Lactation-related dynamics of bacterial and fungal microbiomes in feces of sows and gut colonization in suckling and newly weaned piglets.

Changes in the gut microbial composition of the sow during lactation may influence the gut microbial colonization in their offspring, for which less information was available in the literature. This study aimed to assess: 1) the changes that occur in the bacterial and fungal communities in sow feces during the 28-day lactation period as well as in gastric and cecal digesta of piglets until one week after weaning, and 2) bacterial and fungal taxa in cecal digesta of the piglets postweaning that associate with fecal consistency. Aside from sow milk, piglets had access to creep feed from day of life (DoL) 3. Fecal samples from sows for microbial analysis were collected (n=20) on days postpartum (DPP) 1, 6, 13, 20 and 27, as well as from weaned piglets for fecal scoring on DoL 30 and 34. Gastric and cecal digesta of piglets was collected on DoL3, 7, 14, 21, 28, 31 and 35 (n=5/sex/DoL). Progressing lactation affected bacterial and fungal communities in sow feces, including 10.3- and 3.0-fold increases in the relative abundances of Lactobacillus from DPP1 to 6 and Kazachstania from DPP1 to 13, respectively (P < 0.001). Although time- and gut site-related differences existed, bacterial and fungal taxa found in sow feces were also present in gastric and cecal digesta of piglets, which supports their role for gut colonization in neonatal piglets. In piglets, bacterial and fungal alpha-diversities showed certain fluctuations during the suckling period, whereby weaning affected more the fungal than bacterial diversity at both gut sites (P < 0.05). At both gut sites, Lactobacillus largely increased from DoL3 to 7 and remained a dominating taxon until DoL35 (P < 0.05). Postweaning, plant-glycan fermenters (e.g., Prevotella-9) seemed to replace milk-glycan fermenting Fusobacterium and Bacteroides (P < 0.05). In gastric and cecal digesta, Kazachstania, Tausonia, Candida and Blumeria were dominating fungi from DoL3 to 35, with Kazachstania becoming even more dominant postweaning (P < 0.001). Fecal consistency was softer on DoL34 than 30 (P < 0.05). Correlation analysis identified that softer feces were linked to the relative abundances of plant-glycan and proteolytic bacterial taxa including pathobionts (e.g., Clostridium sensu stricto) in the cecum on DoL34. However, the potential association between cecal mold and plant-pathogenic fungi Talaromyces, Mrakia, and Blumeria and softer feces are worth to investigate in the future in relation to (gut) health of piglets.

An insight into the temporal dynamics in the gut microbiome, metabolite signaling, immune response, and barrier function in suckling and weaned piglets under production conditions.

Little information is available on age- and creep-feeding-related microbial and immune development in neonatal piglets. Therefore, we explored age- and gut-site-specific alterations in the microbiome, metabolites, histo-morphology, and expression of genes for microbial signaling, as well as immune and barrier function in suckling and newly weaned piglets that were receiving sow milk only or were additionally offered creep feed from day of life (DoL) 10. The experiment was conducted in two replicate batches. Creep feed intake was estimated at the litter level. Piglets were weaned on day 28 of life. Gastric and cecal digesta and jejunal and cecal tissue were collected on DoL 7, 14, 21, 28, 31, and 35 for microbial and metabolite composition, histomorphology, and gene expression. In total, results for 10 piglets (n = 5/sex) per dietary group (sow milk only versus additional creep feed) were obtained for each DoL. The creep feed intake was low at the beginning and only increased in the fourth week of life. Piglets that were fed creep feed had less lactate and acetate in gastric digesta on DoL 28 compared to piglets fed sow milk only (p < 0.05). Age mainly influenced the gastric and cecal bacteriome and cecal mycobiome composition during the suckling phase, whereas the effect of creep feeding was small. Weaning largely altered the microbial communities. For instance, it reduced gastric Lactobacillaceae and cecal Bacteroidaceae abundances and lowered lactate and short-chain fatty acid concentrations on DoL 31 (p < 0.05). Jejunal and cecal expression of genes related to microbial and metabolite signaling, and innate immunity showed age-related patterns that were highest on DoL 7 and declined until DoL 35 (p < 0.05). Weaning impaired barrier function and enhanced antimicrobial secretion by lowering the expression of tight junction proteins and stimulating goblet cell recruitment in the jejunum and cecum (p < 0.05). Results indicated that age-dependent alterations, programmed genetically and by the continuously changing gut microbiome, had a strong impact on the expression of genes for gut barrier function, integrity, innate immunity, and SCFA signaling, whereas creep feeding had little influence on the microbial and host response dynamics at the investigated gut sites.

