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Effects of butyrate, avilamycin, and a plant extract combination on the intestinal equilibrium of early-weaned pigs¹

E. G. Manzanilla^*,2, M. Nofrarías, M. Anguita^*, M. Castillo^*, J. F. Perez^*, S. M. Martín-Orúe^*, C. Kamel and J. Gasa^*

^* Departament de Ciència Animal i dels Aliments; and Departament de Sanitat i Anatomia Animals, CReSA (Centre de Recerca en Sanitat Animal), Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain; and Pancosma, 01200 Bellegarde-sur-Valserine Cedex, France

	Abstract

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION IMPLICATIONS LITERATURE CITED

 
We evaluated the effects of 3 additives, sodium butyrate (AC), avilamycin (AB), and a combination of plant extracts (XT), on the productive performance and the intestinal environment of the early-weaned pig. The XT was a standardized mixture with 5% (wt/wt) carvacrol (from Origanum spp.), 3% cinnamaldehyde (from Cinnamonum spp.), and 2% capsicum oleoresin (from Capsicum annum). Pigs (n = 32) weaned at 18 to 22 d of age with an initial BW of 6.0 ± 0.10 kg were allocated to 8 pens that, in turn, were allocated to 4 treatments. The treatments included a basal diet (CT) or the basal diet supplemented with 0.3% of AC, 0.04% of AB, or 0.03% of XT. Productive performance was determined during the initial 14 d postweaning. On d 19 and 21 of the experiment, the pigs were killed to allow collection of digesta and intestinal tissue to evaluate variables indicative of aspects of the gastrointestinal environment. Treatments AB and AC improved G:F (P = 0.012 and 0.003, respectively) compared with the CT. Butyrate included in the diet was only detected in the stomach but not in cranial jejunum. When compared with CT, AC produced a lower ileal starch digestibility (P = 0.002) and a lower whole-tract OM and starch digestibility (P = 0.001 and 0.003, respectively), related to a lower VFA concentration in the cranial colon (P = 0.082) and a numerically reduced branched VFA percentage in the rectum. The AB treatment diminished propionate production in caudal colon (P = 0.002) and rectum (P = 0.012) compared with CT. The AC group exhibited deeper crypt depth in the jejunum without variations in villus height compared with CT (P = 0.042). The AC and AB groups also increased goblet cell presence in the colon (P = 0.001 and 0.032, respectively). On the other hand, AB and XT diminished intraepithelial lymphocytes in the jejunum (P = 0.003 and 0.034, respectively). The XT increased lymphocyte presence in the colon (P = 0.003). These results show the important influence of AB and AC on productive performance and on pig gut dynamics. The intestinal modifications observed for AB and AC compared with CT suggest distinct modes of action for each additive.

Key Words: avilamycin • butyrate • epithelium • fermentation • piglet • plant extract

	INTRODUCTION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION IMPLICATIONS LITERATURE CITED

 
Early weaning produces adverse consequences on pig performance and well-being (Pluske et al., 1997). Therapeutic and growth promoter antibiotics (AGP) were effective in improving performance of pigs through a decrease in the detrimental effects of microbiota (Visek, 1978). However, the possible influence of AGP on the development of bacterial resistance has motivated the full ban of these additives from feeds in the European Union beginning in January 2006. This new situation spawned investigations into alternative sources of feed additives (Kamel, 2001).

Acidifiers have become an alternative of choice to replace antibiotics in piglet diets because of their positive effects (Partanen and Mroz, 1999). Most studies have been done using formic, propionic, lactic, citric acids, or the corresponding salts, and to a lesser extent, butyrate despite its importance as a nutrient and trophic factor of the intestinal epithelium (Bach Knudsen et al., 2003).

Plant extracts are the ingredients of many commercial preparations currently used in animal production and present antimicrobial (Cowan, 1999, Dorman and Deans, 2000), antiviral (García et al., 2003), antioxidant (Aruoma et al., 1996), and antitoxin effects (Azumi et al., 1997) and are able to stimulate enzyme activity (Platel and Srinivasan, 1996) and immune function (Middleton and Kandaswami, 1992). The plant extract mixture used in this study has been shown to have effects on intestinal physiology and microbiota in the piglet and the chicken (Jamroz et al., 2003, Manzanilla et al., 2004).

These investigations were performed to elucidate the main effects of avilamycin, sodium butyrate, and a commercial plant extract combination on live performance, gut fermentation patterns, intestinal epithelium characteristics, and local immune response in the postweaning pig. Information about the intestinal microbiota associated with pigs in this experiment is presented by Castillo et al. (2006).

