J. Anim Sci. 2007. 85:3099-3109. doi:10.2527/jas.2007-0110
© 2007 American Society of Animal Science
Effects of yeast culture on performance, gut integrity, and blood cell composition of weanling pigs1,
C. M. C. van der Peet-Schwering*,3,
A. J. M. Jansman*,
H. Smidt
and
I. Yoon
* Animal Sciences Group, Wageningen University and Research Center, 8200 AB Lelystad, the Netherlands;
and
Laboratory of Microbiology, Wageningen University and Research Center, 6700 EY Wageningen, the Netherlands; and and
Diamond V Mills Inc., Cedar Rapids, IA 74570
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Abstract
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An experiment was conducted to determine the effects of yeast culture (YC) and modified yeast culture [YC + cell wall product (CWP) containing mannan oligosaccharides] in pig diets on the performance, gut integrity, and blood cell composition of weanling pigs and to determine whether these dietary supplements could replace antimicrobial growth promoters (AGP) in pig diets. A total of 480 weanling pigs (27 d old and 7.8 ± 0.1 kg of BW) were assigned to 1 of 4 experimental treatments: 1) diets without AGP or YC (control diet); 2) control + AGP; 3) control + 0.125% YC; and 4) control + 0.125% YC + 0.2% CWP. Piglets were fed experimental diets for 5 wk after weaning. Blood samples were collected from 8 piglets at weaning and from 8 piglets per treatment on d 14 and 35 after weaning for blood cell composition. These piglets were slaughtered for measurement of villous length and crypt depth in the jejunal mucosa and microbial profiling on the intestinal digesta. Average daily gain (P = 0.06) and G:F (P = 0.02) were improved for piglets that were fed the supplemented diets compared with piglets that were fed the control diet. Average daily feed intake was unaffected by dietary treatment. Performance was similar in piglets fed diets supplemented with AGP, YC, and YC + CWP. Blood cell composition, villous length, crypt depth, and microbial composition in the gut were unaffected by dietary treatment, but they were affected by time after weaning. Red blood cells, hemoglobin, hematocrit value, mean cell volume, mean cell hemoglobin, percentage of lymphocytes in the leukocyte population, villous length, and crypt depth were greater (P < 0.05) at 5 wk after weaning than at 2 wk after weaning. Eosinophils (P = 0.06) in the leukocyte population tended to be greater at 5 wk after weaning. Concentration of neutrophils in the leukocyte population and percentages of CD4 and CD8 cells were lower (P < 0.02) at 5 wk after weaning. The CD4:CD8 ratio (P = 0.07) tended to be lower at 5 wk after weaning. Results suggest that yeast culture could be an alternative to AGP in the diets of weanling pigs and that addition of CWP to diets containing YC would not improve the performance or health of weanling pigs above that of YC alone. Thus, more insight into the mode of action of YC is needed.
Key Words: gut integrity • blood cell composition • performance • weanling pig • yeast culture
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INTRODUCTION
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Weaned piglets often suffer from postweaning diarrhea and impaired growth rate, which are associated with a damaged intestinal morphology and function (Pluske et al., 1995
). Antimicrobial growth promoters (AGP) have traditionally been used in piglet diets to reduce the problems with diarrhea (Mikkelsen and Jensen, 2004). However, research has indicated that the use of AGP can result in bacterial resistance. This resistance has been reported to be transferable to humans (Van den Bogaard et al., 2000). Therefore, use of AGP in pig diets has been prohibited in the European Union since January 2006. To maintain both the level of production and the health status of pigs, alternatives to AGP are needed.
Yeast culture (YC) and cell wall products (CWP) containing mannan oligosaccharides (MOS) might be potential alternatives to AGP. Yeast culture is a dried fermented product containing small amounts of live yeast cells (Saccharomyces cerevisiae) and metabolic by-products produced by the yeast during fermentation. The performance response of piglets to yeast or YC has been variable. In some studies, supplementation of live yeast (Jurgens et al., 1997; Van Heugten et al., 2003) or YC (Mathew et al., 1998) has improved postweaning performance, whereas others have reported no benefits of YC supplementation (Jurgens, 1995; Kornegay et al., 1995). Live yeast supplementation may improve the performance and health of piglets by stimulating the immune system and maintaining a beneficial intestinal environment (Van Heugten et al., 2003). Cell wall products may improve the performance and health of piglets by preventing bacteria from binding to intestinal epithelial cells (Spring et al., 2000) and by altering the immune function (Davis et al., 2004).
