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Effect of inclusion of fermentable carbohydrates in the diet on fermentation end-product profile in feces of weanling piglets¹

Animal Nutrition Group, Wageningen University and Research Centre, PO Box 338, 6700 AH, Wageningen, the Netherlands

	Abstract

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An in vivo experiment was conducted to monitor the changes in fermentation end products in the feces of weaning piglets due to the inclusion of selected fermentable carbohydrates in the diet. The experiment involved 3 groups of 16 piglets each. Specially raised piglets (neither antibiotics nor creep feeding) were weaned abruptly at 4 wk of age. The piglets were offered 1 of 2 dietary treatments [a control diet (CON), or a fermentable carbohydrate-enriched diet (CHO)] and were subjected to 1 of the 2 fasting treatments (fasting for 2 d at the beginning of the experimental period or nonfasting). Fecal samples were collected per rectum every day during the experimental period. Piglets were slaughtered at the end of the 10-d experimental period, and digesta samples were collected from different parts of the gastrointestinal tract (GIT): the first half of the small intestine, the second half of the small intestine, the cecum, and colon. The DM, VFA profile, and ammonia concentrations were analyzed from the fecal and digesta samples. Daily feed intake was also recorded. There was no difference in concentrations of VFA in feces between the treatment groups. Ammonia concentration was lower (P < 0.05) in piglets fed the CHO diet compared with those fed the CON diet in both feces and digesta from different parts of GIT. Fasting had no effect on fermentation end products in feces. This study demonstrated that the inclusion of fermentable carbohydrates in weanling diets reduces protein fermentation along the GIT and also reduced the fecal concentration of ammonia.

Key Words: fermentable carbohydrate • fermentation • weaning piglet

	INTRODUCTION

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Weaning of piglets leads to dramatic changes in the gastrointestinal tract (GIT) microbiota by exposure to solid feed. This leaves them susceptible to colonization by pathogens and to the "postweaning syndrome" (Hopwood and Hampson, 2003). There may be possibilities to improve pig health by stimulating the commensal microbiota.

The gut microbiota is dependent upon the animal diet as the main source of substrate for its metabolism. Any changes in diet composition may have effects on the intestinal microbiota (Bedford and Apajalahti, 2001). Depletion in carbohydrates as energy source leads to more proteolytic fermentation in the hindgut (Piva et al., 1996). Excess protein fermentation in the large intestine results in increased ammonia concentration in the colon and predisposes early-weaned piglets to diarrhea (Dong et al., 1996). Sometimes temporary post-weaning anorexia is followed by a sudden increase in feed intake, which leads to an upset of the digestive function and to diarrhea (Hopwood and Hampson, 2003).

Inclusion of fermentable carbohydrates in the diet is an effective strategy to control microbial proteolysis (Shi and Noblet, 1993; Piva et al., 1996; Houdijk Jos, 1998). In pigs, substantial fermentation has been observed in the small intestine (Drochner, 1991; Jensen and Jorgensen, 1994; Konstantinov et al., 2004). By selecting ingredients with different rates of fermentation, it should be possible to design a diet that will stimulate fermentation all along the GIT.

The current study aimed to investigate 1) whether the inclusion of fermentable carbohydrates with variable rates of fermentation in weaning diets influences fermentation along the GIT and reduces the protein fermentation, 2) whether enforced fasting at the beginning of the postweaning period has an effect on concentration of fermentation end products in later periods, and 3) whether there are any gradual changes in the fermentation end-product profiles with time after weaning.

	MATERIALS AND METHODS

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All procedures involving animals were conducted in accordance with Dutch law on experimental animals and were approved by the Wageningen University Animal Experimental Committee (Dier Experimenten Commissie).

Experimental Design

An in vivo experiment was designed as 2 x 2 factorial arrangements of treatments. The experimental treatments were the 2 dietary treatments (with and without inclusion of fermentable carbohydrates) and 2 feeding management treatments (with or without enforced fasting at the beginning of the experimental period). The experiment was conducted with 3 identical groups of piglets. Each group was evaluated over a period of 10 d. For each group, 4 piglets from 4 litters were used (16 piglets per group; 48 piglets in total for the experiment). The piglets were chosen randomly.