	MATERIALS AND METHODS

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The experiment was performed at the Experimental Unit of the Universitat Autònoma de Barcelona and received prior approval from the Animal Protocol Review Committee of the institution. The treatment, housing, husbandry, and slaughter conditions conformed to the European Union Guidelines (The Council of the European Communities, 1986).

Animals, Housing, and Dietary Treatments
A total of 32 (mixed male and female) commercial-cross, early-weaned pigs [(Landrace x Large White) x Pietrain; 6.0 ± 0.10 kg of BW and 18 to 22 d of age] from 8 litters and that had not received creep feed were used. Pigs were free of the principal swine infectious agents at the beginning and at the end of the study. The agents evaluated included porcine circovirus type 2, porcine parvovirus, porcine respiratory and reproductive syndrome virus, swine influenza virus, Aujeszky’s disease virus, Mycoplasma hyopneumoniae, and Lawsonia intracellularis.

The pigs were transported to the Universitat Autònoma de Barcelona facilities, where no particular bio-security measures were applied, and were allotted according to their initial weight into 8 pens (4 animals/pen). Pigs were allocated in the same room and separated by solid walls of 60 cm in height with bars into the top up to 80 cm. Each pen had its own feeder and nipple drinker. The weaning room was an iron-slat room equipped with automatic heating and forced ventilation, and the temperature was gradually reduced from 29 to 25°C during the experiment.

The pens were allotted to 1 of the 4 treatments that included a control diet (CT), or the control plus the inclusion of 1 of 3 different feed additives: avilamycin (AB), sodium butyrate (AC), or a plant extract combination (XT; Tables 1 and 2). Avilamycin 100 g/kg (Maxus, Elanco Animal Health Ltd., Madrid, Spain) was chosen as one of the main AGP used in piglet diets in the European Union immediately before the ban of AGP in 2006. It was included in the feed at commercial use dosage, 0.04%. Butyrate (Nature S.A., Barcelona, Spain) was included at 0.03% as recommended by the supplier for early-weaned pigs, based on Gálfi and Bokori (1990). The XT is a plant extract combination (Pancosma, S.A., Geneva, Switzerland) standardized in 5% (wt/wt) carvacrol (Origanum spp.), 3% cinnamaldehyde (Cinnamonum spp.), and 2% capsicum oleoresin (Capsicum annum) and included an inert fatty carrier (that represented the remaining 90%). It was included in the feed at 0.03% based on results obtained in previous experiments (Manzanilla et al., 2004). Chromic oxide was included in all diets as a digestibility marker (0.15% replacing sepiolite).

On d 19 and 21, after the 1200, 1330, 1500, and 1630 ingestion periods, 1 pig/treatment was weighed and killed by intravenous injection of sodium pentobarbitone (Dolethal, Vetoquinol, S.A., Madrid, Spain; 200 mg/kg of BW). Two pigs of each pen were killed daily. Pigs were exsanguinated, the abdomen opened immediately from sternum to pubis, and the whole gastrointestinal tract (GIT) was removed, weighed, and sampled. The pH in 5 segments was measured by insertion of a unipolar electrode through a small incision made in the wall (penetration pHmeter Crison 507, electrode Crison 52-32, Net Interlab S.A.L., Madrid, Spain). The pH measurements were performed in the middle of the caudal portion of the stomach, 15 cm cranial to the ileocecal valve, in the lowest part of the cecum, and in the colon at 20 cm caudal to the cecum and 50 cm from the anus.

Samples for histological study were obtained from the cranial jejunum (100 cm caudal to the stomach), ileum (20 cm cranial to the ileocecal valve), and colon wall (20 cm from the cecum). Immediately after slaughter, the samples (approximately 10-cm long) were opened longitudinally along the mesenteric attachment to 75% of the sample and fixed by immersion in 10% (vol/vol) buffered formalin.

Samples also were taken from homogenized stomach, cranial jejunum, ileum, cecum, cranial and caudal colon, and rectum. Contents were acidified with H₃PO₄ [approximately 4 g of fresh weight/mL of H₃PO₄, 1% (wt/wt) mercuric chloride, and 50 mM 3-methyl valerate as an internal standard], and stored at –20°C for VFA analysis. Total contents of the ileum (approximately 4 g of DM) and rectum (approximately 10 g of DM) were collected, frozen, lyophilized, milled, and stored for subsequent analysis.

