Review: A systematic review of the effects of functional amino acids on small intestine barrier function and immunity in piglets.

The need to reduce the use of antibiotics and zinc oxide at the pharmacological level, while preserving the performance of postweaning piglets, involves finding adequate nutritional strategies which, coupled with other preventive strategies, act to improve the sustainability of the piglet-rearing system. Amino acids (AAs) are the building blocks of proteins; however, they also have many other functions within the body. AA supplementation, above the suggested nutritional requirement for piglets, has been investigated in the diets of postweaning piglets to limit the detrimental consequences occurring during this stressful period. A systematic review was carried out to summarise the effects of AAs on gut barrier function and immunity, two of the parameters contributing to gut health. An initial manual literature search was completed using an organised search strategy on PubMed, utilising the search term " AND ". These searches yielded 302 articles (published before October 2021); 59 were selected. Based on the method for extracting data (synthesis of evidence), this review showed that L-Arginine, L-Glutamine and L-Glutamate are important functional AAs playing major roles in gut morphology and immune functions. Additional benefits of AA supplementation, refereed to a supplementation above the suggested nutritional requirement for piglets, could also be observed; however, data are needed to provide consistent evidence. Taken together, this review showed that supplementation with AAs during the weaning phase supported a plethora of the physiological functions of piglets. In addition, the data reported confirmed that each amino acid targets different parameters related to gut health, suggesting the existence of potential synergies among them.

Effect of an Escherichia coli F4/F18 bivalent oral live vaccine on gut health and performance of healthy weaned pigs.

Oral live vaccines stimulate host immunity, but they could also affect intestinal mucosa development and gut microbiota of piglets during the postweaning. The aim of this study was to determine the effect of an oral vaccine against Escherichia coli F4 and F18 (Coliprotec F4/F18®), on gut functionality and integrity, growth performance and health status of postweaning piglets. A total of 96 weaned piglets (23.30 ±â€¯1.85 days of age; 7334 ±â€¯1039 g BW) were divided into two groups (16 replicates/group; three piglets/replicate) as follows: (1) Control (CO), fed a standard diet (prestarter up to 14 days, then starter feed); (2) Treated (TRT): as CO but vaccinated with Coliprotec F4/F18® at weaning (day 0). Piglets were weighed at day 0 and weekly until day 35. Individual faecal score was recorded daily. Piglets were sacrificed at days 10 (1/3 of total) and 35 (2/3). Samples of jejunum mucosa and of cecum content were collected for morphometric, immunohistochemistry analysis and for microbiota profile analysis, respectively. Data were fitted using a linear model including treatment, class of starting BW as fixed factors and litter as random factor. From days 0 to 7, piglets from the TRT group tended to have a higher average daily gain (+22.6%, P = 0.08) and average daily feed intake compared to the CO group (+13.2%, P = 0.022). Gain to feed ratio was lower in the TRT group from days 14 to 35 (-6.6%, P = 0.011). From days 7 to 14, the TRT group had a higher diarrhoea index (-199%, P < 0.001). Crypt depth was higher in the CO group (+10.9%, P = 0.04) at day 10, but not at day 35. Jejunal expression of Claudin-4 (probability of having a score = 3) was higher in the TRT group at day 10 (CO = 1.50% vs TRT = 2.69%, P < 0.0001) and day 35 (CO = 1.29% vs TRT = 1.92%, P = 0.012). Oral vaccine affected beta diversity at day 10 (P = 0.040; R2 = 0.05) and increased the abundance of specific taxa and genera in the cecum at day 10, including Prevotella (lg2FC = 23.2, FDR < 0.001). The results showed how an Escherichia coli-based vaccine supplied to weaned pigs can promote gut health by controlling symptoms of the postweaning perturbation in the first 2 weeks postweaning. In addition, the vaccine strains showed a probiotic-like effect by modulating gut microbiota favouring the establishment of beneficial bacteria, and by promoting gut barrier integrity.

Assessment of Biolog EcoplateTM method for functional metabolic diversity of aerotolerant pig fecal microbiota.

