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Effect of plant extracts and formic acid on the intestinal equilibrium of early-weaned pigs¹

E. G. Manzanilla^*,2, J. F. Perez^*, M. Martin, C. Kamel, F. Baucells^* and J. Gasa^*

^* Departament de Ciència Animal i dels Aliments and and Departament de Sanitat i Anatomia Animal, CReSA (Centre de Recerca en Sanitat Animal), Universitat Autonoma de Barcelona, 08193, Bellaterra, Spain; and and Axiss France S.A.S., 01205 Bellegardesur-Valserine Cedex, France

	Abstract

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We evaluated the effects of a plant extracts mixture (XT) standardized in 5% (wt/wt) carvacrol, 3% cinnamaldehyde, and 2% capsicum oleoresin (oregano, cinnamon and Mexican pepper), alone or in combination with formic acid (FA), on the productive performance and the intestinal ecosystem of the early-weaned pig. Pigs weaned at 20 ± 1 d of age (n = 216) were allocated in 24 pens and fed a standard medicated prestarter diet for 12 d. Twelve days after weaning, a stress management system based on social and dietary stress factors was applied to the animals, after which, each group was allocated to one of six dietary treatments, which followed a factorial arrangement, with three levels (as-fed basis) of the XT (0, 150, and 300 mg/kg) and two levels of FA (0 and 0.5%). On d 24 and 25 after the stress episode, eight pigs per treatment were killed to examine variables describing some aspects of the gastrointestinal ecology. Two days after the stress episode, an Escherichia coli K88 diarrhea episode occurred, and five casualties were registered. Four of the five deaths occurred in pens of pigs not fed the XT. The FA resulted in better G:F (P = 0.040) in coincidence with shorter villous height (P = 0.073) and lower rectal total microbial mass (P = 0.078). Both XT and FA addition increased stomach content (P = 0.006 and 0.003, respectively) and percentage of DM (P = 0.089 and 0.010, respectively), suggesting an increased gastric retention time; consequently, pH was also increased (P = 0.005 and 0.060, respectively). The XT decreased ileum total microbial mass (P = 0.025) and increased the lactobacilli:enterobacteria ratio (P = 0.002). The VFA profile in the cecum and colon was modified by XT inclusion, increasing the proportion of acetate (P = 0.018 and 0.025, respectively) and diminishing the proportion of butyrate (P = 0.096 and 0.040, respectively) and valerate (P = 0.001 and 0.039, respectively). Both XT and FA were shown to be effective in modifying the gastrointestinal ecosystem, stomach contents, and stomach emptying rate, which are proposed as important aspects in the mechanisms of action for these additives.

Key Words: Acidifiers • Intestinal Health • Microbiota • Piglets • Plant Extracts • Weaning

	Introduction

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Early weaning produces reductions in the performance of the postweaning pig, making it vulnerable to disease (Pluske et al., 1997). For many years, both therapeutic and growth-promoter antibiotics (AGP) have been effective in improving the performance of the piglets through a decrease in the detrimental effects caused by microbiota (Visek, 1978). Currently, the possible development of bacterial resistance has motivated the ban of most AGP in animal nutrition in the European Union, where only avilamycin, flavophospholipol, monensin, and salinomycin are allowed until January 2006. This situation has allowed for the investigation and use of new feed additives such as acidifiers, probiotics, prebiotics, and phytogenic substances (Kamel, 2001).

Acidifiers seem to be the alternative of choice to replace antibiotics in postweaning pig diets because of the large body of evidence regarding their positive effects, as reviewed by Partanen and Mroz (1999). However, the results documented are not yet comparable to those obtained with AGP, and other alternatives should be tested. Plant extracts are another class of alternatives. These have been shown not only to have antimicrobial effects (Didry et al., 1994; Cowan, 1999; Dorman and Deans, 2000) but also to have antitoxin effects, and might stimulate enzyme activity or have immunomodulatory effects (Middleton and Kandaswami 1992; Azumi et al., 1997; Platel and Srinivasan, 2000). Nonetheless, their modes of action are not yet fully understood, and further scientific investigation is needed. Conversely, the use of different additives in combination could be an approach to achieve better performance.

In the present experiment, we evaluated the activity of a commercial plant extract mixture (XT; containing carvacrol, cinnamaldehyde, and capsicum oleoresin) and formic acid (FA), separately or in combination, to elucidate the specific effects of these components on productive performance, digestive variables, intestinal microbiota populations, and fermentation patterns in the postweaning pig.

