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Short-term feeding of defatted bovine colostrum mitigates inflammation in the gut via changes in metabolites and microbiota in a chicken animal model

Short-term feeding of defatted bovine colostrum mitigates inflammation in the gut via changes in... Background Nondrug supplement strategies to improve gut health have largely focused on the effects of individual compounds to improve one aspect of gut homeostasis. However, there is no comprehensive assessment of the repro- ducible effects of oral, short-term, low-level colostrum supplementation on gut inflammation status that are specific to the ileum. Herein, a chicken animal model highly responsive to even mild gut inflammatory stimuli was employed to compare the outcomes of feeding a standard diet (CON) to those of CON supplemented with a centrifuge-defatted bovine colostrum (BC) or a nonfat dried milk (NFDM) control on the efficiency of nutrient use, ileal morphology, gut nitro-oxidative inflammation status, metabolites, and the composition of the microbiota. Results A repeated design, iterative multiple regression model was developed to analyze how BC affected ileal digesta-associated anti-inflammatory metabolite abundance coincident with observed changes in the ileal micro - biome, mitigation of epithelial inflammation, and ileal surface morphology. An improved whole body nutrient use efficiency in the BC group (v CON and NFDM) coincided with the observed increased ileum absorptive surface and reduced epithelial cell content of tyrosine-nitrated protein (NT, biomarker of nitro-oxidative inflammatory stress). Metabolome analysis revealed that anti-inflammatory metabolites were significantly greater in abundance in BC-fed animals. BC also had a beneficial BC impact on microbiota, particularly in promoting the presence of the bacterial types associated with eubiosis and the segmented filamentous bacteria, Candidatus Arthromitus. Conclusion The data suggest that an anti-inflammatory environment in the ileum was more evident in BC than in the other feeding groups and associated with an increased content of statistically definable groups of anti-inflam- matory metabolites that appear to functionally link the observed interactions between the host’s improved gut health with an observed increase in whole body nutrient use efficiency, beneficial changes in the microbiome and immunometabolism. Keywords Anti-inflammation, Colostrum, Ileum, Immunometabolism, Metabolome, Gut microbiome, Protein tyrosine nitration *Correspondence: Institute for Genome Sciences, University of Maryland School Alan Cross of Medicine, Baltimore, MD 21201, USA across@som.umaryland.edu Center for Vaccine Development and Global Health, University Animal Biosciences and Biotechnology Laboratory, USA Department of Maryland School of Medicine, Baltimore, MD 21201, USA of Agriculture (USDA), Agricultural Research Service (ARS), Beltsville, MD 20705, USA © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Elsasser et al. Animal Microbiome (2023) 5:6 Page 2 of 22 Mammals “jump-start” the immune system of their off - Background spring at birth by providing the nutrient-rich and physi- The One Health concept [1] recognizes the interde - ologically stabilizing first milk, the colostrum. Colostrum pendence between food animal and human health and not only contains fat, protein, mineral, and vitamin nutri- includes such issues as nutrition, appropriateness of ents, but also immunoglobulins, antimicrobial peptides, antibiotic use, zoonotic pathogen transfer, the emer- hormones, oligosaccharides, and a plethora of small mol- gence of antimicrobial resistance and alternatives to ecules derived from the diet and physiological processes antimicrobial drug therapies. More specifically, it rec - of the mother. Bovine colostrum is rich in Bifidobacte - ognizes the commonality of physiological systems of rium and Lactobacillus phylotypes and this combination and interdependence between food animal health and of compound constituents along with the beneficial bac - human health [2]. For decades, antibiotic drugs (ABDs) teria promotes gut maturation, mucosal integrity and tis- were administered to animals in their food for the pur- sue repair [12] with these beneficial effects attributed to pose of enhancing their growth [3–5]. This practice has these components being transferred to the young in their possible links to the increased incidence of antimicrobial initial suckling. The composition and volume of colos - resistance, a major public health concern. Although the trum rapidly changes within days following parturition, mechanisms by which ABD impact growth have been as milk production evolves over time [13]. To date, the debated, the diminution of the local inflammatory stress vast majority of studies that concern the beneficial effects in the gut was attributed to the now recognized non- of colostrum have examined either aspects of neonatal antimicrobial, anti-inflammatory effects of select ABDs, organ maturation or the ability to thrive in the extrau- particularly in the tetracycline/doxycycline/minocycline terine environment [14]. Still other studies have explored and macrolide families (4–8). Much of the diminution the potential for vaccine-induced antibodies in colos- of local inflammatory stress in the gut was coincident trum (through immunization of cows prior to parturi- with the improved efficiency with which feed was chan - tion) to mitigate complications of parasitic and bacterial neled into tissue deposition in the ABD-fed animals [6, gut infections [15–18]. Few studies have comprehensively 7]. Through legislative actions, the subtherapeutic level examined the effects of colostrum supplementation on use of ABDs for growth promotion in animals has now gut health with the simultaneous assessment of changes been restricted; consequently, other strategies for pro- in a whole-body parameter such as feed use efficiency, moting growth via reduced gut inflammation in food ani - impact on the gut microbiome, the patterns of intestinal mals were deemed necessary [3, 5] and was highlighted digesta-associated anti-inflammatory metabolites, and by the National Research Council [5]. Our current study host gut cell responses. In the present study, we hypoth- addresses one such alternative with a specific focus on esized that the actions of some metabolite constituents mitigating low-level