In the last decades, gut microbiota and its role in mammal host development and health have been increasingly investigated. Metabolites produced by gut microbiota can affect intestinal homeostasis and immune system maturity and activation, and in turn, they can influence the health and growth performance of livestock. Therefore, a better understanding of the functional metabolic capability of the gut microbiota would be appreciated by the scientific community. In this study, the BiologTM Ecoplates technology was applied for studying the metabolic potential of the aerotolerant microbial community of pig fecal samples, evaluating the interference of different storage conditions and cell concentrations. The length of time for which a fecal sample maintained detectable and unchanged microbial metabolic activity was also investigated. Two assays aimed to evaluate differences in the metabolic activities between fresh and snap-frozen fecal samples at different dilutions and at different lengths of times of preservation at -80°C were carried out. The biodiversity and the predicted functionality of the entire bacterial community through a targeted metagenomic approach were also explored. The results highlighted that snap freezing of fecal samples preserved the metabolic activity of the microbial community when compared to fresh feces. Sample storage at -80 °C did not significantly affect the metabolic activity of the microbial community, which was stable for 150 days. Furthermore, the highest metabolic activity was detected with 1:2 to 1:5 dilutions of the stock suspension. BiologTM Ecoplates technology is a rapid and useful method to explore microbial communities' metabolism in animal fecal samples contributing to investigate host animal physiology. KEY POINTS: • Freezing of samples can preserve the functional activity of the aerotolerant microbial community for 150 days. • The concentration of microbial cells strongly influences metabolic activity detection. • Sequencing coupled with the BiologTM Ecoplates could be a strategy to evaluate the metabolic potential of the microbiota of the fecal sample.

Maternal antibiotic treatment affects offspring gastric sensing for umami taste and ghrelin regulation in the pig.

BACKGROUND: Scarce is knowledge on the process regulating the development of acid secretion, orexigenic signaling, and chemosensing in the stomach of young pigs. Changes of early microbial encounters by suckling pigs can interact with the gut maturation, by the induction of different molecular signaling. Our goal was to assess if the age of offspring and the maternal environment, influenced by sow antibiotic treatment peripartum, could affect gastric morphology and the expression of genes involved in the control of hydrochloric secretion, feed intake, taste, and inflammation in offspring stomach. METHODS: 84 pigs from sows fed a diet with amoxicillin (on -d10 to +d21 from farrowing, ANT) or without (CON) were sacrificed at d14, d21, d28 (weaning) or d42. Samples of oxyntic (OXY), pyloric (PY) and cardiac mucosae close to OXY were collected and parietal and enteroendocrine cells (EECs) were counted. Relative gene expression of a set of 11 key genes (ATP4A, SSTR2, GAST, GHRL, MBOAT4, PCSK1, GNAT1, TAS1R1, TAS1R3, IL8 and TNF) was assessed by qRT-PCR. In addition, 40 offspring obtained from the same ANT and CON sows were offered a normal or a fat-enriched diet for 4 weeks between 140 and 169 d of age, and then OXY and PY were sampled. RESULTS: The number of parietal and EECs increased with age (P < 0.001). ATP4A increased with age (within suckling, P = 0.043, post-weaning vs. suckling, P < 0.001), SSTR2 increased only after weaning (P < 0.001). In OXY, GHRL increased during suckling (P = 0.012), and post-weaning as a trend (P = 0.088). MBOAT4 tended to increase during suckling (P = 0.062). TAS1R1 increased from suckling to post-weaning period (P =0.001) and was lower in ANT offspring (P = 0.013). GNAT1 in PY was higher in ANT offspring (P = 0.041). Antibiotic treatment of sows peripartum increased expression of GHRL and MBOAT4 in OXY of growing-finishing offspring aged 5 months. CONCLUSIONS: Data show that sensing for umami taste and ghrelin regulation can be affected by maternal environment, but the development of acid secretion, orexigenic signaling and taste perception in the stomach are mostly developmentally controlled.

Resistant starch reduces large intestinal pH and promotes fecal lactobacilli and bifidobacteria in pigs.

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%.

Evaluation of Breed and Parity Order Effects on the Lipid Composition of Porcine Colostrum.

Porcine colostrum lipid classes and fatty acids (FA) were characterized in 6 pools (from 69 samples) from 3 sow breeds (Italian Large White, Italian Landrace, and Italian Duroc) and different parity orders (only Large White). Triacylglycerols (TAG; 94.44 expressed as g/100 g of fat) were the most abundant lipid class, followed by diacylglycerols (DAG; 3.36 g/100 g of fat), free fatty acids (FFA; 0.98 g/100 g of fat), and cholesterol (0.84 g/100 g of fat). The main FAs found in swine colostrum were palmitic (27.29%, expressed as g/100 g of total FA), oleic (28.81%), and linoleic (23.39%) acids. Both the breed of sow and parity order affected the FA and lipid composition. The results suggest that the FA composition of swine colostrum is similar to that of human colostrum and could represent a new source of nutrients for human infants, after further assessment of hygienic and quality aspects. The swine model could be an opportunity for a better understanding of colostrum effects on newborns.

