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Growth performance, digestibility, gut environment and health status inweaned piglets fed a diet supplementedwith a complex of lactic acid bacteriaalone or in combination with Bacillus subtilis and Saccharomyces boulardii

Hoang Huong Giang a, Tran Quoc Viet b, Brian Ogle c, Jan Erik Lindberg c,⁎a Department of Livestock Production, Ministry of Agriculture and Rural Development, 2 Ngoc Ha Street, Ba Dinh District, Ha Noi City, Vietnamb Department of Animal Nutrition, National Institute of Animal Science, Thuy Phuong Commune, Tu Liem District, Ha Noi City, Vietnamc Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, P.O. Box 7024, SE-750 07 Uppsala, Sweden

a r t i c l e i n f o a b s t r a c t

Article history:Received 29 October 2010Received in revised form 1 September 2011Accepted 2 September 2011

Growth performance, digestibility, gut environment and health status were studied in weanedpiglets (26–28 days of age) fed a diet without (C) or with supplementation of bacteria and ayeast (diets L, LB and LBS). Diet L was supplemented with a lactic acid bacteria (LAB) complex(Enterococcus faecium 6H2, Lactobacillus acidophilus C3, Pediococcus pentosaceusD7, and L. fermentumNC1), diet LB with the LAB complex and Bacillus subtilis H4, and diet LBS with the LAB com-plex, B. subtilis H4 and Saccharomyces boulardii Sb. Piglets fed diets L, LB and LBS had higherfeed intake and daily gain, lower feed conversion ratio, higher ileal and total tract digestibil-ity, and had a lower incidence of diarrhoea and a lower faecal score than piglets fed diet C(Pb0.01). Diets L, LB and LBS increased the intestinal LAB population and organic acid con-centrations, and reduced E. coli counts. Supplementation with B. subtilis H4 and S. boulardiiSb resulted in higher digestibility than when supplementing with the LAB complex alone.Supplementation with S. boulardii Sb resulted in lower incidence of diarrhoea and lower fae-cal score than supplementation with the LAB complex alone or together with B. subtilis H4.The LAB complexes used in the current study had probiotic properties and could be used toimprove the health status of weaned piglets under Vietnamese conditions.

© 2011 Elsevier B.V. All rights reserved.

Keywords:Lactic acid bacteriaBacillusSaccharomycesProbioticPigletsGut environment

1. Introduction

Antimicrobial feed additives are still allowed and used in pigproduction in Vietnam. Moreover, the use of antimicrobials tosick animals without any veterinary prescription and supervi-sion is not uncommon (Boisseau, 2002). As a result, there ishigh risk for uncontrolled contamination of antimicrobial resi-dues in raw pig meat (Nhiem et al., 2006). There is also highrisk for spreading of antimicrobial residues in the environmentfrom the uncontrolled discharge of manure into public sewagesystems, rivers and lakes (Vu et al., 2007). Thus, the currentsituation constitutes a major risk for transfer of antimicrobialresistance between bacteria (Vo et al., 2010) and justifies the

search for alternative ways of improving the health status ofpigs in order to minimise the need to use antimicrobial drugs.

Probiotics can be used as microbial feed additives and mayhave the potential to replace feed antibiotics (Close, 2000).Commercial probiotic products may contain single strains ormulti-strains of lactic acid bacteria (LAB), bacterial sporeformers and yeasts. LAB are part of the natural gut microbiota,and probiotic LAB can adhere to and colonise the host's intes-tines (Blomberg et al., 1993; Spencer and Chesson, 1994), andcan improve the animal health (Cross, 2002). Bacterial sporeformers of the genus Bacillus are commonly found in the gut ofanimals (Hong et al., 2005). Bacillus subtilis were able to growunder the environmental conditions of the intestine and hadpositivemetabolic effects in the gastrointestinal tract of animalsand man (Mazza, 1994). Although Saccharomyces is normallyfound in plants, studies in vitro and in animals have shown

Livestock Science 143 (2012) 132–141

⁎ Corresponding author. Tel.: +46 18 672102; fax: +46 18 672995.E-mail address: jan.erik.lindberg@slu.se (J.E. Lindberg).

1871-1413/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.livsci.2011.09.003

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that Saccharomyces boulardii is active against various pathogensand can stimulate enzyme activities, and may therefore be usedas a probiotic (Czerucka and Rampal, 2002). However, the effi-cacy of probiotics is strain-specific (Weichselbaum, 2009),which implies that each single strain or combination of strainshas to be tested to evaluate the potential benefits.

In the current study, four LAB strains (Enterococcus faecium6H2, Lactobacillus acidophilus C3, Pediococcus pentosaceus D7and Lactobacillus fermentum NC1) and B. subtilis H4, and yeaststrain S. boulardii Sb were assessed on weaned piglets. Theaim of this work was to study the impact of supplementing apiglet diet with different microbial complexes (LAB complex,the LAB complex with addition of Bacillus alone or togetherwith Saccharomyces) on growth performance, nutrient digest-ibility, health status and gut environment of weaned pigletsunder the conditions of Vietnam. We hypothesised that themicrobial complexes would have probiotic effects in weanedpiglets, and that the inclusion of Bacillus and Saccharomyces inthe LAB complex would improve the efficacy compared withsupplementing with the LAB complex alone.

2. Materials and methods

2.1. Animals and design

Thirty litters of 10–12piglets per litter (Landrace xYorkshire)that farrowed within three days from three 600-sow farmslocated in Bavi district, Hanoi City, Vietnam, were identified atbirth. During the pre-weaning period, the health status of allpiglet litters was monitored daily and any medical treatmentperformed was recorded. The piglets were given free access tocreep feed (meal form) without antibiotics or probiotics from15 days of age.

At weaning, at 26–28 days of age, 128 piglets (64 femalesand 64 males) from 16 litters (among the original 30 litters),whose body weights (BW) were on average 7.7±0.9 kg andthat were healthy and had not been treated by antibiotics,were selected for the 35-day trial. All piglets were vaccinatedagainst pasteurellosis, parathyphoid, asthma and hog cholera.