The present experiment was conducted to determine the effects of YC and modified yeast culture (YC + CWP) in pig diets on the performance, gut integrity, and blood cell composition of weanling pigs and to determine whether these products can replace AGP in pig diets.
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MATERIALS AND METHODS
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Animals, Experimental Design, and Housing
The experiment was approved by the Animal Experiments Committee of Wageningen University, the Netherlands. The experiment was conducted at the Research Station Sterksel of the Animal Sciences Group of Wageningen-UR with 480 weanling pigs [Tempo boar x (Large White x Dutch Landrace) sow] with an average BW of 7.8 ± 0.1 kg. Six groups with 80 piglets each (gilts and barrows) were weaned at 27 d of age and assigned to 1 of 4 experimental treatments: 1) diets without AGP or YC (control diet); 2) control + AGP (40 mg/kg of avilamycin; Eli Lilly and Company Ltd., Indianapolis, IN); 3) control + 0.125% YC (Diamond V XPCLS Yeast Culture, Diamond V Mills, Cedar Rapids, IA); or 4) control + 0.125% YC + 0.2% CWP (Diamond V Mills).
Piglets were fed experimental diets for 5 wk. Piglets weighing less than 6 kg or more than 10 kg at weaning and piglets with physical abnormalities were not used in this study. Piglets were blocked by BW. A block consisted of 4 pens of 10 piglets (5 barrows and 5 gilts) each, with each treatment being represented within a block. Each group of pigs consisted of 2 blocks. Each experimental treatment contained 12 replicates. In groups 1 and 2, 8 pigs at weaning (4 gilts and 4 barrows) and 8 pigs per treatment (4 gilts and 4 barrows) on d 14 and 35 after weaning were slaughtered to obtain intestinal digesta samples and intestinal mucosa biopsies. Because all piglets in the study originated from the same farm (a closed-system farm), piglets in groups 1 and 2 were considered to be representative of all piglets in the experiment. Piglets were housed in 4 rooms containing 8 pens each, with 1 group of pigs per room. Experimental treatments were randomly assigned to pens within a room. Two rooms had 2.2 x 2.2-m pens and were used for groups 1 and 2 and after 6 wk were reused for groups 5 and 6. Rooms were cleaned between groups. The other 2 rooms had 2.30 x 1.22-m pens and were used for groups 3 and 4. All rooms had fully slatted floors and were equipped with a computer-controlled heating system and a mechanical ventilation system. Groups 1 and 2 began at the same time, whereas groups 3 and 4 began 3 wk later.
Diets and Feeding
Weanling pigs were fed ad libitum by single-space dry feeders. During the first 2 wk after weaning, piglets were fed pelleted weanling diets (Tables 1 and 2). Weanling diets were replaced by pelleted prestarter diets (Tables 1 and 2) between d 15 and 17, and fed until the end of the study. Yeast culture and YC + CWP replaced maize starch. Diets were produced by Arkervaart-Twente (Nijkerk, the Netherlands). Drinking water was supplied ad libitum by a drinking trough.
Measurements
Performance.
Individual BW of all piglets were measured at weaning, 2 wk after weaning, and 5 wk after weaning. Feed consumption per pen was recorded at each weighing interval. When mortality occurred, the date of mortality, weight of the pig, reason for mortality, and total amount of feed consumed were recorded. All medication administered to piglets during the experiment was also recorded.
Blood Cell Composition.