Effect of feed supplementation with live yeast on the intestinal transcriptome profile of weaning pigs orally challenged with Escherichia coli F4.

The ability of live yeasts to modulate pig intestinal cell signals in response to infection with Escherichia coli F4ac (ETEC) has not been studied in-depth. The aim of this trial was to evaluate the effect of Saccharomyces cerevisiae CNCM I-4407 (Sc), supplied at different times, on the transcriptome profile of the jejunal mucosa of pigs 24 h after infection with ETEC. In total, 20 piglets selected to be ETEC-susceptible were weaned at 24 days of age (day 0) and allotted by litter to one of following groups: control (CO), CO+colistin (AB), CO+5×1010 colony-forming unit (CFU) Sc/kg feed, from day 0 (PR) and CO+5×1010 CFU Sc/kg feed from day 7 (CM). On day 7, the pigs were orally challenged with ETEC and were slaughtered 24 h later after blood sampling for haptoglobin (Hp) and C-reactive protein (CRP) determination. The jejunal mucosa was sampled (1) for morphometry; (2) for quantification of proliferation, apoptosis and zonula occludens (ZO-1); (3) to carry out the microarray analysis. A functional analysis was carried out using Gene Set Enrichment Analysis. The normalized enrichment score (NES) was calculated for each gene set, and statistical significance was defined when the False Discovery Rate % was <25 and P-values of NES were <0.05. The blood concentration of CRP and Hp, and the score for ZO-1 integrity on the jejunal villi did not differ between groups. The intestinal crypts were deeper in the AB (P=0.05) and the yeast groups (P<0.05) than in the CO group. Antibiotic treatment increased the number of mitotic cells in intestinal villi as compared with the control group (P<0.05). The PR group tended to increase the mitotic cells in villi and crypts and tended to reduce the cells in apoptosis as compared with the CM group. The transcriptome profiles of the AB and PR groups were similar. In both groups, the gene sets involved in mitosis and in mitochondria development ranked the highest, whereas in the CO group, the gene sets related to cell junction and anion channels were affected. In the CM group, the gene sets linked to the metabolic process, and transcription ranked the highest; a gene set linked with a negative effect on growth was also affected. In conclusion, the constant supplementation in the feed with the strain of yeast tested was effective in counteracting the detrimental effect of ETEC infection in susceptible pigs limits the early activation of the gene sets related to the impairment of the jejunal mucosa.

Next generation semiconductor based sequencing of bitter taste receptor genes in different pig populations and association analysis using a selective DNA pool-seq approach.

Taste perception in animals affects feed intake and may influence production traits. In particular, bitter is sensed by receptors encoded by the family of TAS2R genes. In this research, using a DNA pool-seq approach coupled with next generation semiconductor based target resequencing, we analysed nine porcine TAS2R genes (TAS2R1, TAS2R3, TAS2R4, TAS2R7, TAS2R9, TAS2R10, TAS2R16, TAS2R38 and TAS2R39) to identify variability and, at the same time, estimate single nucleotide polymorphism (SNP) allele frequencies in several populations and testing differences in an association analysis. Equimolar DNA pools were prepared for five pig breeds (Italian Duroc, Italian Landrace, Pietrain, Meishan and Casertana) and wild boars (5-10 individuals each) and for two groups of Italian Large White pigs with extreme and divergent back fat thickness (50 + 50 pigs). About 1.8 million reads were obtained by sequencing amplicons generated from these pools. A total of 125 SNPs were identified, of which 37 were missense mutations. Three of them (p.Ile53Phe and p.Trp85Leu in TAS2R4; p.Leu37Ser in TAS2R39) could have important effects on the function of these bitter taste receptors, based on in silico predictions. Variability in wild boars seems lower than that in domestic breeds potentially as a result of selective pressure in the wild towards defensive bitter taste perception. Three SNPs in TAS2R38 and TAS2R39 were significantly associated with back fat thickness. These results may be important to understand the complexity of taste perception and their associated effects that could be useful to develop nutrigenetic approaches in pig breeding and nutrition.

The A0 blood group genotype modifies the jejunal glycomic binding pattern profile of piglets early associated with a simple or complex microbiota.

