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ANIMAL NUTRITION |
* Department of Veterinary Morphophysiology and Animal Production, Università di Bologna, via Tolara di Sopra 50, 40064, Ozzano Emilia, Italy;
and
ISAN, Facoltà di Agraria, Università Cattolica del S. Cuore, via Emilia Parmense 84, 29100, Piacenza, Italy;
and
DSPVPA, Università di Bologna, via Tolara di Sopra 50, 40064, Ozzano Emilia, Italy; and
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Division of Animal Nutrition and Production Physiology, Technical University of Munich, Hochfeldweg 6, 85350, Freising-Weihenstephan, Germany
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Key Words: cecal microflora • gluconic acid • growth performance • organic acid • piglet • short-chain fatty acid
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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). Antibiotics have been widely used to limit the impact of the postweaning period on animal health. Nevertheless, antibiotics fed to farm animals may be responsible for the spreading of bacteria that are resistant to such antimicrobials (Bager et al., 1997; Phillips et al., 2004). This potential risk and the consumer demand for a food chain that is free of drugs has resulted in the decision of the European Union to completely ban antibiotics used as growth promoters as of January 2006. Therefore, nonpharmaceutical feed supplements must be sought to control microbial activity in the gastrointestinal tract of nonruminant animals. Among alternatives are organic acids (Roth and c, 1998), probiotics (Klaenhammer, 2000), botanicals (Greathead, 2003), and prebiotics (Gibson, 1998).
Gluconic acid (GA) derives from the incomplete oxidation of glucose by some Gluconobacter strains (Deppenmeier et al., 2002). When fed to animals, GA acid is poorly absorbed in the small intestine; therefore, it reaches the lower gut (Asano et al., 1997), where it is fermented by the local microflora mainly to butyric acid (Tsukahara et al., 2002). Because butyric acid is the main energy source for epithelial cells of the large intestine (Roediger, 1980), feeding GA could indirectly stimulate epithelial growth in the lower gut of piglets.
The current study was intended to investigate the effects of GA on growth response and metabolism of swine cecal microflora in vitro and to investigate the effects of different levels of GA in the diets of piglets on growth performance, intestinal wall morphology, and intestinal microflora in vivo.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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. The ground feed (2 g; particle size <1 mm) was incubated in 40 mL of pepsin solution (2 g/L, 0.075 N HCl; Sigma Chemical, St. Louis, MO) in a shaking waterbath at 37sC for 4 h. Then, the pH was adjusted to 7.5 with NaOH (1 N), and 40 mL of pancreatin solution [10 g/L in a PBS solution (pH 7.5); Sigma Chemical] were added; the mixture was incubated in a shaking water bath at 37°C for 4 h. The composition of the PBS solution was as follows: 26.2 mmol of Na2HPO4 + 46.7 mmol of NaHCO3 + 3.3 mmol of NaCl + 3.1 mmol of KCl + 1.3 mmol of MgCl2 + 0.7 mmol of CaCl2 in 1 L of distilled water (Martillotti et al., 1987). After enzymatic digestion, the preparation was centrifuged (3,000 x g, 10 min, 4°C), washed twice with distilled water, recentrifuged (3,000 x g, 5 min, 4°C), and dried at 60°C overnight. Diet composition and chemical analyses of the diet before and after predigestion are reported in Table 1.
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). Within 20 min after slaughter (6 pigs, 10 mo old, live weight = 160 kg), samples of cecal contents were removed, pooled, and kept in a sealed nylon bag at 39°C during transfer to the laboratory. Then, the cecal contents were diluted with buffer (ratio = 1:2) and filtered through 4 layers of cheesecloth. The filtered liquid was used as inoculum. The buffer composition (McDougall, 1948) was as follows: 116.7 mmol of NaHCO3 + 7.6 mmol of KCl + 0.4 mmol of CaCl2·6H2O + 26.0 mmol of Na2HPO4·12H2O + 11.5 mmol of NaCl + 0.5 mmol of MgSO4·7H2O in 1 L of distilled water. Buffer pH was adjusted to 6.7 by adding 3 N HCl. The buffer solution was kept at 39°C and flushed with CO2 for 20 min before use. The inoculum was dispensed into five 10-mL glass syringes (5 mL of inoculum in each syringe) and five 50-mL vessels (previously flushed with CO2; 25 mL of inoculum in each vessel) per treatment, containing 20 and 100 mg of predigested diet (used as control diet), respectively. Syringes and vessels were sealed and incubated at 39°C for 24 h. There were 6 treatments: control diet or control diet plus GA (D-gluconic acid sodium salt; Sigma Chemical) at 2,000, 4,000, 6,000, 8,000, or 10,000 ppm. In all GA treatments, GA was added at the start of incubation, before sealing the 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 the cumulative volume of gas produced every 30 min was recorded. Samples of fermentation fluid were collected from each vessel at 0, 4, 8, and 24 h of incubation for ammonia and at 24 h for short-chain fatty acid (SCFA) 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 cages in a Controlled environment for a 6-wk trial period. After a 4-d adaptation period during which all piglets received the same base diet, animals (live weight = 7.44 ± 0.12 kg) were divided into 4 groups (12 animals per group) that were homogeneous for weight, gender, and litter. Then, piglets received the base diet with a) no addition (control diet) or with the addition of GA at b) 3,000, c) 6,000, or d) 12,000 ppm (a 50% GA solution was used; Merck-Schuchardt, 85662 Hohenbrunn, Germany). 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 diet was changed after the first 3 wk. Feed and water were provided ad libitum. Composition and chemical analyses of the experimental diets are reported in Table 2. Animals were individually weighed, and feed consumption was recorded for each pig weekly. The amount of feed wasted was recorded daily. Animal health was monitored throughout the trial.
