (P<0.05). y
21 Page 21 of 24
373 Table 5. Effects of dietary supplementation with a bacteriophage cocktail on fecal microbiota (log10 CFU/g; at d 35) of growing pigs offered feed ad libitum for 35 days commencing at 50 kg (Exp. 1) x 1.0
1.5
SEM
7.92
8.22
Bifidobacterium spp.
8.92
9.37
9.77
Lactobacillus spp.
8.56
8.67
Clostridium spp.
8.14
7.96
Coliforms
8.57
0.228
9.75
Quadratic
0.050
0.810
0.252
0.020
0.360
8.98
0.194
0.071
0.620
7.61
7.50
0.159
0.006
0.829
7.77
7.84
0.213
0.012
0.340
M
9.06
d
8.22
8.33
us
7.69
an
Total anaerobic bacteria
cr
Linear
Data represents mean based on 5 replicate pens per treatment
te
374 375
0.5
Ac ce p
x
0
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P-values
Bacteriophage supplementation (g/kg)
22 Page 22 of 24
375
b
Bifidobacterium spp.
8.78
Lactobacillus spp.
8.27
Clostridium spp.
8.40
Coliforms
8.51
ab
a
7.76
b
8.07
ab
ab
9.42
c
9.18
b
b
8.47
a
ab
7.75
a
c
7.72
x
BP
8.39
ab
7.93
b
8.23
SEM
a
8.29
a
us
7.34
P
an
Total anaerobic bacteria
B
cr
Control
M
Item
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Table 6. Effects of supplementing diet with a probiotics (P), a bacteriophage cocktail (B) and both (BP) on fecal microbiota (log10 CFU/g; at d 35) of growing pigs offered feed ad libitum for 35 days commencing at 50 kg (Exp. 2). x,y
9.77
a
8.75
b
7.59
c
7.55
0.161 0.214 0.238 0.177 0.252
Data represents mean based on 5 replicate pens per treatment Dietary treatments were basal diet without any antimicrobial (Control), basal diet supplemented with 1.0 g/kg bacteriophage cocktail (B), basal diet supplemented with 3.0 g/kg probiotic (P) and basal diet supplemented with combination of 1.0 g/kg bacteriophage and 3.0 g/kg probiotics (BP) abc Values with different superscripts in the same row are different (P<0.05).
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376 377
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23 Page 23 of 24
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Highlights Effects of dietary supplementation with bacteriophage, probiotics and both on performance of barrows. Bacteriophages and probiotics both improve different aspects of grower pig’s performance Bacteriophages are more effective than probiotics Bacteriophage would appear to offer an alternative to antibiotic type growth promoters.
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377 378 379 380 381 382 383 384 385
24 Page 24 of 24
S0377-8401(14)00198-9 http://dx.doi.org/doi:10.1016/j.anifeedsci.2014.06.012 ANIFEE 13106
To appear in:
Animal
Received date: Revised date: Accepted date:
29-1-2014 20-6-2014 21-6-2014
Feed
Science
and
Technology
Please cite this article as: Kim, K.H., Ingale, S.L., Kim, J.S., Lee, S.H., Lee, J.H., Kwon, I.K., Chae, B.J.,Bacteriophage and probiotics both enhance the performance of growing pigs but bacteriophage are more effective, Animal Feed Science and Technology (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.06.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Bacteriophage and probiotics both enhance the performance of growing pigs but
2
bacteriophage are more effective†
3 K.H. Kim a, 1, S.L. Ingale a, 1, J.S. Kim a, S.H. Lee a, J.H. Lee b, I.K. Kwon a, B.J. Chae a,*
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4 5 6
a
7
of Korea b
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CTC Bio, Inc., Seoul, 138-858, Republic of Korea
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9
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8
College of Animal Life Sciences, Kangwon National University, Chuncheon, 200-701, Republic
10
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11
Abbreviations: ADFI, average daily feed intake; ADG, average daily gain; apparent total tract
13
digestibility; BW, body weight; CP, crude protein; DM, dry matter; G:F, gain:feed; GE, gross
14
energy; NC, negative control; PC, positive control; TAB, total anaerobic bacteria;
15
________________________
17
†
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This study was supported by CTC Bio, Inc. and the Institute of Animal Resources at
Kangwon National University, Chuncheon, Republic of Korea.
18
1
These authors contributed equally to this work.