On d 1 of each period, fecal samples were collected before the piglets were subjected to any treatment. Then, piglets from each litter were assigned to 1 of the 4 treatment combinations. Daily fecal samples were subsequently collected. At the end of each period, the piglets were slaughtered and digesta samples were collected from different parts of the GIT.

Animals and Housing

The 48 crossbred piglets (Hypor x Pietrain, mixed group of males and females) were weaned at 4 wk of age and transported to the experimental facility. The piglets had no access to creep feed before weaning, nor any antibiotic treatment before or during the experimental period. During the experimental period of 10 d, the piglets had free access to their diet (except the fasted piglets for the first 48 h) and clean drinking water. Daily feed intake was measured during the experiment. Piglets from the same litter were kept in adjacent individual pens separated by a wire mesh, so that they could have visual contact with their littermates. This arrangement was to prevent cross-contamination between litters. The continued contact with littermates was done to potentially reduce stress.

Dietary Treatments

The control diet (CON) was semipurified and was designed to have very low levels of fermentable carbohydrates (Table 1). The test diet with added fermentable carbohydrates (CHO) was based on this same diet, but had added carbohydrates in the form of unmolassed sugar beet pulp, native wheat starch, lactulose, and inulin. In the current study, 4 fermentable carbohydrates with different rates of fermentation were included in 1 of the dietary treatments.

For both of the diets, the main source of starch used was native cornstarch, with an ileal digestibility of approximately 97% (Martinez-Puig et al., 2003), to have a contrast in the substrate reaching the large intestine. The diets were composed in such a way that total energy and protein contents were comparable. Both diets contained no antibiotics and no copper beyond that of the trace mineral premix. The composition of the diets is shown in Table 1.

Fasting Treatments

The animals with enforced fasting were not offered any feed for 48 h from the moment of arrival at the experimental facility. The nonfasted animals, on the other hand, had free access to their diet from the moment of arrival at the facility. All piglets had free access to water at all times.

Slaughtering and Sampling

Daily fecal samples were collected in the morning with a gloved finger for DM, VFA, and ammonia concentration analyses. The piglets were slaughtered on d 10. First, Ketamin (Sanaket 10%, Anisane B.V., Raamsdonksveer, the Netherlands) was used as a preanesthetic (15 mg/kg of BW); 30 min later, the piglet was euthanized by intracardiac injection of T61 (a combination of embutramide, mebenzoniumiodide, and tetracain hydrochloride; Hoechst Roussel Vet, Frankfurt, Germany).

After dissection into the abdomen, the entire GIT was separated from the abdominal cavity. The GIT was ligated at regular intervals to avoid mixing of the digesta. In the laboratory, the GIT was divided into 4 parts: the first (cranial) half of the small intestine (SI-1), the second half of the small intestine (SI-2), the cecum (CE), and the colon (CO). From each part, digesta were mixed, and samples were collected for DM, VFA, and ammonia analysis within 10 min of slaughter. Samples were stored at –20°C until the analyses were done.

Analyses

Dry matter was determined by drying to a constant weight at 103°C (ISO standard 6496; ISO, 1999) and ash by combustion at 550°C (ISO standard 5984; ISO, 1978). The VFA concentrations in the fermentation liquids were analyzed by gas chromatography (Fisons HRGC Mega 2, CE Instruments, Milan, Italy), using a glass column fitted with Chromosorb 101; N₂ saturated with methanoic acid as the carrier gas, at 190°C; and isocaproic acid as an internal standard. Ammonia was determined according to the method described by Searle (1984).

Calculations

The branched-chain proportion (BCP) was calculated as an indicator of protein fermentation (Macfarlane et al., 1992), as follows:

[1]

The acetic (AP), propionic (PP), and butyric (BP) acid molar proportions (%) were calculated similarly.

