1. P. Trevisi, S. Botti, C. Lauridsen, J.F. Pérez, D. Papadopoulos, M. Roselli, J. Levic and J. Zentek. 2015. COST Action FA1401 “European network on the factors affecting the gastro-intestinal microbial balance and the impact on the health status of pigs (PiGutNet)”. Journal of Animal and Feed Sciences. 24: 166–170.



The “hoped for” reduction in the use of antibiotics in pig by EU producers has not been materialized as they are still being widely used for the control of enteric infectious diseases. This practice can spread antibiotic resistance in the farm environment and poses a threat to consumer health. Whilst it is widely recognized that a diversified gastro-intestinal tract (GIT) microbiota is essential for optimal health and performance, the underlying factors favoring the development and maintenance of a balanced intestinal microbiota are not fully understood. PiGutNet will establish the first European network focused on this topic, joining specialists in all research areas. It will define both environmental and host genetic factors affecting the GIT microbiota and the complex interactions between microbiota and gut maturation, to maintain a healthy gut throughout life. The network will coordinate databases and unravel innovative tools to define the status of intestinal eubiosis in pigs. The most important outcomes will be genome/metabolome-wide association studies and the provision of a road map to increase pig resistance against GIT infections. This will have an important translational potential, being the foundation for European companies to develop strategies in the areas of feed additives and animal husbandry, resulting in improved animal health and welfare, consumer protection and competitive advantage for the European agriculture.



  1. Nadia Everaert, Steven Van Cruchten, Björn Weström, Michael Bailey, Chris Van Ginneken, Thomas Thymann, Robert Pieper. 2017. A review on early gut maturation and colonization in pigs, including biological and dietary factors affecting gut homeostasis. Animal Feed Science and Technology. 233: 89-103.



During the prenatal, neonatal and post-weaning periods, the mammalian gastrointestinal tract undergoes various morphological and physiological changes alongside with an expansion of the immune system and microbial ecosystem. This review focuses on the time period before weaning and summarizes the current knowledge regarding i) structural and functional aspects ii) the development of the immune system, and iii) the establishment of the gut ecosystem of the porcine intestine. Structural and functional maturation of the gastrointestinal tract gradually progress with age. In the neonatal period colostrum induces gut closure, leads to an increase in intestinal weight, absorptive area and brush border enzyme activities. During the first weeks of life, an increased secretion of stomach and pancreatic enzymes and an increased uptake of monosaccharides and amino acids are observed. The development in digestive function coincides with development in both the adaptive and innate immune system. This secures a balanced immune response to the ingested milk-derived macromolecules, and colonizing bacteria. Husbandry and dietary interventions in early life appear to affect the development of multiple components of the mucosal immune system. Furthermore, the composition of the intestinal microbial communities seems to be affected by the early postnatal environment, which might also contribute to gut maturation, metabolic and immune development. Understanding the interplay between morphological, functional and immunological maturation, as influenced by early microbial colonization and ingestion of dietary factors, is of utmost importance to identify management and feeding strategies to optimize intestinal health. We discuss some possible implications related to intrauterine growth restriction, and preterm delivery as these both dramatically increase the risk of mortality and morbidity. In addition, some nutritional interventions during the perinatal period in both sows and piglets will be discussed in the light of possible health consequences early in life and later on.



  1. Marianna Roselli, Robert Pieper, ClaireRogel-Gaillard, Hugo de Vries Mick Bailey, Hauke Smidt, Charlotte Lauridsen. 2017. Immunomodulating effects of probiotics for microbiota modulation, gut health and disease resistance in pigs. Animal Feed Science and Technology. 233: 104-119.