The intestinal epithelium glycocalyx sugar motif is an important determinant of the bacterial-host interaction and may be affected in pigs by gut microbiota and by blood group genotype. The aim was to study the effect of intestinal association with different microbiota and A0 blood group genotypes on the expressed glycomic pattern in the small intestine. Twelve caesarean-derived pigs previously associated with a simple association (SA) or complex association (CA) microbiota were selected at 26 to 37 d of age. In each subject, different jejunal loops were perfused for 8 h with enterotoxigenic K88 (ETEC), ETEC fimbriae (F4), (LAM), or a saline control. The piglets were genotyped for A0 blood group and the glycomic profile was evaluated by microscopic screening of lectin binding: peanut agglutinin (PNA), which is galactose specific; agglutinin I (UEA), which is fucose specific; lectin II (MALii), which is sialic acid specific; concavalin A, which is mannose specific; soybean agglutinin (SBA), which is -acetyl-galactosamine specific; and wheat germ agglutinin (WGA), which is -acetyl-glucosamine specific. A0 pigs had fewer UEA-positive cells, MALii-positive cells ( < 0.001), and SBA-positive cells ( < 0.10) than 00 pigs. Simple association pigs had more SBA positive cells ( < 0.01) than CA pigs. Enterotoxigenic K88-perfused intestinal loops had fewer UEA-positive cells ( < 0.01) and WGA positive cells ( < 0.001) cells and more PNA positive cells (only in SA pigs, < 0.01). No effects of introduction of F4 and LAM in the intestinal lumen were observed. The porcine A0 blood group genotype and the luminal presence of ETEC strongly affected the jejunal mucosa glycomic pattern profile whereas an early oral simple or complex microbial association had limited effects. Pig genetic background has relevance on the cross talk between intestinal epithelium glycocalyx sugar motif and ETEC and, ultimately, on the gut microbial colonization in later life.

Metabolomics evidences plasma and serum biomarkers differentiating two heavy pig breeds.

In pigs, many production traits are known to vary among breeds or lines. These traits can be considered end phenotypes or external traits as they are the final results of complex biological interactions and processes whose fine biological mechanisms are still largely unknown. This study was designed to compare plasma and serum metabolomic profiles between animals of two heavy pig breeds (12 Italian Large White and 12 Italian Duroc), testing indirectly the hypothesis that different genetic backgrounds might be the determining factors of differences observed on the level of metabolites in the analyzed biofluids between breeds. We used a targeted metabolomic approach based on mass spectrometric detection of about 180 metabolites and applied a statistical validation pipeline to identify differences in the metabolomic profiles of the two heavy pig breeds. Blood samples were collected after jugulation at the slaughterhouse and prepared for metabolomics analysis that was carried out using the Biocrates AbsoluteIDQ p180 Kit, covering five different biochemical classes: glycerophospholipids, amino acids, biogenic amines, hexoses and acylcarnitines. A statistical pipeline that included the selection of the most relevant metabolites differentiating the two breeds by sparse Partial Least Squares Discriminant Analysis (sPLS-DA) was coupled with a stability test and significance test determined with leave one out and permutation procedures. sPLS-DA plots clearly separated the pigs of the two investigated breeds. A few metabolites (a total of five metabolites considering the two biofluids) involved in key metabolic pathways largely contributed to these differences between breeds. In particular, a higher level of the sphingomyelins SM (OH) C14:1 (both in plasma and serum), SM (OH) C16:1 (in serum) and SM C16:0 (in serum) were observed in Italian Duroc than in Italian Large White pigs and the inverse was for the biogenic amine kynurenine (in plasma). The level of another biogenic amine (acetylornithine) was higher in Italian Large White than in Italian Duroc pigs in both analysed biofluids. These results provided biomarkers that could be important to understand the biological differences between these two heavy pig breeds. In particular, according to the functional role played by sphingomyelins in obesity-induced inflammatory responses, it could be possible to speculate that a higher level of sphingomyelins in Italian Duroc might be related to the higher interrmuscular fat deposition of this breed compared with the Italian Large White. Additional studies will be needed to evaluate the relevance of these biomarkers for practical applications in pig breeding and nutrition.

Deconstructing the pig sex metabolome: Targeted metabolomics in heavy pigs revealed sexual dimorphisms in plasma biomarkers and metabolic pathways.

