Individual differences in behaviour and gut bacteria are associated in collared peccary (Mammalia, Tayassuidae).

AIMS: We tested the hypothesis that the behaviour of an individual is associated with the diversity of its gut bacteria, using the collared peccary (Pecari tajacu) as a model. METHODS AND RESULTS: In all, 24 adult male collared peccaries received either low- (n = 12) or high-fibre diet (n = 12) to induce contrasting gut fermentation profiles. They were submitted to three short-term challenges, allowing us to rate the animals in a coping-style dimension named 'calmness'. At the end of the experimental period, we collected samples of peccaries' forestomach contents to characterize bacterial diversity. We found a significant positive association between individual 'calmness' z-scores and the bacterial evenness index in gut bacteria (and a similar trend with the Simpson's diversity index), suggesting a more homogeneous bacterial community of calmer individuals. We also found a positive association between fibres digestibility and gut bacterial diversity in the peccaries' forestomach, but no effect of the dietary fibre level. CONCLUSIONS: Gut bacteria evenness increases with 'calmness' z-scores, suggesting a more homogeneous bacterial community of calmer individuals, compared with the more heterogeneous of the most distressed ones. Our results also suggest associations between the digestibility of ADF with the gut bacterial diversity indices and with the relative abundance of the Actinobacteria phylum. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data showed that the hosts' individual behavioural differences are potentially aligned with gut bacterial diversity. The behaviour-microbiota link is correlated with host feed efficiency and, ultimately, may have implications for animal health and welfare of farm animals.

Individual differences in behavior affect total tract fiber digestibility: the example of collared peccary.

Differences in how individuals cope with stressful conditions (e.g. novel/unfamiliar environment, social isolation and increases in human contact) can explain the variability in data collection from nutrient digestibility trials. We used the collared peccary (Pecari tajacu), which is under process of domestication and shows high individual behavioral distinctiveness in reactions toward humans, to test the hypothesis that behavioral differences play a role in nutrient digestibility. We assessed the individual behavioral traits of 24 adult male collared peccaries using both the 'behavioral coding' and the 'subjective ratings' approaches. For the behavioral coding assessment, we recorded the hourly frequency of behaviors potentially indicative of stress during the 30-day habituation period to the experimental housing conditions. The subjective ratings were performed based on the individuals' reactions to three short-term challenge tests (novel environment, novel object and threat from a capture net) over a period of 56 days. During the last 26 days, the collared peccaries were fed diets either high (n = 12) or low (n = 12) in dietary fiber levels, and we determined the total tract apparent digestibility of nutrients. The individual subjective ratings showed consistency in the correlated measures of 'relaxedness', 'quietness' and 'satisfaction' across the three challenge tests, which were combined to produce z score ratings of one derived variable ('calmness'). Individual frequency of BPIS/h and calmness scores were negatively correlated and both predicted the total tract digestibility of acid detergent fiber (ADF), which ranged from 0.41 to 0.79. The greater the calmness z scores (i.e. calmer individuals), the greater the total tract digestibility of ADF. In contrast, the higher the frequency of BPIS/h, the lower the total tract digestibility of ADF. Therefore, our results provide evidence that by selecting calmer collared peccaries, there will be an increase in their capacity to digest dietary fiber.

Review on the effects of potential prebiotics on controlling intestinal enteropathogens Salmonella and Escherichia coli in pig production.

Salmonella enterica serotypes (Salmonella sp.) are the second cause of bacterial foodborne zoonoses in humans after campylobacteriosis. Pork is the third most important cause for outbreak-associated salmonellosis, and colibacillosis is the most important disease in piglets and swine. Attachment to host cells, translocation of effector proteins into host cells, invasion and replication in tissues are the vital virulence steps of these pathogens that help them to thrive in the intestinal environment and invade tissues. Feed contamination is an important source for Salmonella infection in pig production. Many on-farm feeding strategies intervene to avoid the introduction of pathogens onto the farm by contaminated feeds or to reduce infection pressure when pathogens are present. Among the latter, prebiotics could be effective at protecting against these enteric bacterial pathogens. Nowadays, a wide range of molecules can potentially serve as prebiotics. Here, we summarize the prevalence of Salmonella sp. and Escherichia coli in pigs, understanding of the mechanisms by which pathogens can cause disease, the feed related to pathogen contamination in pigs and detail the mechanisms on which prebiotics are likely to act in order to fulfil their protective action against these pathogens in pig production. Many different mechanisms involve the inhibition of Salmonella and E. coli by prebiotics such as coating the host surface, modulation of intestinal ecology, downregulating the expression of adhesin factors or virulence genes, reinforcing the host immune system.