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Animal housing and care were conducted under supervision of the veterinarian’s office of the Bavarian government. The handling protocol ensured proper care and treatment of all animals in conformity with the German law for animal protection.
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) and Van Soest et al. (1991) for NDF and ADF determinations. Gross energy of the predigested diet was measured by bomb calorimetry (model 1261, Parr Instrument, Moline, IL). Ammonia in fermentation fluid and in intestinal chyme was measured according to Searcy et al. (1967) using a commercial kit (Urea/BUN – Color, BioSystems S.A., Barcelona, Spain). The SCFA in fermentation fluid were analyzed by gas chromatography (Varian 3400, Varian Analytical Instruments, Sunnyvale, CA) with Carbopack B-DA/4% CW 2M and 80/120 packed column (Supelco, Sigma Aldrich s.r.l., 20151 Milano, Italy). The fermentation liquid was centrifuged (3,000 x g, 15 min), and 2 mL of the supernatant was added with pivalic acid as an internal standard (Fussel and McCalley, 1987) before injection.
For determination of SCFA in the intestinal chyme, the digesta were diluted 1:2 with distilled water and centrifuged (Hermle, Gosheim, Germany) in microfuge tubes (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). The concentration of SCFA in the supernatant was determined by a high performance liquid chromatograph equipped with a Polyspher OA KC column (Merck). The mobile phase consisted of 0.005 N H2SO4 with a flow rate of 0.4 m/min and a 70°C column temperature.
Bacterial Counts
Immediately after collection of feces and chyme samples, 1 g of sample was diluted with 9 mL of a 1% peptone solution and homogenized. Counts of viable bacteria in feces and chyme samples were determined by plating serial 10-fold dilutions (in 1% peptone solution) onto Lactobacillus Medium III agar plates (Medium 638, DSMZ, Germany) for lactic acid bacteria (LAB), Difco DRCM agar plates (Becton, Dickinson and Company, Franklin Lakes, NJ) for clostridia, and Mac-Conkey agar plates (N. 1.05465, Merck) for enterobacteriaceae. There were 6 feces and 4 chyme replicates per treatment. Lactobacillus Medium III and DRCM agar plates were incubated for 48 h at 39°C under anaerobic conditions (BBL GasPak Plus Anaerobic System Envelopes, Becton, Dickinson and Co., Sparks, MD). MacConkey agar plates were incubated for 24 h at 39°C under aerobic conditions.
Morphological Evaluations
Mucosal samples from jejunum, ileum, and cecum of each subject were fixed in 10% buffered formalin (formalin 40% 100 mL, monobasic sodium phosphate 18.16 g, sodium hydroxide 4.125 g, distilled water 1 L) and embedded in paraffin; histological sections were obtained from tissue blocks, cut perpendicular to the mucosal surface, and stained with haematoxylin and eosin. Histomorphometric measurements were performed using a computer-assisted, image-analysis system (Cytometrica, Byk Gulden, Milan, Italy) to assess the height of 10 villi and the depth of 10 crypts in each section on random-selected microscopic fields (Mitjans and Ferrer, 2004).
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; 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 of SAS (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 GA. 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 (SAS Inst., Inc.) in a completely randomized design. Linear and quadratic contrasts were used to determine the nature of the response to the feeding of GA. 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 METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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to 5, respectively. Gas production curves were accurately described by the modified Gompertz model (r2 = 0.90). Total gas production was increased by GA (linear, P < 0.001). Compared with control, the higher gas production determined by GA ranged from +28% when GA was added at 2,000 ppsm to +290% when GA was added at 10,000 ppm. Gluconic acid also increased the maximum rate of gas production (from +80% to +593% with GA at 2,000 and 10,000 ppm, respectively; linear, P < 0.001). The addition of GA shortened the exponential phase (linear and quadratic, P < 0.001) and increased the lag time (linear, P < 0.001) relative to control.