Probiotics are live microorganisms that can confer a health benefit on the host, and amongst various mechanisms probiotics are believed to exert their effects by production of antimicrobial substances, competition with pathogens for adhesion sites and nutrients, enhancement of mucosal barrier integrity and immune modulation. Through these activities probiotics can support three core benefits for the host: supporting a healthy gut microbiota, a healthy digestive tract and a healthy immune system. More recently, the concept of combining probiotics and prebiotics, i.e. synbiotics, for the beneficial effect on gut health of pigs has attracted major interest, and examples of probiotic and prebiotic benefits for pigs are pathogen inhibition and immunomodulation. Yet, it remains to be defined in pigs, what exactly is a healthy gut. Because of the high level of variability in growth and feed conversion between individual pigs in commercial production systems, measuring the impact of probiotics on gut health defined by improvements in overall productivity requires large experiments. For this reason, many studies have concentrated on measuring the effects of the feed additives on proxies of gut health including many immunological measures, in more controlled experiments. With the major focus of studying the balance between gut microbiology, immunology and physiology, and the potential for prevention of intestinal disorders in pigs, we therefore performed a literature review of the immunomodulatory effects of probiotics, either alone or in combination with prebiotics, based on in vivo, in vitro and ex vivo porcine experiments. A consistent number of studies showed the potential capacity in terms of immunomodulatory activities of these feed additives in pigs, but contrasting results can also be obtained from the literature. Reasons for this are not clear but could be related to differences with respect to the probiotic strain used, experimental settings, diets, initial microbiota colonization, administration route, time and frequency of administration of the probiotic strain and sampling for analysis. Hence, the use of proxy measurements of enteric health based on observable immunological parameters presents significant problems at the moment, and cannot be considered robust, reliable predictors of the probiotic activity in vivo, in relation to pig gut health. In conclusion, more detailed understanding of how to select and interpret these proxy measurements will be necessary in order to allow a more rational prediction of the effect of specific probiotic interventions in the future.



  1. B.U. Metzler-Zebeli, P. Trevisi, J.A.M. Prates, S. Tanghe, P. Bosi, N. Canibe, L. Montagne, J. Freire, Q. Zebeli. 2017. Assessing the effect of dietary inulin supplementation on gastrointestinal fermentation, digestibility and growth in pigs: A meta-analysis. Animal Feed Science and Technology. 233: 120-132.



Inulin has been reported to improve the homeostasis in the gastrointestinal tract (GIT) of pigs by modulating the intestinal microbiota and fermentation. The present study aimed to quantify the relationship between dietary inulin and microbial response variables in digesta from the GIT and feces of weaned, growing and finishing pigs using a meta-analytical approach. We further examined the effect of dietary inulin on the coefficients of ileal (CIAD) and total tract apparent digestibility (CTTAD) of nutrients and ADG. Pig’s starting body weight was considered the main inclusion criterion. Missing information about explanatory variables and few values available for response variables reduced the number of studies included. From the 33 included articles published between 2000 and 2016, individual sub-datasets for fermentation metabolites, bacterial abundances, CIAD, CTTAD and performance were built. Prediction models on the effect on inulin were computed accounting for inter- and intra-study variability. Dietary inulin levels ranged from 0.1 to 25.8%, whereby the median and mean inulin levels were 0.1–2% and 3–4%, respectively. Few of the investigated fermentation response variables were influenced by dietary inulin. Strong negative relationships were found between dietary inulin and gastric pH in weaned pigs (R2 = 0.81; P < 0.001; n = 12), colonic enterobacteria (R2 = 0.50; P < 0.001; n = 19) and fecal lactobacilli (R2 = 0.41; P < 0.001; n = 26) throughout all production phases, whereas observed negative relationships between inulin and colonic bifidobacteria and fecal enterobacteria and Escherichia coli were of minor physiological relevance (P < 0.05). Moreover, increasing inulin levels negatively correlated with the CTTAD of crude protein (R2 = 0.83; P < 0.001; n = 15), but they did not influence average daily gain of pigs. Best-fit models indicated that dietary crude protein amplified the effect of inulin on CTTAD of crude protein and gastric pH, but counteracted the inulin effect on fecal E. coli (P < 0.05). Accordingly, both pig’s body weight and inulin decreased gastric pH and fecal lactobacilli but counteracted the inulin effect on colonic bifidobacteria and fecal E. coli (P < 0.05). In conclusion, this study supported a stimulatory effect of dietary inulin on gastric acid secretion, which may be favorable GIT health in weaned pigs. However, due to limiting information provided in the original studies, like dietary fructans or fibers, low numbers of observation and low inulin levels, relationships should be regarded as trends.



