Variations in colostrum metabolite profiles in association with sow parity.

Information about the full spectrum of metabolites present in porcine colostrum and factors that influence metabolite abundances is still incomplete. Parity number appears to modulate the concentration of single metabolites in colostrum. This study aimed to 1) characterize the metabolome composition and 2) assess the effect of parity on metabolite profiles in porcine colostrum. Sows (n = 20) were divided into three parity groups: A) sows in parity 1 and 2 (n = 8), B) sows in parity 3 and 4 (n = 6), and C) sows in parity 5 and 6 (n = 6). Colostrum was collected within 12 h after parturition. A total of 125 metabolites were identified using targeted reversed-phase high-performance liquid chromatography-tandem mass spectrometry and anion-exchange chromatography-high resolution mass spectrometry. Gas chromatography additionally identified 19 fatty acids (FAs). Across parities, colostrum was rich in creatine and creatinine, 1,3-dioleyl-2-palmitatoylglycerol, 1,3-dipalmitoyl-2-oleoylglycerol, and sialyllactose. Alterations in colostrum concentrations were found for eight metabolites among parity groups (P < 0.05) but the effects were not linear. For instance, colostrum from parity group C comprised 75.4% more valine but 15.7%, 34.1%, and 47.9% less citric, pyruvic, and pyroglutamic acid, respectively, compared to group A (P < 0.05). By contrast, colostrum from parity group B contained 39.5% more spermidine than from group A (P < 0.05). Of the FAs, C18:1, C16:0, and C18:2 n6 were the main FAs across parities. Parity affected four FAs (C18:3n3, C14:1, C17:0ai, and C17:1), including 43.1% less α-linolenic acid (C18:3n3) in colostrum from parity group C compared to groups A and B (P < 0.05). Signature feature ranking identified 1-stearoyl-2-hydroxy-sn-glycero-3-phosphatidylcholine and the secondary bile acid hyodeoxycholic acid as the most discriminative metabolites, showing a higher variable importance in the projection score in colostrum from parity group A than from groups B and C. Overall, results provided a comprehensive overview about the metabolome composition of sow colostrum. The consequences of the changes in colostrum metabolites with increasing parity for the nutrient supply of the piglets should be investigated in the future. The knowledge gained in this study could be used to optimize feeding strategies for sows.

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.

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.

Postnatal development of gut microbial activity and their importance for jejunal motility in piglets.

Despite their anti-inflammatory properties, role in barrier function, absorption and microbial balance in the gut, knowledge on maturational and dietary effects on intestinal short-chain fatty acids (SCFA) in neonatal piglets is scarce. Moreover, little information exists whether SCFA and lactic acid (LA) modulates gut motility at this age. The present study aimed (1) to investigate the maturational changes in the SCFA profile with and without creep feeding of piglets in the first 3 wk of life; and (2) to examine the effects of SCFA and LA on muscle contractibility in jejunal tissue from neonatal piglets ex vivo. SCFA concentrations were measured in fecal samples of 52 piglets from 10 litters collected on days 2, 6, 13, and 20 of life using gas chromatography. Half of the litters were fed a commercial creep feed from day 10 of life. The organ bath system was used to test the effect of SCFA (acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, and caproate) as well as of LA and the combination of LA and SCFA on muscle contractibility in piglet's jejunum. Average daily gain of piglets was similar between groups before and after introduction of creep feed. SCFA were detectable in feces in relevant concentrations from day 2 of life and increased on day 6 in males by 3.0-fold and on day 13 in females by 1.6-fold but decreased again on day 20 in both sexes compared with day 2 (P < 0.05). Creep feeding reduced fecal SCFA by 0.6-fold on day 13 without largely modifying molar proportions, whereas it increased fecal SCFA by 0.8-fold on day 20 of life compared with the sow-reared only piglets (P < 0.05). Applying SCFA ex vivo increased the muscle contraction of the jejunum by 30% (P < 0.05). Likewise, addition of LA and the combination of LA and SCFA increased the jejunal muscle contractibility by 34.9% and 32.2%, respectively, compared with the muscle tension preaddition (P < 0.05). In conclusion, the present results for fecal SCFA in first days of life suggest high bacterial activity on milk components and emphasize the importance of SCFA for intestinal development and function. After a lag phase, creep feeding promotes fermentation in the distal colon, which may be beneficial for the gut homeostasis. Results further demonstrate the stimulating effect of SCFA and LA for jejunal motility, suggesting a role for mixing of digesta (segmentation) and digestion and absorption of nutrients as well as passage in the jejunum of neonatal piglets.

Physicochemical stressors and mixed alkaloid supplementation modulate ruminal microbiota and fermentation in vitro.

The drop of ruminal pH and heat are common physicochemical stressors challenging ruminal microbiota, nutrient digestion and cattle performance. We characterized the ruminal microbiota and digestive activity in response to different pH (6.0 and 6.6) and temperature (39.5 and 42 °C), as well as established the effective dose of alkaloid supplementation (0, 0.088 and 0.175% of feedstock DM) to modulate ruminal fermentation under these conditions. The acidotic condition decreased microbial diversity and abundances of minor bacterial families whereas most of the highly abundant families like Lactobacillaceae, Prevotellaceae, and Bifidobacteriaceae thrived under the stress. Abundances of all three methanogenic archaea taxa detected increased with heat, as did methane production. However, while Methanomassiliicoccaceae benefited from the low pH, Methanomicrobiaceae diminished and methane production decreased. The low dose of alkaloid addition shifted the fermentation to more propionate and less acetate and the high dose decreased methane and ammonia concentration under the low pH. In conclusion, physicochemical stressors shape the microbial community and function. Mixed alkaloid supplementation facilitates the activity of rumen microbial community under acidotic stress.