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* DIMORFIPA, Università di Bologna, 40064 Ozzano Emilia, Italy;
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ISAN, Facoltà di Agraria, Università Cattolica del S. Cuore, 29100 Piacenza, Italy;
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DSPVPA, Università di Bologna, 40064 Ozzano Emilia, Italy; and
and
Division of Animal Nutrition and Production Physiology, Technical University of Munich, 85350 Freising-Weihenstephan, Germany
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: butyrate • cecal microflora • growth performance • organic acid • swine
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Boudry et al., 2004; Hedemann and Jensen, 2004). These problems were counteracted with widespread use of antibiotic substances that may as a side-effect select antibiotic-resistant genes in the intestinal flora with the possibility of a transfer to human pathogens (Bager et al., 1997; Phillips et al., 2004). Because of these concerns, the European Union decided to completely ban the antibiotics used as growth promoters as of January 2006.
Adding organic acids to piglet diets is known to be helpful in overcoming problems occurring in the post-weaning period (Tsiloyiannis et al., 2001) and improving animal growth performances (Partanen and Mroz, 1999). In particular, sodium butyrate (SB) has been shown to improve growth performance of weaned piglets (Piva et al., 2002b). Moreover, butyric acid is the main energy source for the epithelial cells of the large intestine (Roediger, 1980) and the terminal ileum (Chapman et al., 1995). In a trial with growing pigs, SB not only improved animal growth but also increased the length of the ileal microvilli and depth of the cecal crypts on intestinal mucosa (Gálfi and Bokori, 1990).
The purpose of the current study was to investigate the possible dose of SB effective in modulating swine cecal microflora and in vivo dose-response of SB fed to piglets on growth performances, intestinal wall morphology, and microflora.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In Vitro Fermentation
The in vitro study was conducted at the Department of Veterinary Morphophysiology and Animal Production, University of Bologna, Italy, following the procedures described in Biagi et al. (2006a).
A diet for pigs was predigested in vitro to simulate the ileal digestion as described by Vervaeke et al. (1989). This is a 2-step procedure in which feed is first incubated in a pepsin-HCl solution and then in a pancreatin solution. Diet composition and chemical analyses of the diet before and after predigestion are reported in Table 1. The predigested diet was used as the substrate in the in vitro fermentation study (Biagi et al., 2006a).
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) and filtered. The filtered liquid was used as the inoculum. The inoculum was dispensed into five 10-mL glass syringes (5 mL of inoculum in each syringe) and five 50-mL vessels (25 mL of inoculum in each vessel) per treatment, containing 20 and 100 mg of the predigested diet (used as control diet), respectively. Syringes and vessels were sealed and incubated at 39°C for 24 h. There were 4 diet treatments: control diet, or control diet plus SB (butyric acid sodium salt; Sigma Chemical, St. Louis, MO) at 60, 120, or 240 mM. In all SB treatments, SB was added at the beginning of incubation, prior to sealing syringes and vessels. The pH of the inoculum was adjusted to 6.7.
Gas production was measured as described by Menke et al. (1979), using 10-mL glass syringes and recording the cumulative volume of gas produced every 30 min. Samples of fermentation fluid were collected from each vessel at 0, 4, 8, and 24 h of incubation for ammonia determinations.
In Vivo Feeding Trial
The in vivo feeding trial was conducted at the Division of Animal Nutrition and Production Physiology, Technical University of Munich, Germany. Forty-eight crossbred piglets (German Landrace x Pietrain) were weaned at 28 d and transported from the piggery to the barn where they were housed in individual crates (60 x 100 cm, on slatted plastic floor with free access to feed and drinking water provided by nipple drinkers placed in one corner of the crate) in a controlled environment for a 6-wk trial period. The current study was done reproducing the experimental design that had been previously used for a trial with another group of weanling pigs (Biagi et al., 2006a). After a 4-d adaptation period during which all piglets received the same base diet, the pigs (6.68 ± 0.13 kg of BW) were divided into 4 groups (12 animals per group) that were homogenous for weight, sex, and litter. Then the pigs received 1 of 4 diet treatments, consisting of the base diet with a) no addition (control diet), or with the addition of SB at b) 1,000 ppm, c) 2,000 ppm, or d) 4,000 ppm (spray dry n-butyric acid sodium salt complexed with vegetal protein, Adimix CP, INVE Nutri-Ad, Kasterlee, Belgium; containing SB at 85%). All diets were formulated to provide the same amount of energy, protein, essential amino acids, calcium, and phosphorus. No antimicrobial agents were added to the diets. The composition of the base control diet was changed after the first 3 wk. Feed and water were provided on an ad libitum basis. Composition and chemical analyses of the experimental diets are reported in Table 2.