Reducing agent can be omitted in the incubation medium of the batch in vitro fermentation model of the pig intestines.

Over the past decade, in vitro methods have been developed to study intestinal fermentation in pigs and its influence on the digestive physiology and health. In these methods, ingredients are fermented by a bacterial inoculum diluted in a mineral buffer solution. Generally, a reducing agent such as Na2S or cysteine-HCl generates the required anaerobic environment by releasing metabolites similar to those produced when protein is fermented, possibly inducing a dysbiosis. An experiment was conducted to study the impact of two reducing agents on results yielded by such in vitro fermentation models. Protein (soybean proteins, casein) and carbohydrate (potato starch, cellulose) ingredients were fermented in vitro by bacteria isolated from fresh feces obtained from three sows in three carbonate-based incubation media differing in reducing agent: (i) Na2S, (ii) cysteine-HCl and (iii) control with a mere saturation with CO2 and devoid of reducing agent. The gas production during fermentation was recorded over 72 h. Short-chain fatty acids (SCFA) production after 24 and 72 h and microbial composition of the fermentation broth after 24 h were compared between ingredients and between reducing agents. The fermentation residues after 24 h were also evaluated in terms of cytotoxicity using Caco-2 cell monolayers. Results showed that the effect of the ingredient induced higher differences than the reducing agent. Among the latter, cysteine-HCl induced the strongest differences compared with the control, whereas Na2S was similar to the control for most parameters. For all ingredients, final gas produced per g of substrate was similar (P>0.10) for the three reducing agents whereas the maximum rate of gas production (R max) was reduced (P0.10) after 24 h of fermentation with Na2S and in the control without reducing agent. Molar ratios of branched chain-fatty acids were higher (P<0.05) for protein (36.5% and 9.7% for casein and soybean proteins, respectively) than for carbohydrate (<4%) ingredients. Only fermentation residues of casein showed a possible cytotoxic effect regardless of the reducing agent (P<0.05). Concerning the microbial composition of the fermentation broth, most significant differences in phyla and in genera ascribable to the reducing agent were found with potato starch and casein. In conclusion, saturating the incubation media with CO2 seems sufficient to generate a suitable anaerobic environment for intestinal microbes and the use of a reducing agent can be omitted.

Health relevance of intestinal protein fermentation in young pigs.

The physiological role of the gastrointestinal microbiota has become an important subject of nutrition research in pigs in the past years, and the importance of intestinal microbial activity in the etiology of disease is doubtless. This review summarizes the recent knowledge related to the microbial ecology of protein fermentation and the appearance of protein-derived metabolites along the pig intestine. The amount of fermentable protein depends on factors such as dietary protein concentration, protein digestibility due to secondary or tertiary structure, the interaction with dietary compounds or anti-nutritional factors, and the secretion of endogenous proteins into the gut lumen. High protein diets increase the luminal concentrations and epithelial exposure to putatively toxic metabolites and increase the risk for post-weaning diarrhea, but the mechanisms are not yet clarified. Although the use of fermentable carbohydrates to reduce harmful protein-derived metabolites in pigs is well-established, recent studies suggest that the inclusion of fermentable carbohydrates into diets with low protein digestibility or high dietary protein level may not ameliorate all negative effects with regard to epithelial response. Based on the current knowledge, the use of diets with low levels of high-quality protein may help to reduce the risk for intestinal disease in young pigs.

Adding mucins to an in vitro batch fermentation model of the large intestine induces changes in microbial population isolated from porcine feces depending on the substrate.

Adding mucus to in vitro fermentation models of the large intestine shows that some genera, namely lactobacilli, are dependent on host-microbiota interactions and that they rely on mucosal layers to increase their activity. This study investigated whether this dependence on mucus is substrate dependent and to what extent other genera are impacted by the presence of mucus. Inulin and cellulose were fermented in vitro by a fecal inoculum from pig in the presence or not of mucin beads in order to compare fermentation patterns and bacterial communities. Mucins increased final gas production with inulin and shifted short-chain fatty acid molar ratios (P < 0.001). Quantitative real-time PCR analyses revealed that Lactobacillus spp. and Bifidobacterium spp. decreased with mucins, but Bacteroides spp. increased when inulin was fermented. A more in-depth community analysis indicated that the mucins increased Proteobacteria (0.55 vs 0.25%, P = 0.013), Verrucomicrobia (5.25 vs 0.03%, P = 0.032), Ruminococcaceae, Bacteroidaceae and Akkermansia spp. Proteobacteria (5.67 vs 0.55%, P < 0.001) and Lachnospiraceae (33 vs 10.4%) were promoted in the mucus compared with the broth, while Ruminococcaceae decreased. The introduction of mucins affected many microbial genera and fermentation patterns, but from PCA results, the impact of mucus was independent of the fermentation substrate.