19
*
Corresponding author: Tel.:+82 33 250 8616; Fax: +82 33 244 4946
20
E-mail address: [email protected] (B.J. Chae)
21
1 Page 1 of 24
ABSTRACT
22
Two experiments were conducted to determine the effects of dietary supplementation with
23
bacteriophage, probiotics and their combination on growth performance, apparent total tract
24
digestibility (ATTD), fecal bacterial populations and serum immunoglobulins in growing pigs. In
25
both experiments, 200 barrows (Landrace × Yorkshire × Duroc) were randomly allotted to 4
26
treatments on the basis of BW. There were 5 replicate pens in each treatment with 10 pigs per
27
pen. Experimental diets were fed in meal form for 35 d. In Exp. 1, dietary treatments included
28
basal diet supplemented with 0 (control diet without any antimicrobials), 0.5, 1.0 and 1.5 g/kg
29
commercial bacteriophage product. Bacteriophage product contained a cocktail of
30
bacteriophages of Salmonella (S. typhimurium, S. enteritidis, S. cholerasuis and S. derby),
31
Staphylococcus aureus, Escherichia coli and Clostridium perfringens types A and C. Dietary
32
increasing levels of bacteriophage linearly improved (P<0.05) the ADG, ADFI and ATTD of
33
DM. At d 35 of the experiment, pigs fed diets supplemented with increasing levels of
34
bacteriophage had greater (linear, P<0.05) fecal TAB, Bifidobacterium spp. and Lactobacillus
35
spp. and fewer (linear, P<0.05) fecal Clostridium spp. and coliforms. Dietary treatments had no
36
effect (P>0.05) on serum immunoglobulin concentrations at d 35 of experiment. In Exp. 2,
37
dietary treatments were basal diet without any antimicrobials (Control) and basal diets
38
supplemented with 3.0 g/kg fermented probiotic product (P), 1.0 g/kg bacteriophage (B) and
39
combination of 1.0 g/kg bacteriophage and 3.0 g/kg fermented probiotic product (BP). Probiotic
40
products used herein contained Lactobacillus acidophilus, Bacillus subtilis and Saccharomyces
41
cerevisiae. Pigs fed the B and BP diets had greater (P<0.05) ADG, ADFI, G:F and ATTD of
42
DM, CP and GE than that of pigs fed the control and P diets. Pigs fed the P diet had greater
43
(P<0.05) ADG, ADFI and ATTD of CP than that of pigs the fed control diet. At d 35, pigs fed
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2 Page 2 of 24
the BP diet had greater (P<0.05) fecal TAB, Bifidobacterium spp. and Lactobacillus spp. and
45
fewer (P<0.05) Clostridium spp. and coliforms than pigs fed the control diet. Also, pigs fed the P
46
and B diets had greater (P<0.05) Lactobacillus spp and fewer (P<0.05) coliforms at d 35 than
47
that of pigs fed the control diet. Dietary treatments had no effect (P>0.05) on serum
48
immunoglobulin concentrations (d 35). The present results suggest that bacteriophages and
49
probiotics both improve different aspects of grower pig’s performance but that bacteriophages
50
are more effective than probiotics and would appear to offer an alternative to antibiotic type
51
growth promoters.
us
cr
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44
an
52
Key words: Bacteriophage, Fecal bacterial populations, Growing pigs, Immunoglobulins,
54
Performance, Probiotics.
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53
1. Introduction
te
56
d
55
In South Korea, use of antibiotics as growth promoters in animal feeds has been forbidden
58
since 2011 (Global Agricultural Information Network, 2011). Therefore, the search continues for
59
non-antibacterial growth promoters that are active in vivo, are fast acting, possess a broad
60
spectrum in activity, do not induce bacterial resistance and subsequently promote growth
61
performance of pigs. A number of research findings on the use of alternatives like probiotics,
62
oligosaccharides, organic acids and antimicrobial peptides to replace antibiotics in feed have
63
been documented with varying success (Choi et al., 2011a; Yan et al., 2011; Yoon et al., 2012,
64
2013; Lee et al., 2014). In this context, bacteriophages are believed to be an ideal candidate, due
65
to their natural antibacterial properties (Jamalludeen et al., 2009; Yan et al., 2012; Wang et al.,
66
2013).
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3 Page 3 of 24
67
Bacteriophages are obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery (McGrath et al., 2004). Bacteriophages are
69
amongst the most abundant living entities on earth playing important roles in maintaining the
70
natural abundance and distribution of microorganisms (Sulakvelidze, 2011) and have been used
71
to both prevent and treat bacterial diseases in human and animals. Most of the previous studies
72
on bacteriophages evaluated their therapeutic effects on disease challenged pigs (Barrow, 2001;
73
Jamalludeen et al., 2009; Wall et al., 2010) and poultry (Huff et al., 2002; Toro et al., 2005;
74
Atterbury et al., 2007). Previous studies with dietary supplementation of anti- Salmonella
75
bacteriophage reported improved performance and reduced bacterial shedding in growing pigs
76
(Gebru et al., 2010; Yan et al., 2012). Recently it has been reported that supplementation with
77
bacteriophages to laying hens diets resulted in greater feed efficiency, egg production and
78
improved excreta microbiota (Zhao et al., 2012; Wang et al., 2013). Previous studies in the
79
authors laboratory reported that a multimicrobe probiotic products had potential to improve the
80
performance and gut health and could be used as an alternative to antibiotics growth promoters
81
in pigs and broilers (Choi et al., 2011b; Kim et al., 2012). The present study was designed to
82
investigate the effects of dietary supplementation with bacteriophage, probiotics and
83
combination of bacteriophage and probiotics on growth performance, ATTD of nutrients, fecal
84
bacterial populations and serum immunoglobulins of growing pigs.