The piglets were randomly divided into four groups, bal-anced for sex, weight and litter origin, and each group wasgiven one of four treatments. There were 4 replicate pens foreach treatment and 8 pigs (4 females and 4 males) in eachpen. The pens had concrete floors with no litter, and each penwas equipped with a feeder and nipple drinker. During the ex-perimental period, feed and water were provided ad libitum.

The trial was divided into 2 periods: the first 2 weeks post-weaning (Per I), and the subsequent 3-week (week 3–5) periodpost-weaning (Per II). On day 14 and 35, four piglets per treat-ment (one from each pen), resulting in a total of 16 piglets ineach period, were killed (euthanized by intra-venous injectionof sodium pentobarbitone, Lethabarb Virbac, Australia with0.5 ml/kg BW) for sampling to determine the ileal and totaltract digestibility, organic acid concentrations and bacterianumbers in different intestinal segments. Thus, for Per II, therewere 7 piglets remaining in each pen.

2.2. Feed and supplementation

The starter diet was in meal form, and consisted of ex-truded maize, extruded rice, soybean meal, soybean protein

concentrate (53% CP), milk replacer, sweet whey, and avitamin–mineral premix, and was formulated to meet NRC(1998) recommendations (Table 1). The four dietary treatmentsincluded: the basal diet without any addition of probiotic or an-tibiotic (C), the basal dietwith addition of a LAB complex (L), thebasal diet with addition of the LAB complex and B. subtilis H4(LB), and the basal diet with addition of the LAB complex, andB. subtilis H4 and S. boulardii Sb (LBS). The bacteria strains andSaccharomyces were prepared in culture form every three daysand stored in a fridge at 4 °C in separate bottles. The colony den-sities were: B. subtilis H4 (4×1011–8×1011 CFU/ml), S. boulardiiSb (3×1010–9×1010 CFU/ml) and the LAB complex consistedof E. faecium 6H2 (4×109–8×109 CFU/ml), L. acidophilus C3(3×109–7×109 CFU/ml), Pediococcus pentosaceus D7 (1.3×109–8.5×109 CFU/ml), and L. fermentum NC (5×109–7×109 CFU/ml). Each day, the supplements (2 ml of each bacteriastrain, and of Saccharomyces, per kg basal diet) were mixedmanually with a portion of the basal diet prior to feeding.

2.3. Bacteria and yeast sources

E. faecium 6H2, L. acidophilus C3 and P. pentosaceus D7were isolated from ileal digesta from healthy fattening pigs,and L. fermentum NC1 was isolated from a Vietnamese tradi-tional fermented food (nem chua). The LAB strains were se-lected in previous tests based on their resistance in vitro toheat, lowpH, bile salts, and antagonismwith pathogenic bacteriasuch as Salmonella and Escherichia coli (Viet et al., 2006).

B. subtilis H4 was also isolated from gut digesta of healthyfattening pigs, and the yeast strain S. boulardii Sb was obtainedfrom the Vietnam Type Culture Collection, Institute of Microbi-ology and Biotechnology, Vietnam National University Hanoi,Vietnam. They were tested in vitro for resistance to heat, low

Table 1Ingredient and chemical composition of basal diet.

Ingredient composition (%, as fed basis)

Extruded maize 24.38Extruded broken rice 25.00Soybean meal (46% CP) 22.20Soybean protein concentrate (53% CP) 5.00Milk replacer (42% CP) 10.00Sweet whey (11% CP) 10.00Vitamin–mineral premix 0.25Lysine HCl 0.10Methionine 0.05Limestone 1.29Monocalcium phosphate 1.13Salt 0.30Cr2O3 (indicator) 0.30

Chemical composition, % of dry matterCrude protein 23.51Crude fibre 3.29Lysine 1.48Methionine 0.45Methionine+cystine 0.83Threonine 0.96Tryptophan 0.28Calcium 0.98Available phosphorusa 0.49ME, MJ/kg dry mattera 15.00

a Calculated based on Composition and Nutritive Value of Animal Feeds inVietnam (Agricultural Publishing House, Hanoi, Vietnam, 2001).

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pH, bile salts, and enzyme activities (for B. subtilis H4) and an-tagonism with the pathogenic bacteria Salmonella, Shigella andE. coli (for S. boulardii Sb), and the tests revealed that they hadprobiotic potential (Viet et al., 2009).

2.4. Recordings and sampling

The piglets were weighed individually at the beginning ofthe experiment (at weaning), on day 14 and on day 35, andthe feed offered and refused was weighed daily.

The health status of piglets during the first 14 days afterweaning was assessed by faecal consistency scoring using afour-grade system, where 0 corresponded to firm and dry; 1to pasty; 2 to thick and fluid; and 3 to watery (Cupere et al.,1992). The incidence of diarrhoea (%) was calculated as thesum of the total number of diarrhoeal piglets over the perioddivided by the number of piglet days in the period multipliedby 100. The faecal score was calculated as the sum of thediarrhoea score over theperiod divided by thenumber of pigletdays in the period.

Immediately after slaughter, the gastrointestinal tract wasremoved, and the digesta collected from stomach, ileum(1.2 m of intestine before the ileo-caecal ostium), mid-colon(divided into 3 equal parts, with the digesta in the middlepart being collected), and faeceswas collected from the rectum.The samples were stored in separate sterile tubes on ice con-tainers and transported to the laboratory shortly afterwards.

2.5. Nutrient digestibility

The coefficients of apparent digestibility (CAD) of dietarycomponents at the ileum (CIAD) and total tract (CTTAD)were determined using chromium oxide (3 g kg−1 diet) asindigestible marker and calculated according to the followingequation (Sauer et al., 2000):

CADD ¼ 1− DCF × IDð Þ= DCD× IFð Þ

where CADD is the coefficient of apparent digestibility of a die-tary component,DCF is the dietary component concentration indigesta or faeces, ID is the indicator concentration in diet,DCD isthe dietary component concentration in diet, and IF is the indi-cator concentration in digesta or faeces.