Temporal Microbial Dynamics in Feces Discriminate by Nutrition, Fecal Color, Consistency and Sample Type in Suckling and Newly Weaned Piglets.

Feces enable frequent samplings for the same animal, which is valuable in studies investigating the development of the gut microbiome in piglets. Creep feed should prepare the piglet's gut for the postweaning period and shape the microbiome accordingly. Little is known about the variation that is caused by differences in fecal color and consistency and different sample types (feces versus swab samples). Therefore, this study evaluated the age-related alterations in the microbiome composition (16S rRNA gene) in feces of suckling and newly weaned piglets in the context of nutrition and fecal consistency, color and sample type from day 2 to 34 of life. Feces from 40 healthy piglets (2 each from 20 litters) were collected on days 2, 6, 13, 20, 27, 30 and 34. Weaning occurred on day 28. Half of the litters only drank sow milk during the suckling phase, whereas the other half had access to creep feed from day 10. Creep feeding during the suckling phase influenced the age-related total bacterial and archaeal abundances but had less of an influence on the relative bacterial composition. Results further showed different taxonomic compositions in feces of different consistency, color and sample type, emphasizing the need to consider these characteristics in comprehensive microbiome studies.

Plant-oriented microbiome inoculum modulates age-related maturation of gut-mucosal expression of innate immune and barrier function genes in suckling and weaned piglets.

In the immediate time after weaning, piglets often show symptoms of gut inflammation. The change to a plant-based diet, lack of sow milk, and the resulting novel gut microbiome and metabolite profile in digesta may be causative factors for the observed inflammation. We used the intestinal loop perfusion assay (ILPA) to investigate jejunal and colonic expression of genes for antimicrobial secretion, oxidative stress, barrier function, and inflammatory signaling in suckling and weaned piglets when exposed to "plant-oriented" microbiome (POM) representing postweaning digesta with gut-site specific microbial and metabolite composition. Two serial ILPA were performed in two replicate batches, with 16 piglets preweaning (days 24 to 27) and 16 piglets postweaning (days 38 to 41). Two jejunal and colonic loops were perfused with Krebs-Henseleit buffer (control) or with the respective POM for 2 h. Afterward, RNA was isolated from the loop tissue to determine the relative gene expression. Age-related effects in jejunum included higher expression of genes for antimicrobial secretions and barrier function as well as reduced expression of pattern-recognition receptors post- compared to preweaning (P < 0.05). Age-related effects in the colon comprised downregulation of the expression of pattern-recognition receptors post- compared to preweaning (P < 0.05). Likewise, age reduced the colonic expression of genes encoding for cytokines, antimicrobial secretions, antioxidant enzymes, and tight-junction proteins post- compared to preweaning. Effect of POM in the jejunum comprised an increased the expression of toll-like receptors compared to the control (P < 0.05), demonstrating a specific response to microbial antigens. Similarly, POM administration upregulated the jejunal expression of antioxidant enzymes (P < 0.05). The POM perfusion strongly upregulated the colonic expression of cytokines and altered the expression of barrier function genes, fatty acid receptors and transporters, and antimicrobial secretions (P < 0.05). In conclusion, results indicated that POM signaled via altering the expression of pattern-recognition receptors in the jejunum, which in turn activated the secretory defense and decreased mucosal permeability. In the colon, POM may have acted pro-inflammatory via upregulated cytokine expression. Results are valuable for the formulation of transition feeds for the immediate time after weaning to maintain mucosal immune tolerance towards the novel digesta composition.