86
cr
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2. Materials and methods
87
The project underwent proper ethical standards and the experiments were approved by the
88
Institutional Animal Care and Use Committee of Kangwon National University, Chuncheon,
89
Republic of Korea. These experiments were conducted at the facility of Kangwon National
4 Page 4 of 24
90
University farm and the pigs (Landrace × Yorkshire × Duroc) were housed in partially slotted
91
and concrete floor pens with a pen size of 2.80 m × 5.00 m. All pens were equipped with a self-
92
feeder and nipple drinker to allow ad libitum access to feed and water.
95
2.1. Bacteriophage
Bacteriophage product was obtained from a commercial feed company (CTC Bio, Inc., Seoul,
cr
94
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93
Republic of Korea). In short, the product contained a cocktail of bacteriophages of Salmonella
97
(S. typhimurium, S. enteritidis, S. cholerasuis and S. derby), Staphylococcus aureus, Escherichia
98
coli (k88, k99 and f41) and Clostridium perfringens types A and C. The product contained 109
99
plaque-forming units (pfu)/g bacteriophages.
d
102
2.2. Preparation of probiotic product
Lactobacillus acidophilus isolated from faces of weaned pigs, Bacillus subtilis isolated from
te
101
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100
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natto (fermented soybeans), and Saccharomyces cerevisiae isolated from koji (malted wheat)
104
were maintained in the laboratory as stock culture. A culture broth (CB) medium containing 60.0
105
ml corn steep liquor, 40.0 ml molasses, 3.0 g/l yeast extract, 5.0 g/l KH2PO4 and 2.5 g/l K2HPO4
106
in distilled water was prepared and autoclaved before being used.
107
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Two litres of autoclaved CB were inoculated with 2.0 mL of culture of each microbe
108
separately and subjected to fermentation for 48 h. L. acidophilus and B. subtilis were incubated
109
at 37 oC at pH 7.0, whereas S. cerevisiae was incubated at 32 oC at pH 4.0. The microbes grown
110
on CB were directly sprayed on corn-soybean meal (1:1) followed by drying at 40 oC for 72 h.
111
The microbes grown on CB were used as starter and pasteurized corn: soybean meal (1:1) was
112
used as the substrate for carrying out fermentation as described previously by Shim et al. (2010).
5 Page 5 of 24
113
Then the substrates (13.0 kg) were inoculated with 2.0 litres of starter and fermented for 7 d at 32
114
o
115
72 h and mixed to obtain the fermented probiotic product. The counts of L. acidophilus, B.
116
subtilis and S. cerevisiae in fermented probiotic product were 4.0 × 108, 4.8 × 109 and 1.0 × 104
117
cfu/g respectively.
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120
2.3. Animals, housing and treatments
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In Exp. 1, 200 barrows (average initial BW: 50.9 ± 0.530 kg) were randomly allotted to 4 treatments on the basis of initial BW. There were 5 replicate pens in each treatment with 10 pigs
122
per pen. Dietary treatments consisted of a basal diet supplemented with 0 (control diet without
123
any antimicrobial), 0.5, 1.0 and 1.5 g/kg commercial bacteriophage product (109 pfu/g). In Exp.
124
2, 200 barrows (average initial BW: 50.5 ± 0.928 kg) were randomly allotted to 4 treatments on
125
the basis of initial BW. There were 5 replicate pens in each treatment with 10 pigs per pen.
126
Dietary treatments consisted of a basal diet without any antimicrobial (Control), basal diet
127
supplemented with 1.0 g/kg bacteriophage (B; 109 pfu/g), basal diet supplemented with 3.0 g/kg
128
fermented probiotic product (P) and basal diet supplemented with combination of 1.0 g/kg
129
bacteriophage and 3.0 g/kg fermented probiotic product (BP). Fermented probiotic products used
130
herein contained Lactobacillus acidophilus, Bacillus subtilis and Saccharomyces cerevisiae.
131
Corn was replaced with bacteriophage (Exp. 1 and 2), probiotics and combination of
132
bacteriophage and fermented probiotic product (Exp. 2) on an equal percentage basis. In both
133
experiments, all diets met or exceeded the nutrient requirements as suggested by NRC (1998)
134
and fed in meal form for 35 d (Tables 1 and 2).
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136 137
2.4. Experimental procedures, measurements, and analyses Pigs were weighed individually, and feed consumption was calculated at the end of each experiment to calculate ADG, ADFI and G:F. To evaluate the effects of dietary treatments on the
139
ATTD of energy and nutrients, 2.50 g/kg chromic oxide (an inert indigestible indicator) was
140
included in each diet from d 28 to 35 of each experiment. Fecal grab samples were collected
141
from the floor of each pen during last 4 days of each experiment to determine the ATTD of DM,
142
GE and CP. The fecal samples were pooled within pen and dried in a forced air oven at 60°C for
143
72 h, and ground in a Wiley mill (Thomas Model 4 Wiley Mill, Thomas Scientific, Swedesboro,
144
NJ) using a 1-mm screen and used for chemical analysis.