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On d 42, 6 animals per treatment were killed by electrical stunning followed by complete bleeding. Within 20 min after death, the content and the mucosa from jejunum, ileum, and cecum were sampled for pH, ammonia, and short-chain fatty acids (SCFA) determination and for intestinal mucosal morphology analysis (Biagi et al., 2006a). Samples from the jejunum and the cecum were also cultured for viable counts of bacteria.
Chemical Analyses of Feed, Fermentation Fluid, and Intestinal Contents
Analyses of the diets (CP, crude fiber, ether extract, ash, and starch) were performed according to AOAC standard methods (AOAC, 2000; method 954.01 for CP, method 962.09 for crude fiber, method 920.39 for ether extract, method 942.05 for ash, and method 920.40 for starch) and Van Soest et al. (1991) for NDF and ADF determinations. Neutral detergent fiber was assayed with a heat-stable amylase (Sigma Chemical, St. Louis, MO) and expressed inclusive of residual ash; acid detergent fiber was expressed inclusive of residual ash; lignin was determined by solubilization of cellulose with sulphuric acid.
Ammonia in fermentation fluid and intestinal chyme was measured according to Searcy et al. (1967) using a commercial kit (Urea/BUN – Color, BioSystems S.A., Barcelona, Spain). For the determination of SCFA in the intestinal chyme, the digesta were diluted 1:2 (vol/ vol) with distilled water and centrifuged (14,000 x g, 10 min), and 1 mL of the supernatant was transferred to microfuge tubes and deproteinized with 50 µL of perchloric acid (Merck, Darmstadt, Germany). After 3 h, the samples were centrifuged again (14,000 x g, 10 min). Concentration of SCFA in the supernatant was determined by gas chromatography (Biagi et al., 2006a).
Bacterial Counts
Immediately after collection of the chyme samples, a 1-g sample was diluted with 9 mL of a 1% peptone (Becton Dickinson, Franklin Lakes, NJ) solution and homogenized. Viable counts of bacteria in chyme samples (n = 6) were measured by plating serial 10-fold dilutions (in 1% peptone solution) onto Lactobacillus Medium III agar plates (Medium 638, DSMZ, Germany) for lactic acid bacteria, Difco DRCA agar plates (Becton, Dickinson and Company, Franklin Lakes, NJ) for clostridia, and MacConkey agar plates (No. 1.05465, Merck) for enterobacteriaceae. Lactobacillus Medium III and DRCA agar plates were incubated for 48 h at 39°C under anaerobic conditions (H2 + approximately 4 to 10% CO2; BBL GasPak Plus Anaerobic System Envelopes, Becton, Dickinson and Company). MacConkey agar plates were incubated for 24 h at 39°C under aerobic conditions.
Morphological Evaluations
The height of villi and depth of crypts on mucosal samples from jejunum, ileum, and cecum were assessed as described in Biagi et al. (2006a).
Statistical Analyses
In Vitro Fermentation.
A modified Gompertz bacterial growth model (Zwietering et al., 1992) was used to fit gas production data. This model assumes that substrate levels limit growth in a logarithmic relationship (Schofield et al., 1994), as follows:
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where V = volume of gas produced at time t; t = fermentation time; VF = maximum volume of gas produced; µm = maximum rate of gas production, which occurs at the point of inflection of the gas curve; e = Napier’s constant (2.718); and 
= the lag time, as the time-axis intercept of a tangent line at the point of inflection (Zwietering et al., 1990).