After weaning, piglets often show symptoms of gut inflammation and reduced performance. The plant-based diet, lack of sow milk, and the resulting novel gut microbiome and metabolite composition in digesta may be causative. However, the acute response of the gut mucosa when exposed to the novel digesta composition has not been fully elucidated. Here, we used the intestinal loop perfusion assay to characterize the immediate effect of a plant-oriented microbiome inoculum (POM) representing postweaning digesta composition on gene expression related to innate immune pathways and barrier function at the jejunal and colonic mucosa in suckling and weaned piglets. Results showed that the recognition of microbial components and barrier function changed in the jejunal and colonic mucosa from pre- to postweaning, indicating age-related maturation and priming by digesta compounds prior to the intestinal loop perfusion assay. In the jejunum, exposure to POM increased expression of receptors recognizing microbial components. In the colon, POM exposure upregulated the expression of genes for pro-inflammatory cytokines and other components of the first line of defense. Results have implications for the formulation of transition feeds for the immediate time after weaning. Inclusion of bioactive porcine milk components may help maintain mucosal immune tolerance towards the novel digesta composition.

Creep Feeding and Weaning Influence the Postnatal Evolution of the Plasma Metabolome in Neonatal Piglets.

Data on the evolution of blood metabolites and metabolic markers in neonatal piglets are scarce, although this information is vital to detect physiological aberrations from normal development. We aimed to characterize age- and nutrition-related changes in the plasma metabolome and serum biochemistry of suckling and newly weaned piglets and assess metabolite patterns as physiological markers for the two phases. In two replicate batches (n = 10 litters/group), piglets either received sow milk alone or were additionally offered creep feed from day 10 until weaning (day 28). Blood was collected from one piglet/litter on days 7, 14, 21, 28, 31 and 35 of life, totaling five females and five males/group/day. Signature feature ranking identified plasma triglycerides (TG) as discriminative for age and nutrition during the suckling phase. Influential TG 20:4_36:5, TG 17:0_34:2 and TG 18:2_38:6 were higher in creep-fed piglets on days 14, 21 and 28 of life, respectively, compared to only sow milk-fed piglets. Metabolites belonging to pathways within histidine, D-glutamine and D-glutamate metabolism as well as hippuric acid were distinctive for the postweaning compared to the suckling period. In conclusion, plasma lipid profiles especially corresponded to the type of nutrition in the suckling phase and showed a strong weaning effect.

Available phosphorus levels modulate gene expression related to intestinal calcium and phosphorus absorption and bone parameters differently in gilts and barrows.

OBJECTIVE: Dietary phytase increases bioavailability of phytate-bound phosphorus (P) in pig nutrition affecting dietary calcium (Ca) to P ratio, intestinal uptake, and systemic utilization of both minerals, which may contribute to improper bone mineralization. We used phytase to assess long-term effects of two dietary available P (aP) levels using a one-phase feeding system on gene expression related to Ca and P homeostasis along the intestinal tract and in the kidney, short-chain fatty acids in stomach, cecum, and colon, serum, and bone parameters in growing gilts and barrows. METHODS: Growing pigs (37.9±6.2 kg) had either free access to a diet without (Con; 75 gilts and 69 barrows) or with phytase (650 phytase units; n = 72/diet) for 56 days. Samples of blood, duodenal, jejunal, ileal, cecal, and colonic mucosa and digesta, kidney, and metacarpal bones were collected from 24 pigs (6 gilts and 6 barrows per diet). RESULTS: Phytase decreased daily feed intake and average daily gain, whereas aP intake increased with phytase versus Con diet (p<0.05). Gilts had higher colonic expression of TRPV5, CDH1, CLDN4, ZO1, and OCLN and renal expression of TRPV5 and SLC34A3 compared to barrows (p<0.05). Phytase increased duodenal expression of TRPV5, TRPV6, CALB1, PMCA1b, CDH1, CLDN4, ZO1, and OCLN compared to Con diet (p<0.05). Furthermore, phytase increased expression of SCL34A2 in cecum and of FGF23 and CLDN4 in colon compared to Con diet (p<0.05). Alongside, phytase decreased gastric propionate, cecal valerate, and colonic caproate versus Con diet (p<0.05). Phytase reduced cortical wall thickness and index of metacarpal bones (p<0.05). CONCLUSION: Gene expression results suggested an intestinal adaptation to increased dietary aP amount by increasing duodenal trans- and paracellular Ca absorption to balance the systemically available Ca and P levels, whereas no adaption of relevant gene expression in kidney occurred. Greater average daily gain in barrows related to higher feed intake.