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). At 8 and 24 h, GA reduced ammonia concentration (linear, P < 0.001; quadratic, P < 0.01) from 12 to 83% and from 12 to 28% for GA at 2,000 and 10,000 ppm, respectively.
Production of total SCFA after 24 h of fermentation was increased by GA (linear, P < 0.001; Table 5). The greater total SCFA production ranged from +12% when GA was added at 2,000 ppm to +81% when GA was added at 10,000 ppm. Compared with the control, increasing GA concentrations from 2,000 to 10,000 ppm resulted in linear increases of acetic acid (+16 to +98%, respectively; P < 0.001), n-butyric acid (+25 to +148%, respectively; P < 0.001), and propionic acid (+2 to +26%, respectively; P < 0.001). Compared with the control, GA also increased n-valeric acid by 23% (linear, P < 0.001). The production of isobutyric and isovaleric acid was not affected by GA. The acetic to propionic acid and the acetic + butyric to propionic acid ratios were linearly increased by GA (P < 0.001).
In Vivo Feeding Trial
Performance of animals and intestinal SCFA concentrations are shown in Tables 6 and 7, respectively. All animals remained in good health throughout the duration of the trial. Compared with the control, GA tended to increase ADG (P of the model = 0.11; quadratic, P < 0.02), resulting in a 13 and 14% higher ADG when added at 3,000 and 6,000 ppm, respectively. Daily feed consumption and feed to gain ratio were not influenced by GA.
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Ammonia concentration in chyme sampled from the jejunum, ileum, and cecum was not influenced by the diet and averaged 34.4, 32.5, and 40.0 mmol/L, respectively.
Lactic acid bacterial counts in the jejunum were not influenced by treatments and averaged 6.2 log10 cfu/mL. Enterobacteriaceae and clostridia concentrations in the jejunum were under the detection limit and could not be determined. Clostridia, enterobacteriaceae, and LAB cecal counts were not significantly influenced by GA and averaged 7.3, 5.8, and 8.3 log10 cfu/mL, respectively.
Gluconic acid tended to increase total SCFA in the jejunum (+174, +87, and +74% for GA at 3,000, 6,000, and 12,000 ppm, respectively; P of the model = 0.07; quadratic, P = 0.07).
Morphological evaluations of intestinal mucosal samples from jejunum, ileum, and cecum did not show any significant differences among treatments. The length of villi in the jejunum and ileum averaged 374 and 342 µm, respectively. Average depth of crypts in jejunum, ileum, and cecum was 311, 270, and 330 µm, respectively.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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), the in vitro growth rate of cecal microflora was increased by an organic acid blend containing citric, fumaric, and malic acids. The current results further suggest that some organic acids may have a stimulating rather than an inhibiting effect on intestinal microflora.
In this study, in vitro production of total SCFA was linearly increased by GA. Tsukahara et al. (2002) incubated GA at 7,000 ppm for 24 h with cecal inoculum collected from pigs and observed that GA was mainly fermented to acetic, propionic, and n-butyric acid, the latter accounting for about 26% of total SCFA. In our study, GA was not the only energy source available to the bacteria because the cecal inoculum was added with 4 g of a predigested diet for growing pigs/L. This difference may be the reason for the different pattern of observed SCFA. In particular, the increased n-butyric acid in GA-containing vessels ranged from 9.6 to 11.7% of total SCFA, a much lower value compared with that reported by Tsukahara et al. (2002). In both studies, the cecal inoculum was collected from animals that had not been adapted to GA; however, it is likely that animals receiving different diets show differences in the composition of the cecal microflora that could affect the GA fermentation pattern.
In our study, the acetic + butyric to propionic acid ratio was progressively increased by increasing concentrations of GA. Overall, GA had only little effect on the production of propionic acid. Increasing butyric acid production in the hindgut may result in improved animal health because 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). Luciano et al. (2001) showed that within 2 h after perfusion of the colon of guinea pigs with a solution without butyrate, colon epithelial cells underwent apoptosis. 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). In a trial with weaned piglets, van Beers-Schreurs et al. (1998) observed that weaned piglets had shorter intestinal villi but greater hindgut concentrations of SCFA than unweaned animals. Those researchers speculated that SCFA could represent an important energy source during the postweaning period. Feeding GA to piglets may, therefore, not only stimulate the growth of the hindgut mucosa but also provide animals with energy in the form of SCFA. Nevertheless, in our in vivo study, GA failed to produce any significant changes in the piglet intestinal wall morphology.