inflammation. of the ileal digesta as modified by bovine colostrum Over the last 10 years the traditional concept of “nutri- intake can combine in an additive manner or synergize tion” has evolved from feeding the body to now include to mitigate inflammation resulting more efficiently in feeding the microbes that inhabit microenvironments improved nutrient use. We used mass spectroscopic and niches throughout the digestive tract [8, 9]. The metabolomics and S16 rRNA microbial analysis on the nuances of nutrition now include not only the composi- same luminal digesta samples along with quantitative tion of the foods and supplements ingested and the way measurement of intestinal morphometrics and a defined the digestive system processes these nutrients, but also epithelial cell nitro-oxidative stress inflammation marker must address the interactions of the gut microbiota with (3′-tyrosine-nitrated proteins, NT) to study the interac- both the ingested nutrients and the host’s gastrointesti- tions between gut-modifying metabolites of the bovine nal cells. The importance of the interaction between the colostrum diet and the mitigation of low-level inflamma - microbes and the host cells, and the nutrient-derived tion in the ileum. A positive association between a BC- metabolites that function as signaling molecules between supplemented diet and reduced gut inflammation would the two” compartments” is now encompassed in the term be consistent with the outcomes on growth and nutrient “immunometabolism” [10, 11]. The model developed in use reported with ABDs but devoid of the issues of drug our laboratory which was aimed at integrating multiple residues and antimicrobial resistance [19]. To explore this physiological systems [11] suggests that the regulation of further, we examined the effect of short-term feeding of a immunometabolism occurs within the interactive rela- diet containing semi-purified, centrifuge-defatted bovine tionships between endocrine, immune, metabolic, and colostrum (BC) to a commercial line of meat chickens microbial inputs to result in the preferential partitioning (broilers). While these animals are characterized as rap- of nutrients to different tissue beds in times of health or idly growing with high nutrient use efficiency under ideal stress. management conditions, they also are highly sensitive Elsasser  et al. Animal Microbiome (2023) 5:6 Page 3 of 22 to even mild stresses that negatively impact nutrient use efficiency as associated with increased gastrointes - tinal perturbation. Though less studied than oxidative stress (cell stress responses that mainly generate post- translational carbonyl modifications to proteins), nitro- oxidative stress (i.e., cell responses that result in aberrant intracellular post-translational protein tyrosine nitration) is a significant additional component of such gut distur - bances [20, 21]), indicative of sustained gut inflammation Fig. 1 BC-supplemented diet improved feed efficiency. Mean body [22–24]). We report that the refined colostrum supple - weight gain ± SEM (grams/day) and feed efficiency (grams feed ment improved feeding efficiency consistent with the consumed per gram body weight gained) in chickens fed the base observed increase in the absorptive surface area of the diet (Control, n = 10) or diets supplemented with either nonfat dry ileum and alterations in the gut microbiota and digesta milk (NFDM, n = 10) or a processed bovine colostrum preparation (BC, n = 20) metabolites that reflect an overall increase in the anti- inflammatory capacity of the luminal environment high - lighted by a significant decrease in gut epithelial protein NT content. or at the least, a sensed perturbation. Presented as an expression of the density of villi, the number of villi per Results linear unit of ileum was significantly increased in animals Short-term BC supplementation of the standard diet sig- fed the BC diet in contrast to CON (P < 0.03) and NFDM nificantly improved feeding efficiency and consistently (P < 0.004, Panel A). When the relative villus area (RVA, modified the metabolic and bacteriological milieu of the mid-villus width x the villus length x number of villi per gut compared to CON and in a manner superior to that unit villus length) was calculated for each animal and the of NFDM. To examine these effects in greater detail, we mean group RVAs compared, the calculated area for BC particularly focused on the ileum. We found diet-asso- was 42 percent (P < 0.05) and 24 percent (P < 0.06) greater ciated changes in epithelial anatomy, anti-inflammatory than the mean areas calculated for CON and NFDM, metabolites and gut microbiota of the ileum. respectively (Panel B). Mean villus lengths were 265, 280 and 250  µm for CON, NFDM and BC, respectively Feeding efficiency was significantly improved in short‑term (P = NS, data not shown). By comparison, corresponding BC diet‑fed chickens mean crypt depths, associated with enterocyte cell pro- The impact of feeding diets supplemented with BC or liferation and digestion capacity of the small intestine, of the nonfat dry milk-supplement (NFDM, a macronutri- 28, 31 and 37  µm were observed for CON, NFDM and ent balanced control to BC) compared to CON on weight BC, respectively, yielding the resulting villus-to-crypt gain and feeding efficiency is summarized in Fig.  1. ratios (Fig.  2, Panel C). Though the mean values calcu - For chickens fed the BC diet, the feed efficiency was lated for villus length and crypt depths alone across the improved approximately 9 percent (P < 0.05) over CON dietary treatments did not attain statistical significance, and NFDM diets. Although the lowest and highest mean the mean villus:crypt ratio for chickens in BC was 27 per- weight gain responses were observed in NFDM and BC cent lower (P < 0.03) and 24 percent lower (P < 0.05) than chickens, respectively, the differences between the means that calculated for CON and NFDM, respectively (Panel of these treatment groups as well as the difference from B). The value for the calculated estimate of villus area per CON were only trends (P > 0.05 < 0.1). unit length was significantly increased in BC compared to the other fed diets (Panel C). Collectively, the increase Intestinal measurements indicate significant biological in estimated villus surface area with BC is consistent with impacts of the BC diet on gut morphology an effect of BC to stimulate a more efficient use of