Forage plants as an alternative feed resource for sustainable pig production in the tropics: a review.

Globally, pressure on concentrate feed resources is increasing, especially in the tropics where many countries are net importers of food. Forage plants are a possible alternative, but their use as feed ingredients for pigs raises several issues related to their higher fibre and plant secondary metabolites contents as well as their lower nutritive value. In this paper, the nutritive value of several forage species and the parameters that influence this nutritive value in relationship to the plant family, the physiological stage, the plant part and the preservation method (fresh, hay and silage) are reviewed. The influence of the breed and the physiological status of the animal on animal voluntary intake of fibre-rich ingredients, digestibility as related to gastrointestinal volume and transit time and growth performances are also discussed. The final section highlights the advantages and drawbacks of forage plants in pig diets and stresses the need for proper economic evaluation to conclude on the benefits of the use of forage plants in pig feed.

Influence of fermentable carbohydrates or protein on large intestinal and urinary metabolomic profiles in piglets.

It was recently shown that variations in the ratio of dietary fermentable carbohydrates (fCHO) and fermentable protein (fCP) differentially affect large intestinal microbial ecology and the mucosal response. Here we investigated the use of mass spectrometry to profile changes in metabolite composition in colon and urine associated with variation in dietary fCHO and fCP composition and mucosal physiology. Thirty-two weaned piglets were fed 4 diets in a 2 × 2 factorial design with low fCP and low fCHO, low fCP and high fCHO, high fCP and low fCHO, and high fCP and high fCHO. After 21 to 23 d, all pigs were euthanized and colon digesta and urine metabolite profiles were obtained by mass spectrometry. Analysis of mass spectra by partial least squares approach indicated a clustering of both colonic and urinary profiles for each pig by feeding group. Metabolite identification and annotation using the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways revealed increased abundance of metabolites associated with arachidonic acid metabolism in colon of pigs fed a high concentration of fCP irrespective of dietary fCHO. Urinary metabolites did not show as clear patterns. Mass spectrometry can effectively differentiate metabolite profiles in colon contents and urine associated with changes in dietary composition. Whether metabolite profiling is an effective tool to identify specific metabolites (biomarkers) or metabolite profiles associated with gut function and integrity needs further elucidation.

Use of medium without reducing agent for in vitro fermentation studies by bacteria isolated from pig intestine.

Over the past decade, several in vitro methods have been developed to study intestinal fermentation in pigs and its influence on health. In these methods, samples are fermented by a bacterial inoculum diluted in a mineral buffer solution. Generally, a reducing agent such as Na(2)S or cysteine HCl generates the required anaerobic environment by release of H(2)S inducing an imbalance among bacterial species by the production of toxic metabolites. Therefore, an experiment was conducted to study the impact of reducing agent on fermentation patterns. Protein (soybean protein and/or casein) and carbohydrate (potato starch and/or cellulose) ingredients were fermented in vitro by pig intestinal bacteria from fresh feces obtained from 3 sows fed an antibiotic-free commercial diet in 3 incubation media differing in reducing agent: (i) Na(2)S, (ii) cysteine HCl, or (iii) without reducing agent. Gas fermentation kinetics were monitored over 72 h (pressure was measured every 2 min). Short-chain fatty acid (SCFA) production after 24 and 72 h were compared among ingredient and reducing agents (n = 2). Gas production was higher (P < 0.05) when fermenting carbohydrate than protein ingredients. Except for soybean protein, total SCFA production after 24 and 72 h was similar (P > 0.05) for each ingredient regardless the incubation medium. The SCFA molar ratios did not differ (P > 0.05) between Na(2)S and without reducing agent. In conclusion, saturation of incubation media with CO(2) seems sufficient to generate an anaerobic environment. So incubation media could be simplified by omitting the reducing agent without influencing the fermentation kinetics and SCFA production.

Discrepancies in microbiota composition along the pig gastrointestinal tract between in vivo observations and an in vitro batch fermentation model.