  1. Đuro Vukmirović, Radmilo Čolović, Slađana Rakita, Tea Brlek, Olivera Đuragić, David Solà-Oriol. 2017. Importance of feed structure (particle size) and feed form (mash vs. pellets) in pig nutrition – A review. Animal Feed Science and Technology. 233: 133-144.



Pigs are monogastric animals with simple, single-chambered stomach and require easily digestible, high quality feed. One of the most important factors that determine feed utilization by pigs is the particle size distribution. The reduction of particle size of feed improves pigs’ performance due to increased specific surface of feed particles allowing better contact with digestive enzymes. In this respect, fine grinding could be recommended in production of pig feed. Additionally, in modern pig production dry feed is predominantly used in pelleted form, which is mainly due to improved (i.e. decreased) feed conversion ratio (FCR) of pigs fed pelleted feed, but also due to other advantages of pelleted over mash feed.

Size of feed particles is strongly reduced during pelleting process. Consequently, digestibility of nutrients in pig feed could be improved. On the other hand, presence of high quantities of fine particles in pig feed (both mash and pelleted) negatively affects the health of gastro-intestinal tract (GIT) leading to higher incidence of stomach ulceration and other negative alterations of gastric mucosa (keratization, erosions). Gastric ulcers are one of the most important causes of sudden death on farm that can result in large economic losses in pig production. Concerning that the animal therapy is expensive, labor-intensive, and mostly non-effective due to late recognition of ulceration, prophylactic recommendations are required. Thus, according to literature data, decreasing the quantity of fine particles in pig feed is strongly recommended.

Particle size distribution of the pigs’ feed has a strong influence on presence of pathogen bacteria in GIT of pigs. Feeding pigs with coarse mash feed decreases pH value of stomach content compared to pigs fed finely ground mash feed and compared to pigs fed pelleted feed. This can be explained by slower passage rate, increased dry matter, and more dense consistency of stomach content in pigs fed coarse mash diets. Consequently, feed acidification in stomach is better, number of lactic acid bacteria and concentration of organic acids is higher, and pH of stomach content is lower. These conditions create additional “barrier” against pathogen bacteria.

According to available data, optimal particle size of diets for pigs is in the range between 500 and 1600 μm, while particles smaller than 400 μm are considered as undesirable with high ulcerogenic capacity. Optimal particle size could be designed in the grinding process, and it was shown that the most convenient grinding method is to combine roller mill and hammer mill. Concerning that nowadays pigs are mainly fed pelleted feed, and that pelleting causes strong additional grinding of feed particles, particle size distribution (PSD) obtained within the grinding process would be dramatically changed during pelleting. The possibilities to decrease the intensity of grinding of particles during pelleting, by variation of parameters of pelleting process, are very limited. Modified extrusion process (i.e. processing using expander) followed by shaping element, is suggested in the literature as an alternative for pelleting in order to obtain agglomerated pig feed with preserved PSD, but this process is not extensively studied so far.



  1. A. Torres-Pitarcha, D. Hermans, E.G. Manzanilla, J. Bindelle, N. Everaert, Y. Beckers, D. Torrallardona, G. Bruggeman, G.E. Gardiner, P.G. Lawlor. 2017. Effect of feed enzymes on digestibility and growth in weaned pigs: A systematic review and meta-analysis. Animal Feed Science and Technology. 233: 145-159.