Blood samples were collected from 8 randomly chosen piglets at weaning and from 8 piglets per treatment on d 14 and 35 after weaning. Blood samples were analyzed for blood cell composition, which consisted of red blood cells, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, hemoglobin, hematocrit value, white blood cells, platelets, and leukocytes (lymphocytes, neutrophils, basophils, eosinophils, and monocytes). Blood cell composition was determined by using a Sysmex semiautomatic blood cell meter (model F-800, OA Medical Electronics Co. Ltd., Kobe, Japan). Moreover, plasma intestinal fatty acid-binding protein (I-FABP) was determined by using a human commercial ELISA test kit (HyCult Biotechnology BV, Uden, the Netherlands, Niewold et al., 2004). Plasma I-FABP concentration is a parameter used to determine damage to the intestinal mucosa in pigs. In addition, CD4 (T helper cells) and CD8 lymphocytes (cytotoxic-suppressor T cells), as well as the CD4:CD8 ratio were measured by flow cytometry (FACS Calibur 3CS, BD Biosciences, San Jose, CA) after incubation for 30 min at 4°C with CD4 monoclonal antibody, clone 74/12/4, isotype m IgG2b and CD8 monoclonal antibody, clone 11/295/33, isotype m IgG2a (Saalmüller et al., 1994), and incubation for 30 min at 4°C with the secondary antibodies fluorescein isothiocyanate goat anti-mouse IgG2a, and R-PE IgG2b (SouthernBiotech, Birmingham, AL).
Gastrointestinal Tract.
After blood samples were collected, piglets were euthanized by i.v. injection of T61 [Intervet Nederland BV, Boxmeer, the Netherlands, containing 200 mg/mL of N-[2-(m-methoxyphenyl)-2-ethylbutyl-(1)]-hydroxy-butyramide, 50 mg/mL of 4,4'-methylene-bis-(cyclohexyl-trimethyl)-ammonium iodide, and 5 mg/mL of tetracaine hydrochloride in aqueous solution with dimethyl-formamide as a preservative] to obtain samples from ileal digesta and biopsies from the jejunal mucosa. Microbial profiling was performed by using denaturing gradient-gel electrophoresis (DGGE) to determine major shifts in microbial composition in the ileal digesta. Mucosa samples were collected from the jejunum approximately 2 m caudal to the Treitz ligament. Samples were rinsed in saline, pinned to a piece of dental wax, fixed in 10% phosphate-buffered formalin, and embedded in paraffin wax. Two sections (4 to 6 µm) were removed from each sample. Measurements of villous length (distance from the crypt opening to the tip of the villous) and crypt depth were performed in hematoxylin and eosin-stained sections at 100x magnification (Nabuurs et al., 1993). The mean of 10 values was calculated per section and used for further analysis.
Microbiological Analysis.
The intestinal digesta samples were immediately cooled on dry ice and stored at –80°C until further processing. Ileal digesta samples taken from 4 animals at the start of the experimental period and from 4 animals per treatment on d 14 and 35 (total of 36 samples) were analyzed by DGGE. Deoxyribonucleic acid was isolated directly from samples of gut contents by using the Fast DNA SPIN Kit for Soil (Q BIOgene, Cambridge, UK) according to the manufacturer’s instructions and as described by Konstantinov et al. (2004). Primers S-D-Bact-0968-a-S-GC and S-D-Bact-1401-a-A-17 (Nübel et al., 1996) were used to amplify the V6 to V8 region of the 16S rRNA gene with the Taq DNA Polymerase Kit from Life Technologies (Gaithersburg, MD). Polymerase chain reaction mixtures (50 µL) contained 0.5 µL of Taq polymerase (1.25 U), 20 mM Tris-HCl (pH 8.5), 50 mM KCl, 3.0 mM MgCl2, 200 µM each deoxynucleotide 5'-triphosphate, 5 pmol of the primers, 1 µL of DNA diluted to approximately 1 ng/µL and UV-treated sterile water. Samples were amplified in a thermocycler (T1, Whatman Biometra, Göttingen, Germany), and the cycling consisted of 94°C for 5 min; 35 cycles of 94°C for 30 s, 56°C for 20 s, and 68°C for 40 s; and finally 68°C for 7 min of final extension. Aliquots of 5 µL were analyzed by electrophoresis on 1.2% (wt/vol) agarose gels containing ethidium bromide to determine product size and quantity. Polymerase chain reaction products obtained from the ileal lumen-extracted DNA were separated by DGGE according to the procedure of Konstantinov et al. (2004). All gels were scanned at 400 dpi and analyzed by using the Bionumerics software package, version 4.5 (Applied Maths, Kortrijk, Belgium).