The GA treatments affected in vitro production of ammonia. Ammonia concentrations were reduced by GA after 4, 8, and 24 h of fermentation. Protein digestion in young pigs is limited by the fact that gastric acid secretion does not reach appreciable levels until 3 to 4 wk after weaning (Cranwell and Moughan, 1989), which may lead to an insufficient activation of pepsinogen that occurs rapidly at pH 2 and very slowly at pH 4 (Taylor, 1962). As a result, feed proteins could enter the small intestine essentially intact with reduction in the efficiency of the protein digestion and greater amounts of protein reaching the hindgut. Findings from this in vitro experiment suggest that the presence of GA in the hindgut may prevent the energy limitation that leads to increased proteolysis and consequent release of toxic substances, such as ammonia and amines (Russell et al., 1983). Ammonia has also been shown to reduce the rate at which colonocytes oxidize butyric acid (Cremin et al., 2003).
Feeding GA to piglets tended to improve growth performance compared with piglets fed the control diet. In particular, the animals that received GA at 3,000 and 6,000 ppm showed the highest ADG, but the feed to gain ratio was not influenced by the GA. In fact, GA at 3,000 and 6,000 ppm increased the ADFI, but this effect was not significant. The relationship between the positive effect of organic acids on growth of piglets and the increase of feed intake is known (Kirchgeßner et al., 1995; Partanen and Mroz, 1999). At the same time, feeding high doses of organic acids may result in reduced feed intake and poor growth performance because of reduced feed acceptance (Eckel et al., 1992; Roth and Kirchgeßner, 1998).
Organic acids in their undissociated forms can cross membranes and be absorbed in the small intestine and hardly reach the large intestine if they are not microencapsulated (Piva et al., 1997). On the contrary, GA is only poorly absorbed in the small intestine, and it can reach the lower gut, where it is fermented (Asano et al., 1997). When large amounts of prebiotics (nondigestible oligosaccharides) are fed, their rapid fermentation in the hindgut may result in intestinal distension and abdominal pain and reduce animal feed intake (Houdijk et al., 1997; Mul, 1997). In our study, feeding levels of GA up to 1.2% of the diet to piglets did not result in reduced feed intake.
Intestinal bacterial counts were not affected by GA. In humans, GA is known to be fermented mainly by Lactobacillus spp. and bifidobacteria (Asano et al., 1994). Fermentation responses of LAB and bifidobacteria differ in relation to the substrate that is fermented and to the bacterial strains (Palframan et al., 2003). It is known that bifidobacteria and LAB do not produce butyrate (Cummings and Macfarlane, 1991), but bifidobacteria can produce, in addition to lactate, high amounts of acetate (Marounek et al., 1998). In our study, GA tended to increase total SCFA in the jejunum, but SCFA in the cecum were not affected. Although we killed a limited number of animals (4 per treatment), these data may suggest that GA is already fermented to some extent in the small intestine. Despite the fact that acetic acid was dramatically increased by GA in vitro, no acetic acid increase was observed in the cecal chyme collected from the piglets at the end of the in vivo trial. In vivo, organic acids are absorbed through the intestinal wall, and SCFA accumulate in the chyme only if there is a saturation of the intestinal absorption capacity. Therefore, SCFA total production determined by the diet may not be observable because of the intestinal absorption that occurs in vivo.
In a study with weaned piglets (Piva et al., 2002), feeding tributyrin and lactitol as precursors of butyric acid improved animal growth and reduced the cecal crypt depth; the cecal butyric acid concentration was not affected. Gálfi and Bokori (1990) observed that feeding sodium butyrate to growing pigs increased the length of ileal microvilli and the depth of cecal crypts. In our study, morphological evaluations of intestinal villi and crypts did not show any significant differences among treatments. One reason for the absence of major effects of GA on intestinal morphology and microflora could be that mucosal and digesta samples were collected at the end of the trial, 6 wk after weaning; therefore, from piglets with an almost fully developed digestive system (Gabert and Sauer, 1994).
This study showed that GA modulated the cecal microflora in an in vitro system, increasing the SCFA production and reducing the ammonia concentration. When fed to piglets after weaning, GA tended to improve animal growth and increase intestinal SCFA concentration. These results suggest that GA is worthy of further investigation as a potential alternative to antibiotics to improve growth performance and intestinal health status of piglets in the postweaning phase.
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IMPLICATIONS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Footnotes |
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2 Corresponding author: gbiagi{at}vet.unibo.it
Received for publication February 28, 2005. Accepted for publication September 19, 2005.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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This article has been cited by other articles:
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  G. Biagi, A. Piva, M. Moschini, E. Vezzali, and F. X. Roth Performance, intestinal microflora, and wall morphology of weanling pigs fed sodium butyrate J Anim Sci, May 1, 2007; 85(5): 1184 - 1191. [Abstract] [Full Text] [PDF]
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