Exposure to plant-oriented microbiome altered jejunal and colonic innate immune response and barrier function more strongly in suckling than in weaned piglets.

Weaning often leaves the piglet vulnerable to gut dysfunction. Little is known about the acute response of a gut mucosa primed by a milk-oriented microbiome before weaning to a plant-oriented microbiome (POM) after weaning. We evaluated the epithelial structure, secretory response and permeability in the small and large intestines of piglets receiving a milk-based (i.e., preweaning) or plant-based diet (i.e., postweaning) to POM inocula using intestinal loop perfusion assays (ILPA). The POM were prepared from jejunal and colonic digesta of four 7 week-old weaned (day 28 of life) piglets, having gut-site specific microbial and metabolite composition. Two consecutive ILPA were performed in 16 piglets pre- (days 24 to 27) and 16 piglets postweaning (days 38 to 41) in two replicate batches. Two jejunal and colonic loops per piglet were perfused with Krebs-Henseleit buffer (control) or the respective POM. The outflow fluid was analyzed for antimicrobial secretions. Jejunal and colonic loop tissue were collected after each ILPA for histomorphology and electrophysiology using Ussing chambers. ANOVA was performed using the MIXED procedure in SAS. The POM stimulated the secretory response by increasing mucin in the jejunal and colonic outflow by 99.7% and 54.1%, respectively, and jejunal IgA by 19.2%, whereas colonic lysozyme decreased 25.6% compared to the control (P < 0.05). Fittingly, the POM raised the number of goblet cells by 96.7% in jejunal and 56.9% in colonic loops compared to control loops (P < 0.05). The POM further flattened jejunal villi by 18.3% and reduced crypt depth in jejunal and colonic loops by 53.8% and 9.0% compared to the control (P < 0.05); observations typically made postweaning and indicative for mucosal recognition of 'foreign' compounds. The POM altered the jejunal and colonic net ion flux as indicated by 22.7% and 59.2% greater short-circuit current compared to control loops, respectively; the effect being stronger postweaning (P < 0.05). Colonic barrier function improved with age (P < 0.05), whereas POM perfusion compromised the mucosal barrier as suggested by 17.7% and 54.1% greater GT and mucosal-to-serosal flux of fluorescein-isothiocyanate dextran, respectively, compared to the control (P < 0.05). In conclusion, results demonstrated that the preweaning gut epithelium acutely responds to novel compounds in postweaning digesta by upregulating the first line of defense (i.e., mucin and lysozyme secretion) and impairment of the structural integrity.

Creep feed is offered during the suckling period to prepare the piglet's gut for the dietary transition from a milk- to a plant-based diet at weaning. Nevertheless, the discontinuation of sow milk consumption after weaning can lead to disturbed interactions between the host mucosa and the gut microbiota. Little information is available on the immediate mucosal response towards the altered microbial and metabolite composition in digesta. Therefore, the main objective of this study was to evaluate the immediate effect of the exposure of the jejunal and colonic mucosa to a plant-oriented microbiome (POM), prepared from intestinal digesta of weaned pigs, on the mucosal structure, secretory response, and permeability in piglets before and after weaning using the intestinal loop perfusion assay. The perfusion with POM stimulated the host's secretory response, altered the gut structure and decreased the epithelial integrity before and after weaning. Effects were less strong postweaning, indicating that adaptation processes at the gut epithelium occurred from pre- to postweaning which increased the tolerance towards the POM inoculum.

Short-Chain Fatty Acids Modulate Permeability, Motility and Gene Expression in the Porcine Fetal Jejunum Ex Vivo.