Metabolomics has opened new possibilities to investigate metabolic differences among animals. In this study, we applied a targeted metabolomic approach to deconstruct the pig sex metabolome as defined by castrated males and entire gilts. Plasma from 545 performance-tested Italian Large White pigs (172 castrated males and 373 females) sampled at about 160 kg live weight were analyzed for 186 metabolites using the Biocrates AbsoluteIDQ p180 Kit. After filtering, 132 metabolites (20 AA, 11 biogenic amines, 1 hexose, 13 acylcarnitines, 11 sphingomyelins, 67 phosphatidylcholines, and 9 lysophosphatidylcholines) were retained for further analyses. The multivariate approach of the sparse partial least squares discriminant analysis was applied, together with a specifically designed statistical pipeline, that included a permutation test and a 10 cross-fold validation procedure that produced stability and effect size statistics for each metabolite. Using this approach, we identified 85 biomarkers (with metabolites from all analyzed chemical families) that contributed to the differences between the 2 groups of pigs ( < 0.05 at the stability statistic test). All acylcarnitines and almost all biogenic amines were higher in castrated males than in gilts. Metabolites involved in tryptophan catabolism had the largest differences (i.e., delta = 20% for serotonin) between castrated males (higher) and gilts (lower). The level of several AA (Ala, Arg, Gly, His, Lys, Ser, Thr, and Trp) was higher in gilts (delta was from approximately 1.0 to approximately 4.8%) whereas products of AA catabolism (taurine, 2-aminoadipic acid, and methionine sulfoxide) were higher in castrated males (delta was approximately 5.0-6.0%), suggesting a metabolic shift in castrated males toward energy storage and lipid production. Similar general patterns were observed for most sphingomyelins, phosphatidylcholines, and lysophosphatidylcholines. Metabolomic pathway analysis and pathway enrichment identified several differences between the 2 sexes. This metabolomic overview opened new clues on the biochemical mechanisms underlying sexual dimorphism that, on one hand, might explain differences in terms of economic traits between castrated male pigs and entire gilts and, on the other hand, could strengthen the pig as a model to define metabolic mechanisms related to fat deposition.

Comparison of three patterns of feed supplementation with live Saccharomyces cerevisiae yeast on postweaning diarrhea, health status, and blood metabolic profile of susceptible weaning pigs orally challenged with Escherichia coli F4ac.

The development of effective feeding strategies to reduce the detrimental effect of enterotoxigenic F4ac (ETEC) plays a crucial role in reducing the occurrence of therapeutic intervention with antibiotics in livestock. The ability of CNCM I-4407 (SCC), supplied in different patterns to counteract ETEC infection in weaned pigs, was evaluated. Fifty pigs weaned at 24 d were then divided into 5 groups: control (CO), CO + colistin (AB), CO + 5 × 10(10) cfu of SCC/ kg feed, from d 0 to 21 (PR), CO + 5 × 10(10) cfu of SCC/ kg feed from d 7 to 11 (CM), and CO + 1 shot of 2 × 10(11) cfu of SCC when the first diarrhea appeared (CU). On d 7 postweaning, all the pigs were orally challenged with 10(8) cfu of ETEC. Blood samples were taken from the pigs (d 7, 8, 12, and 21) while the fecal excretion of ETEC was assessed on d 7 and 10. Fecal consistency was scored from 12 h before infection to 144 h postinfection (p.i.). On d 21, the pigs were sacrificed. The in vitro adhesion test on the intestinal villi confirmed individual susceptibility to ETEC, excluding the presence of resistant pigs. Growth performance did not differ between the treatments. Mortality was reduced in the AB group (P< 0.01) and, marginally, in the PR group (P = 0.089) when compared to the CO group. The CO group had a higher fecal score than AB in the period of observation (from P = 0.01 to P< 0.001). Yeast administration reduced the fecal score when compared to the CO group 12 and 48 h p.i. (P = 0.04). Total IgA never differed among the treatments, but the ETEC-specific IgA concentration was lower in the AB group than in CO (P = 0.04) at d 12. Four days p.i., the pigs fed live yeast had reduced ETEC excretion compared with the CO pigs (P = 0.05). Blood concentrations of dodecenoyl-L-carnitine (P < 0.01), glutaryl-L-carnitine/hydroxyhex¬anoyl-L-carnitine, phosphatidylcholine diacyl and phosphatidylcholine diacyl (P = 0.01 and P< 0.01, respectively), and α-amino adipic acid (P < 0.01) were reduced in the AB group compared to the CO group; PR + CM reduced the concentration of sphingomyelin-ceramide (P = 0.02) and increased the concentration of decadienyl-L-carnitine (C10:2; P= 0.02) vs. CO. The CM group had an increased concentration of C10:2 (P < 0.01) compared to the PR group. In conclusion, the administration of live yeast, even in concomitance with ETEC infections, reduces pig illness and mortality. The strain of SCC tested did not show a therapeutic effect.