2.6. Microbiological determinations

The digesta and faecal samples were dissolved in sterile sa-line (0.9%) in a 1:10 dilution. The stomach content sampleswere diluted further from 10−2 to 10−4 for E. coli counts andfrom 10−4 to 10−6 for total LAB counts. The further dilutionsof the samples of ileal and colon digesta and of faeces takenfrom rectumwere from 10−4 to 10−6 to estimate E. coli countsand were from 10−5 to 10−7 to estimate total LAB population.E. coli was cultured on MacConkey agar (MAC), under aerobiccondition for 24 h at 37 °C, and colonies with smooth, convex,circles, entire edges, and pink colour were counted as E. coli.LAB were cultured on MRS agar (Mann, Rogosa and Sharpe),under anaerobic conditions for 48 h at 37 °C, and all colonieswere counted as LAB.

2.7. Chemical analysis

Samples of feed, ileal digesta and faeces were analysed fordry matter (DM), crude protein (CP) and crude fibre (CF)according to standard AOAC (1990) methods. The chromiumcontent in feed, digesta and faeces was analysed using anatomic absorption spectrophotometer (Spectr AA PerkinElmer™, USA) (National Institute for Occupational Safetyand Health, 1994). Acetic, propionic, butyric and lactic acidin samples from the stomach, ileum, colon and rectum wereanalysed with high-performance liquid chromatography(HPLC Shimadzu, Japan) (Food Components Adhoc Committee,2002).

2.8. Statistical analysis

The datawere analysed statistically using theGLMofMinitabSoftware Version 14.1. Treatment means which showed signifi-cant differences at Pb0.05 were compared using Tukey's pair-wise comparison procedure. The Chi-Square Test was used tocompare the diarrhoea incidence between the treatments.

For data of LAB and E. coli counts (transformed as log10 beforestatistical analysis) and organic acid concentration, the statisticalmodel included main effects (treatments and segments) andtheir interactions, and was as follows:

Yijk ¼ μ þ ai þ bj þ abð Þij þ eijk

where i is segment (stomach, terminal ileum, mid-colon andrectum), j is treatment (C, L, LB and LBS), k is replicate (4 rep-licates); Yijk is LAB or E. coli counts, and concentration of lacticacid or volatile fatty acids in each segment; μ is overall mean; aiis effect of segment; bj is effect of treatment; (ab)ij is interactionbetween segment and treatment; eijk is random error.

3. Results

3.1. Growth performance

The piglets fed diets L, LB and LBS had higher averagedaily feed intake (ADFI), average daily weight again (ADG)and lower feed conversion ratio (FCR) (Pb0.01) than pigletsfed diet C in both experimental periods (Per I and II), andoverall (Table 2).

In Per I, piglets fed diet LBS had higher ADFI than those feddiets L and LB (Pb0.05), while there was no significant differ-ence in ADFI between piglets fed diets L and LB (PN0.05). TheADG of piglets fed diet LBS was higher (Pb0.05) than of pigletsfed diet L, but no differences in ADG were observed betweenpiglets fed diets L and LB (PN0.05) or between piglets fed dietsLB and LBS (PN0.05). Therewas no difference in FCR among pig-lets fed diets L, LB and LBS (PN0.05).

In Per II and overall, no difference in ADFI among pigletsfed diets L, LB and LBSwas observed (PN0.05). However, pigletsfed diets LB and LBS had higher ADG (Pb0.05) and lower FCR(Pb0.05) than piglets fed diet L, while no differences werefound between piglets fed diets LB and LBS (PN0.05).

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3.2. Nutrient digestibility

The CIAD and CTTAD of organic matter (OM) and CP werehigher (Pb0.01) in piglets fed diets L, LB and LBS than in pigletsfed diet C in both experimental periods (Table 3). However,there was no difference in the CIAD and CTTAD of CF amongthe treatment groups (PN0.05).

In Per I, the CIAD of CP was higher (Pb0.05) in piglets feddiet LBS than in those fed diet L, while there were no differ-ences between piglets fed diets L and LB (PN0.05) or betweenpiglets fed diets LB and LBS (PN0.05). However, the CIAD ofOM and the CTTAD of CP and OM were higher (Pb0.05) inpiglets fed diets LB and LBS than in piglets fed diet L, whilethere were no differences between piglets fed diets LB andLBS (PN0.05).

In Per II, the CIAD and CTTAD of OM and CP were higher inpiglets fed diets LB and LBS than in those fed diet L (Pb0.05),while there were no differences between piglets fed diets LBand LBS (PN0.05).

3.3. Diarrhoea

None of the pigs had a diarrhoea score of 3 (watery faeces)during the first two weeks post-weaning. However, some pig-lets in all the treatment groups showed diarrhoea symptoms(Table 4). During the first week post-weaning, none of thethree microbial complexes tested (L, LB and LBS) had an effecton the incidence of diarrhoea compared with diet C (PN0.05).There were lower faecal scores (Pb0.05) in piglets fed dietLBS than in piglets fed diet C (Pb0.05).

During the second week post-weaning and overall, all pigletsgiven microbial complexes had a lower incidence of diar-rhoea and a lower faecal scores than piglets fed the diet C(Pb0.01). Diet LBS was more effective (Pb0.05) than diets Land LB, while there was no difference between diets L andLB (PN0.05).

3.4. Gut bacterial counts

No interactionwas found between treatment and intestinalsegment for bacterial counts (Tables 5 and 6). The counts ofLAB and E. coliwere lower in stomach than in the other intesti-nal segments on all diets (Pb0.05) and increased along thedigestive tract at both day 14 and 35. Across all intestinal seg-ments, the average LAB count was higher (Pb0.01) in pigletsfed diets L, LB and LBS than in piglets fed diet C on day 14 and35.