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At d 35 of both experiments, fresh fecal samples were collected from 2 pigs in each pen and used for measuring fecal bacterial populations. The samples collected for bacterial population
147
analysis were immediately placed on ice until analyses were conducted later on the
148
corresponding day. On d 35 of each experiment, a 10.0-mL blood sample was collected by
149
jugular vein puncture from 2 randomly selected pigs in each pen using a disposable vacutainer
150
tube without anticoagulant (Becton Dickinson, Franklin, NJ). After centrifugation (3,000 × g for
151
15 min at 4°C), serum samples were separated and stored at -20°C and later analyzed for
152
concentrations serum immunoglobulins (IgG, IgA and IgM).
154 155
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2.5. Chemical and microbial analyses Experimental diets and excreta samples were analyzed in triplicate for DM (Method 930.15)
156
and CP (Method 990.03) using AOAC (2007) methods. Gross energy of diets and feces were
157
measured by a bomb calorimeter (Model 1261, Parr Instrument Co., Moline, IL), and chromium
158
concentration was determined with an automated spectrophotometer (Jasco V-650; Jasco Corp.,
7 Page 7 of 24
159
Tokyo, Japan) according to the procedure of Fenton and Fenton (1979). Serum IgG, IgA and
160
IgM were determined using radial immune-diffusion kit (Tripple J farm, Bellingham, WA,
161
USA). The microbiological assay of fecal samples was carried out by culturing in different media as
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suggested by Choi et al. (2011a). For the determination of total anaerobic bacteria (Tryptic soy
164
agar), Lactobacillus spp. (using MRS agar + 0.200 g/l NaN3 + 0.500 g/l L-cystine hydrochloride
165
monohydrate), Bifidobacterium spp. (MRS-NPNL: MRS agar + nalidixic acid, paromomycin +
166
neomycin sulphate + lithium chloride), Clostridium spp. (TSC agar) and coliforms (violet red
167
bile agar) were used. The microbiological assay of potential probiotic products was also carried
168
out by culturing technique. Lactobacillus acidophilus was enumerated using MRS agar + 0.200
169
g/l NaN3 + 0.500 g/l L-cystine hydrochloride monohydrate, B. subtilis by using plate count agar,
170
S. cerevisiae by potato dextrose agar. The anaerobic conditions during the assay of total
171
anaerobic bacteria and Clostridium spp. were created by using gas pak anaerobic system (BBL,
172
No. 260678, Difco, Detroit, MI). The tryptic soy agar (No. 236950), MRS agar (No. 288130),
173
violet red bile agar (No. 216695), plate count agar (No. 247940), and potato dextrose agar (No.
174
213400) used were purchased from Difco Laboratories (Detroit, MI), and TSC agar (CM0589)
175
was purchased from Oxoid (Hampshire, UK). The bacterial concentrations were transformed
176
(log) before statistical analysis.
178 179
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2.6. Statistical Analysis In Exp. 1, statistical analysis was conducted using the GLM procedure of SAS (SAS Institute
180
Inc., Cary, NC) as a randomized complete block design. Orthogonal polynomials were used to
181
evaluate linear and quadratic effects of dietary bacteriophage supplementation (0, 0.5, 1.0 and
8 Page 8 of 24
1.5 g/kg). In Exp. 2, statistical analysis was conducted using the one way ANOVA procedure
183
(SAS Institute Inc., Cary, NC), and when significant differences were identified among treatment
184
means, they were separated using Tukey’s Honestly Significant Difference test. In both
185
experiments, the pen was used as the experimental unit for all analysis of all the parameters.
186
Probability values of ≤0.05 were considered as significant in both experiments.
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3. Results
189
3.1. Growth performance
In experiment 1, ADG and ADFI increased linearly (P<0.05) with increasing dietary
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bacteriophage supplementation but there was no treatment effect on F: G (Table 3). In
192
experiment 2, pigs fed the B and BP diets exhibited similar performance and had significantly
193
better ADG, ADFI and F: G than those on the other two treatments. Pigs on the P treatment also
194
grew faster and ate more than their control counterparts (Table 4).
197
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3.2. Apparent total tract digestibility
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In experiment 1, ATTD of DM increased linearly (P<0.05) with increasing dietary
198
bacteriophage supplementation but there was no treatment effect on ATTD of CP and GE (Table
199
3). In experiment 2, pigs fed the B and BP diets exhibited similar ATTD of nutrients and had
200
significantly greater (P<0.05) ATTD of DM, CP and GE than those on the other two treatments.