Analytical Procedures
Chemical analysis of the diet was performed according to AOAC (1995) standard procedures. The GE was determined by adiabatic calorimetry, and Cr concentration in the diet, ileum, and feces was analyzed by atomic absorption spectrophotometry following the procedure described by Williams et al. (1962). Total starch of feed and digesta samples was measured by the method of Theander (1991). Briefly, total starch was determined by colorimetry as glucose liberated after enzymatic incubation of 0.2 g of sample with 0.1 mL of thermostable -amylase (Ref. A-4551, Sigma, Madrid, Spain) diluted 1/10 with distilled water for 1 h at 100°C, and amyloglucosidase (Ref. A-3514, Sigma) for 6 h at 60°C.

Tissue samples for histological study were dehydrated and embedded in paraffin, sectioned at 3 µm, and stained with hematoxylin and eosin. Morphometric measurements were performed with a light microscope (BHS, Olympus, Spain). Villus height (VH) and crypt depth (CD), intraepithelial lymphocyte (IEL) number in the villi, the index of mitosis in the crypt, intravillus lamina propria cell density, and goblet cell number in villi and crypts were measured. Measurements were performed on 10 well-oriented villi and crypts from each intestinal section of each animal. The VH and CD were measured using a linear ocular micrometer (Olympus, Ref. 209-35040, Microplanet, Barcelona, Spain). The VH/CD ratio was calculated by dividing VH by CD. The same villus and crypt columns were used to determine the number of IEL, goblet cells, and mitoses (meta- and anaphases); these variables were expressed per 100 enterocytes. On the basis of the cellular morphology, differences between the nuclei of enterocytes, mitotic figures, goblet cells, and lymphocytes were clearly distinguishable at 400x magnification. Intravillus lamina propria cell density was determined by counting total visibly stained nuclei and total lymphocytes in 10 fields (total area of 4,000 µm²) from each section using a grid ocular (Olympus, Ref. 209-35046, Microplanet). Cell density was expressed as the number of total stained cells and the number of lymphocytes per 1,000 µm² area. Number of lymphocytes in relation to the number of total cells was also calculated. All morphometric analysis was done by the same person, who was blinded to treatment.

The VFA concentration (µmol/g of fresh matter) in deproteinized cecal and colonic digesta was determined following the method of Jouany (1982) using 4-methylvaleric acid as the internal standard. The analysis was performed by GLC (HP-6890 Series, Hewlett Packard, Palo Alto, CA) with a flame ionization detector using a polyethylene glycol TPA-treated capillary column (30 m x 0.25 mm; BP21, SGE, Europe Ltd., Milton Keynes, UK).

Calculations and Statistical Analysis
Ileal and rectal apparent digestibility of each nutrient fraction (Nf) was calculated by the marker (Cr) ratio method between diet (D) and digestive content (dc) and using the equation

All results were analyzed by ANOVA with the GLM procedure (v. 9.1., SAS Inst. Inc., Cary, NC), using treatment as the classification factor. In productive performance analysis, pen was used as the experimental unit for ADG, ADFI, and G:F. In slaughter data analysis, pig was used as the experimental unit because the treatments were qualitative, and any potential differences in feed intake among pigs could not obscure those distinct treatment differences. When pig was used as the experimental unit, the effects of sex, litter, pen, and day and period of sacrifice were initially studied in the model but were not significant for any variable (P = 0.43 to 0.94); thus, they were ultimately excluded from the model. Preplanned comparisons of each of the treatment groups to the control treatment were made using Tukey’s test. The alpha level used for determination of significance for all analyses was set at 0.05 with statistical tendencies reported when the alpha level was less than 0.10.

	RESULTS

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Weight Gain and Digestibility
Table 3 shows the ADFI, ADG, and G:F observed during the 14-d experimental period, and the ileal and whole tract digestibilities of diets on d 19 and 21 after weaning. No differences were noted among treatments for ADFI. From 7 to 14 d, animals fed AB compared with CT had greater ADG (P = 0.095), and animals fed AB and AC had greater G:F (P = 0.015 and 0.015, respectively) compared with CT. It was reflected in the whole experimental period where AB and AC compared with CT resulted in a greater total G:F (P = 0.012 and 0.003, respectively). The AC decreased starch digestibility both in ileum (P = 0.002) and rectum (P = 0.003) and also decreased whole tract digestibility of OM (P = 0.001).

View larger version (15K): [in this window] [in a new window]   Figure 1. Total VFA concentration [µmol/g of fresh matter (FM)] in samples from the cecum, cranial and caudal colon, and rectum of early-weaned pigs receiving a control diet (CT) or the same diet with 0.04% avilamycin (AB), 0.3% sodium butyrate (AC), or 0.03% plant extract mixture (XT). The AC group tended to show lower VFA concentration in cranial colon compared with CT (P = 0.082).