On day 14, higher (Pb0.05) LAB counts were found in allfour segments (stomach, ileum, colon and rectum) in pigletsfed diets L, LB and LBS than in piglets fed diet C. On day 14,LAB counts in the colon of piglets fed diet L was higher(Pb0.05) than in piglets fed diet LBS.

On day 35, LAB countswere higher (Pb0.05) in the stomach,ileum and colon of piglets fed diets L, LB and LBS than in pigletsfed diet C, while therewere no differences (PN0.05) among pig-lets fed diet L, LB and LBS (PN0.05). The LAB count in the rectumwas higher (Pb0.05) in piglets fed diet L than in piglets fed diet

Table 2Effects of lactic acid bacteria (LAB) and Bacillus and Saccharomyces com-plexes on average daily feed intake (ADFI, g/pig/day), average daily weightgain (ADG, g/pig/day), and feed conversion ratio (FCR, kg feed/kg gain) ofweaned pigs*.

Item Treatment SEM P value

C L LB LBS

Number of pigs 32 32 32 32Body weight(at weaning), kg

7.65 7.72 7.80 7.60 0.08 0.312

0–2 weeksADFI 293a 323b 324b 335c 2.07 b0.001ADG 223a 262b 274bc 281c 3.78 b0.001FCR 1.31a 1.23b 1.18b 1.19b 0.02 0.001

3–5 weeksNumber of pigs 28 28 28 28

ADFI 709a 779b 765b 759b 7.36 b0.001ADG 332a 398b 418c 419c 3.44 b0.001FCR 2.13a 1.96b 1.83c 1.81c 0.02 b0.001

OverallADFI 543a 597b 589b 589b 4.76 b0.001ADG 291a 345b 362c 366c 2.10 b0.001FCR 1.87a 1.73b 1.63c 1.61c 0.02 b0.001

* Piglets were weaned at 26–28 days of age.a, b, c Means within a row with different superscripts are significantlydifferent (Pb0.05).C: Basal diet without antibiotics or probiotics.L: Basal diet+LAB complex (Enterococcus faecium 6H2, Lactobacillusacidophilus C3, Pediococcus pentosaceus D7 and Lactobacillus fermentumNC1).LB: Basal diet+LAB complex+Bacillus subtilis H4.LBS: Basal diet+LAB complex+Bacillus subtilis H4+Saccharomyces boulardiiSb.

Table 3Effects of lactic acid bacteria (LAB) and Bacillus and Saccharomyces com-plexes on the coefficient of ileal apparent digestibility (CIAD) and the coeffi-cient of total tract apparent digestibility (CTTAD) in weaned pigs1.

Items Treatment SEM P value

C L LB LBS

Period 12

CIADCrude protein 0.71a 0.79b 0.81bc 0.83c 0.005 b0.001Organic matter 0.78a 0.81b 0.83c 0.84c 0.004 b0.001Crude fibre 0.59 0.61 0.60 0.61 0.010 0.604

CTTADCrude protein 0.83a 0.87b 0.89c 0.90c 0.003 b0.001Organic matter 0.83a 0.85b 0.87c 0.88c 0.003 b0.001Crude fibre 0.65 0.65 0.66 0.66 0.007 0.292

Period 23

CIADCrude protein 0.75a 0.79b 0.82c 0.84c 0.004 b0.001Organic matter 0.73a 0.79b 0.81c 0.81c 0.004 b0.001Crude fibre 0.59 0.61 0.61 0.62 0.013 0.294

CTTADCrude protein 0.84a 0.86b 0.88c 0.89c 0.004 b0.001Organic matter 0.85a 0.88b 0.90c 0.90c 0.003 b0.001Crude fibre 0.69 0.69 0.70 0.70 0.006 0.904

a, b, c Means within a row with different superscripts are significantlydifferent (Pb0.05).C: Basal diet without antibiotics or probiotics.L: Basal diet+LAB complex (Enterococcus faecium 6H2, Lactobacillus acidophilusC3, Pediococcus pentosaceus D7 and Lactobacillus fermentum NC1).LB: Basal diet+LAB complex+Bacillus subtilis H4.LBS: Basal diet+LAB complex+Bacillus subtilis H4+Saccharomyces boulardiiSb.1 Piglets were weaned at 26–28 days of age.2 Digesta samples were taken at day 14 following weaning.3 Digesta samples were taken at day 35 following weaning.

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C, while no differences (PN0.05) were found among the pigletsfed diets L, LB and LBS.

The average E. coli count across all intestinal segments waslower (Pb0.01) in piglets fed diets L, LB and LBS than on diet C

on days 14 and 35. Similarly, E. coli counts were lower in pigletsfed diets L, LB and LBS than in piglets fed diet C in the stomachand ileum on day 14 and in ileum only on day 35, while therewere no treatment effects (PN0.05) in the colon and rectumon days 14 and 35. On day 35, the E. coli count in stomach waslower (Pb0.05) in piglets fed diet LBS, while there was no effect(PN0.05) in piglets fed diet L and LB.

3.5. Gut organic acids

There were interactions between treatment and intestinalsegment for the content of lactic and acetic acid at days 14and 35 (Pb0.001), and for propionic acid content at day 35(Pb0.05) (Tables 7 and 8). On both days 14 and 35, lacticacid content was highest (Pb0.01) in the ileum, followed bythe colon and stomach, andwas lowest (Pb0.01) in the rectum.Total VFA was highest in the colon (Pb0.01), decreased in therectum and ileum, and was lowest in the stomach (Pb0.01).Overall, across all intestinal segments, piglets fed diets L, LBand LBS had higher (Pb0.01) contents of lactic acid and totalVFA than piglets fed diet C on both days 14 and 35.

On day 14, across all intestinal segments, there were nodifferences (PN0.05) among piglets fed diets L, LB and LBSin the content of individual VFA and total organic acids.