The duration of the exponential phase was calculated from the parameters of the modified Gompertz equation, as suggested by Zwietering et al. (1992), with the following:
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Curve fitting was performed using the program GraphPad Prism 4.0 (GraphPad Software, San Diego, CA). Total gas production, maximum rate of gas production, lag time, duration of the exponential phase, ammonia, and SCFA data were analyzed by ANOVA using the GLM procedure (SAS Inst. Inc., Cary, NC) in a completely randomized design. Linear and quadratic contrasts were used to determine the nature of the response exhibited to the addition of SB. Each syringe and vessel formed the experimental unit. Differences were considered statistically significant at P < 0.05.
In Vivo Feeding Trial.
Data were analyzed by ANOVA using the GLM procedure of SAS in a completely randomized design. Linear and quadratic contrasts were used to determine the nature of the response exhibited to the feeding of SB. Each piglet formed the experimental unit. Differences were considered statistically significant at P < 0.05.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Compared with control, total gas production was reduced by SB (linear, P < 0.001; Table 3), and this reduction ranged from 10% when SB was added at 60 mM to 33% with SB used at 240 mM. Sodium butyrate also reduced the maximum rate of gas production (from –22 to –56% with SB at 60 and 240 mM, respectively; linear and quadratic, P < 0.001). The addition of SB resulted in a longer exponential phase than control (linear, P < 0.001), whereas the lag time was not influenced by SB.
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). The increase in ammonia ranged from 5 to 53% at 4 h and from 18 to 56% at 8 h for SB at 60 and 240 mM, respectively. At 24 h, ammonia was increased by 20 and 42% when SB was used at 120 and 240 mM, respectively.
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). However, animals receiving SB at 4,000 ppm reached a 2 kg numerically greater final BW than control animals (28.8 vs. 26.8 kg).
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).
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).
Sodium butyrate did not affect SCFA concentrations in the jejunum and ileum (Table 7). In the cecum, isobutyric acid concentration was higher (linear, P < 0.01) in animals fed SB at 2,000 and 4,000 ppm than in the control animals (+11 and +41%, respectively).
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Morphological evaluations of intestinal mucosa samples from jejunum, ileum, and cecum did not show any significant differences among treatments. The length of villi in jejunum and ileum averaged 465 and 340 µm, respectively. Average depth of crypts in jejunum, ileum, and cecum was 346, 279, and 366 µm, respectively (data not shown in tables).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), the use of an acid blend (containing fumaric, malic, citric, and phosphoric acid at a final concentration of 0.75 g/L) enhanced bacterial cecal fermentation and reduced ammonia concentrations. In this experiment, we used SB at concentrations (60, 120, and 240 mM are equivalent to 5.3, 10.6, and 21.2 g/L) that were much higher than those previously used by Piva et al. (2002a). Thus, organic acids may have different effects on cecal gas production and ammonia concentration depending on the organic acids and concentration being used.
In the in vivo experiment, SB did not significantly improve animal growth performance. However, pigs receiving the diet containing SB at 4,000 ppm showed a numerical greater final BW (28.8 vs. 26.8 kg) and ADFI (835 vs. 773 g) than control animals. During another feeding trial, Piva et al. (2002b) showed that feeding SB at 800 ppm to piglets in the first 8 wk after weaning improved ADG and ADFI during the first 2 wk, whereas no additional benefits were observed in the third week and onward of treatment. In a previous study, Gálfi and Bokori (1990) observed that feeding SB at 1,700 ppm improved ADG, ADFI, and feed efficiency of growing pigs. The incoherent effects of SB on pig growth may be primarily associated with the different dietary composition and gut maturation status.