nutri - A visual depiction of the ileum structure attributes as ents and the observed improved feed efficiency in BC. associated with the three diets as well as measured and estimated morphological attributes of the ileum are sum- marized in Fig.  2. Where nuclei are depicted as white BC‑ and to a lesser extent nonfat dry milk objects against the black/grey background, the increased (NFDM)‑supplemented diets are associated with less ileal number of nuclei in the lamina propria of villi of ani- epithelial cell nitro‑oxidative stress mals in the CON and NFDM groups, higher than that We measured the cellular pixel density associated with in observable in BC, is consistent with a larger number immunofluorescence localization of NT, on the exte - of infiltrating immune cells, a hallmark of inflammation rior, epithelial layer of the villi, thereby excluding lamina Elsasser et al. Animal Microbiome (2023) 5:6 Page 4 of 22 Fig. 2 The BC-supplemented diet increases the villus surface abundance. The effects of inclusion of nonfat dry milk (NFDM) or the bovine colostrum fraction (BC) to the standard feed (Control) on the morphology of the ileum. Data represent the means ± SEM of the number of villi counted per standardized length of tissue (Panel A) or the calculated area of the villi as associated with the diet treatments fed to the chickens (Panel B), or the ratio of the villus height to crypt depth (Panel C). Panel D depicts representative images of the diet-associated ileal architecture. (Control, basal diet, n = 6. NFDM. n = 6. BC, n = 10). For reference, “V” = villi, “C” = crypts, and “SM” = submucosa propria and interior layers of the villi that contain infil - measure of the number of cells specified in the demar - trating immune inflammatory cells. Though nitrated cated area of interest, was significantly decreased by 37% proteins were observed in the gut epithelial cells of in BC-fed chickens compared to that present in chicken all chickens, the highest levels were detected in those tissues from either the CON (P < 0.05) or the NFDM chickens fed the CON and NFDM diets (Fig.  3G, H, I). (P < 0.04) diets (Fig. 3J). The overall decrease in NT pixel The epithelial cell NT protein content, characterized density per epithelial cell was attributable to “clusters” of as pixel density as normalized by nuclear counting as a nitration elements that were both fewer in number and Elsasser  et al. Animal Microbiome (2023) 5:6 Page 5 of 22 Fig. 3 Decreased protein tyrosine nitration (NT ) in epithelial cells of villi in samples of ileum from chickens fed a BC-supplemented diet. Immunofluorescence is presented in Panels A–F and Image analysis depicted in Panels G–I. Epithelial cell (anti-cytokeratin 18 epithelial cell marker) presence is indicated by green fluorescence pixels (A, C). NT, as detected using an anti-nitrotyrosine antibody marker, is presented as red (cell presence, B and D–F, for isolated single channel image analysis quantification) or orange (colocalized in green cytokeratin 18-positive cells, C Representative patterns of image analysis-selected nitrotyrosine pixels (G–I, yellow pixels) were summated and processed to yield the statistical analysis summarized in the lower panel bar graph(J). Values are group means ± SEM for Control (n = 6), NFDM (n = 6), and BC (n = 10) smaller in the contained number of pixels. The lower a literature-documented capacity to mitigate tissue level of nitrated proteins in the ileal epithelial cells sug- inflammation. Thus, the goal was to identify those anti- gests that these cells were less subject to nitro-oxidative inflammatory compounds derived from NFDM or BC stress in the presence of BC-derived diet than were the present in amounts over and above those contributed ileal cells of chickens fed the Con and NFDM diets. through consumption of the basal CON diet that had a potential to lower the NT proteins and then test them Short‑term feeding of BC‑ and NFDM‑supplemented in a statistical model for association with any impact on diets alters the anti‑inflammatory metabolite profile ileum inflammation status. Across dietary treatments, of the ileum metabolomic profiling of the ileal digesta revealed 649 We focused on the identified metabolite digesta com - named and 300 unnamed compounds (Additional file  8: pounds derived from the animal treatment diets Data S1). Quantified levels of the identified compounds themselves or as further derived from host and/ or in the metabolite data base in our third-party analyti- microbial enzymatic action on these compounds with cal contract (Metabolon, Inc., Morrisville, NC) were Elsasser et al. Animal Microbiome (2023) 5:6 Page 6 of 22 reported to us as “scaled imputed values (SIV, see “Meth- The effects of the NFDM and BC diets on the changes ods” and Additional file  9: Data S2 for full definition and in these compounds as well as those identified in the derivation). other relevant seven pathway metabolite groups are pre- The digesta of BC was significantly more complex in sented in Table  1. We identified numerous compounds metabolite richness (number of detected compounds) with potential anti-inflammatory activity (as cited in the than that of CON or NFDM. Plant-derived compounds literature) in the ileal digesta. Comparing the levels of the are well-known for having anti-inflammatory and anti- identified metabolites in the ileum digesta between the nitro-oxidative properties and as such were of significant diet treatments, data revealed, perhaps not unexpect- interest here. Of seventy-seven compounds in the ileal edly, that the mean levels of many individual metabolites digesta classified as xenobiotics of plant origin identified varied numerically, sometimes greatly, among the three at or higher than levels stated as the threshold limits of diet treatments and the two replications of the experi- detection (indicated as “DETECTED” in the accompa- ment. Further analysis revealed that fewer individual nying table), 40 compounds were in CON, 46 in NFDM metabolites consistently differed in the two experimental and all 77 in BC (Additional file  5: Table  S1). Interest- replications between CON and BC or CON and NFDM. ingly, though beyond the scope of the present paper, This became apparent with respect to the animal-to-ani - data in Additional file  5: Table  S1 are also presented for mal variation and replication variation for many of the the sake of relative comparison of the