	Materials and Methods

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This experiment was performed at the Experimental Unit of the Universitat Auto `noma de Barcelona and received prior approval from the Animal Protocol Review Committee of the institution. The treatment, housing, husbandry, and slaughtering conditions conformed to current European Union guidelines.

Animals, Housing, Management, and Dietary Treatments
Preexperimental Period.
The preexperimental period lasted 12 d. Two hundred forty weaning pigs ([Landrace x Large white] x Pietrain) excluded from receiving creep feed, 6.0 ± 0.40 kg live weight (BW) and 20 ± 1 d old, were allocated to 24 pens (10 animals per pen) in an environmentally controlled room. Pens were organized in four lines of six pens, and each line was considered to be a replicate of pens for the slaughter of the pigs. During the preexperimental period, pigs were fed ad libitum with a standard medicated (400 mg/kg of colistin sulfate 10% and oxytetracycline 20%) prestarter diet based on cereals, 50%; milk by-products, 20%; soybean meal (SBM) 44, 5%; and soy concentrate, 5% (analyzed CP on a DM basis = 18.7%, and calculated lysine on a DM basis = 1.30%).

Experimental Period.
Twelve days after weaning, a stress-management system (adapted from Kyriakis, 1989) based on social and dietary stress factors was applied to the animals. Specifically, the lightest animals (n = 24) were removed from the experiment, and the remaining animals (216 animals; 8.1 ± 0.20 kg BW) were mixed in the same room (social stress) for 2 h. Then animals were reorganized at random in the same 24 pens, nine animals each, and a starter nonmedicated diet containing 21.5% SBM (17% CP, 1.29% Lys; Tables 1 and 2) was fed (dietary stress). The starter diet was supplemented with six different treatments following a 2 x 3 arrangement, resulting from the combination of three levels (0, 150, and 300 mg/kg) of XT standardized in 5% (wt/wt) carvacrol (Origanum spp.), 3% cinnamaldehyde (Cinnamonum spp.) and 2% capsicum oleoresin (Capsicum annum) with two levels of FA (0 and 5 g/kg). Plant extracts were included in an inert fatty carrier before including them in the feed. The treatments were distributed at random in each replicate of pens. Chromic oxide was included as an indigestible marker.

Analytical Procedures
Chemical analysis of the diet was performed according to AOAC (1995) standard procedures. The GE was determined by adiabatic calorimeter and Cr concentration in diet, ileum, and feces was analyzed following the procedure described by Williams et al. (1962) by atomic absorption spectrophotometry.

Tissue samples for histological study were dehydrated and embedded in paraffin wax, sectioned at 3 µm, and stained with haematoxylin and eosin. Ten villous height (VH) and 10 crypt depth (CD) measurements were taken from each section. The average of the 10 VH and CD measurements was treated as an experimental unit. For bacterial counts, one gram of sample was weighed, serially diluted, and 100-µL aliquots were plated in agar MacConkey (Oxoid, Ref. CM 115, Oxoid S.A., Madrid, Spain) for enterobacteria counts (dilutions 10^–3 to 10^–7) and in agar rogosa (Oxoid, Ref. CM 627) for lactobacilli counts (dilutions 10^–5 to 10^–9). Enterobacteria were counted after a 24-h incubation (37°C) and lactobacilli were counted after a 48-h incubation (37°C, 5% CO₂). The VFA concentration (µmol/g of fresh matter) in deproteinized caecal and colonic digesta was determined by GLC, following the procedures of Jouany (1982). Purine bases (PB; adenine and guanine) in lyophilized ileal and rectal contents (60 mg) were determined by HPLC (Agilent 1100 Series), according to Makkar and Becker (1999), after their acid hydrolysis with 2 mL of 2 M perchloric acid at 100°C for 1 h, including 0.5 mL of 1 mM allopurinol as internal standard.