According to the in vitro results, in the in vivo study, cecal ammonia concentrations tended to show a linear increase when pigs were fed increasing amounts of SB. Moreover, cecal concentrations of isobutyric acid were linearly increased by SB. Because isoacids are formed from the deamination of valine and leucine (Van Soest, 1982) and are indicative of protein catabolism extent, our results seem to suggest that cecal proteolysis was increased by SB. Furthermore, whereas pH of stomach and small intestine was not affected by treatment, the cecal pH was linearly increased by SB. Because undissociated organic acids can cross membranes and be absorbed in the small intestine and hardly reach the large intestine unless they are microencapsulated (Piva et al., 1997), a direct effect on cecal pH by SB is very improbable. In this study, intestinal butyric acid concentrations were not increased in SB-fed animals in any of the sampled segments. Experimental diets contained the same amount of protein and fiber and slightly differed only for the starch content because cornstarch was used as a substitute for SB. Nevertheless, it is very unlikely that small amounts of dietary starch could justify the observed differences in the ammonia cecal concentrations. We do not have explanation of how SB increased cecal pH and cecal concentrations of ammonia and isobutyric acid considering that clostridia cecal counts were unchanged by SB.
The antimicrobial properties of organic acids may be the result of the ability of organic anions to build up into the bacterial cells (Russell and Diez-Gonzalez, 1998). In the in vitro study, SB only partially inhibited the in vitro bacterial fermentation, despite the fact that SB was added to the fermentation vessels at relatively high concentrations. In vivo, bacterial counts in jejunum, ileum, and cecum were not affected by feeding SB to piglets that were slaughtered at 10 wk of age. Conversely, Gálfi and Bokori (1990) reported that feeding SB reduced ileal counts of coliforms in growing pigs slaughtered at 241 d of age. It is possible that the different age at which animals were slaughtered together with environmental and dietary differences between the 2 studies might explain the different effect of SB on intestinal microflora.
The butyric acid is the main energy source for the epithelial cells of the large intestine (Roediger, 1980) even in presence of glucose and glutamine (Darcy-Vrillon et al., 1993). There is also evidence that in humans butyric acid rather than glucose and glutamine is the preferred fuel substrate of the terminal ileal mucosa (Chapman et al., 1995). It is well known that weaning has a dramatic negative impact on the intestinal mucosal morphology of piglets (Gu et al., 2002). Feeding butyrate may reduce some of the negative effects of weaning by providing the ileal and the hindgut mucosa with the preferred energy source. In this study, morphological evaluations of intestinal villi and crypts did not show any significant differences among treatments. Results do not agree with Gálfi and Bokori (1990) where SB increased the length of the ileal microvilli and depth of the cecal crypts on intestinal mucosa of growing pigs (241 d of age). Wang et al. (2005) reported that feeding weaning piglets with 1,000 ppm of SB increased the height of the intestinal villi. In another study, when weaned piglets were fed tributyrin and lactitol as precursors of butyric acid (Piva et al., 2002c) animal growth was improved, whereas cecal crypt depth was reduced. In the present trial, one reason for the absence of major effects of SB on intestinal morphology could be that the SB concentrations along the gut were not increased by any tested dose, suggesting a very rapid metabolization of SB prior to reaching the jejunum. Furthermore, intestinal mucosa samples have been collected at the end of the trial, 6 wk after weaning, therefore from piglets with almost a fully developed digestive system (Gabert and Sauer, 1994). When gluconic acid was fed at 0.3% for 42 d to weanling pigs (Biagi et al., 2006a) as a potential source of butyric acid (Tsukahara et al., 2002) in the piglet intestine, animal growth tended to be improved, but the morphology of the intestinal wall was not affected by treatment. Conversely, when piglets fed with 0.3% sodium gluconate were killed 32 d after weaning, ileal villi tended to be longer and cecal crypts were significantly shorter than in control animals (Biagi et al., 2006b).
This study showed that SB influenced the activity of the cecal microflora in an in vitro system, reducing the total gas production but increasing ammonia concentrations. When fed to piglets, SB did not improve the animal growth performance but indirectly increased cecal pH and cecal ammonia concentrations. Further studies will be needed to clearly understand the mechanisms underlying the effects observed when SB is fed to piglets.
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Footnotes |
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2 Corresponding author: gbiagi{at}vet.unibo.it
Received for publication June 13, 2006. Accepted for publication January 29, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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