Supplementation of post-weaning diets with exogenous enzymes has been suggested to control post-weaning syndrome, by compensating for the under-developed endogenous enzyme secretory capacity and increasing nutrient digestibility in newly weaned pigs. The effect of in-feed enzymes in improving gut maturation, growth and/or health in weaned piglets is not always consistent. A systematic review and a meta-analysis were therefore conducted to determine which exogenous enzymes are most consistent in improving piglet growth and digestibility when supplemented to post-weaning diets. The mean difference effect of enzyme supplementation on growth and digestibility of dry matter (DM), gross energy (GE), crude protein (CP), and P digestibility was calculated for each study and this was used as the effect size estimate in the meta-analysis. The impact of feed enzyme supplementation on intestinal health and bone mineralization was also discussed where information was available in the literature. From a total of 90 studies included in the meta-analysis, gain to feed ratio (G:F) was improved in 55, remained un-changed in 28 and deteriorated in 7, in response to enzyme supplementation. Average daily gain, average daily feed intake and G:F was improved when phytase was supplemented in the diet. Phosphorous digestibility was increased with phytase supplementation while GE digestibility was reduced. Dietary phytase supplementation increased bone mineralization in pigs fed diets with a reduced P content. Supplementation with multi-enzyme complexes increased DM and CP digestibility. In conclusion, the most consistent improvements in piglet growth, P digestibility and bone mineralization were found due to exogenous phytase supplementation. Supplementation with xylanase alone or in combination with β-glucanase had inconsistent effects on piglet growth and nutrient digestibility. The most consistent improvements in growth and nutrient digestibility due to supplementation with multi-enzyme complexes were found when mannanase and/or protease were included in the complex.



  1. B. U. Metzler-Zebeli, N. Canibe, L. Montagne, J. Freire, P. Bosi, J. A. M. Prates, S. Tanghe and P. Trevisi. 2018. Resistant starch reduces large intestinal pH and promotes fecal lactobacilli and bifidobacteria in pigs. Animal, 1-10. doi:10.1017/S1751731118001003.



Dietary resistant starch (RS) may have prebiotic properties but its effects on fermentation and the microbial population are inconsistent. This meta-analysis aimed to quantify the relationship between RS type 2 (RS2) and intestinal short-chain fatty acids (SCFA) and pH as well as certain key bacterial taxa for intestinal health in pigs. From the 24 included articles with sufficient information about the animal, and dietary and physiological measurements published between 2000 and 2017, individual sub-data sets for fermentation metabolites, pH, bacterial abundances and apparent total tract digestibility were built and used to parameterize prediction models on the effect of RS2, accounting for inter- and intra-study variability. In addition, the effect of pig’s BW at the start of the experiment and duration of the experimental period on response variables were also evaluated using backward elimination analysis. Dietary RS levels ranged from 0% to 78.0% RS, with median and mean RS levels of 28.8% and 23.0%, respectively. Negative relationships could be established between dietary RS and pH in the large intestine (P<0.05), with a stronger effect in the mid and distal colon, and feces (R 2=0.64 to 0.81; P<0.001). A dietary level of 15% RS would lower the pH in the proximal, mid-, distal colon and feces by 0.2, 0.6, 0.4 and 0.6 units, respectively. Increasing RS levels, however, did not affect SCFA concentrations in the hindgut, but enhanced the molar proportion of propionate in mid-colon and reduced those of acetate in mid-colon and of butyrate in mid- and distal colon (R 2=0.46 to 0.52; P<0.05). Backward elimination indicated an age-related decrease in mid-colonic propionate proportion and increase in mid- and distal colonic butyrate proportion (P<0.05), thereby modulating RS2 effects. In feces, increasing RS levels promoted fecal lactobacilli (R 2=0.46; P<0.01) and bifidobacteria (R 2=0.57; P<0.01), whereby the slope showed the need for a minimal RS level of 10% for a 0.5 log unit-increase in their abundance. Best-fit equations further supported that a longer experimental period increased fecal lactobacilli but decreased fecal bifidobacteria (P<0.05). In conclusion, dietary RS2 seems to effectively decrease digesta pH throughout the large intestine and increase lactic acid-producing bacteria in feces of pigs which may limit the growth of opportunistic pathogens in the hindgut. To achieve these physiologically relevant changes, dietary RS should surpass 10% to 15%.