composition of metabolites. However, when multiple related metabo- digesta in the ileum compared to that in the duodenum lites for a given common pathway and mechanism of and cecum. These data clearly demonstrate that the avail - anti-inflammatory action were grouped and concentra - ability of metabolites changes with the host-microbiome tion levels considered additively as a singular entity (for interactions that process the digesta as it flows down the example, if several anti-inflammatory compounds like alimentary canal. This finding is consistent with the con - flavonoids/isoflavones or active metabolites like glu - cept that anti-inflammatory compounds available in one tathione and its major precursors), the abundance of a gut segment might not be available in other gut segments select set of compounds/groups were significantly greater to provide their beneficial effects and that bacterial popu - for BC than those measured in CON or NFDM. The liter - lations differ between gut segments. Our considerations ature-supported anti-inflammatory pathway metabolite for inclusion of a metabolite compound in a list of com- categories included tryptophan indole-conjugates, toco- pounds with diet-derived anti-inflammatory character pherols (α-, γ-, δ-tocopherols), flavonoids/isoflavones stemmed from a PubMed (https:// pubmed. ncbi. nlm. (apigenin, daidzein, genistein, and their conjugates), pri- nih. gov/) search for literature citations of studies inves- mary and secondary bile acids (chenodeoxycholic acid, tigating such biochemical modes of action of qualifying 7-ketolithocholic acid), glutathione (N-acetylglycine, compounds with specific reference to compounds in the γ-glutamylcysteine, cysteine,), polyunsaturated fatty acid digesta identified by the metabolomic separation and (docosahexaenoic acid, arachidonic and eicosapentaenoic analysis (Additional file  10: Online References S1). The acids), oligosaccharides (3′- and 6′-sialyllactose), and output from a Boolean search using the terms “polyphe- mitochondrial TCA cycle compounds (itaconate). (see nol, flavonoids, bile acids, amino acids, fatty acids, or oli - Additional file  10: Anti-inflammatory Metabolite Refer - gosaccharides along with small intestine, metabolite, and ences in the online material), inflammation or anti-inflammation” yielded 1968 cita - We also observed some significant effects of diet on tions and formed the basis of our assessments. the potent tripeptide anti-inflammatory compound glu - With focus on the ileum, ten xenobiotic compounds tathione and its precursors in the luminal digesta which with or without anti-inflammatory character were found are critical to the anti-nitro-oxidative capacity of the gut. to be significantly different in abundance between CON The metabolomic report contained data on the reduced and BC and reproducibly across both treatment trials. form of glutathione, GSHr. Overall, the mean level of Apigenin, 2,3-dihydroxyisovalerate, carotenediol, daid- GSHr in BC was half that present in CON (P < 0.04) and zein, daidzein sulfate, genistein, gluconate, naringenin, 70 percent of that present in the digesta of NFDM (NS). and 2-piperidinone were higher in BC than CON and However, the digesta from chickens in BC had a sum- panose (a purported prebiotic) lower, respectively. Simi- mated glutathione precursor pool of compounds sig- larly, digesta from NFDM chickens contained six more nificantly increased over that measured in CON. This is compounds than were present in the digesta from CON consistent with the need for an abundance of precursors with 2,8-quinolinediol sulfate, 2-oxindiole-3-acetate, (cystine, glycine, glutamate, and associated dipeptides) 1-kestose, 1,1-kestotetraose higher and homostachydrine being available to the synthesis pathway in the gut epi- lower. thelial cells to replenish glutathione as it is consumed in countering nitro-oxidative processes. When considered Elsasser  et al. Animal Microbiome (2023) 5:6 Page 7 of 22 Table 1 The effects of inclusion of nonfat dry milk (NFDM) or the bovine colostrum fraction (BC) on ileal digesta components with literature-supported anti-inflammatory effects on tissues varied according to the involved biochemical pathway components Data represent the means ± SEM of the Scaled Imputed Values of metabolites that were increased by either BC or NFDM identified by the metabolomic analysis to be consistently changes across the two experimental replicates and the source(s) of the colostrum. Control, basal diet, n = 11. NFDM. N = 11. BC, n = 20. General Linear Model (GLM) contrast statement coding and weighting indicated in line 2 under GLM Specific Treatment Contrasts. The statistical comparisons of Scaled Imputed Values between control (#1), NFDM (#2) and BC (#3) treatments are shown in the far-right columns with the shades of red colors indicating increases or shades of green colors indicating decreases in significance (or trends). Comparisons highlighted in grey are not significant. as a precursor group, the summated mean of glycine, For some pathways, for example tryptophan or toco- cystine, N-acetylglycine, and γ-glutamylcysteine digesta pherols, only a single pathway compound with pur- abundances averaged 158 percent greater levels in BC ported anti-inflammatory potential was significantly than in CON (3.46 ± 0.46 v 1.34 ± 0.22, P < 0.001). Both different between one or among all diets. Only indole - the difference in summated abundance (P < 0.001) as lactic acid in the tryptophan pathway or γ-tocopherol well as the ratio (P < 0.002) of GSH -to-GSHr may in the tocopherol pathway, respectively, were affected precursors reflected the potential for BC to increase the anti-nitro- by inclusion of nonfat dry milk or the colostrum matrix oxidative capacity of the gut over that present in CON into the basal diet. Surprisingly, the effect amounted by contributing to the metabolites the gut needs to make to a reduction in relative mean ileal SIV. The overall glutathione. impact of this reduction in ileal gamma-tocopherol Statistical evaluation of the pathway components in content was sufficient to account for the majority of the Table  1 showed that polyunsaturated fatty acid (PUFA) pathway change in total tocopherols. compounds, and especially metabolites of eicosapen- The inclusion of BC to the basal diet consistently taenoic and arachidonic acids, were not significantly increased the ileal digesta content of