Statistical Analysis
Performance, Blood Cell Composition, and Gut Integrity Data.
Average daily gain, average daily feed intake, and G:F were analyzed by GLM (GenStat 8, VSN International, 2005), with pen as the experimental unit. The following model was used:
where Yijk is the dependent variable, µ is the overall mean, group is the fixed effect of group (i = 1 to 6), block within group is the fixed effect of block j nested within group i, experimental treatment is the fixed effect of experimental treatment (k = 1 to 4), and eijk is the error term. There was no interaction of group with experimental treatment; therefore, the interaction was omitted from the model. Blood cell composition, I-FABP, and gut integrity data were analyzed by GLM (GenStat 8, VSN International, 2005), with piglet as the experimental unit. The following split-plot model was used:
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where Yijklm is the dependent variable; µ is the overall mean; group is the fixed effect of group (i = 1, 2); experimental treatment is the fixed effect of experimental treatment (k = 1 to 4); age is the fixed effect of age [time point of the section, 2 or 5 wk after weaning (l = 1, 2)]; experimental treatmentk x agel is the interaction between both factors; sex is the fixed effect of sex [gilt or barrow (m = 1, 2)]; and pen within group, age within pen, and eijklm are random errors. Experimental treatment was tested against pen within batch. Age and the interaction of age x experimental treatment were tested against age within pen. Sex and the interaction of sex x experimental treatment were tested against eijklm. The following contrasts were tested: 1) control vs. supplemented diets (AGP, YC, and YC + CWP); 2) AGP vs. YC supplementation (YC and YC + CWP); 3) YC vs. YC + CWP. The number of pigs that died and the number of pigs that were veterinary treated were analyzed by means of a 
2 test (GenStat 8, VSN International, 2005). There were no interactions between group and experimental treatment for any of the response variables measured. Therefore, only the main effect is presented.
DGGE Data.
After normalization, bands were defined for each sample by using the band-searching algorithm within Bionumerics. A manual check was conducted by using the corresponding densitometric curves, and the DGGE bands constituting less than 1% of the total area of all bands were omitted from further analysis (Konstantinov et al., 2003). The similarity among DGGE profiles was determined by calculating similarity indices of the densitometric curves of the profiles, compared by using a Pearson product-moment correlation (Häne et al., 1993; Zoetendal et al., 2001). The unweighted pair group method with arithmetic mean algorithm was used as implemented in the analysis software for the construction of dendrograms. Differences in intragroup similarity coefficients (similarity in microbial profile in animals from the same treatment), among groups (dietary treatments), between different ages, and the interactions among them were tested for significance by 2-way ANOVA with the following model:
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where Yij is the dependent variable, µis the overall mean, experimental treatment is the fixed effect of the experimental treatment (i = 1 to 4), age is the fixed effect of age [time point of the section, 2 or 5 wk after weaning (j = 1, 2)], experimental treatmenti x agej is the interaction between both factors, and eijk is the error term. The effects of replicate and litter were tested separately and were not significant for any of the parameters; therefore, they were removed from the statistical model. Differences among treatment least squares means were evaluated by using Tukey’s multiple comparisons test. Differences were considered significant when P < 0.05.