  1. Ann-Sofie Riis Poulsen, Diana Luise, Mihai Victor Curtasu, Sugiharto Sugiharto, Nuria Canibe, Paolo Trevisi, Charlotte Lauridsen. 2018. Effects of alpha-(1,2)-fucosyltransferase genotype variants on plasma metabolome, immune responses and gastrointestinal bacterial enumeration of pigs pre- and post-weaning. PLoS ONE 13(8): e0202970.



In pigs, the alpha-(1,2) fucosyltransferase (FUT1) gene has been highlighted for its properties in controlling the intestinal expression of enterotoxigenic E. coli (ETEC) F18 receptors; a pathogen causing edema disease and post-weaning diarrhoea. In this study, we hypothesized that pigs with different genotypes (ETEC F18 resistant (FUT1AA) versus susceptible (FUT1AG)) differed in following systemic and enteric responses: growth performance, plasma metabolic profiles, expression of candidate genes for intestinal mucosal homeostasis and immunity, number of selected bacteria and the concentration of short-chain fatty acids (SCFA) in faeces and digesta in piglets pre and post-weaning, and on the ETEC F18 adherence ex vivo. Genotype had the strongest impact on plasma metabolomic profile on day 7 and 28 of age. FUT1AG piglets had higher level of N-methyl-2-pyrrolidinone, hippuric acid, oxindole, and 3-oxo-5-beta-chol-7-en-24-oic acid on day 7, and a higher level of guanosine on day 28 than that in the FUT1AA piglets. FUT1AA piglets had a higher level of betaine on day 7 and 3-methylguanine on day 28. On day 34 of age, the FUT1AA pigs had higher levels of S-2-hydroxyglutarate, L-phenylalanine, tauroursodeoxycholic acid and an undetermined PC/LysoPC, while Ile Glu Phe Gly peptide and genistein 5-O-glucuronide, and PC (18:0/0:0) were at higher levels in the FUT1AG piglets. FUT1 genotype did not affect the growth performance and expression of candidate genes. FUT1AG piglets had a higher number of haemolytic bacteria in faeces and in digesta than that in FUT1AA at 34 days of age. The colonic acetic acid concentration was highest in FUT1AG piglets. FUT1 genotype may influence not only the expression of E. coli F18 receptors but could potentially impact the gut homeostasis and metabotype of piglets pre and post-weaning. Further investigations on the relation between FUT1 genotype and these aspects including the intestinal commensal microbiota will expand the knowledge on factors affecting the intestinal ecosystem.



  1. Paolo Trevisi, Davide Priori, Alfons J. M. Jansman, Diana Luise, Sietse-Jan Koopmans, Ulla Hynönen, Airi Palva, Jan van der Meulen, Paolo Bosi. 2018. Molecular networks affected by neonatal microbial colonization in porcine jejunum, luminally perfused with enterotoxigenic Escherichia coli, F4ac fimbria or Lactobacillus amylovorus. PLoS ONE 13(8): e0202160.



The development of an early complex gut microbiota may play an important role in the protection against intestinal dysbiosis later in life. The significance of the developed microbiota for gut barrier functionality upon interaction with pathogenic or beneficial bacteria is largely unknown. The transcriptome of differently perfused jejunal loops of 12 caesarian-derived pigs, neonatally associated with microbiota of different complexity, was studied. Piglets received pasteurized sow colostrum at birth (d0), a starter microbiota (Lactobacillus amylovorus (LAM), Clostridium glycolicum, and Parabacteroides) on d1-d3, and a placebo inoculant (simple association, SA) or an inoculant consisting of sow’s diluted feces (complex association, CA) on d3-d4. On d 26–37, jejunal loops were perfused for 8 h with either enterotoxigenic Escherichia coli F4 (ETEC), purified F4 fimbriae, LAM or saline control (CTRL). Gene expression of each intestinal loop was analyzed by Affymetrix Porcine Gene 1.1_ST array strips. Gene Set Enrichment Analysis was performed on expression values. Compared to CTRL, 184 and 74; 2 and 139; 2 and 48 gene sets, were up- and down-regulated by ETEC, F4 and LAM, respectively. ETEC up-regulated networks related to inflammatory and immune responses, RNA processing, and mitosis. There was a limited overlap in up-regulated gene sets between ETEC and F4 fimbriae. LAM down-regulated genes related to inflammatory and immune responses, as well as to cellular compound metabolism. In CA pigs, 57 gene sets were up-regulated by CA, while 73 were down-regulated compared to SA. CA up-regulated gene sets related to lymphocyte modulation and to cellular defense in all loop perfusions. In CA pigs, compared to SA pigs, genes for chemokine and cytokine activity and for response to external stimuli were down-regulated in ETEC-perfused loops and up-regulated in CTRL. The results highlight the importance of the nature of neonatal microbial colonization in the response to microbial stimuli later in life.