On day 35, across all intestinal segments, piglets fed dietLBS had higher contents of lactic acid and total VFA than pig-lets fed diet L (Pb0.05). Total VFA concentrations in the ileumwere different (Pb0.05) among treatments (L, LB and LBS),with lowest values in treatment L.

On both day 14 and 35, piglets fed diets L, LB and LBS hadhigher contents of total VFA (acetic, propionic and butyric

Table 4Effects of lactic acid bacteria (LAB) and Bacillus and Saccharomyces com-plexes on diarrhoea incidence1 and faecal score2 in weaned pigs†.

Items Treatment SEM P value

C L LB LBS

Week 1Incidence,% 13.4 9.8 8.9 6.7 0.114Faecal score 0.18a 0.12ab 0.11ab 0.08b 0.016 0.008

Week 2Incidence,% 17.0a 8.9b 9.8b 4.5c b0.001Faecal score 0.23a 0.12b 0.13b 0.05c 0.014 b0.001

Weeks 1–2Incidence,% 15.2a 9.4b 9.4b 5.6c b0.001Faecal score 0.21a 0.12b 0.12b 0.07c 0.012 b0.001

a, b, c Means within a row with different superscripts are significantly different(Pb0.05).C: Basal diet without antibiotics or probiotics.L: Basal diet+LAB complex (Enterococcus faecium 6H2, Lactobacillus acidophilusC3, Pediococcus pentosaceus D7 and Lactobacillus fermentum NC1).LB: Basal diet+LAB complex+Bacillus subtilis H4.LBS: Basal diet+LAB complex+Bacillus subtilis H4+Saccharomyces boulardiiSb.† Piglets were weaned at 26–28 days of age.1 The incidence of diarrhoea (%)=the sum of the total number of diarrhoealpiglets over the period divided by the number of piglet days in the periodmultiplied by 100.2 Faecal score=the sum of the diarrhoea score over the period divided bythe number of piglet days over the period (score 0: firm, dry faeces; score1: pasty faeces; score 2: thick, fluid faeces; and score 3: watery faeces).

Table 5Effects of lactic acid bacteria (LAB) andBacillus and Saccharomyces complexes on LAB and E. coli counts1 in different intestinal segments of piglets2 on day 14 post-weaning.

Item Stomach Ileum Colon Rectum Mean SEM3 P value3

LAB⁎

C x4.71a x6.08b x6.33bc x6.67c x5.95 0.14 b0.001L y5.32a y6.90b y7.38c y7.63c y6.81 0.08 b0.001LB y5.28a y6.84b yz7.11bc y7.39c yz6.65 0.10 b0.001LBS y5.17a y6.78b z6.93bc y7.29c z6.54 0.09 b0.001Mean 5.12a 6.65b 6.94c 7.24d

SEM4 0.07 0.10 0.09 0.14P value4 b0.001 b0.001 b0.001 0.004

E. coli⁎⁎

C x4.78a x6.82b 7.18b 7.42b x6.55 0.20 b0.001L y3.86a y6.18b 6.73bc 7.18c y5.99 0.17 b0.001LB y3.80a y6.17b 6.79b 6.83b y5.90 0.19 b0.001LBS y3.81a y6.24b 7.06c 6.69bc y5.95 0.13 b0.001Mean 4.06a 6.35b 6.94c 7.03c

SEM4 0.15 0.12 0.16 0.24P value4 0.001 b0.001 0.21 0.19

a, b, c Means within a row with different superscripts are significantly different (Pb0.05).x, y, z Means within a column with different superscripts are significantly different (Pb0.05).⁎Pooled SEM=0.05; P (segment) b0.001; P (treatment) b0.001; P (segment×treatment)=0.671.⁎⁎Pooled SEM=0.09; P (segment) b0.001; P (treatment) b0.001; P (segment×treatment)=0.305.C: Basal diet without antibiotics or probiotics.L: Basal diet+LAB complex (Enterococcus faecium 6H2, Lactobacillus acidophilus C3, Pediococcus pentosaceus D7 and Lactobacillus fermentum NC1).LB: Basal diet+LAB complex+Bacillus subtilis H4.LBS: Basal diet+LAB complex+Bacillus subtilis H4+Saccharomyces boulardii Sb.1 Bacterial number is expressed as log10 CFU/g fresh digesta.2 Piglets were weaned at 26–28 days of age.3 SEM and P values when comparing segments within treatment.4 SEM and P values when comparing treatments (microbial complex supplementation) within segment.

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acid) and organic acids (VFA plus lactic acid) in the ileum andcolon (Pb0.01) than piglets fed diet C, while no treatmenteffects on the contents of organic acids and total VFA werefound in the stomach and rectum (PN0.05).

4. Discussion

4.1. Growth performance, nutrient digestibility and health

Compared to the basal diet (C), supplementing the diet ofpost-weaning piglets with different complexes of bacteria andyeast (diets L, LB and LBS) increased ADFI (9.9, 8.5 and 8.5%, re-spectively), improved ADG (18.5, 24.4 and 25.8%, respectively)and FCR (7.5, 12.8 and 13.9%, respectively).Weaning is a stress-ful period in the life of piglets, associated with changes of diet,and in gut environment and gut morphology. This may resultin low feed intake, imbalanced intestinal microflora and im-paired health (Pluske et al., 1997). Supplementing the dietwith probiotic microorganisms may be a means of reducingthese problems, and thereby of improving health and perfor-mance. In this context, multiple-strain probiotic lactobacilliare believed to be active in a wider range of conditions in thegut compared to single-strain lactobacillus probiotics (Fuller,1989; Gardiner et al., 2004). Earlier studies have shown thatsupplementation of the diet with LAB complexes improvedfeed intake (11.8%–31.45%), weight gain (9.7%–43.3%) andfeed efficiency (4.2%–21.4%) in weaned piglets (Guerra et al.,2007; Huang et al., 2004; Lessard and Brisson, 1987; Mallo etal., 2010; Pollmann et al., 1980).