All statistical analyses were performed with PROC GLM (SAS Inst. Inc., Cary, NC). Gini coefficients, as a measure of community evenness, were calculated based on the method of Lorenz (1995), as described by Mertens et al. (2005). Multivariate statistical analysis was performed with Canoco 4.5 software (Ter Braak and 
milauer, 2002) to assess the extent to which dietary treatment, age, and litter (further referred to as environmental variables) affected the ileal microbiota as measured by DGGE profiling. Redundancy analysis was chosen, because it explains the structure of the "species" data table (in this case, band position and relative intensity) by environmental variables, assuming a linear distribution of species (Ter Braak, 1987; Salles et al., 2004). Community similarities were graphed by using ordination plots with scaling focused on intersample difference (Marschner and Baumann, 2003). To determine the significance of the relationship between community response and environmental variables, a Monte Carlo permutation test was performed with 499 random permutations, and a significance level of 0.05 was chosen. The permutation was unrestricted. In 3 subsequent analyses, either 1) all environmental variables (treatment, sex, litter) were used as variables, 2) treatments were used as covariables, or 3) litter and sex were used as covariables to test for the significance of these.
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RESULTS
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Performance
Average daily feed intake was not affected (P > 0.05) by dietary treatment during all phases of the experiment (Table 3). During phase 1 (d 1 to 14), ADG was not affected by dietary treatment (Table 3). Gain to feed ratio in phase 1 was improved (P = 0.05) in weanling pigs fed the diets supplemented with AGP, YC, or YC + CWP compared with weanling pigs fed the control diet. During phase 2 (d 15 to 35), piglets fed the control diet tended to gain less (P = 0.07) and had a poorer (P = 0.07) G:F than piglets fed the supplemented diets. Performance was similar in piglets fed the diets supplemented with AGP, YC, or YC + CWP in phase 2. During the total experimental period, BW (P = 0.05) and G:F (P = 0.02) were poorer in piglets fed the control diet compared with piglets fed the supplemented diets. Average daily gain (P = 0.06) tended to be lower in piglets fed the control diet compared with piglets fed the supplemented diets. During the overall experimental period, all performance parameters were similar in piglets fed the diets supplemented with AGP, YC, or YC + CWP.
Numbers of culled (8, 6, 8, and 6 piglets in the control, AGP, YC, and YC + CWP group, respectively) and individually morbid, antibiotic-treated piglets (19, 17, 23, and 12 piglets in the control, AGP, YC, and YC + CWP group, respectively) were not affected (P > 0.05) by dietary treatment. Of these piglets, most piglets died and were morbid because of an infection with Streptococcus suis. In groups 3 and 4, all piglets were treated for 3 d with Depomycin (Intervet, Boxmeer, the Netherlands) because of an infection with Strep. suis. In piglets in groups 1, 2, 5, and 6, clinical cases of Strep. suis infection were absent. There were no differences (P > 0.05) in the number of culled and antibiotic-treated piglets per specific cause. Yeast culture + CWP reduced (P = 0.04) the number of morbid and antibiotic-treated piglets compared with YC alone.
Blood Cell Composition
Dietary treatments had no effect (P > 0.05) on blood cell composition for most parameters measured, except for the numbers of platelets and CD4 and CD8 lymphocytes (Table 4). The number of platelets was less (P = 0.04) in piglets fed the control diet compared with piglets fed the supplemented diets. Piglets fed YC or YC + CWP increased (P < 0.05) both CD4 and CD8 lymphocytes, resulting in no change in the CD4:CD8 ratio compared with the AGP treatment. There was, however, an age effect for most of the parameters measured (Table 5; P < 0.01). At 5 wk after weaning, piglets had greater values (P < 0.01) for red blood cells, hemoglobin, hematocrit value, mean cell volume, mean cell hemoglobin, and percentage of lymphocytes in the leukocyte (white blood cell) population than at 2 wk after weaning. Eosinophils (P = 0.06) in the leukocyte population tended to be greater at 5 wk after weaning. Concentration of neutrophils in the leukocyte population, and the percentages of CD4 and CD8 cells were less (P < 0.05) at 5 wk after weaning than at 2 wk after weaning. The CD4:CD8 ratio (P = 0.07) tended to be lower at 5 wk after weaning. There was no age x dietary treatment interaction, except for the hematocrit value (P > 0.05). Two weeks after weaning, the hematocrit values did not differ among dietary treatments (P > 0.05), whereas they were greater in pigs fed YC (40.9%) and lowest in pigs fed the diet with AGP (38.1%) at 5 wk after weaning. Hematocrit values of the other 2 treatments were 39.1 and 39.4%, respectively, in piglets fed the control diet and the diet with YC + CWP.