	DISCUSSION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION IMPLICATIONS LITERATURE CITED

Productive Performance and Digestion
The most relevant and intriguing results were those observed with the AC treatment, which showed an improved feed efficiency in spite of a lower total tract digestibility of DM and starch. The higher content of starch in the feces samples of AC-treated animals (mean = 147 mg/g of DM) compared with CT (means = 72 mg/g of DM; SEM = 11.3 and P = 0.001), associated with lower VFA concentration in the cranial colon, and numerically lower proportion of branched VFA in caudal colon support the lower digestibility values observed. Volatile fatty acids are the major end products of bacterial metabolism in swine large intestine (Bergman, 1990) and may be used as an index of the quantity and composition of substrates being fermented in the hindgut. The results of VFA concentrations along the hindgut in animals fed on the AC diet (Figure 1) showed the most stable values with small variations between cecum and rectum. Similarly, the concentration of purine bases presented by Castillo et al. (2006) as an index of microbial activity showed slight changes along the large intestine compared with other treatments as CT or AB that showed the highest variations. Changes in the microbial community of the GIT of animals receiving the AC diet may have promoted a more stable ecosystem that may have prevented the proliferation of some particular bacterial groups as could be amilolytic bacteria. The slower fermentation rate of starch along hindgut could explain the lower digestibility values observed and that resistant starch remained available.

Given the better productive performance of animals fed the AC treatment, it suggests that AC could have improved health status of the animals and the efficiency of the use of nutrients for growth. Butyrate is considered an important nutrient for the epithelium integrity along the intestinal tract (Scheppach et al., 1996) and a likely protection against pathogenic bacteria (Gardiner et al., 1995). However, butyrate concentration was increased in the stomach of animals fed on the AC treatment but not in the small intestine or the hindgut. So, no direct effect of butyrate could be expected on intestinal epithelium. Gálfi and Bokori (1990) obtained productive improvements in nursery and fattening pigs using sodium butyrate, and they also showed that butyrate was present in the stomach but not in the intestine. They proposed 2 explanations for this fact; conversion of the butyrate to substances like acetic acid (Bugaut, 1987) or absorption of the butyrate in the oesophagal part of the stomach (Bugaut, 1987). No conversion to acetic acid was showed in our experiment, but other substances should be analyzed to rule out this possibility. Concerning butyrate absorption, we could expect some interesting systemic effect like the increase in plasma insulin found by Gálfi and Bokori (1990). In particular, butyrate is involved in the regulation of the IGF axis in different tissues (Nishimura et al., 1998; Tsubaki et al., 2001). Hathaway et al. (1999) defined increases in IGF-I as a consequence of AGP usage, and they proposed this mechanism as a possible mechanism of action for AGP. The study of this mechanism could also be of interest for butyrate.

Animals fed on the AB treatment had improved growth rates and feed efficiency with no changes in the whole-tract digestibility of OM. Animals fed this diet also showed a lower concentration of propionic acid in the hindgut, likely associated with the fermentation of different fractions of the OM (Bergman, 1990). However, it also has been shown that different bacteria fermenting the same substrate may be able to produce differences in fermentation products (Jiang and Savaiano, 1997). The changes observed on total VFA concentration with the AB treatment were associated with the highest variation of purine bases concentration along the hindgut; that evolved from the highest values in the cecum to the lowest values in the caudal colon (Castillo et al., 2006). These results on fermentation products likely reflect a higher microbial growth in the cecum with AB that may exhaust fermentable carbohydrates and promote fermentation of protein as digesta flows through the colon.

The improvement in productive performance observed with AB, as that observed with the AC diet, could be related with changes on microflora and their likely interaction with the nutritional requirements of animals. In the results presented by Perez de Rozas et al. (2004) and Castillo et al. (2006) using molecular techniques, different intestinal segments were studied and some bacterial groups identified. Bacterial profile (Perez de Rozas et al., 2004; Castillo et al., 2006) showed important divergences among treatments, with the AB and AC diets promoting the highest bio-diversity in the GIT microflora, particularly in the caudal parts of the gut. The biodiversity is a measure of the quantity of different microorganisms detected, and it has been proposed as an indicator of stability of the intestinal microbiota (Zoetendal et al., 2004). A higher biodiversity may produce a beneficial effect on animal performance because it avoids the proliferation of a simple bacterial group and a likely disbiosis (Jensen et al., 2003). This benefit may have appeared even in the case that digestibility was reduced (AC treatment) or not modified (AB treatment), raising the importance of different factors affecting the energy and protein requirement for growing of early-weaned pigs. How these additives produce this higher biodiversity and modify growth, and how the changes in microbial populations, and especially biodiversity, are related to changes in digestion and fermentation of the substrates remains to be elucidated.