Effect of added dietary threonine on growth performance, health, immunity and gastrointestinal function of weaning pigs with differing genetic susceptibility to Escherichia coli infection and challenged with E. coli K88ac.

Threonine (Thr) is important for mucin and immunoglobulin production. We studied the effect of added dietary Thr on growth performance, health, immunity and gastrointestinal function of weaning pigs with differing genetic susceptibility to E. coli K88ac (ETEC) infection and challenged with ETEC. Forty-eight 24-day-old weaned pigs were divided into two groups by their ETEC susceptibility using mucin 4 (MUC4) gene as a marker (2 MUC4(-/-) , not-susceptible, and 2 MUC4(+/+) , susceptible, pigs per litter). Within genotype, pigs were fed two different diets: 8.5 (LThr) or 9.0 (HThr) g Thr/kg. Pigs were orally challenged on day 7 after weaning and slaughtered on day 12 or 13 after weaning. Before ETEC challenge, HThr pigs ate more (p < 0.05). The diet did not affect post-challenge growth, but HThr tended to increase post-challenge feed efficiency (p = 0.087) and overall growth (p = 0.087) and feed efficiency (p = 0.055). Before challenge, HThr pigs excreted less E. coli (p < 0.05), while after challenge, diet did not affect the number of days with diarrhoea and ETEC excretion. MUC4(+/+) pigs responded to the challenge with more diarrhoea, ETEC excretion and anti-K88 IgA in blood and jejunal secretion (p < 0.001). HThr pigs had a higher increase of anti-K88 IgA values in jejunal secretion (p = 0.089) and in blood (p = 0.089, in MUC4(+/+) pigs only). Thr did not affect total IgA and IgM values, morphometry of jejunum, goblet cells count in colon, total mucin from jejunum and colon, but varied jejunal goblet cells counts (p < 0.05). In the first two post-weaning weeks, 8.5 g Thr/kg diet may be not sufficient to optimize initial feed intake, overall feed efficiency and intestinal IgA secretion and to control the gut microbiota in the first post-weaning week, irrespective of the pig genetic susceptibility to ETEC infection.

Effect of fasting and refeeding on expression of genes involved in the gastric nutrient sensing and orexigenic control of pigs.

Knowledge on orexigenic signals in the pig stomach is poor. Gastric amino acid sensing by taste receptor type 1 member 3 (T1R3) and calcium-sensing receptor (CASR), and active ghrelin release, controlled by preproghrelin, proprotein convertase (PC1/3) and ghrelin O-acyltransferase (GOAT) genes, may be affected by fasting or refeeding. Twelve pigs (12.0 kg LW) were adapted to a base diet and assigned to three individual feeding schedules: Control (C), fed twice a day; Fasting (F), fasted for 24 h; Refeeding (R), fasted for 24 h and refed before slaughtering. Gastrointestinal segments were collected for histology and molecular biology analyses. Total RNA isolated from oxyntic and pyloric mucosae was reverse transcribed, specific porcine primers were designed and transcript quantification was performed by real-time RT-PCR. F decreased villus height in duodenum (p < 0.01) and ileum (p < 0.05) vs. C and R. R increased oxyntic PC1/3 (p < 0.05) and tended to increase oxyntic preproghrelin (p = 0.06), and pyloric GOAT (p = 0.07) gene expression vs. C. PC1/3 gene expression was higher in pyloric mucosa. Ghrelin-positive cells numbers were not different between the two gastric mucosae. Gastrin expression tended to be higher in R than in C and F (p = 0.068 and p = 0.055). CASR was higher in pyloric than in oxyntic mucosa, and pyloric CASR expression tended to be higher in R than in C (p = 0.072). T1R1 was not affected by treatment. Our results indicate that the pool of genes involved in the secretion of active ghrelin is active both in oxyntic and pyloric mucosa of pig. Refeeding can significantly affect the expression of genes that control octanoyl-ghrelin production and partially the amino acid sensing by CASR gene, while the absence of effect of fasting on the expression of ghrelin-related genes needs further confirmations.