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Table 4. Blood cell composition, concentration of intestinal fatty acid-binding protein (I-FABP), and villous length and crypt depth in the jejunum as affected by dietary treatment1
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Table 5. Effects of piglet age on blood cell composition, concentration of intestinal fatty acid-binding protein (IFABP), and villous length and crypt depth in the jejunum
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Gut Morphology
Villous length and crypt depth were not affected by dietary treatment (Table 4
). Villi were longer (P = 0.04) and crypts were deeper (P < 0.001) at 5 wk of age than at 2 wk of age (Table 5).
Gut Microbiology
16S rRNA gene-targeted PCR-DGGE bacterial community profiling was used to assess the effect of the dietary treatments on ileal microbiota of weaned piglets (Figure 1). In general, time of sampling showed the strongest effect on microbiota, as evidenced by an un-weighted pair group method with arithmetic mean cluster analysis based on a Pearson product-moment correlation. Nearly all samples taken at wk 5 formed a separate cluster (sample numbers 42 to 72, indicated by the vertical black bar on the far right in Figure 1), whereas no further subclustering by treatment could be observed. This was also supported by the least squares means of similarity values of samples within dietary treatments, which were increased for samples taken at wk 5 as compared with wk 2 (63.82 vs. 31.12; P < 0.001; Table 6). Overall, samples from AGP and YC showed greater intratreatment similarities than samples from the control and YC + CWP groups (Table 6; control vs. YC, P = 0.03; YC + CWP vs. AGP, P < 0.001; YC + CWP vs. YC, P = 0.01).
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Figure 1. 16S rRNA gene-targeted PCR-denaturing gradient-gel electrophoresis fingerprint analysis of the total bacterial community in ileal samples. Similarity among samples is indicated by the tree structure at the left side and the scale at the top left side of the figure. W0 = sample numbers 1 to 5 (4 samples) from week (W) 0 (at the start of the experimental period); W2 = sample numbers 5 to 30 (16 samples); and W5 = sample numbers 42 to 72 (16 samples). The vertical black bar on the far right indicates a cluster of samples taken almost exclusively at wk 5. Treatments: control = T1, antimicrobial growth promoter = T2, yeast culture (YC) = T3, and YC + cell wall product = T4.
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Table 6. Similarities of bacterial community profiles among samples within dietary treatment, based on a Pearson product-moment correlation1
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Community evenness of individual samples, as measured by the Gini coefficient on a scale of 0 (low evenness) to 1 (high evenness), did not differ among dietary treatments (0.64, 0.62, 0.67, and 0.62 in control, AGP, YC, and YC + CWP, respectively) or sampling time (0.64 and 0.64 in wk 2 and 5, respectively), indicating that neither treatment nor time caused dramatic changes in bacterial evenness.
To further substantiate these observations, multivariate statistical analysis was performed by using the Canoco software package. In line with the above analyses, time was the only factor that affected (P = 0.002) the ileal bacterial community structure (Figure 2), whereas sex, litter, or dietary treatment had no effect. This is also visualized in Figure 2, where centroids representative of the dietary treatments are close to the center of the ordination plot, and time is strongly correlated with the first canonical axis.
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Figure 2. Redundancy analysis of community profiles. For the analysis shown, sex and litter were used as covariables, whereas treatment and time were taken as environmental (Env.) variables. In the analysis, the denaturing gradient-gel electrophoresis community profiles are presented in a 2-dimensional way, using variables (e.g., treatment and time) as possible discriminatory parameters. The percentages provided at the first 2 canonical axes indicate to what extent the environmental variables are able to explain the observed variability in community profiles. In total, 2.3 + 10.6% (12.9%) of the variation could be explained by the indicated variables in this analysis. The different treatments are plotted relatively close to the crossing of both dotted lines, indicating that treatments could hardly be differentiated on the basis of known variables. The arrow in the plot indicates the direction of the effect of time of sampling (0, 2, or 5 wk). The covariables sex and litter together were able to explain 4.2% of the observed variation, whereas treatment alone could explain only 1.1% of the observed variation. Treatment 1 (T1) = control diet; T2 = control + antimicrobial growth promoter; T3 = control + 0.125% yeast culture (YC); T4 = control + 0.125% YC + 0.2% cell wall product.