  1. L. Blavi, J.F. Perez, C. Villodre, P. López, S. M. Martín-Orúe, V. Motta, D. Luise, P. Trevisi, D. Sola-Oriola. 2018. Effects of limestone inclusion on growth performance, intestinal microbiota, and the jejunal transcriptomic profile when fed to weaning pigs. Animal Feed Science and Technology. 242: 8-20.



Plant ingredients contain low Ca levels and 1/3 may be bound to P phytate. Therefore, Ca has to be supplemented with animal or mineral sources, such as limestone or calcium phosphates. However, growth performance and feed intake has been reported by other authors to decrease linearly with increasing dietary Ca. To better understand the possible mechanisms underlying detrimental effect of including limestone in weanling diets, two experimental trials were performed to evaluate its possible impact on growth performance, colonic microbiota and, jejunal gene expression. In Trial 1, a total of 240 piglets (26 ± 2 d old, average body weight (BW) = 7.7 ± 1.04 kg) were distributed into 3 dietary treatments: 0% limestone, 0.8% limestone, and 1.6% limestone to provide 0.6, 0.9 and 1.2% total Ca, respectively. Feed intake and individual BW were registered during the pre-starter phase (d 0–14 post-weaning). Pigs fed diet with 1.6% of limestone (1.2% of total Ca) had lower BW, average daily gain (ADG), and gain to feed ratio (G:F) compared with pigs fed diets with 0.8 and 0% limestone (0.9 and 0.6% total Ca, respectively; P < 0.05). In Trial 2, a total of 18 pigs (28 ± 0 d old, average BW = 7.2 ± 0.24 kg) were allocated individually in cages and assigned to 2 dietary treatments: 0% limestone and 1.4% limestone to provide 0.6 and 1.2% total Ca, respectively. Piglets were fed for 14 d and then euthanized to obtain jejunum tissue for gene expression and colon digesta for 16SRNA gene microbiota analyses. Pigs fed 1.4% limestone (1.2% of total Ca) showed a higher beta-diversity and an increase on the Bacteroides genera in colon (P < 0.05). These animals also showed an up regulation of gene sets related to the cell cycle regulation, DNA and RNA transcription and inflammatory response in the jejunum. In conclusion, high levels of limestone inclusion in weaning diets, decreases growth performance without affecting feed intake in weaned pigs. Also, addition of limestone to diets for 14 d after weaning can upregulate the expression of genes related to the inflammatory response, and enlarge colonic beta-diversity with an increased Bacteroides genera.


   11.  R. Pieper, C. Villodre Tudela, M. Taciak, J. Bindelle, J. F. Pérez and J. Zentek. 2016. Health relevance of intestinal protein fermentation in young pigs. Animal Health Research Reviews. 17(2) :137-147


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.

12.   Burbach K, Seifert J, Pieper DH, Camarinha-Silva A. Evaluation of DNA extraction kits and phylogenetic diversity of the porcine gastrointestinal tract based on Illumina sequencing of two hypervariable regions.  Microbiologyopen. 2016 Feb;5(1):70-82. doi: 10.1002/mbo3.312. Epub 2015 Nov 5.