Piglets fed diet LB and LBS improved ADG and FCR duringthe second period post-weaning (week 3–5) compared topiglets fed diet L, indicating positive effects of B. subtilis H4

on the performance. These improvements could be due tothe higher nutrient digestibility in the Bacillus-fed piglets.These findings are supported by earlier studies, showing pos-itive effects on feed intake, growth performance and FCR inweaned piglets fed diets supplemented with Bacillus probio-tics (Kirchgessner et al., 1993; Kyriakis et al., 1999; Taras etal., 2005; Zani et al., 1998). Kirchgessner et al. (1993) foundthat supplementing the diet of piglets with Bacillus cereusresulted in higher ADFI (+11%) and higher ADG (+11%)during a 21-day post-weaning period.

The current results also showed that the inclusion ofS. boulardii Sb in diet (LBS) increased ADFI and ADG of the pig-lets. These effects could be linked to lower diarrhoea incidenceand faecal score of the Saccharomyces-fed piglets. Similarly, in-clusion of Saccharomyces has resulted in positive effects onfeed intake, growth performance and FCR in weaned piglets inearlier studies (Bontempo et al., 2006; Jurgens et al., 1997;Mathew et al., 1998). Mathew et al. (1998) reported that addi-tion of a Saccharomyces cervisiae culture to a pelleted diet fedto piglets during 25 days post weaning increased ADFI (+17%)and improved ADG (+16%).

In the current study, piglets fed diets supplemented withmicrobial complexes (diets L, LB and LBS) showed higher nutri-ent digestibility than piglets fed the un-supplemented basaldiet. Similar results were found by Huang et al. (2004) whoreported that supplementation of the diet with a LAB complex(Lactobacillus gasseri, Lactobacillus reuteri, L. acidophilus andL. fermentum) increased digestibility in piglets after weaning.Our data shows that the LAB-fed piglets had higher LAB popu-lation in the intestine, which could imply higher hydrolytic en-zyme activity (Fuller, 1989; Nousiainen and Setälä, 1998) inthese piglets. Moreover, gut function may have been improved

Table 6Effects of lactic acid bacteria (LAB) and Bacillus and Saccharomyces on LAB and E. coli counts1 in different intestinal segments of piglets2 on day 35 post-weaning.

Item Stomach Ileum Colon Rectum Mean SEM3 P value3

LAB⁎

C x6.08a x6.48b x6.72b x7.24c x6.63 0.09 b0.001L y6.55a y7.31b y7.47bc y7.84c y7.29 0.10 b0.001LB y6.48a y7.10b y7.25b xy7.69c yz7.13 0.10 b0.001LBS y6.52a y7.13b y7.18bc xy7.50c z7.08 0.09 b0.001Mean 6.41 a 7.00 b 7.15 b 7.57 c

SEM4 0.09 0.08 0.082 0.12P value4 0.014 b0.001 b0.001 0.018

E. coli⁎⁎

C x4.25a x6.48b 6.61b 6.73b x6.02 0.11 b0.001L xy3.75a y5.56b 6.35c 6.54c y5.55 0.18 b0.001LB xy3.74a y5.54b 6.31c 6.50c y5.52 0.13 b0.001LBS y3.31a y5.27b 6.25c 6.39b y5.31 0.11 b0.001Mean 3.76a 5.71b 6.38c 6.54c

SEM4 0.18 0.13 0.14 0.08P value4 0.027 b0.001 0.293 0.079

a, b, c Means within a row with different superscripts are significantly different (Pb0.05).x, y, z Means within a column with different superscripts are significantly different (Pb0.05).⁎Pooled SEM=0.05; P (segment) b0.001; P (treatment) b0.001; P (segment×treatment)=0.532.⁎⁎Pooled SEM=0.07; P (segment) b0.001; P (treatment) b0.001; P (segment×treatment)=0.066.C: Basal diet without antibiotics or probiotics.L: Basal diet+LAB complex (Enterococcus faecium 6H2, Lactobacillus acidophilus C3, Pediococcus pentosaceus D7 and Lactobacillus fermentum NC1).LB: Basal diet+LAB complex+Bacillus subtilis H4.LBS: Basal diet+LAB complex+Bacillus subtilis H4+Saccharomyces boulardii Sb.1 Bacterial number is expressed as log10 CFU/g fresh digesta.2 Piglets were weaned at 26–28 days of age.3 SEM and P values when comparing segments within treatment.4 SEM and P values when comparing treatments (microbial complex supplementation) within segment.

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as piglets fed diet supplemented probiotic LAB showed in-creased lactase and sucrase activities in the small intestinemu-cosa (Collington et al., 1990). The CIAD and CTTAD of CP andOM were improved by supplementing diets with B. subtilis H4(diet LB and LBS) in both periods post-weaning. One explana-tion could be higher production of hydrolytic enzymes in thegut, as it has been shown that B. subtilis can produce a numberof hydrolytic enzymes (such as α-amylase, arabinase, cellulase,dextranase, levansucrase, maltase, alkaline protease, neutralprotease and β-glucanase) (Priest, 1977). Moreover, B. cereusvar. toyoi was able to stimulate the rate of glucose transportthroughout brush border vesicles from porcine jejunum (Breveset al., 2000). In addition, the lower scouring frequency in pigletsfed diets supplemented with microorganisms (diets L, LB andLBS) could have contributed to the higher digestibility.

In the current study, some piglets in all treatment groupswere affected by diarrhoea during the first 2 weeks post-

weaning. However, piglets fed diets supplemented with micro-bial mixtures (diets L, LB and LBS) had lower diarrhoea frequen-cy and lower faecal scores than those fed the un-supplementedcontrol diet. In piglets fed diets supplemented with microbialmixtures, E. coli counts were lower, and the content of organicacids in the intestine was higher, than in piglets fed the controldiet. The higher content of organic acidswill acidify the intestinalcontent and exert antibacterial effects. Moreover, the highercontent of VFA in the colon will be a powerful driving force forwater and sodium absorption from the colonic lumen, whichcan reduce scouring (Argenzio and Whipp, 1979; Cummings,1983; Cummings and Macfarlane, 1991).