four anti-inflam - affected by diet (all grey). This lack of change with BC matory flavonoid compounds: apigenin, daidzein, feeding may relate to the defatting process used to genistein, and naringin. In contrast, none of the com- generate the BC matrix and the NFDM supplemented pounds was affected by adding NFDM to the basal feeds. However, seven other identified pathways were diet. The effect of adding BC to the diet resulted in a significantly affected by diet either as single compounds doubling of the amount of flavonoid present in the gut or, as summed components of an individual pathway. digesta based on summated SIVs. Data from the larger Elsasser et al. Animal Microbiome (2023) 5:6 Page 8 of 22 metabolite profiling indicated that the levels of the fla -adjusted R (terms shaded in green) they were dropped vonoids in their conjugated forms (glucoside, glucuro- from the model (terms shaded in pink). Consequently, we nide) did not contribute to the gut levels of the parent excluded gamma-tocopherol and the two sialyloligosac- compound’s anti-inflammatory status suggesting that charides from the cumulative Ai-i, based on the fact that the parent compounds alone were to be considered the the adjusted R resolved with these metabolites included major components of interest. in the model was lower than that resolved before either The summated values of the primary and secondary metabolite was added to the model. Therefore, in the final bile acids with anti-inflammatory effects, chenodeoxy - assessment, the combination of chenodeoxycholic acid, cholic acid and 7-ketolithocholic acid content, respec- total flavonoids, glutathione precursors, 7-ketolithocho - tively, were significantly elevated in digesta from BC and late, itaconate and indolelactate accounted for approxi- NFDM compared to CON diets. However, digesta from mately 76 percent of the observed decline of cellular NT BC-fed chickens tended to have higher levels of chenode- protein content. Second, BC feeding yielded mean Ai-i oxycholic acid than that from NFDM (Table 1). values (derived from data in Panel A) significantly dif - Only two oligosaccharides were identified by the mass ferent from and higher than the average Ai-i’s from both spectroscopic analysis, 3′-sialyllactose and 6′-sialyllac- NFDM and Con groups (Panel B). Third, the variability tose. Levels of total oligosaccharides in BC digesta were in the magnitude of the derived NT protein pixel quan- 5.7-fold higher (P < 0.002) than those measured in CON tification data among treatment groups could be better digesta and 2.8-fold (P < 0.003) higher than those in analyzed with respect to a linear model after perform- digesta from NFDM. ing a logarithmic transformation of the pixel data. When The cellular energetics generated by subcellular mito - this was done, the regression analysis showed a signifi - chondrial processes is a fundamental requisite of cell cant (P < 0.005) negative linear correlation (R = 0.694) stability, immune system function, and the efficiency of wherein the higher the Ai-i, the lower the NT protein in nutrient use. Of the eleven mitochondrial compounds villus epithelial cells (Panel C). associated with energy production identified in the digesta, only one, itaconate, derived from the decar- BC and NFDM added to a standard diet altered boxylation of cis-aconitate in the mitochondrial matrix, specific populations of bacteria in the ileum rapidly was an anti-inflammatory metabolite and found to be and specifically. increased in digesta in NFDM and BC relative to CON Using 16S rRNA gene amplicon sequencing, we per- (P < 0.03). formed a community survey of the gut microbiota of 41 chickens under 3 different dietary conditions (stand - Short‑term colostrum extract supplementation (BC) ard or CON feeding, and CON feeding supplemented is associated with a significantly higher Anti‑inflammatory by NFDM or BC). We collected intraluminal digesta index (A‑i‑i) than found in CON or NFDM diets. from the proximal and distal intestine including jeju- The relative contribution of each of these metabo - num, duodenum, and cecum for a total of 156 samples lites to our model was assessed according to whether as well as scrapings from ileal epithelial cells washed free the inclusion of a particular metabolite or metabolite of digesta. We obtained a total of 25,838,078 high-qual- group improved the adjusted R in the multiple regres- ity 16S rRNA V4 amplicon sequences corresponding to sion model, and not all did. To facilitate our analysis of 51,165 (± 2,267, μ ± s.e.m.) sequences per sample (Addi- the impact of dietary-derived metabolites on chicken tional file  6: Table  S2). Overall, we found distinct differ - gut inflammation, we devised an anti-inflammatory ences in the digesta and mucosal-associated microbiota index and found associations arising from the gut pro- among animals fed the three different diets. Across diets, cessing of the ingested feed components of the spe- five distinct types or groupings of intestinal microbiota, cific diets that correlated with protein nitration (Fig.  4). Type I–Type V, were observed and summarized in the First, the data used to construct Panel A of Fig.  4 were heat map and bar graph (Fig.  5, Panels A and B); Type I derived from the SIV values in the metabolomic data- is enriched in Lactobacillus spp., Type II is enriched in base. As indicated by the terms “Yes” and “No” in the Lachnospiraceae spp., Type III is enriched in both Lach- table line “Model Inclusion”, as each successive variable nospiraceae spp. and Bacteroides fragilis, Type IV is was added to the regression model, that variable was only characteristic of Candidatus Arthromitus, and Type V is retained in the model if it added a positive incremen- enriched in Bifidobacterium, Coriobacteriaceae spp. and tal increase to the adjusted R . While some metabolites Lactobacillus spp. Type I and IV microbiota had the low- present in the digesta of chickens may have had a pur- est community diversity (Additional file  1: Figure S1) and ported anti-inflammatory character, if they did not have were highly enriched with Lactobacillus and Candidatus added a benefit in the regression model to increase the Arthromitus, respectively (Fig.  5 Panel B). In particular, Elsasser  et al. Animal Microbiome (2023) 5:6 Page 9 of 22 Fig. 4 Correlation between anti-inflammatory index (A-i-i) and epithelial cell protein nitration (NT ). Multiple regression