201
Pigs fed the control and P diets exhibited similar ATTD of all nutrients.
202 203
3.3. Fecal bacterial populations
9 Page 9 of 24
In experiment 1, at d 35, pigs fed diets supplemented with increasing levels of bacteriophage
205
had greater (linear, P<0.05; Table 5) fecal TAB, Bifidobacterium spp. and Lactobacillus spp. and
206
fewer (linear, P<0.05) fecal Clostridium spp. and coliforms. In experiment 2, pigs fed the BP
207
diets had greater (P<0.05; Table 6) fecal TAB, Bifidobacterium spp. and Lactobacillus spp. and
208
fewer (P<0.05) Clostridium spp. and coliforms than that of pigs fed the control diet. Pigs fed the
209
P and B diets had greater (P<0.05) Lactobacillus spp. and fewer (P<0.05) coliforms than that of
210
pigs fed the control diet. Moreover, pigs fed the BP diets had greater (P<0.05) Lactobacillus spp.
211
and fewer (P<0.05) coliforms than that of pigs fed the B diet.
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At d 35 of experiment 1 and 2, serum IgG, IgA and IgM levels averaged 6.53, 0.417 and
215
0.956 respectively. They were similar in both experiments and not significantly affected by the
216
treatments investigated
4. Discussion
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Altering and enhancing normal gut bacterial populations by targeting intestinal pathogens
220
through nonantibiotic approaches can improve the gut health, immunity and performance of pigs
221
and poultry (Choi et al., 2011 b; Sen et al., 2012; Jo et al., 2012; Kim et al., 2012; Yoon et al.,
222
2012). Among various nonantibiotic alternatives, bacteriophages have received attention due to
223
their natural antimicrobial properties (Gebru et al., 2010; Wang et al., 2013).
224
Results obtained in the current study indicated that dietary supplementation with probiotics
225
and bacteriophages but particularly with bacteriophages has the potential to improve the
226
performance of growing pigs. Our results are in agreement with Gebru et al. (2010), who
10 Page 10 of 24
observed improvement in overall ADG and G:F of Salmonella challenged pigs fed diets
228
supplemented with 3× 109 pfu/kg diet Salmonella typhimurium bacteriophage. A recent study in
229
author’s laboratory also reported that dietary supplementation with a bacteriophage cocktail
230
improved the growth performance of weanling pigs (Kim et al., 2013). In contrast Yan et al.
231
(2012) reported that dietary supplementation with anti-Salmonella bacteriophage had no effect
232
on ADG and G:F of growing pigs. The differences between experiments are likely associated
233
with differences in the level and type of bacteriophage investigated, health status within herds,
234
farm hygiene, diets composition, feed forms and interactions with other dietary feed additives.
235
The results of our second study confirmed the growth promoting potential of the bacteriophage
236
product and showed there were no synergistic effects of combining the probiotics with the
237
bacteriophages. This suggests there was no additional benefit of addition of probiotic product to
238
bacteriophage diets of growing pigs. In contrast Kim et al. (2013) working with weanling pigs
239
reported that a combination of probiotics and bacteriophages supported better growth
240
performance than bacteriophage alone.
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In the preset study, pigs fed diets supplemented with a bacteriophage cocktail, probiotics and
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242
both had greater ATTD of DM (Exp. 1 and 2), GE and CP (Exp. 2). In agreement with present
243
results, Yan et al. (2012) reported improved ATTD of DM, nitrogen and energy of growing pigs
244
fed diets supplemented with 0.5 g/kg diet anti-Salmonella bacteriophage. The results of
245
Experiment 2 are in agreement with Kim et al. (2013), who observed greater ATTD of DM, CP
246
and GE in weaning pigs fed diet supplemented with combination of bacteriophage and
247
probiotics. The improved growth rate elicited in both experiments and in feed efficiency in
248
experiment 2 by bacteriophage supplementation was likely associated with the concomitant
249
changes in nutrient digestibility and in the balance of gut microbiota. The probiotic supplement
11 Page 11 of 24
250
elicited small changes in the gut microbiota, had no effect on nutrient digestibility and
251
marginally improved rate compared to the control.
252
The reduced populations of Clostridium spp. and coliform and increased Bifidobacterium and Lactobacillus found in pigs fed the diet supplemented with bacteriophage (Exp. 1) and a
254
combination of bacteriophage and probiotics (Exp. 2) observed in the present study are in
255
agreement with the results of other studies involving growing pigs, broilers and laying hens fed
256
diets supplemented with anti-Salmonella bacteriophage (Yan et al., 2012; Zhao et al., 2012;
257
Wang et al., 2013). Gebru et al. (2010) observed reduced bacterial shedding score in Salmonella
258
typhimurium challenged pigs fed diets supplemented with bacteriophage. It should be noted
259
however that the changes in bacterial populations reported here for pigs fed diets supplemented
260
with bacteriophage and/or probiotics were not large and unlikely to contribute entirely to the
261
improvement in growth performance elicited by the technologies and in particular by the
262
bacteriophage product. Further research is required to better understand the mechanisms behind
263
the quite significant improvement in performance associated with supplementing the diets with
264
bacteriophages.