Calculations and Statistical Analyses
Ileal and rectal apparent digestibility of OM was calculated by the marker (Cr) ratio method, using the following equation:

Persistence of diarrhea and registered casualties where analyzed by ² test of FREQ procedure of SAS (Version 8.1; SAS Inst., Inc., Cary, NC) for XT and FA factors. Other results were analyzed by ANOVA with the GLM procedure of SAS, using XT and FA included as classification factors. The XT x FA interaction was included in the model. In productive performance analysis, pen was used as experimental unit, and initial live weight was used as covariate. In slaughter data analysis, pig was used as experimental unit, and weight of the animals at sacrifice was included as covariate in physical digestive measurements. Day and period of sacrifice were initially included in the model but were not significant for any variables (P = 0.37 to 0.97); thus, they were ultimately excluded from the model. As suggested by Lowry (1992) for this factorial arrangement, the interaction was studied when the P-value was significant or when it was less than 0.15 without any significant principal effect. In particular, the linear and quadratic trend of the XT factor was studied for equally spaced levels within each level of FA, and the FA effect was studied within XT at the 0 ppm rate. Both were studied by orthogonal contrasts. The alpha level used for determination of significance for all analyses and contrasts was 0.05. Additionally, the REG procedure of SAS was used for regression determinations of pH with various other responses.

	Results

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Productive Performance and Digestibility
First, it should be noted that on d 14 after weaning, 2 d after the starter diet introduction, a diarrhea episode occurred. The presence of liquid feces was observed in all pens. An enterohemolytic E. coli K88 was identified as the etiological agent, and animals were immediately treated thorough intramuscularly administered amoxicylin (Hipramox, Laboratorios Hipra, S.A., Girona, Spain) during 3 d. The diarrhea episode persisted over 2 d and five casualties were registered; all casualties occurred in different pens. Four casualties were in the 0 ppm XT group (5.6% mortality; Table 3) and one animal belonged to the 150 ppm XT group (1.4% mortality). No casualties were registered in animals treated with 300 ppm XT (0% mortality).

pH and Morphology of the Small Intestine
No differences were observed between treatments on digestive tract weight or content weight except for stomach content. The XT increased stomach content linearly (P = 0.006) when 0% of FA was included (Table 4). The FA also increased stomach content (P = 0.003) and the DM of this content (P = 0.010) when no XT was included (Table 4).

Morphology of the Epithelium
Means for VH and CD in the jejunum were 366 ± 10.3 and 286 ± 6.3 (µm), respectively. Proximal jejunum villi tended to be shorter in the FA groups (Table 5, P = 0.073), whereas crypt depth was unaffected.

The XT increased linearly lactobacilli (P = 0.019) when no FA was added (Table 5). As a consequence, the lactobacilli:enterobacteria ratio showed an increase (P = 0.002) due to the inclusion of XT (Table 6).

No differences were observed between the cecum and colon for the profile of acetic (55.3 ± 0.62 mol/100 mol), butyric (13.7 ± 0.33 mol/100 mol), and valeric acids (2.3 ± 0.13 mol/100 mol). On the other hand, the proportion of branched-chain VFA increased from the cecum to colon (0.66 ± 0.121 vs. 2.28 ± 0.124 mol/100 mol; P = 0.001), whereas the proportion of propionic decreased (28.2 ± 0.49 vs. 25.9 ± 0.50 mol/100 mol; P = 0.011).

Comparing treatments, XT inclusion increased acetate in the cecum (P = 0.018) and in the colon (P = 0.025), simultaneous to a decrease in butyrate (P = 0.096 in cecum; P = 0.040 in colon) and valerate (P = 0.001 in cecum; P = 0.039 in colon). Acidification with FA did not affect the VFA proportions.

	Discussion

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Postweaning anorexia has been described as the leading detrimental factor for the piglet health; however, other factors that also occur at weaning, such as the hypersensitivity induced by the presence of soybean meal in diets (Li et al., 1990, 1991) and the regrouping of animals (Blecha et al., 1985), may increase the incidence of digestive disturbances. In the current study, a stress episode was incorporated after weaning, so that it was independent of postweaning anorexia, to evaluate the effect of the studied additives under a programmed adverse situation.

Stress management and/or nonmedicated diet SBM level on d 12 probably induced the diarrhea episode that occurred on d 14. Pens from treatments without XT registered four of the five casualties independent of the formic FA inclusion. With the mixing method used (Kyriakis, 1989), we assumed an equal distribution of diarrhea between pens, and our observations support this assumption. However, an assurance of the homogeneity of the exposure to the pathogenic agent cannot be provided because no experimental infection was carried out. In any case, our results are encouraging for future studies. In this experiment, the benefits obtained from the inclusion of both plant extracts and FA were additive; lower casualties appeared in coincidence with better feed conversion. On the other hand, changes on digestive tract variables were nonadditive, as noted below.