was used to assess the relationship between the level of NT content (Log10, pixels/cell) present in intestinal epithelial cells and the independent metabolite variables. Inclusion of a metabolite (class) to the model was valid if the resulting adjusted R was increased over that of the prior iterative assessments (Panel A). The effects of dietary treatment on the A-i-i (the sum of the Scaled Imputed Values of the individual metabolites retained in the model, Panel B). Linear regression of the NT pixel content on the A-i-i suggested a significant negative correlation where the greater the A-i-i- the lower the nitration stress (Panel C) the different physiological sections of the intestine dem - consistent with the nature of the cell attachment mecha- onstrated distinct microbiota (Additional file  2: Figure nism this organism employs to persevere in its growing S2), and the Type IV microbiota was enriched in the microenvironmental niche largely specific to the ileum. ileal microbiota. The other types of microbiota were sig - Consolidating across all data comprising Fig. 5 and Addi- nificantly more diverse and had relatively more abun - tional file  2: Figure S2 and Additional file  4: Figure S4, dant Lachnospiraceae spp., Bacteroides fragilis and a the patterns, distribution, and abundance of microbes wide array of strict and facultative anaerobic bacterial is consistent with BC having greater microbial diversity species (Additional file  7: Table  S3). Statistical mod- than that in the digesta from animals in the other two eling using Bayesian Poisson model (details in Methods diets. Increases in this microorganism in the colon might and (Additional file  3: Figure S3) showed the intestinal reflect remaining pass-through organisms originating in microbiota in the digesta of BC-fed chickens was signifi - the ileum and perhaps simply shed into the digesta going cantly enriched in Type I and IV microbiota. We further into the large intestine in association with the naturally- performed LDA effect size (LEfSe) analyses to quantita - occurring programmed epithelial cell losses from the tips tively characterize the phylotypes that could explain the of the villi associated with apoptosis. In addition, Candi- differences observed under different conditions (Fig.  5C, datus Arthromitus was minimally detected in the cecum (Additional file  1: Figure S4). Candidatus Arthromi- or duodenum digesta or scrapings (Fig. 5C, left side). tus was shown to be the most differentially abundant In order to determine whether the presence of Can- phylotype in ileal mucosal scrapping samples of BC-fed didatus Arthromitus might have arisen in the BC animals but not in ileal digesta (Fig.  6A). This finding is chickens as a result of feeding the actual processed Elsasser et al. Animal Microbiome (2023) 5:6 Page 10 of 22 Fig. 5 Chicken intestinal microbiota analyses. A Heatmap of the 50 most abundant phylotypes of 156 samples collected. Five types of intestinal microbiota were revealed based on clustering patterns. Ward linkage clustering is used to cluster samples based on their Jensen-Shannon distance calculated in vegan package in R [116]. Identified microbiota types are labeled as I–V. B Distribution of different community type by cluster as shown in A in diet treatment groups of Control, colostrum and NFDM. Type I is significantly lowered in control than in other two groups while type IV is significantly higher in BC and NFDM. Type II is borderline significantly higher in BC and NFDM than control, based on statistical modeling using Bayesian Poisson model. C Phylotype biomarkers for digesta and scrapings generated using program LEfSe [105]. Vertical bars represent the relative abundance of Bacteriodes in each sample. Dotted line represents mean, solid line represents median relative abundance. The alpha value for the non-parametric factorial Kruskal–Wallis (KW ) sum-rank test was 0.05 and the threshold for the logarithmic LDA model (43) score for discriminative features was set at 2.0 colostrum fraction that contained a live Candidatus Arthromitus genomes or using marker gene-based Arthromitus, we characterized the composition of approach (Additional file  7: Table  S3). This result indi - the microbiota associated with various samples of the cated that the Candidatus Arthromitus was not likely bovine colostrum used in the diets, and showed that derived from the bovine colostrum diet. these samples contained very low abundance of bacte- Other than Candidatus Arthromitus, Streptococcus ria in general and revealed no Lactobacillus nor Candi- was another phylotype that was shown to be enriched datus Arthromitus. While fresh colostrum is known to in BC-fed animals, irrespective of the sub-anatomi- contain many microorganisms beneficial to the nursing cal location (digesta vs. mucosa-associated) (Fig.  5C). infant, the lack of such microbial abundance in our BC This result again emphasizes the importance of sub- diet preparations is consistent with the effects of the anatomical location, anatomic site, and dietary condi- 1–h centrifugation of the preparations at 20,000×g to tions for specific groups of bacteria in stark contrast sediment bacteria into the bottom of the tube. Further to data more commonly generated from fecal collec- analyses confirmed no detected Candidatus Arthro - tion sampling. Other phylotypes that demonstrated mitus via reads mapping to published Candidatus varied enrichment under different feeding conditions Elsasser  et al. Animal Microbiome (2023) 5:6 Page 11 of 22 Fig. 7 Sialyllactose content of ileal digesta is correlated with presence of SFBs. Ileal digesta content of the combined mean (± SEM) levels of the 3′-sialyllactose and 6’-sialyllactose represented as the metabolomic Scaled Imputed Values as measured in samples Fig. 6 Candidatus Arthromitus is present in the ileal scraping from chickens fed the three diets (Panel A). Summated 3′-sialyllactose and increased by BC. Mean (± SEM) abundance of Candidatus and 6′-sialyllactose values were regressed on the Log10 [OTU of the Arthromitus represented as Log10 [OTU] in the three different SFB] yielding a significant positive linear correlation. (Panel B) segments of the gut and further differentiated by digesta vs. scraping (Panel A). Mean (± SEM) abundance of Candidatus Arthromitus represented as Log10 [OTU] in the ileum as measured in digesta or cell scrapings (attached) as affected by diet. *P < 0.04 (Panel B) the combined oligosaccharides versus the lo g of the abundance of total Candidatus Arthromitus indicate a significant positive correlation wherein higher levels of the oligosaccharides in the digesta were associated with were also included (Fig.  5C). For example, Bifidobac - increased numbers of the Candidatus Arthromitus with terium were more enriched with the NFDM diet, and its potential impact on the immune capacity of the gut. Bacteroides fragilis, Bacteroides ovatus, and E. coli, was particularly enriched in cecal samples. Together, we observed that Candidatus Arthromitus was only Discussion in the mucosa-associated microbiota of the ileum in Significant increases in growth rate of domestic food ani - these chickens and its presence was amplified signifi - mals have been linked to some of the nonantibiotic, anti- cantly when BC was incorporated into the basal diet inflammatory effects of ABDs [5, 6], with more recent (Fig. 6A, B). The higher relative abundance of Candida - trends towards increased popularity of the use of sup- tus Arthromitus is consistent with a localized prolifera- plements with purported anti-inflammatory effects [25]. tion of the organism, most likely due to factors in the Therefore, we addressed whether the anti-inflammatory environment of the organism rather than being sourced gut benefits of ABDs could be mimicked by the feeding from the fed colostrum itself. of defatted colostrum and further, whether a model could While the oligosaccharide content of the various diets be developed to better define what aspects of colos - did not affect the anti-inflammatory index, the 3′- and trum consumption fostered these beneficial impacts. 6′-sialyl-oligosaccharides did lead to a dramatic site- Our results were based on the simultaneous assessment specific increase in Candidatus Arthromitus. The oli - of how a diet enriched in a partially refined BC prepa - gosaccharide content of the ileal digesta was increased ration differed from other diets in their impact on the approximately 4- and 2.5-fold in BC compared to that host gut morphology, the gut metabolite milieu, and the measured in digesta from CON and NFDM, respectively gut microbiota with particular emphasis on the ileum. (Fig.  7). Further regression analysis between the SIVs for The terminal ileum segment of the gut was focused on Elsasser et al. Animal Microbiome (2023) 5:6 Page 12 of 22 because of its role as the transition segment between enzymes such as JAK-2 [34, 35] or mitochondrial pro- the small and large intestine, the unique presence of the teins thereby causing dysfunction and ATP shortage in immune-modulating segmented filamentous bacteria, cells [28, 36, 37]. Additionally, epithelial cells can have Candidatus Arthromitus, and further in light of the rela- levels of NT proteins with cell function deficits that tive paucity of research compared to that of the cecum vary by diet in the absence of detectable infectious dis- and colon on integrated host, metabolome, and microbi- ease [26, 27]. Oxidative/nitration protein and lipid dam- ome interactions. age has been generated in chickens simply by low levels Research from our laboratory has demonstrated that of partially oxidized corn oil or polyunsaturated fatty nitro-oxidative stress occurs in the intestine as observed acids being in the diet [38]; similarly, that oxidized oil- with the increased tissue content of nitrated proteins in induced inflammation was relieved with the addition of conjunction with parasitic infection and correlated with the flavonoid antioxidant quercetin to the chicken diet the severity of infection [26, 27]. In comparison to exten- [39]. Mitigation of gut dysfunction by reducing inflam - sive research done on oxidative stress in the gut, there is mation is a goal of both human and veterinary medicine. less research on nitration stresses in the gut, even though In the present study the short-term feeding of the refined nitration stress and oxidative stress share a pool of cell BC matrix was significantly associated with the observed response-induced common oxygen- and nitrogen-based reduction in ileal epithelia-specific NT protein content. reactants (nitric oxide and superoxide anion [20, 24]). Gut metabolism in health can account for up to 20 per- With peroxynitrite (ONOO ) substantively being the cent of maintenance energy expenditure [40–42]. Gut principal effector relating to this form of inflammation, inflammation increases energy demands by as much the term nitro-oxidative stress therefore encompasses as 10 percent of the body’s metabolic energy need [43, the result of nitric oxide (the “nitro” component) and 44] with a significant portion of the caloric substrates superoxide anion (the “oxidative” component) react- diverted from growth and muscle accretion to support ing to generate the ONOO insult [22, 23, 28, 29]. The immune function particularly in young animals [22, 24]. present study demonstrates that the fractionated prepa- Microbial populations in the gut, and more specifically ration of BC developed in this study, when added to a changes in such populations, significantly impact host standard feed and consumed by animals for 8  days, is immune and metabolic gene expression [45] and there- associated with a significant reduction of the amount of fore the energetic needs of the host’s physiological sys- nitro-oxidative inflammation in the epithelial cells of the tems. Signals from the gut microbiome can prioritize the ileum, denoted as a reduction in the content of intracel- flux of nutrients between the nervous, endocrine, and lular NT proteins. In concert with this effect, feeding BC immune systems and shape their interactions [22, 24, was strongly associated with an increase in the absorp- 46, 47]. Since oxidative and nitration stress impacts the tive surface area of the ileum. The rapidity with which energy utilization of affected cells, even marginal changes this occurred is consistent with an increased capacity to in gut health result in energetic deficiencies [48, 49] that generate epithelial cells [30] as reflected in the calculated rapidly translate into growth and nutrient use efficiency crypt-to-villus ratio. These observations on the increased deficits but improved significantly in BC. These observa - ileal surface area are also consistent with the observed tions are consistent with the regulatory model proposed improvement in nutrient absorption