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265
In the current study, supplementation of growing pigs diet with bacteriophage, probiotics or
266
their combination had no effects on serum immunoglobulins. These findings are consistent with
267
results of Kim et al. (2013), who reported no effects of supplementation of bacteriophage,
268
probiotics or their combination to weanling pig’s diet. Previous studies with dietary
269
supplementation with probiotics (Bacillus or Lactobacillus spp. alone or in combination)
270
reported enhanced immune response of the piglets (European Food Safety Authority, 2010; Lee
271
et al., 2014).
272
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273 274
5. Conclusions The present results suggest that bacteriophages and probiotics both improve different aspects of grower pig’s performance but that bacteriophages are more effective than probiotics and
276
would appear to offer an alternative to antibiotic type growth promoters.
277
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References
279
AOAC, 2007. Official Methods of Analysis of the Association of Official Analytical Chemists
us
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Atterbury, R.J., Van Bergen, M.A., Ortiz, F., Lovell, M.A., Harris, J.A., De Boer, A., Wagenaar,
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J.A., Allen, V.M., Barrow, P.A., 2007. Bacteriophage therapy to reduce Salmonella
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Barrow, P., 2001. The use of bacteriophages for treatment and prevention of bacterial disease in
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Choi, J.Y., Kim, J.S., Ingale, S.L., Kim, K.H., Shinde, P.L., Kwon, I.K., Chae, B.J., 2011a. Effect of potential multimicrobe probiotic product processed by high drying temperature and
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substrate fermentation and antibiotics in weaning pigs. Livest. Sci. 138, 144–151.
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European Food Safety Authority, 2010. Scientific opinion on the safety and efficacy of Calsporin (Bacillus subtilis) as feed additive for piglets. The EFSA J. 8, 1426-1437. Fenton, T.W., Fenton, M., 1979. An improved method for chromic oxide determination in feed and feces. Can. J. Anim. Sci. 59, 631-634.
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Gebru, E., Lee, J.S, Son, J.C., Yang, S.Y., Shin, S.A., Kim, B., Kim, M.K., Park, S.C., 2010. Effect of probiotics, bacteriophage, or organic acid supplemented feeds or fermented soybean
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Global Agricultural Information Network: Korea Phases Out Antibiotic Usage in Compound
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Huff, W.E., Huff, G.R., Rath, N.C., Balog, J.M., Donoghue, A.M., 2002. Prevention of
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Jamalludeen, N., Johnson, R.P., Shewen, P.E., Gyles, C.L., 2009. Evaluation of bacteriphages for
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2012. Effects of exogenous enzyme supplementation to corn-soybean based or complex diets
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Kim, J.S., Ingale, S.L., Kim, Y.W., Kim, K.H., Sen, S., Ryu, M.H., Lohakare, J.D., Kwon, I.K.,
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Chae, B.J., 2012. Effect of supplementation of multi-microbe probiotic product on growth
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performance, apparent digestibility, cecal microbiota and small intestinal morphology of
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Kim, K.H., Ingale, S.L., Lee, S.H., Kim, J.S., Noh, H.S., Lee, J.H., Kwon, I.K., Chae, B.J., 2013. Effects of dietary supplementation with bacteriophage on the growth performance, nutrient
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digestibility, fecal and intestinal microflora and intestinal morphology of weanling pigs. In:
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Lee, S.H., Ingale, S.L., Kim, J.S., Kim, K H., Lokhande, A., Kim, E.K., Kwon, I.K., Kim, Y.H., Chae, B.J., 2014. Effects of dietary supplementation with Bacillus subtilis LS 1-2
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fermentation biomass on growth performance, nutrient digestibility, cecal microflora and
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intestinal morphology of weanling pig. Anim. Feed Sci. Technol. 188, 102-110.
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Ryu, M.H., Kwon, I.K., Chae, B.J., 2012. Effect of supplementation of Bacillus subtilis LS
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Shim, Y.H., Shinde, P.L., Choi, J.Y., Kim, J.S., Seo, D.K., Park, J.I., Chae, B.J., Kwon, I.K.,
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Sulakvelidze, A., 2011. Bacteriophage: A new journal for the most ubiquitous organisms on
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infected chickens. Avian Dis. 49, 118-124. Wall, S.K., Zhang, J., Rostagno, M.H., Ebner, P.D., 2010. Phage therapy to reduce pre-
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344
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characteristics and excreta microbial shedding in broilers. Asian-Aust. J. Anim. Sci. 26, 573-
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Yan, L., Meng, Q.W., Kim, I.H., 2011. The effects of an herb extract mixture on growth
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performance, nutrient digestibility, intestinal and fecal microflora and intestinal morphology
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in weanling pigs. Anim. Feed Sci. Technol. 177, 98-107.