Postnatally, short-chain fatty acids (SCFA) are important energetic and signaling agents, being involved in host nutrition, gut imprinting and immune and barrier function. Whether SCFA exert similar effects during the late fetal phase has been insufficiently elucidated. This study aimed to evaluate whether the fetal jejunum senses SCFA and whether SCFA modify the muscle tension and epithelial permeability and related signaling in jejunal tissue from the porcine fetus in late gestation. Exposure of fetal jejunal tissue to a mix of SCFA (70 µmol/mL) in an organ bath for 20 min lowered the muscle tension. Moreover, SCFA decreased the transepithelial conductance while increasing the short-circuit current in the Ussing chamber, indicating reduced permeability and increased SCFA absorption. Gene expression in the tissues harvested from the Ussing chamber after 30 min indicated downregulation of the expression of receptors (i.e., FFAR2 and TLR2), MCT1 and tight-junction and adherens proteins, which may be a negative feedback response to the applied high SCFA concentration compared with the micromolar concentration detected in fetal gastric fluid. Taken together, our data demonstrate that the fetal jejunum senses SCFA, which trigger electrophysiological, muscle contraction and related gene transcription responses. Hence, SCFA may play a role in prenatal gut nutrition and imprinting.

Short-, medium-, and long-chain fatty acid profiles and signaling is responsive to dietary phytase and lactic acid treatment of cereals along the gastrointestinal tract of growing pigs.

Dietary and microbially derived fatty acids (FA) play important roles in gut mucosal inflammatory signaling, barrier function, and oxidative stress response. Nevertheless, little information is available about gastrointestinal FA profiles and receptor distribution in pigs, especially for long-chain FA (LCFA). Therefore, the present pilot study aimed to (1) investigate the gastrointestinal FA profiles; (2) link the luminal FA profiles to the mucosal expression of genes related to FA sensing and signaling; and (3) assess potential dietary effects on gut and systemic lipid metabolism in pigs. Gut, liver, and serum samples were obtained from barrows (13.1 ± 2.3 kg) fed diets containing either phytase (500 phytase units/kg diet) or cereals treated with 2.5% lactic acid (LA; n = 8/diet) for 18 d. Results showed gut regional and diet-related differences in luminal FA profiles and mucosal receptor expression, whereas diet little affected hepatic expression levels and serum lipids. Short-chain fatty acids (SCFA) increased from stomach, jejunum, and ileum to the cecum (P < 0.05), whereas LCFA were higher in stomach, cecum, and colon than in jejunum and ileum (P < 0.05). LA-treated cereals enhanced cecal acetate and butyrate, whereas phytase and LA treated cereals decreased the LCFA by 35.9% and 14.4%, respectively (P < 0.05). Gut regional differences suggested stronger signaling via FFAR1 expression in the ileum, and via FFAR2, FFAR4, and HCAR1 expression in cecum and colon (P < 0.05). Expression of AMPK, FASN, PPARG, SREBP1, and SREBP2 was higher in the cecum and colon compared with the small intestine (P < 0.05), with stronger sensing via FASN and SREBP2. Phytase decreased expression of FFAR2 and FFAR4, whereas it increased that of FFAR3 and MCT1 in the cecum (P < 0.05). LA-treated cereals raised cecal expression of FFAR3 and HCAR1 (P < 0.05). Pearson's correlations (|r| > 0.35; P < 0.05) supported that FA receptor- and nuclear transcription factor-dependent pathways were involved in the mucosal regulation of gut incretin expression but differed across gut regions. In conclusion, results support regional differences in SCFA, lactate and LCFA sensing and absorption capacities in the small and large intestines of pigs. Effects of phytase and the LA-treated cereals on intestinal FA levels and signaling can be explained by differences in nutrient flows (e.g., phosphorus and carbohydrate fractions). This overview provides a solid basis for future intestinal FA sensing in pigs.

Dietary Phytase- and Lactic Acid-Treated Cereals Caused Greater Taxonomic Adaptations than Functional Adaptations in the Cecal Metagenome of Growing Pigs.