Encapsulation and modified-release of thymol from oral microparticles as adjuvant or substitute to current medications.

The aim of this study was to encapsulate, thymol, in natural polymers in order to obtain (i) taste masking effect and, then, enhancing its palatability and (ii) two formulations for systemic and local delivery of herbal drug as adjuvants or substitutes to current medications to prevent and treat several human and animal diseases. Microspheres based on methylcellulose or hydroxypropyl methylcellulose phthalate (HPMCP) were prepared by spray drying technique. Microparticles were in vitro characterized in terms of yield of production, drug content and encapsulation efficiency, particle size, morphology and drug release. Both formulations were in vivo orally administered and pharmacokinetic analysis was carried out. The polymers used affect the release and, then, the pharmacokinetic profile of thymol. Encapsulation into methylcellulose microspheres leads to short half/life but bioavailability remarkably increases compared to the free thymol. In contrast, enteric formulation based on HPMCP shows very limited systemic absorption. These formulations could be proposed as alternative or adjuvants for controlling pathogen infections in human or animal. In particular, methylcellulose microspheres can be used for thymol systemic administration at low doses and HPMCP particles for local treatment of intestinal infections.

Effect of free thymol on differential gene expression in gastric mucosa of the young pig.

Thymol is the most common molecule in thyme and has been proposed as an oral alternative to antibiotics in the feed of pigs and broilers. The knowledge of the in vivo physiological effects of thymol on tissues is limited, particularly its impact on the gastric mucosa, where it is primarily absorbed when it is orally supplied. In this study, thymol (TH, 50 mg/ kg BW) or a placebo (CO) was introduced directly into the stomach of 8 weaned pigs that were slaughtered 12 h later and sampled for gastric oxyntic and pyloric mucosa. The analysis of whole transcript expression was performed using Affymetrix© Porcine Gene 1.1 ST array strips. Affymetrix Transcripts IDs were associated with 13 406 human gene names based on Sus scrofa Ensemble. Gene Set Enrichment Analysis was performed, comparing TH and CO pigs. For each gene set, the normalized enrichment score (NES) was defined as significant when the false discovery rate % was <25 and the P-value of NES was <0.05. In response to TH, 72 and 19 gene sets were significantly enriched in the oxyntic and pyloric mucosa, respectively. Several gene sets involved in mitosis and its regulation ranked near the top, primarily in the oxyntic mucosa; the gene set DIGESTION ranked first and ninth in the pyloric and oxyntic mucosa, respectively. Within this group, somatostatin (SST), SST receptors, peptide transporter 1 (SLC15A1) and calpain 9 (gastrointestinal tract-specific calpain) were the most strongly upregulated genes. Thymol reduced the enrichment of 120 and 59 gene sets in the oxyntic and pyloric mucosa, respectively. Several gene sets related to ion transport and channeling and aqueous pores across membranes, including short transient receptor potential (TRP) channel 4, potassium voltage-gated channel members 1 and 2, and ryanodine receptors 2 and 3, were less enriched. The downregulation of these genes sensitive to thymol in vitro could depend on the thymol dose and contact with the gastric tissues that causes an adaptive response with their reduced activation. Conversely, the activation of the TRPA1 gene (ranked 1072 and 128 among all the genes in the oxyntic and pyloric mucosa, respectively) indicates the involvement of another TRP-regulating cellular calcium storage. In conclusion, the stimulation of gastric proliferative activity and the control of digestive activity by thymol can influence positively gastric maturation and function in the weaned pigs. These properties should be considered in addition to thymol's antimicrobial properties when supplementation of this molecule in feed is evaluated.

Polymorphisms in an obesity-related gene (PCSK1) are associated with fat deposition and production traits in Italian heavy pigs.