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17 Page 17 of 24
366 Table 1 Ingredient and chemical composition of experimental diets (as-fed basis; Exp. 1) 0.5
1.0
462.6
462.1
461.6
461.1
Wheat
110
110
110
110
Soybean meal (450 g/kg)
340
340
340
340
Animal fat
45.0
45.0
45.0
45.0
Molasses
10.0
10.0
10.0
10.0
Choline chloride (500 g/kg)
0.200
0.200
0.200
0.200
9.00
9.00
9.00
9.00
3.20
3.20
3.20
6.00
6.00
6.00
8.00
8.00
8.00
L-Lysine
(230 g/kg) (880 g/kg)
3.20 6.00
Dicalcium phosphate
8.00
Salt
3.00
3.00
3.00
3.00
Vitamin premix
1.50
1.50
1.50
1.50
Mineral premix2
1.50
1.50
1.50
1.50
-
0.500
1.00
1.50
13.8
13.8
13.8
13.8
Crude protein, g/kg
200
200
200
200
Lysine, g/kg
10.0
10.0
10.0
10.0
Methionine + Cysteine, g/kg
5.90
5.90
5.90
5.90
Calcium, g/kg
7.00
7.00
7.00
7.00
Available phosphorus, g/kg
3.00
3.00
3.00
3.00
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Limestone
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DL-Methionine
Bacteriophage Chemical composition
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Ingredient, g/kg
1.5
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Bacteriophage supplementation (g/kg)
Supplied per kilogram of diet: 16,000 IU vitamin A, 3,000 IU vitamin D3, 40 IU vitamin E, 5.0 mg vitamin K3, 5.0 mg vitamin B1, 20 mg vitamin B2, 4 mg vitamin B6, 0.08 mg vitamin B12, 40 mg pantothenic acid, 75 mg niacin, 0.15 mg biotin, 0.65 mg folic acid. 2 Supplied per kilogram of diet: 45 mg Fe, 0.25 mg Co, 50 mg Cu, 15 mg Mn, 25 mg Zn, 0.35 mg I, 0.13 mg Se. 367 368 369
18 Page 18 of 24
Table 2 Ingredient and chemical composition of experimental diets (as-fed basis; Exp. 2) Treatments1
Control
P
B
BP
462.6
459.6
461.6
458.6
Wheat
110
110
110
110
Soybean meal (450 g/kg)
340
340
340
340
Animal fat
45.0
45.0
45.0
45.0
Molasses
10.0
10.0
10.0
10.0
Choline chloride (500 g/kg)
0.200
0.200
0.200
0.200
9.00
9.00
9.00
9.00
3.20
3.20
3.20
6.00
6.00
6.00
8.00
8.00
8.00
(230 g/kg)
DL-Methionine
(880 g/kg)
3.20
3.00
3.00
3.00
3.00
2
1.50
1.50
1.50
1.50
3
1.50
1.50
1.50
1.50
-
3.00
-
3.00
-
-
1.00
1.00
ME, MJ/kg
13.8
13.8
13.8
13.8
Crude protein, g/kg
200
200
200
200
Lysine, g/kg
10.0
10.0
10.0
10.0
Methionine + Cysteine, g/kg
5.90
5.90
5.90
5.90
Calcium, g/kg
7.00
7.00
7.00
7.00
Available phosphorus, g/kg
3.00
3.00
3.00
3.00
6.00
Dicalcium phosphate
8.00
Salt
M
Limestone
Vitamin premix
d
Mineral premix Bacteriophage
Ac ce p
Chemical composition
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Probiotics
1
cr
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L-Lysine
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Corn
ip t
Ingredient, %
Dietary treatments were basal diet without any antimicrobial (Control), basal diet supplemented with 1.0 g/kg bacteriophage cocktail (B), basal diet supplemented with 3.0 g/kg probiotic (P) and basal diet supplemented with combination of 1.0 g/kg bacteriophage and 3.0 g/kg probiotics (BP) 2 Supplied per kilogram of diet: 16,000 IU vitamin A, 3,000 IU vitamin D3, 40 IU vitamin E, 5.0 mg vitamin K3, 5.0 mg vitamin B1, 20 mg vitamin B2, 4 mg vitamin B6, 0.08 mg vitamin B12, 40 mg pantothenic acid, 75 mg niacin, 0.15 mg biotin, 0.65 mg folic acid. 3 Supplied per kilogram of diet: 45 mg Fe, 0.25 mg Co, 50 mg Cu, 15 mg Mn, 25 mg Zn, 0.35 mg I, 0.13 mg Se. 370 19 Page 19 of 24
370 Table 3 Effects of dietary supplementation with a bacteriophage cocktail on the performance and apparent total tract digestibility of nutrients in pigs offered feed ad libitum for 35 days commencing at 50 kg (Exp. 1) x 1.0
1.5
SEM
737
764
815
822
Average daily feed intake, g
2,079
2,129
2,240
2,222
Feed:Gain
2.82
2.79
2.75
19. 5
0.001
0.592
us
Quadratic
61.2
0.043
0.544
0.103
0.318
0.941