Phosphorus (P) is an essential nutrient for the gut bacteria and the host. Nevertheless, little information exists that indicates to what extent an improved level of P availability in the small intestine leads to functional adaptations in bacterial metabolic pathways in the large intestine. Therefore, we investigated the changes in the taxonomic and functional bacterial metagenome in cecal digesta of growing pigs fed diets containing phytase and/or cereals treated with 2.5% lactic acid (LA) for 19 days (n = 8/diet) using shotgun metagenome sequencing. The phytase supplementation resulted in strikingly distinct bacterial communities, affecting almost all major bacterial families, whereas functional changes were less dramatic among the feeding groups. While phytase treatment decreased predominant Prevotellaceae levels, it seemed that Clostridiaceae, Ruminococcaceae, and Lachnospiraceae filled the opening metabolic niches (P < 0.05). The LA-treated cereals mediated reduced levels of Bacteroidaceae and increased levels of Veillonellaceae, but those results were mainly seen when the cereals were fed as a single treatment (P < 0.05). In association with the taxonomic alterations, phytase caused changes within the major functional pathways corresponding to amino acid metabolism; translation; membrane transport; folding, sorting, and degradation; and energy metabolism, whereas the LA treatment of cereals resulted in decreased enzymatic capacities within the carbohydrate metabolism and energy metabolism pathways (P < 0.05). Metabolic dependencies corresponding to the starch and sucrose metabolism, glycolysis/gluconeogenesis, and citrate cycle pathways were indicated by diet-associated changes in enzymatic capacities related to short-chain fatty acid, methane, vitamin, and bacterial antigen synthesis. Accordingly, the present results support the idea of the importance of the availability of intestinal P for bacterial metabolism. However, the functional profiles were less different than the taxonomic profiles among the dietary treatment results, indicating a certain degree of metabolic plasticity within the cecal metagenome.IMPORTANCE Dietary strategies (e.g., phytase supplementation and lactic acid [LA] treatment of cereals) used to improve the availability of phytate-phosphorus (P) from pig feed reduce the amount of P flowing into the large intestine, whereas LA treatment-induced changes in nutrient fractions alter the substrate being available to the microbiota. In ruminants, lower intestinal P availability compromises the fibrolytic activity of the microbiome. Here, we report that the functional capacities were less dramatically affected than the taxonomic composition by phytase-supplemented and LA-treated cereals. The bacterial community appeared to be partly capable of functionally compensating for the altered flow of P by replacing taxa with higher P needs by those with lower P needs. Therefore, by acting as mucosal immune stimulants, alterations in microbiota-associated molecular patterns (MAMPs) due to the taxonomic shifts may play a greater role for host physiology and health than functional differences caused by differing intestinal P availabilities, which merits further research.

Shifts in the Fecal Microbial Community of Cystoisospora suis Infected Piglets in Response to Toltrazuril.

The protozoan parasite Cystoisospora suis causes diarrhea and reduced weight gain in suckling piglets. Infections occur in the first days of life; it is transient but can lead to dysbiosis, exacerbating disease and increasing mortality. Cystoisosporosis is effectively controlled by toltrazuril treatment; however, alterations of the gut microbial composition upon infection and treatment have not been investigated. This study evaluated the development of fecal microbiota of C. suis infected piglets in response to treatment with toltrazuril. Thirty-eight conventional piglets were infected with C. suis on the first day of life (dol 1). Twenty-six of them received either parenteral or oral toltrazuril 2 days later. Fecal samples were collected pre- and post-weaning (dol 1-15 and 31-38) for microbiota analysis using 16S rRNA amplicon sequencing and during dol 5-18 to determine fecal consistency and parasite excretion. All control animals shed parasites at least once and the majority developed diarrhea, while toltrazuril-treated piglets did not excrete parasites and only had low levels of diarrhea. Age-related shifts in the fecal microbiota composition and increase in diversity and species richness were seen until after weaning. Parasite infection disrupted bacterial maturation 2 weeks after infection. Irrespective of the route of administration, fecal communities of piglets in the treated groups clustered separately and were more diverse compared to that of control piglets during the acute phase of infection on dol 11. Control piglet feces showed higher levels of Fusobacteriaceae and Veillonellaceae, while Ruminococcaceae, Lachnospiraceae, S24-7, Clostridiaceae, and Erysipelotrichaceae were more abundant in feces of treated piglets on dol 11. Thereafter, treatment-related effects on the microbial communities were small and mainly detectable on dol 34 (5 days post-weaning), potentially indicating that the oral toltrazuril treatment might have had long-term effects on host physiological responses post-weaning. Irrespective of the administration route, toltrazuril prevented C. suis-related dysbiosis and maintained species richness and diversity on dol 11. In addition to cystoisosporosis prevention, toltrazuril seems to contribute to the stabilization of the gut microbial development during the suckling phase and thus may reduce the need for antibiotics to control infections with secondary bacterial enteropathogens in C. suis-infected suckling piglets.