The proprotein convertase subtilisin/kexin type 1 (PCSK1) gene encodes the prohormone convertase 1/3 enzyme that processes prohormones into functional hormones that, in turn, regulate central and peripheral energy metabolism. Mutations in the human PCSK1 gene cause severe monogenic obesity or confer risk of obesity. We herein investigated the porcine PCSK1 gene with the aim of identifying polymorphisms associated with fat deposition and production traits in Italian heavy pigs. By re-sequencing about 5.1 kb of this gene in 21 pigs of different breeds, we discovered 14 polymorphisms that were organized in nine haplotypes, clearly distributed in two clades of putative European and Asian origin. Then we re-mapped this gene on porcine chromosome 2 and analysed its expression in several tissues including gastric oxyntic mucosa of weanling pigs in which PCSK1 processes the pre-pro-ghrelin into ghrelin, which in turn is involved in the control of feed intake and energy metabolism. Association analyses between PCSK1 single-nucleotide polymorphisms (SNPs) and production, carcass and several other traits were conducted on five groups of pigs from three different experimental designs, for a total of 1221 animals. Results indicated that the analysed SNPs were associated (P < 0.01 or P < 0.05) with several traits including backfat thickness and visible intermuscular fat in Italian Duroc (ID) and growth performances in Italian Large White (ILW) and in ILW × Italian Landrace pigs. However, the effects estimated in the ILW were opposite to the effects reported in the ID pigs. Suggestive association (P < 0.10) was observed with muscle cathepsin B activity, opening, if confirmed, potential applications to reduce the excessive softness defect of the green hams that is of particular concern for the processing industry. The results obtained supported the need to further investigate the PCSK1 gene to fully exploit the value of its variability and apply this information in pig breeding programmes.

Assessment of the presence of chemosensing receptors based on bitter and fat taste in the gastrointestinal tract of young pig.

Knowledge on porcine bitter and fat taste receptors and on their expression in gastrointestinal tract of pigs is scarce. We searched for the presence of porcine homologous sequences for 13 human transcripts of bitter and fat taste receptors in ENSEMBL and National Center for Biotechnology Information databases. For taste 2 receptor (TAS2R) 8, alignment was not observed; for TAS2R13 and TAS2R46 the porcine predicted sequence aligned with several other human bitter genes. For 7 genes for bitter taste (TAS2R1, TAS2R3, TAS2R7, TAS2R9, TAS2R10, TAS2R16, and TAS2R38) and for 3 genes for fat taste (GPR40, GPR43, and GPR120), a full homology for exon sequences was found and primers were designed by Primer3. These 7 genes were amplified with real-time PCR and verified on agarose gel in 5 gastrointestinal segments of weaned pigs: oxyntic (ST1), pyloric (ST2), and cardiac to oxyntic transition mucosa (ST3), jejunum (JEJ), and colon (COL). Suitability of mRNA was verified by amplifying RPL4 and HMBS2 genes. Each bitter taste gene was detectable on agarose gel in at least 1 subject of all the gastrointestinal segments except for TAS2R3 and TAS2R38 that were never detected in ST1 and COL, respectively. The inspection of bitter taste genes amplification curve indicated that the expression was in general very low. GPR43 and GPR120 were present in all segments from all pigs. Expression was not detected for GPR40. Data also indicate that colon is the preeminent tract where fat detection by GPR120 takes place (P < 0.001). The presence of gene expression for several chemosensing receptors for bitter and fat taste in different compartments of the stomach confirms that this organ should be considered a player for the early detection of bolus composition.

In vitro test on the ability of a yeast cell wall based product to inhibit the Escherichia coli F4ac adhesion on the brush border of porcine intestinal villi.

The ability of a yeast cell wall (YCW)-based product (SENTIGUARD C; Nutriad) to inhibit the enterotoxigenic Escherichia coli F4ac (ETEC) adhesion on the brush border of porcine intestinal villi was tested. The ETEC suspensions were preincubated with 2 batches of the product (A and B) at different concentrations (10, 5, and 0.5%, wt/vol) or with their filtrates (AF and BF) and then with intestinal villi susceptible to ETEC adhesion. In all the trials, ETEC suspensions were also preincubated with egg yolk (E) immunized against ETEC to assess the maximum inhibition of the adhesiveness or directly with the villi [control group (Con)] to verify the maximum adhesiveness of the pathogen. For each treatment, 20 different villi were observed, brush border measured, and the adherent pathogens counted. A scanning electron microscope analysis was used to confirm the ability of ETEC to adhere on the YCW. The E treatment significantly reduced the pathogen adhesion on the villi compared with the C group in all the trials (P < 0.001). Both batches of SENTIGUARD C significantly reduced the pathogen adhesion on the villi compared with the C group at the concentration of 10 and 5% (P < 0.001) but not at the concentration of 0.5%. The BF did not significantly reduce the ETEC adhesion whereas the AF significantly increased bacterial adhesion (P = 0.015). The microscopy results confirm the ability of ETEC to adhere on YCW. Taken together, our results indicate the ability of the SENTIGUARD C to contain the intestinal infection from ETEC in young pigs with the affinity of ETEC to YCW.