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.
Abstract
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 Escherichia coli K88 (ETEC), ETEC fimbriae (F4), Lactobacillus amylovorus (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; Ulex europaeus agglutinin I (UEA), which is fucose specific; Maackia amurensis lectin II (MALii), which is sialic acid specific; concavalin A, which is mannose specific; soybean agglutinin (SBA), which is N-acetyl-galactosamine specific; and wheat germ agglutinin (WGA), which is N-acetyl-glucosamine specific. A0 pigs had fewer UEA-positive cells, MALii-positive cells (P < 0.001), and SBA-positive cells (P < 0.10) than 00 pigs. Simple association pigs had more SBA positive cells (P < 0.01) than CA pigs. Enterotoxigenic E. coli K88–perfused intestinal loops had fewer UEA-positive cells (P < 0.01) and WGA positive cells (P < 0.001) cells and more PNA positive cells (only in SA pigs, P < 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.
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.
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. PUBLICATION ABSTRACT
Summary
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.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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.
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Supplementary l-tryptophan (l-Trp) limits the decrease of feed intake and growth in Enterotoxigenic Escherichia coli F4 (ETEC) susceptible pigs upon oral challenge with this pathogen. Susceptibility ...to ETEC is genetically controlled and related to the presence of receptors for the F4 fimbriae (F4R). We aimed to assess if dietary Trp affects genes involved in the intestinal barrier of healthy pigs carrying or not the F4R. Thirty-six littermate weaning pigs were selected to have potentially eighteen ETEC-susceptible and eighteen ETEC-non-susceptible subjects, based on a Mucin 4 gene polymorphism. For 21 days they were fed a diet with 0.17 or 0.22 ileal digestible Trp:Lys ratio. Using the test of ETEC adhesion to the intestinal villi, the pigs were divided into F4R negative (no bacteria adhering, F4R−), F4R positive (F4R+), and F4R mildly positive (F4Rm+). A preliminary test (GeneChip® Porcine Genome Array) highlighted the differentially expressed genes in the jejunum of 3 F4R− and 3 F4R+ pigs. The expression of the most interesting genes was assessed on the whole sample. In F4R+ pigs, Trp reduced the mRNA of four genes involved in the intestinal barrier and/or induced by several bacteria-associated molecular patterns, like lipopolysaccharide (LPS) (REG3G, Regenerating islet-derived 3 gamma; SFTPD, Surfactant pulmonary-associated protein D; CFB, Complement factor B; LBP, LPS-binding protein) (P<0.05). In pigs fed the low-Trp diet, REG3G, SFTPD and LPB mRNA increased with F4R presence (P<0.05). Interleukine-8 tended to be less expressed with the higher Trp level whatever the F4R presence (P=0.09). No DNA from ETEC was detected in the jejunum contents. Trp favorably interacts to reduce the bacterial induction of some genes involved in the intestinal barrier in ETEC susceptible pigs, but the causative mechanism is yet to be established.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Reports highlighting the positive effects of probiotics on the performance of pigs or on in vitro traits are now quite
frequent, but the use of probiotics in feed compounds has not been widespread. ...Prerequisites for the healthy and
efficient growth of young pigs are the rapid maturation of the gut mucosa and the mucosa-associated lymphoid
tissue, and the formation of a local stable and complex bacterial community. In neonatal pigs, suckling and the
maternal environment shape the gut microbiota. Later, when weaning stress causes a transient drop in favourable
bacteria, the oral supply of microbes could contribute to re-establish the microbiota balance. Some strains isolated
from piglets were tested for their ability to settle in the intestine. After weaning, piglets experience new and often
unfavourable bacteria. Probiotics have been investigated to contrast the enteropathogens, owing to their properties
(production of antibacterial molecules, competition on adhesion sites, stimulation of immune response, etc.). Data
in general show that their oral administration can be favourable or, at least, innocuous. However, two cases are
presented here, where a probiotic given to pigs already combating enteropathogens impaired pig health, and this
could be explained by their effect on the immune response. A more tolerogenic response of the host is expected
when beneficial bacteria directly contrast the pathogens, probiotics are claimed to directly modulate or even activate
the immune system. For one probiotic divergent effects on growth and health are presented, and these differences
may be due to different experimental details or different starting microbiological environments. Scarce data are
available on specific immune responses induced by commensal microbes in pigs, and on the interaction of resident
microbiota with orally supplied probiotics. Increased knowledge of the role of commensal microbiota in the gut and
in the pig metabolism, helps in selecting the best bacteria and in designing the best feeding strategies for improving
the efficacy and the reliability of their oral use.
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