In vitro fermentation models are increasingly used to assess prebiotic potential of novel indigestible carbohydrates (CHO). A trial was performed to assess the validity of such approaches by comparing the influence of fermentation of inulin and cellulose on microbiota in vivo and in vitro. Two semipurified diets based on 5% inulin or 5% cellulose were fed to 2 groups of four 25-kg pigs. After 3 wk, the pigs were slaughtered and digesta was sampled from jejunum, ileum, cecum, and 3 parts of the colon to measure pH and microbiota population. An in vitro gas fermentation test was also performed on inulin and cellulose using fresh feces of the experimental pigs as bacterial inoculum. The gas production kinetics were modeled and fermentation broth sampled after 5, 8, 12, 24, and 72 h. Bacterial DNA was extracted and quantitative PCR was performed to quantify total bacteria, lactobacilli, bifidobacteria, Bacteroides, Clostridium cluster I, and Escherichia coli. Total bacteria quantification was similar between both systems. In vivo, total bacteria increased (P < 0.001) along the gut until the second part of the colon (from 10.5(7) to 10(10) cfu/mg) and then decreased (P < 0.05) to 10(9) cfu whereas in vitro, it increased (P < 0.05) until 12 to 24 h of fermentation (from 10(9) to 10.5(9) cfu/mL) and then decreased (P < 0.05) to initial level (10(9) cfu/mL). This evolution was consistent with fermentation kinetics. In both models, inulin increased (P < 0.05) the ratio of bifidobacteria and E. coli populations in the total microflora compared to cellulose. However, in vivo this was observed only in the first parts of the gut whereas in vitro the effect lasted for 72 h. Inulin also increased (P < 0.001) Bacteroides genus in vitro but not in vivo where the evolutions of Bacteroides were similar (P > 0.05) for both CHO. Evolutions of lactobacilli and Clostridium populations in both systems were also not consistent. This can be ascribed to specific bacterial properties as, for example, adhesive properties or sensitivity to sulfur reducing agent used in the in vitro model. As is, the in vitro model does not reflect properly changes in microbiota along the digestive tract induced by specific feed ingredients compared to in vivo observations.

Nutritive value of unconventional fibrous ingredients fed to Guinea pigs in the Democratic Republic of Congo.

The energy and protein value for Guinea pigs (GP) of 9 forages (7 dicots and 2 grasses) and 5 hay-based diets was determined. The apparent faecal digestibility of dry matter, organic matter, crude protein and energy was measured on GP housed in metabolic cages. The forages and the diets were digested in vitro using pepsin and pancreatin hydrolysis and gas fermentation test to simulate stomach, small intestine and large intestine, respectively. Most of the dicots had high digestible crude protein content (152-201 g/kg DM) and the 2 grasses showed lower values (80-85 g/kg DM). Digestible energy content of the forages ranged between 5.79 to 13.08 MJ/kg DM. None of the forage species or hay-based diets provided sufficient energy to supply the 11.7 MJ/kg metabolic energy requirements. The influence of intestinal fermentation on energy and protein values was highlighted by correlations (P<0.05) between in vivo and in vitro data, including gas fermentation. It is the first time that such relationships are reported in single-stomach animals.

Influence of source and concentrations of dietary fiber on in vivo nitrogen excretion pathways in pigs as reflected by in vitro fermentation and nitrogen incorporation by fecal bacteria.

The inclusion of dietary fiber (DF) in diets has been suggested as a way to reduce NH(3) emission in pig barns because it contributes to a shift in N excretion from urine to feces owing to enhanced bacterial growth in the intestines. This study compared an in vitro method to measure bacterial protein synthesis during fermentation with an in vivo N excretion shift induced by diets differing in DF concentrations and solubility. The first experiment measured the effect of graded concentrations of sugar beet pulp (SBP; 0, 10, 20, and 30%) in corn- and soybean meal-based diets on in vivo N excretion partitioning between the urine and feces. A second experiment investigated the replacement of SBP, rich in soluble DF, with oat hulls (OH), rich in insoluble DF (20:0, 10.5:10.5, and 0:22%, respectively). In parallel, the fermentation characteristics of the dietary carbohydrates not digested in the small intestine were evaluated in an in vitro gas test, based on their incubation with colonic microbiota, using a mineral buffer solution enriched with (15)N. The N originating from the buffer solution incorporated into the bacterial proteins (BNI) was measured when half the final gas volume was produced (8.5 to 14.5 h of fermentation) and after 72 h of fermentation. Short-chain fatty acids were determined in the liquid phase. In the first experiment, the inclusion of SBP linearly decreased urinary N excretion from 0.285 to 0.215 g of N excreted in the urine per gram of N ingested and decreased the urinary-N:fecal-N excretion ratio from 2.171 to 1.177 (P < 0.01). In the second experiment, substituting SBP with OH linearly increased the urinary-N:fecal-N excretion ratio (P = 0.009). Unlike short-chain fatty acid production, BNI was greater at half-time to asymptotic gas production than at 72 h of fermentation. Sugar beet pulp enhanced BNI linearly (P < 0.001), 2.01, 2.06, and 2.35 mg g(-1) of diet with 10, 20, and 30% SBP, respectively, as compared with 1.51 mg for the control diet. The substitution of SBP with OH decreased BNI (P < 0.01). With the exception of final gas production, all in vitro kinetic characteristics and BNI were correlated with in vivo N excretion parameters, and regression equations for the prediction of N excretion pathways from in vitro data were identified. Even if the presence of resistant starch in the diet might alter the composition of the fibrous residue that is fermented, the in vitro method is a possible useful tool for the formulation of diets, reducing the effects of pig production on the environment.