Small Intestine Effects
Treated animals showed a higher total content and a percentage of DM of this content in the stomach. Higher stomach content in animals in which we assumed a similar feed intake may reflect a lower emptying rate and consequently a more homogeneous digesta flow to the duodenum. A decrease in emptying rate due to acidifiers has been described earlier (Hunt and Knox, 1972) to be associated with the effects of the low pH of the lumen on the duodenum receptors (Partanen and Mroz, 1999). In this sense, the retention time in the stomach, as affected by acidifiers, has been suggested as a possible mechanism to improve the protein digestion in the stomach and to increase the barrier effect of the stomach against pathogenic bacteria (Partanen and Mroz, 1999). However, in the present experiment, pH was higher with the inclusion of the FA, and showed a positive correlation with the stomach content (r = 0.83). This fact suggests that measured pH in the stomach was mainly affected by the buffering capacity of solid meal and water and was probably indirectly affected by the gastric emptying rate. In fact, when including stomach contents as a covariate in the statistical model, the effect of the additives on pH was not significant. Concerning plant extracts, influences on the gastric emptying have been described previously, as in the case of capsaicin present in the capsicum (Debreceni et al., 1999; Kang et al., 1999). In fact, increased mean retention time has been described using capsaicin in doses similar to those used in our experiment (Chang et al., 1999). This slower emptying is due to the direct effect of capsaicin on gastric motility (Gonzalez et al., 1998). In the present experiment, the addition of the XT as well as the FA promoted an increase of this retention time; however, no additive effect was found. One explanation for this lack of additivity can be found in another effect of the capsaicin. The same capsaicin receptors that decrease gastric motility are present in duodenal receptors responsible of acid brake, and when capsaicin is administered, acid brake is abolished (Raybould and Hölzer, 1993). Due to the important influence of the stomach on the predigestion of the diet and as a barrier for pathogens, increasing gastric retention time without affecting ingestion could produce important beneficial effects on the digestive ecosystem, and more efforts should be made to study the likely influence and mechanisms of the plant extracts and acidifiers on gastric emptying.

Both VH and CD are important indicators of the digestive health of the pig and directly related to the absorptive capacity of the mucous membrane (Buddle and Bolton, 1992). From a theoretical point of view, VH reflects a balance between the mitotic activity of the crypt enteric cells (Cera et al., 1988) and the desquamation produced principally by external aggressors (Nabuurs, 1995). In the current study, VH and CD measurements were in a range comparable with data obtained by Cera et al. (1988), Zijlstra et al. (1996), and Pluske et al. (1996). However, FA led to shorter VH without CD variation. The exact cause of this shorter VH, and whether it is related to the better conversion, remains unclear but could be a consequence of an increased desquamation and/or diminished mitotic rate.

It is generally accepted that the largest microbial population of the pig is localized in the large intestine; however, it has also been established that the microbial population of the small intestine is the most important factor in determining diarrhea (Buddle and Bolton, 1992) and can affect animal immune function (Anderson et al., 1999). Lactobacilli represent the largest group of microorganisms in the small intestine, followed by enterobacteria (from which some strains of E. coli may contribute to diarrhea; Ewing and Cole, 1992). It is generally accepted that lactobacilli are important to maintain good intestinal health because of their ability to control potentially pathogenic groups, such as E. coli (Blomberg et al., 1993; Canibe and Jensen, 2003), and their other positive effects, as reviewed by Perdigon et al. (2001). In this sense, we used the ratio of lactobacilli and enterobacteria (Lact:Ent) as an index of intestinal equilibrium. The XT inclusion increased lactobacilli counts in nonacidified diets and tended to decrease enterobacteria counts, resulting in a increase of the Lact:Ent ratio. Similar results have been previously obtained in our laboratory (Manzanilla et al., 2001). Despite the nonexistence of a direct correlation, it is interesting to see how Lact:Ent ratio followed the same pattern showed by stomach content and pH. These results may indicate some influence of the gastric emptying rate and pH on the microbiota in caudal segments. Canibe and Jensen (2003) indicated that changes in gastric contents that decrease survival of pathogens or proliferation in the stomach also seem to decrease the presence of pathogens along the remaining digestive tract. This suggests that the stomach acts as a barrier against colonization of pathogens in the gastrointestinal tract, and may be modulated by feeding/management strategies that are expected to influence gastric function. In any case, a direct modulator effect of FA or XT on the microbiota cannot be ruled out.