Supplementation with XT did not promote better productive performance or whole-tract digestibility of OM and did not affect the VFA profile.

Intestinal Morphology
Enterocytes show a secretory function when they are in the crypt and absorptive function when they migrate to the villi, which implies that net absorption in the small intestine depends on the VH/CD ratio (Buddle and Bolton, 1992). Decreases in this ratio after weaning impair absorption and increases the diarrhea risk (Pluske et al., 1997). In this experiment, morphology measurements of intestinal mucous membrane (VH and CD) were in a range comparable with data obtained by Cera et al. (1988), Zijlstra et al. (1996), and Pluske et al. (1996). However, AB and AC decreased VH/CD ratio through an increase in crypt depth. This lower ratio was not associated with changes in ileal OM digestibility and was present in the groups with better productive performance. As shown by Vente-Spreeuwenberg et al. (2003), VH is directly correlated to productive performance and fecal consistency but not crypt depth. So the change in VH/CD produced here only by increased crypts could not be considered as a risk factor for the piglets.

In the AB-treated animals, the lower VH/CD ratio appeared in coincidence with a decreased number of IEL and numerically decreased number of lymphocytes and nuclei in the lamina propria. In contrast, IEL were not altered in the AC-treated animals, and goblet cells were increased in ileum. Jiang et al. (2000) related a higher number of nuclei in lamina propria to a higher activation of the immune system. Lymphocytes also have been shown to have important functions in the regulation of the epithelium renewal growth and differentiation (Matsumoto et al., 1999), and in this experiment could be related to an upregulation of the epithelial renewal. The causal relation of these higher goblet cell number and deeper crypts is not clear. Tsukahara et al. (2003) showed both changes because of the presence of luminal butyrate, but we did not find this change in this experiment. On the other hand, the coincidence of these changes in AB, AGP, and AC treatments could support the hypothesis of the effects on the IGF axis because this axis is involved in the regulation and differentiation of the epithelial cells (Nishimura et al., 1998).

The XT supplementation promoted a decrease in IEL in jejunum and ileum, but this variation was accompanied by an increase of lymphocyte presence on the lamina propria, which was also present in colon. The IEL and lamina propria lymphocytes are in constant dialogue in the intestine, and they can migrate from lamina propria to IEL during the early maturation of the intestine (Stokes et al., 2002). The different disposition of the lymphocytes in epithelium and lamina propria may indicate differences of the luminal stimuli among treatments, which produce different maturation patterns. The changes observed on histology with the 3 additives could be related to different luminal stimuli. It is known that luminal bacteria can produce variations in epithelial morphology and immune responses (McCracken and Lorenz, 2001). For instance, IEL number and goblet cells variations may be a response of the epithelium to changes in microbiota (Deplancke and Gaskins, 2001; McCracken and Lorenz, 2001). The results presented on microflora by Castillo et al. (2006) showed important divergences among treatments. However, other mechanisms interfering with epithelium instead of microbiota should not be ruled out. For instance, cinnamaldehyde has been described to have effects on lymphocyte proliferation and maturation (Koh et al., 1998) and could have caused the change observed in epithelium immune cells.

	IMPLICATIONS

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION IMPLICATIONS LITERATURE CITED

 
Results presented here introduce the existence of different mechanisms of action for various in-feed additives, in particular avilamycin and sodium butyrate, and the distinct changes produced in the intestinal mucosa by these additives. It is important to highlight the action of sodium butyrate for future investigations not only due to its acid properties but also due to effects on epithelial parameters and digestibility. Microbial biodiversity and its likely influence on epithelia are proposed as an important factor for a successful weaning process.

	Footnotes

 
¹ EGM received of a grant from the Departament d’Universitats, Recerca i Societat de la Informació (DURSI) of the Generalitat de Catalunya for this study. This work was financed by Ministerio de Ciencia y Tecnología (project AGL2001-2621-C02-01) and Pancosma.

² Corresponding author: edgar.garcia{at}uab.es

Received for publication September 9, 2005. Accepted for publication July 3, 2006.

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Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION IMPLICATIONS LITERATURE CITED

 


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