Addition of B. subtilis H4 to the LAB complex did not resultin any improvement in the diarrhoea frequency or in faecalscore in this study. The reported effects of Bacillus on the in-cidence of diarrhoea in weaned piglets are inconsistent. Forexample, Kyriakis et al. (1999) and Taras et al. (2005) foundthat Bacillus supplements reduced the incidence and severity

Table 7Effects of lactic acid bacteria (LAB) and Bacillus and Saccharomyces on concentrations of volatile fatty acids (VFA— acetic, propionic and butyric acids) and organicacids (lactic acid and VFA) (mmol/kg dry matter) in different intestinal segments of piglets† on day 14 post-weaning.

Item Segment Mean PooledSEM

P value

Stomach Ileum Colon Rectum Segment (S) Treatment (T) S×T

VFAC 108a x230b x514c 277d x282L 110a y298b y722c 297b y357LB 108a y300b y720c 301b y357LBS 111a y308b y757c 299b y369Mean 109a 284b 678c 293b 4.94 *** *** ***

Organic acidC 233a x386b x632c 334b x396L 254a y548b y860c 358d y505LB 252a y548b y854c 356d y502LBS 255a y556b y891c 354d y514Mean 248a 465b 809c 350d 6.20 *** *** ***

Lactic acidC x125a x156b x118a 57.2c x114L y144a y250b y138a 60.6c y148LB y144a y248b y134a 55.4c y145LBS y144a y248b y134a 54.2c y145Mean 139a 226b 131a 57.0c 2.53 *** *** ***

Acetic acidC 91a x157b x367c 220d x209L 93a y237b y575c 239b y286LB 90a y241b y568c 241b y285LBS 93a y247b y603c 240b y296Mean 92a 221b 528c 235b 4.95 *** *** ***

Propionic acidC 12.4a 51.2b 122c 38.1d 56.0L 12.6a 52.7b 121c 39.6d 56.4LB 12.8a 53.2b 125c 40.1d 57.8LBS 12.9a 65.3b 124c 40.4d 60.7Mean 12.7a 51.3b 123.2c 39.6d 1.38 *** NS NS

Butyric acidC 4.5a 7.4a 24.7b 18.8c 13.8L 4.5a 7.6a 26.4b 18.7c 14.3LB 4.8a 7.8a 27.1b 19.8c 14.9LBS 5.1a 8.1a 30.3b 19.3c 15.8Mean 4.7a 7.7b 27.1c 19.1d 0.51 *** NS NS

† Piglets were weaned at 26–28 days of age.a, b, c, d Means within row with different superscripts are significantly different (Pb0.05).x, y, z Means within column with different superscripts are significantly different (Pb0.05).*** Pb0.001; NS is non-significant difference (PN0.05).C: Basal diet without antibiotics or probiotics.L: Basal diet+LAB complex (Enterococcus faecium 6H2, Lactobacillus acidophilus C3, Pediococcus pentosaceus D7 and Lactobacillus fermentum NC1).LB: Basal diet+LAB complex+Bacillus subtilis H4.LBS: Basal diet+LAB complex+Bacillus subtilis H4+Saccharomyces boulardii Sb.

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of diarrhoea in post-weaned piglets, while Kirchgessner et al.(1993) did not find any effect of B. cereus on the diarrhoeafrequency in post-weaned piglets.

Interestingly, addition of S. boulardii to the LB complex re-duced the incidence of diarrhoea during the first week post-weaning, although the E. coli counts were not affected. Theanti-pathogenic effects of S. boulardii may not primarily bedue to reduction of the pathogen populations in the intestine(Czerucka and Rampal, 2002), but could also be due to the pro-duction of other anti-diarrhoeal factors. For example, S. boulardiihas been shown to inhibit the toxicity of E. coli surface endo-toxins (Buts et al., 2006), to stimulate intestinal immunityand to inhibit binding of bacterial toxins to enterocyte re-ceptors (Buts et al., 1990; Pothoulakis et al., 1993). In addition,S. boulardii can secrete a protease which digested Clostridumdifficile toxins (Castagliuolo et al., 1999) and substances thatmay interfere with Ca-mediated chloride secretion (Krammer

and Karbach, 1993). Furthermore, S. cerevisiae ssp. boulardii re-duced themucus layer in the ileumofweaned piglets, confirm-ing decreased levels of potential pathogens in the gut lumen(Bontempo et al., 2006).

4.2. Bacteria counts and gut environment

At weaning, there will be a decrease in the LAB populationin the gut, while the population of E. coliwill increase (Jensen,1998). In addition, the decreased lactobacilli population is coin-ciding with decreased VFA concentration together with in-creased L+ and D-lactate concentration in the ileum contentof piglets subsequent to weaning (Mathew et al., 1994, 1998).In the current study, supplementation of the basal dietwith dif-ferent microbial complexes resulted in an increase in the intes-tinal LAB population in all gut segments (stomach, ileum, colonand rectum) and lowered E. coli counts (stomach and ileum) at

Table 8Effects of lactic acid bacteria (LAB) and Bacillus and Saccharomyces on concentrations of volatile fatty acids (VFA— acetic, propionic and butyric acids) and organicacids (lactic acid and VFA) (mmol/kg dry matter) in different intestinal segments of piglets† on day 35 post-weaning.