Voluntary intake, chemical composition and in vitro digestibility of fresh forages fed to guinea pigs in periurban rearing systems of Kinshasa (Democratic Republic of Congo).

The daily voluntary intake (DVI) of Guinea pigs (GP) fed 15 fresh forages used in periurban rearing systems of Kinshasa (Democratic Republic of Congo) was investigated. In order to determine the best forages combination for GP diet, DVI was compared to their nutritional value measured in vitro using (1) a pepsin-pancreatin hydrolysis, (2) an gas fermentation test on the hydrolysed residues with an inoculum prepared from GP faeces, and (3) the chemical composition of the offered feeds and the hydrolysis residues. The forages ranking based on the DVI was correlated to the NDF content, but not to their nutritional values determined in vitro. According to their high DVI (from 4.23 to 7.75 g/kg liveweigth), and their valuable in vitro nutritional values (crude protein ranging from 261 to 279 g crude protein kg(-1) DM, pepsin-pancreatin digestibilities of the dry matter from 0.55 to 0.59 and final gas production from 170 to 196 1 kg(-1) DM), Desmodium intortum, Euphorbia heterophylla or Amaranthus hybridus, can be suggested to the farmers to complement the usual diet distributed to the GP based on Panicum maximum.

The source of fermentable carbohydrates influences the in vitro protein synthesis by colonic bacteria isolated from pigs.

Two in vitro experiments were carried out to quantify the incorporation of nitrogen (N) by pig colonic bacteria during the fermentation of dietary fibre, including non-starch polysaccharides and resistant starch. In the first experiment, five purified carbohydrates were used: starch (S), cellulose (C), inulin (I), pectin (P) and xylan (X). In the second experiment, three pepsin-pancreatin hydrolysed ingredients were investigated: potato, sugar-beet pulp and wheat bran. The substrates were incubated in an inoculum, prepared from fresh faeces of sows and a buffer solution providing 15N-labelled NH4Cl. Gas production was monitored. Bacterial N incorporation (BNI) was estimated by measuring the incorporation of 15N in the solid residue at half-time to asymptotic gas production (T/2). The remaining substrate was analysed for sugar content. Short-chain fatty acids (SCFA) were determined in the liquid phase. In the first experiment, the fermentation kinetics differed between the substrates. P, S and I showed higher rates of degradation (P < 0.001), while X and C showed a longer lag time and T/2. The sugar disappearance reached 0.91, 0.90, 0.81, 0.56 and 0.46, respectively, for P, I, S, C and X. Among them, S and I fixed more N per gram substrate (P < 0.05) than C, X and P (22.9 and 23.2 mg fixed N per gram fermented substrate v. 11.3, 12.3 and 9.8, respectively). Production of SCFA was the highest for the substrates with low N fixation: 562 and 565 mg/g fermented substrate for X and C v. 290 to 451 for P, I and S (P < 0.01). In the second experiment, potato and sugar-beet pulp fermented more rapidly than wheat bran (P < 0.001). Substrate disappearance at T/2 varied from 0.17 to 0.50. BNI were 18.3, 17.0 and 10.2 fixed N per gram fermented substrate, for sugar-beet pulp, potato and wheat bran, respectively, but were not statistically different. SCFA productions were the highest with wheat bran (913 mg/g fermented substrate) followed by sugar-beet pulp (641) and potato (556) (P < 0.05). The differences in N uptake by intestinal bacteria are linked to the partitioning of the substrate energy content between bacterial growth and SCFA production. This partitioning varies according to the rate of fermentation and the chemical composition of the substrate, as shown by the regression equation linking BNI to T/2 and SCFA (r2 = 0.91, P < 0.01) and the correlation between BNI and insoluble dietary fibre (r = -0.77, P < 0.05) when pectin was discarded from the database.