The antimicrobial properties of some plant extracts have been reported in numerous in vitro studies (Dorman and Deans, 2000), and some selective antimicrobial effect also has been shown depending on the extract used and the dose of inclusion (Zaika, 1988; Smith-Palmer et al., 1998). In the current study, PB concentration in ileal digesta was diminished by the XT inclusion. The manner by which microbial mass was decreased is difficult to explain because the principal group of microbes in the small intestine (i.e., lactobacilli) was increased. This fact could be a consequence of the importance of other bacterial groups in the gut equilibrium that have not been well described until now because of the technological limitations (Anderson et al., 1999). In agreement with this supposition, Collier et al. (2003) found lower total microbial mass in coincidence with increased lactobacilli in animals treated with antibiotics, using PCR techniques.

The dose of carvacrol and cinnamaldehyde used in this experiment was approximately 10-fold under the antimicrobial dose determined by Dorman and Deans (2000). No previous data have been reported regarding the antimicrobial dose of these plant extracts in vivo. However, this dose could be effective or inactive depending on the media, especially pH and or presence of fats, as demonstrated in vitro by Juven et al. (1994). On the other hand, higher doses of these compounds should be studied specially to avoid negative effects on the palatability, accumulation of some compounds in fat depots, or toxic effects for the animals.

Hindgut Effects
Volatile fatty acids are the major end products of bacterial metabolism in swine large intestine (Bergman, 1990). In the present experiment, we used VFA concentration and profile as an index of the changes on the microbial population and of the quantity and source of products being fermented in the hindgut. Present results indicate changes in contents of the cecum and colon from the VFA profile. The most important factor affecting VFA production is the quantity and source of substrate arriving in the hindgut (Bergman, 1990). From in vitro studies, it has been demonstrated that fermentation of different polysaccharides produce distinct patterns of VFA production (Macfarlane and Macfarlane, 2003). In our experiment, no differences in total OM ileal digestibility were found, but with only these data, we cannot rule out possible differences in fermentable substrates arriving to hindgut. Increases in acetic acid normally indicate a higher proportion of easily fermentable carbohydrate arriving to the hindgut. The lower microbial mass described in the ileum could produce lower consumption of these substrates, allowing their arrival to hindgut. How this could happen without affecting ileum digestibility is difficult to understand. On the other hand, probiotic development investigations carried out over the last few years have shown that for a specific substrate, changes in microbiota can result in different fermentation products due to different metabolism by the bacteria (Jiang and Savaiano, 1997). In these studies, most bacteria studied have been lactic acid bacteria, and increases in total VFA and in the proportion of acetate have been reported (Sakata et al., 2003). Important increases in the lactobacilli arriving from the small intestine have been described in the present experiment; however, it is difficult to demonstrate an influence of small intestine microbiota on the bacterial populations inhabiting the hindgut, and as such, the beneficial or detrimental effect produced by these changes in fermentation.

	Implications

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The incorporation of carvacrol, cinnamaldehyde, and capsicum oleoresin, or formic acid, promotes changes in the digestive function and microbial ecology and fermentation of weaned pigs. The results presented in this article should encourage the scientific community to continue investigations of plant extracts as alternatives to antibiotics, but not only through direct antimicrobial mechanisms. Stomach-emptying variables are not usually included in additive test experiments concerning pig intestinal health because they are difficult to measure, although important variations on gastric emptying due to additive inclusion were demonstrated herein. Conversely, interactions between different additives as found in this study must been taken into account in future experiments to avoid possible negative effects.

	Footnotes

 
¹ E. G. Manzanilla received a grant from the Departament d’Universitats, Recerca i Suport a la Investigació (DURSI) of the Generalitat de Catalunya for this study. The authors are grateful to J. Segalés for assistance with preparation of histological slides and Pinsos Baucells S.A. for allowing us to use their animal installations. AXISS S.A.S. France provided all financial support for these investigations.

² Correspondence: Edifici V, Nutrició i Alimentació Animal (phone: +34-93-5811556; fax: +34-93-5811494; e-mail: edgar.garcia{at}uab.es).

Received for publication January 19, 2004. Accepted for publication August 4, 2004.

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