Statistics

All statistical analyses were performed using the PROC GLM procedure of SAS (SAS Inst., Inc., Cary, NC). Because concentrations of the fermentation end product in feces and daily feed intake of the animal were measured repeatedly during the experiment, the statistical analysis was done for repeated measurements. In short, for the separation of time from treatment effects, a straight line was fitted through the end-product concentrations or daily feed intake values against time, beginning with the measurement from d 2. The existence of a quadratic component in this relationship was tested, but because it failed to improve the statistical fit over the linear, the latter was preferred. The slope of the line () represented the effect of time (t), and the intercept (at t = t) represented the average end-product concentration or daily feed intake during the experiment. Both the slope and intercept were treated as dependent variables, and treatment effects on slope and intercept were analyzed by ANOVA (see Eq. 2).

The values from d 1 feces were not considered in the analysis because on d 1, fecal samples were collected before the piglets were exposed to the treatments. The difference between the intercept for different treatment combinations and d 1 values for end-product concentrations was considered the postweaning change. This change was analyzed for significant difference among treatment combinations by ANOVA, using the following model:

[2]

where Y is the parameter to be tested, µ is the overall mean, D_i represents the effect of the diet i (i = 1, 2), F_j represents the effect of the fasting treatment j (j = 1, 2), (D x F)_ij represents the interaction of diet and fasting, and _ijk is the error term. The effect of period and litter was tested separately, and was removed from the model because it had no effect on any of the parameters tested.

For the digesta samples on d 10, the effects of diet, fasting, and the site within the GIT (where feces on d 10 were also considered as an additional site), and the interaction between diet and site within GIT, were analyzed by ANOVA, as follows:

[3]

where Y was the parameter to be tested; µ represented the overall mean; D_i represented effect of the diet I; F_j represented the effect of the fasting treatment; (D x F)_ij represented the effect of the interaction of diet and fasting; 1_ijk was the error term 1, which represented the random effect of animal within diet i, and fasting treatment j; G_l represented the effect of site within the GIT l; (D x G)_il, (F x G)_jl, and (D x F x G)_ijl denoted the respective interactions; and 2_ijklm was the error term 2, which represented the overall error. Differences were considered significant when P < 0.05.

	RESULTS

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Effect of Time

The changes in ADFI and fermentation end-product concentration in feces with time are presented in Table 2. Most of the responses, including ADFI and fecal DM content, changed with time (P < 0.001) except ammonia and total VFA concentration. This effect of time was independent of the experimental treatments with the exception that the effect of time on ADFI was affected by fasting (P = 0.018).

The effect of experimental treatments on ADFI, fermentation end-product concentration in feces, and fecal DM content are presented in Table 3. Fasting had no effect on any of the responses nor was there any interaction with diet. Diet had an effect on the ammonia concentration (P = 0.03) and BCP (P = 0.04), which were both lower for the CHO diet compared with the CON diet. Diet had an effect on the DM content of the feces (P = 0.05) but no effect on the total VFA concentrations.

The postweaning change; that is, the difference in fecal concentrations of different fermentation end products between d 1 and the average of the first 10 d on the treatments and dietary effect on this postweaning change are shown in Figure 1. For both dietary groups, there was a postweaning increase in the total VFA concentrations (for CON diet, P = 0.019 and for CHO diet, P = 0.015). However, ammonia concentration (P < 0.001 for both diets), AP (P = 0.007 for CON diet and P = 0.017 for CHO diet), and BCP (P = 0.002 for CON diet and P < 0.001 for CHO diet) were decreased postweaning. The diets had an effect on the postweaning change in terms of BCP and ammonia concentration. The CHO diet was observed to be more effective in lowering BCP (P = 0.04) and ammonia (P < 0.01) concentrations compared with the CON diet, but failed to increase total VFA concentration.

View larger version (12K): [in this window] [in a new window]   Figure 1. Postweaning changes (difference between d 1 and the postweaning average) in the fermentation end-product profile of feces as affected by dietary treatments in weaning piglets. Ammonia (mmol/L of fecal water), total VFA (mmol/L of fecal water), acetic acid proportion (AP, %), propionic acid proportion (PP, %), butyric acid proportion (BP, %), branched-chain fatty acid proportion (BCP, %). CON = control diet; CHO = diet with the inclusion of fermentable carbohydrates (**P < 0.01 represents the effect of diet on postweaning changes in the respective variable).