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DISCUSSION
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Performance
Addition of AGP, YC, or YC + CWP to weanling pig diets improved BW, ADG, and G:F compared with weanling pigs on the control diet. Positive effects of AGP in piglet diets have frequently been reported (Anderson et al., 1999; Freitag et al., 1999), although the exact mechanism of action is not well defined. Piglets fed diets with YC performed similarly to those fed the diet supplemented with AGP. These results agree with those from a study conducted by Van Krimpen and Binnendijk (2001), in which piglets fed diets supplemented with AGP (40 mg/kg of avilamycin) or live yeast cells (Saccharomyces cerevisiae CS47) performed better than piglets fed the negative control diet. However, the performance response of piglets to live yeast cells or to YC has been variable. In studies by Jurgens (1995) and Kornegay et al. (1995), the performance of piglets was not improved by supplementing the diet with YC, whereas in a study by Mathew et al. (1998), postweaning performance did improve. The exact mechanism of action of live yeasts cells and of YC is not clear. However, stimulating the immune system, maintaining a beneficial intestinal environment (Van Heugten et al., 2003), and improving intestinal immunity (Jurgens et al., 1997) have all been suggested as potential modes of action of yeast products.
Piglets fed the diet supplemented with YC + CWP performed similarly to piglets fed the diet with YC alone. Several studies have indicated that addition of MOS to diets improves the performance of piglets (Davis et al., 2004; Rozeboom et al., 2005). However, LeMieux et al. (2003) reported no effect of MOS on piglet performance. From our study, CWP containing MOS, supplied at a level of 2 g/kg, seemed to have no additional effect on performance when it was added to a diet that contained YC. A possible improvement in performance as a result of CWP may have been masked by inclusion of YC in the diet, because both MOS (Spring et al., 2000; Davis et al., 2004) and yeast (Van Heugten et al., 2003) may alter immune function and microbiota in the intestinal tract.
Blood Cell Composition, Gut Integrity, and Gut Microbiology
To obtain more insight into the mechanism of action of YC, we measured blood cell composition, gut integrity, and intestinal microflora in our study. Yeast or yeast cell walls may be able to modulate the immune system. This could contribute to the positive effect of yeasts or YC as feed additives in piglet diets. Davis et al. (2004) found an improvement in performance and a modulation of the immune system after supplementing the diet with phosphorylated mannans derived from S. cerevisiae in piglets. They found a tendency toward a lower level of neutrophils and a greater proportion of lymphocytes in blood of the mannan-fed group compared with an unsupplemented control group. Because there is often an increase in blood neutrophils as the first line of defense associated with subclinical and clinical infections, the authors concluded that the observed tendency for neutrophil proportions to decrease in the blood of mannan-fed piglets was due to a reduced inflammatory challenge imposed on the piglets. They also observed a lower CD3+CD4+ (T helper cells):CD3+CD8+ (cytotoxic-suppressor T cells) T lymphocyte ratio in jejunal lamina propria tissue. In general, an increase in blood neutrophils is the first line of defense associated with clinical and subclinical infection. In the current study, however, there was no effect of dietary treatment on the concentration of white blood cells and the percentage of lymphocytes or neutrophils within the white blood cell population.
The functional status of the small intestine is characterized in part by the villous length and crypt depth. Villous length is reduced in the first days after weaning. Villous atrophy is caused by an increased rate of cell loss and decreased rate of renewal. Increased cell loss can be due to apoptosis or programmed cell death. Apoptosis can be affected by endogenous stressors, such as oxidative stress, and by extrinsic pathways activated by binding of special ligands, such as tumor necrosis factor-
, on the cell surface (Fleck and Carey, 2005). The current study did not show effects of dietary treatments on villous length, crypt depth, or the villous:crypt ratio, although the dietary treatments were applied during the period when gut morphology was likely compromised as a result of the process of weaning.