Item Segment Mean PooledSEM

P value

Stomach Ileum Colon Rectum Segment (S) Treatment (T) S×T

VFAC 103a x172b x540c 444d x315L 105a y273b y701c 465d y387LB 105a z297b y702c 468d yz393LBS 106a k315b y729c 481d z408Mean 105a 266b 668c 465d 4.29 *** *** ***

Organic acidsC 159a x268b x571c 447d x361L 168a y413b y753c 469d y451LB 170a yz439b y755c 472b y459LBS 170a z465b y788c 485b z477Mean 166a 396b 717c 468d 4.13 *** *** ***

Lactic acidC 55.4a x97b x30.5c 3.4d x46.5L 63.3a y134b y51.4c 3.5d y63.2LB 64.1a y141b y52.9c 3.5d yz65.4LBS 64.6a y150b y59.5a 3.5c z69.3Mean 61.9a 130b 48.6c 3.4d 1.58 *** *** ***

Acetic acidC 89.3a x144b x410c 347d x248L 89.3a y236b y561c 359d y311LB 90.4a yz246b y553c 355d y311LBS 89.9a z262b y573c 360d y321Mean 89.8a 222b 525c 355d 4.31 *** *** ***

Propionic acidC 10.3a x21.0b x103c x81.0d x53.8L 11.1a y36.4b xy112c xy87.2d y61.7LB 11.0a yz45.2b y119c yz95.8d z67.8LBS 11.6a z46.6b y124c z102.7d z71.3Mean 11.0a 37.2b 115c 91.7d 1.32 *** *** **

Butyric acidC 3.9a 6.3a 26.9b 15.9c 13.3L 4.2a 6.4a 27.7b 18.7c 14.2LB 4.0a 6.6a 29.5b 17.5c 14.4LBS 4.1a 6.8a 30.8b 18.5c 15.0Mean 4.0a 6.5b 28.7c 17.7d 0.67 *** NS NS

†Piglets were weaned at 26–28 days of age.a, b, c, d Means within row with different superscripts are significantly different (Pb0.05).x, y, z, k Means within column with different superscripts are significantly different (Pb0.05).** Pb0.01; *** Pb0.001; NS is non-significant difference (PN0.05).C: Basal diet without antibiotics or probiotics.L: Basal diet+LAB complex (Enterococcus faecium 6H2, Lactobacillus acidophilus C3, Pediococcus pentosaceus D7 and Lactobacillus fermentum NC1).LB: Basal diet+LAB complex+Bacillus subtilis H4.LBS: Basal diet+LAB complex+Bacillus subtilis H4+Saccharomyces boulardii Sb.

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both day 14 and 35. Parallel with the change in LAB and E. colicounts, lactic acid concentration increased (stomach, ileumand colon at day 14, and ileum and colon at day 35) andtotal VFA concentrations increased (ileum and colon at day14 and 35) along the gut. Sakata et al. (1999) found that pro-duction of lactic acid and VFA was increased by addition ofprobiotic preparations (containing Bifidobacterium, Enterococcusor Lactobacillus) in pig caecal contents in vitro. It appears mostlikely that the increase in intestinal Lactobacilli counts was dueto the supplementation of the feed in the present study. Thelower counts of E. coli may be due to inhibited growth throughthe production of organic acids by the LAB (Jin et al., 2000)and through the release of proteinaceous components that willinhibit the adhesion of E. coli in themucus in the ileum of piglets(Blomberg et al., 1993). Similarly, other studies found that LAB-fedweaned piglets had higher Lactobacilli populations and lowerE. coli counts along the intestine (Huang et al., 2004; Mallo et al.,2010; Tortuero et al., 1995). However, Huang et al. (2004) didnot find any difference in the intestinal VFA concentrations ofpiglets fed un-supplemented and LAB supplemented feed.

The reported effects of dietary supplementationwith Bacillusor Saccharomyces on organic acid concentration in pig's intes-tines are inconsistent. For example, Jadamus et al. (2002)found that supplementation of the diet with B. cereus var. toyoiincreased lactic acid concentrations in the middle and lastparts of jejunum and increased VFA concentrations at the endof jejunum, and in ileum, caecum and colon of piglets at day 4post weaning. However, Kirchgessner et al. (1993) reportedthat B. cereus decreased lactate and VFA concentrations in thesmall intestine, and acetic and propionic acid in the caecum ofpost-weaned piglets. Marinho et al. (2007) found that inclusionof S. cerevisiae in the diet of weaned piglets decreased VFA con-centration in the colon, while Mathew et al. (1998) did not findany effects of supplementing the basal diet with a commercialS. cerevisiae culture on Lactobacilli and E. coli counts, and VFAconcentrations along the intestinal tract (stomach, duodenum,ileum, caecum and colon) of weaned piglets. In the currentstudy, B. subtilis H4 and S. boulardii Sb did not have any effecton the lactic acid and total VFA concentrations.

5. Conclusions

The LAB complexes used in the current study had probioticproperties and could be used to improve the health status ofweaned piglets under Vietnamese conditions. All threemicrobialmixtures (L, LB and LBS) increased LABpopulation, increased lac-tic acid and total VFA concentrations, and reduced E. coli countsin the piglets' intestine. Moreover, addition of B. subtilis H4resulted in improved diet digestibility and addition of S. boulardiiSb resulted in a lowered incidence of diarrhoea. This improvedpiglet growth performance during the 5-week period post-weaning. These findings suggest that a multi-bacterial mixtureincluding bacteria other than LABmay have benefits over amix-ture of LAB only, and that inclusion of yeast may have additionalbenefits, in terms of probiotic effects in weaned piglets.

Acknowledgements

Theauthors thank the Sida-SARECMekongBasinAnimal Re-search Network (MEKARN) for financial support, researchers atthe Department of Animal Nutrition and the laboratory staff

of the National Institute of Animal Science, Hanoi, Vietnam,for assistance in carrying out the study and chemical ana-lyses. Thanks also to researchers at the Department of Micro-biology, National Institute of Veterinary Research, Hanoi,Vietnam, for assistance in slaughtering piglets, digesta sam-pling and bacteria quantification, and researchers and staffat the Institute of Microbiology and Biotechnology, VietnamNational University, Hanoi, Vietnam, for assistance in preparingthe microbial cultures.

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