The CHO diet reduced the ammonia concentration and BCP in different sites within the GIT (Figure 2). Although the total VFA concentrations were greater for the CHO group (Awati, 2005), the difference between dietary groups did not reach significance. The GIT segment had effects (P < 0.001) on all responses including DM content, VFA, and ammonia concentrations. However, by d 10, fasting had no effect on the fermentation end products in the different sites within GIT, nor any interaction with diet.

View larger version (24K): [in this window] [in a new window]   Figure 2. Effect of diet on (a) ammonia concentration, and (b) branched-chain proportions, in the first (cranial) half of the small intestine (SI 1), the second half of the small intestine (SI 2), the cecum, the colon, and feces on d 10 postweaning. CON = control diet; CHO = diet with inclusion of fermentable carbohydrates (**P < 0.01, ***P < 0.001, for diet within a variable).

	DISCUSSION

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View larger version (7K): [in this window] [in a new window]   Figure 3. Average daily feed intake of weaning piglets without (nonfasted) or with enforced fasting (fasted) for 2 d at the beginning of the weaning period (**P < 0.01, ***P < 0.001, for nonfasted vs. fasted within day).

Time after weaning plays a major role in fermentation end-product profiles in the feces (Table 2). The AP, PP, and BP were time dependent (P < 0.001), although the effects of time on ammonia and total VFA concentration were less clear. The PP and BP were increased and the AP decreased (P < 0.001) with time after weaning. This might be due to postweaning development of microbiota, both in terms of numbers as well as in diversity. Furthermore, with the increase in ADFI with time (Table 2), availability of the substrate for the large intestinal microbiota was also increased. With diverse microbiota and high substrate availability, there is greater PP and BP, whereas AP decreases considerably (Macfarlane and Macfarlane, 2003). The DM content of the feces was increased with time (Table 2). Absorption of short-chain fatty acids stimulates sodium absorption from the intestinal lumen, which adds to the efficient reabsorption of the water in the large intestine (Roediger, 1980). Furthermore, the colon increases in size in the postweaning period (Pluske et al., 2003), which adds to the absorptive capacity of the colon, resulting in increasing DM content of feces.

Although, in comparison with the CON diet, the CHO diet resulted in lower ammonia concentrations (P = 0.03) and BCP (P = 0.04), which are mainly products of protein fermentation (Yokoyama et al., 1982; Russell et al., 1983; Macfarlane et al., 1992), there were no differences for VFA concentrations in the feces due to diet (Table 3). It was expected that cornstarch with approximately 97% ileal digestibility (Martinez-Puig et al., 2003) would reach the large intestine in negligible amounts. But the decrease in amylase activity during the first week after weaning (Lindemann et al., 1986) might have led to the escape of some cornstarch from enzymatic digestion in the small intestine and becoming available to microbial fermentation in the large intestine. This may therefore have masked the contrast in the 2 dietary treatments in the current study.

Postweaning change in fermentation characteristics showed a decrease in ammonia (P < 0.001 for both diets) and BCP (P = 0.002 for CON diet and P < 0.001 for CHO diet). This decrease was facilitated by the CHO diet (Figure 1). In the current study, both diets contained native cornstarch as the main starch source, along with fermentable carbohydrates in the CHO diet (Table 1). This clearly demonstrates a shift from protein fermentation toward carbohydrate fermentation.

In different parts of the GIT, a significant reduction in ammonia (cecum, colon, and feces) and BCP (SI1) on d 10 was also observed with the CHO diet compared with the CON diet (Figure 2). Similar results had been confirmed in an earlier in vivo study in our laboratory (Awati, 2005) for animals fed the same diets. This supports the hypothesis that inclusion of fermentable carbohydrates with different fermentation rates in weaner diets improves carbohydrate fermentation along the GIT with a reduction in harmful protein fermentation.

	Footnotes

 
¹ This research was funded by the EU grant "Healthy Pigut" (QLK5-LT2000-00522). Appreciation is expressed to Meijke Booij, Jane-Martine Muijlaert, Dick Bongers, and Huug Boer of the Animal Nutrition Group for their assistance with the laboratory analyses. Tamme Zandstra and personnel of the animal experimental facilities at "De Haar" are thanked for their cooperation during this study.

² Corresponding author: a.awati{at}massey.ac.nz

Received for publication December 7, 2004. Accepted for publication March 23, 2006.

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