0.852
0.012
0.984
2.71
Dry matter
0.841
0.846
0.025
Gross energy
0.874
0.875
0.875
0.879
0.048
0.458
0.754
Crude protein
0.831
0.832
0.838
0.845
0.046
0.088
0.371
0.847
d
M
Apparent total tract digestibility of nutrients
te
Data represents mean based on 5 replicate pens per treatment
Ac ce p
371 372
Linear
an
Average daily gain, g
ip t
0.5
Growth performance
x
P-values
0
cr
Bacteriophage supplementation (g/kg)
20 Page 20 of 24
372 Table 4. Effects of supplementing diet with a probiotics (P), a bacteriophage cocktail (B) and both (BP) on the performance and apparent total tract digestibility of nutrients in pigs offered feed ad libitum for 35 days commencing at 50 kg (Exp. 2). x Control
B
P
PB
a
Average daily feed intake, g
2,032
Feed:Gain
2.77
c
a
a
b
0.835
Gross energy
0.871
Crude protein
0.822
a
0.839
M
Dry matter
b
826
b
2,089
a
2.72
an
2.63
a
768
2,121
Apparent total tract digestibility of nutrients
b
a
0.874
b
d
x
b
809
us
c
733
cr
Growth performance Average daily gain, g
a
0.829
SEM
ip t
Item y
b
0.836
b
0.871
b
0.825
a
2,137
b
2.59
a
0.840
a
0.876
a
0.832
17.8 46.9 0.052
0.057 0.046 0.053
Ac ce p
373
te
Data represents mean based on 5 replicate pens per treatment Dietary treatments were basal diet without any antimicrobial (Control), basal diet supplemented with 1.0 g/kg bacteriophage cocktail (B), basal diet supplemented with 3.0 g/kg probiotic (P) and basal diet supplemented with combination of 1.0 g/kg bacteriophage and 3.0 g/kg probiotics (BP) abc Means with different superscripts in the same row are different (P<0.05). y
21 Page 21 of 24
373 Table 5. Effects of dietary supplementation with a bacteriophage cocktail on fecal microbiota (log10 CFU/g; at d 35) of growing pigs offered feed ad libitum for 35 days commencing at 50 kg (Exp. 1) x 1.0
1.5
SEM
7.92
8.22
Bifidobacterium spp.
8.92
9.37
9.77
Lactobacillus spp.
8.56
8.67
Clostridium spp.
8.14
7.96
Coliforms
8.57
0.228
9.75
Quadratic
0.050
0.810
0.252
0.020
0.360
8.98
0.194
0.071
0.620
7.61
7.50
0.159
0.006
0.829
7.77
7.84
0.213
0.012
0.340
M
9.06
d
8.22
8.33
us
7.69
an
Total anaerobic bacteria
cr
Linear
Data represents mean based on 5 replicate pens per treatment
te
374 375
0.5
Ac ce p
x
0
ip t
P-values
Bacteriophage supplementation (g/kg)
22 Page 22 of 24
375
b
Bifidobacterium spp.
8.78
Lactobacillus spp.
8.27
Clostridium spp.
8.40
Coliforms
8.51
ab
a
7.76
b
8.07
ab
ab
9.42
c
9.18
b
b
8.47
a
ab
7.75
a
c
7.72
x
BP
8.39
ab
7.93
b
8.23
SEM
a
8.29
a
us
7.34
P
an
Total anaerobic bacteria
B
cr
Control
M
Item
ip t
Table 6. Effects of supplementing diet with a probiotics (P), a bacteriophage cocktail (B) and both (BP) on fecal microbiota (log10 CFU/g; at d 35) of growing pigs offered feed ad libitum for 35 days commencing at 50 kg (Exp. 2). x,y
9.77
a
8.75
b
7.59
c
7.55
0.161 0.214 0.238 0.177 0.252
Data represents mean based on 5 replicate pens per treatment Dietary treatments were basal diet without any antimicrobial (Control), basal diet supplemented with 1.0 g/kg bacteriophage cocktail (B), basal diet supplemented with 3.0 g/kg probiotic (P) and basal diet supplemented with combination of 1.0 g/kg bacteriophage and 3.0 g/kg probiotics (BP) abc Values with different superscripts in the same row are different (P<0.05).
Ac ce p
376 377
te
d
y
23 Page 23 of 24
te
d
M
an
us
cr
ip t
Highlights Effects of dietary supplementation with bacteriophage, probiotics and both on performance of barrows. Bacteriophages and probiotics both improve different aspects of grower pig’s performance Bacteriophages are more effective than probiotics Bacteriophage would appear to offer an alternative to antibiotic type growth promoters.
Ac ce p
377 378 379 380 381 382 383 384 385
24 Page 24 of 24