Plasma I-FABP can be considered as a parameter indicating mucosal cell damage in the gut. Intestinal fatty acid-binding protein is found only in the epithelial cells of the stomach and the small and large intestines and can be detected in plasma and urine after cell damage (Niewold et al., 2004). Reduction of the intestinal oxygen supply in pigs as a way to induce metabolic stress to the intestinal mucosa was previously shown to induce intestinal cell damage and increase plasma I-FABP concentrations (Niewold et al., 2004). The concentration of I-FABP in blood was not affected by the dietary treatments in the current study, suggesting that the dietary treatments did not affect small intestinal integrity.
Microbiota in the digestive system of piglets contributes to the defense mechanisms of the animal in different ways. One of the important abilities of stable microbiota in the gastrointestinal tract is to prevent colonization of pathogenic bacteria (colonization resistance). Microbial composition in various parts of the gastrointestinal tract of pigs can be affected by dietary manipulation (Anderson et al., 1999). Both feed ingredient composition and dietary additives, such as pro- and prebiotics, could potentially influence the composition of microbiota in the gastrointestinal tract. The current study did not reveal clear effects of the dietary treatments on microbial composition at the ileal level, based on the DGGE analysis. Although this technique provides a fast, semiquantitative comparison of the overall bacterial community structure in a large number of samples, relative changes in minor populations with relative abundance below 1% cannot be detected. Diet composition was largely the same in terms of the quantitative ingredient composition. The amount of CWP containing MOS included in the YC + CWP diet (2 g/kg), which could be considered as a substrate for microbial growth in the gastrointestinal tract, might have been too low to affect the microbial community at the ileal level to a substantial extent.
In all treatment groups, the proportion of neutrophils was greater at 2 wk compared with 5 wk after weaning. This could be related to the shorter interval between weaning (and its associated metabolic, physiological, and immunological effects) and to the first time point of blood sampling. The weaning process could have triggered the immune system and induced inflammation of the gut tissue related to increased levels of proinflammatory and inflammatory cytokines (Verdonk, 2005). In addition, piglets exposed to stress, such as in weaning, show an increased proportion of neutrophils in the blood (Morrow-Tesch et al., 1994). A subclinical infection of the piglets with Strep. suis may also have contributed to an increase in the proportion of neutrophils in the blood, although no clinical cases of such an infection were observed in the first part of the study when the blood samples were taken. In contrast, some clinical cases of such an infection were observed in the middle part of the study. At 2 and 5 wk after weaning, there were no differences in I-FABP concentrations among dietary treatments. On average, I-FABP levels were numerically less in piglets 5 wk after weaning compared with 2 wk after weaning. Results suggest that intestinal cell damage was not affected by dietary treatments at these 2 time points. This does not exclude the existence of differences in this respect more closely after the moment of weaning (e.g., in the first week). The time effect appeared to be an important factor in explaining the differences among bacterial profiles in the samples of the current experiment, suggesting that the microbiota in the gastrointestinal tract is not yet stable in the first weeks after weaning. This result is in agreement with previous studies of microbiota development in young animals (Konstantinov et al., 2006).
The current study suggests that YC might be an alternative to AGP in diets for weanling pigs. In addition, supplementing CWP containing MOS at a rate of 2 g/ kg to a diet containing YC did not show additional benefits to weanling pigs. More research is warranted to better understand the mode of action of YC in piglet diets with or without the addition of prebiotic substrates, such as CWP.
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Footnotes
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1 This research was financially supported by Diamond V Mills Inc. 
2 The technical assistance of A. H. A. A. M. van Bussel-van Lierop, G. P. Binnendijk, and R. A. Dekker is gratefully acknowledged. We thank O. N. Perez-Gutierrez and W. F. Pellikaan for their help with microbial community analyses.
3 Corresponding author: carola.vanderpeet{at}wur.nl
Received for publication February 2, 2007.
Accepted for publication June 27, 2007.
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Abstract
INTRODUCTION