as reflected in feed for the controlling inputs to immunometabolism [50] and utilization efficiency. The presence of naturally-occur - further impacts on nutrient use in the young growing ring, low-level gut stressors such as changes in environ- animal. ment and social structure, present in our select, high The enhanced genetic lines of commercial production growth rate animal model, has been identified previously chickens with a high growth rate used in our chicken as having the capability of significantly altering the archi - model required no direct disease or chemical challenge tecture of the villus absorptive area as well as the mor- to trigger gut nitrooxidative stress. Further, low levels of phogenesis of the crypts [31–33]. nitrated proteins were shown to be generated by entero- Elevated levels of intracellular tyrosine-nitrated cytes in response to non-infectious perturbations such (3’-nitrotyrosine-) NT proteins are a cause of protein as changes in feed composition, allergens, endogenous dysfunction in non-immune cells, as documented in bacterial endo- and exotoxins, mold toxins, and even many tissue inflammation pathologies [23, 24, 29]. Epi- metabolic ketosis [51–53]. In the past, this stress sensi- thelial cells generate highly reactive nitrogen interme- tivity was managed in part through the extensive use of diates (i.e., peroxynitrite, ONOO ) from nitric oxide antibiotics added to the diet [3, 5]. However, herein we and superoxide anion [23, 24, 28], which can react with report that consumption of components of BC can ame- critical tyrosine(s) in phosphorylation sites of many criti- liorate inflammation in this gastrointestinal situation and cal intracellular proteins including signal transduction improve nutrient use. Elsasser  et al. Animal Microbiome (2023) 5:6 Page 13 of 22 Dietary factors are potent modulators of the micro- the tryptophan pathway metabolite, indolelactate, also biota composition and its interaction with the host [54]. identified in our metabolomic screening, concurrent with Reciprocally, microbes metabolizing ingested nutrients the enhanced presence of bifidobacteria, and their ben - and generating nutrient-derived metabolites play a criti- eficial effects on the balance between inflammatory and cal role in regulating the host immune response and gut anti-inflammatory states [63]. The capacity for microbe- cell function, as documented in the newly emerging area derived indolelactate to function as an anti-inflammatory of immunometabolism [55]. Active microbial modifica - metabolite [64, 65] is consistent with our findings of the tion of feed-derived nutrients was evident (Additional reduction in nitrated proteins, the increased nutrient use file  8: Data S1) as observed in the presence of microbe- efficiency, and favorable microbial populations in BC- derived compounds like p-cresol sulfate and hippurate as compared to CON- or NFDM-fed chickens. These previ - well as deconjugation of flavonoid compounds differen - ously published data lend support to the validity of our tially affecting their bioavailability [56–59] and levels of regression analysis that identified indolelactate among the various secondary bile acids consistent with litera- the many metabolites in the digesta. Collectively, the pre- ture reports [57]. From the metabolomic analysis of the sent data demonstrate the potential for diet composition ileal digesta of test animals fed the various dietary treat- to significantly impact gut health through changes in the ments, we were able to identify and statistically model a microbiota, the gut metabolite milieu and host responses. defined set of nutrient molecules and microbially-derived Specific metabolites generated by the localized bacte - metabolites that correlated with the reduction in level rial populations acting on ingested dietary components of nitrooxidative stress present in the ileal epithelial facilitate processes needed for healthy eubiotic regula- cells. The positive effects of the colostrum feeding were tion of the gut epithelia. In studies of the murine colonic reflected in the increased portion of bacteria with anti- microbiota, Tiffany and Baumler [66] proposed that dur - inflammatory/ immunomodulatory properties in the ing gut homeostasis, bacteria of the phyla Firmicutes and microbiota profiles, and the generation of anti-inflamma - Bacteroidetes, obligate anaerobic bacteria, fermented tory metabolites bathing the cells of the lumen. dietary fiber and maintained a hypoxic environment We determined that classes of related microbially- that could reduce colonization with Enterobacteriaceae derived metabolites should be considered for their (phylum Proteobacteria). In the absence of hypoxia, Pro- anti-inflammatory effect in addition to the more com - teobacteria gained a foothold. This deviation from an monly literature-cited approach of identifying single obligate anaerobic microbiota was defined as gut “dysbio - compounds. For example, the polyphenol flavonoids sis”. Dysbiosis in the ileal microbiome stems from lower identified in the mass spectroscopic analysis revealed levels of microbial diversity with imbalances between that as a class they were significantly increased in BC both beneficial and pathogenic organisms as well as dis - and NFDM feeding in comparison to CON. If, however, proportionate imbalances in commensal bacteria that only one flavonoid, apigenin, of the identified group was can change the local microenvironments downstream assessed, the larger group effect would have been missed. thus facilitating a bacterial overgrowth syndrome or Similarly, while the effect of adding NFDM or BC to the pathogen emergence [67, 68]. Thus, in the present study, basal diet on the individual components identified in the the increased diversity and favorable ratios in microbial anti-inflammatory glutathione pathway may have been populations in the BC animals would be consistent with a insignificant (i.e., P > 0.1), collectively the sum of the pre- greater opportunity for eubiosis and sound gut health to cursor pool and the glutathione components improved be maintained. the regression model (Table  1). This is consistent with The microbiome of the small intestine, the primary the need for an abundance of precursors (cystine, glycine, site for the absorption of nutrients, differs substantially glutamate, and associated dipeptides) being available to from that of the colon both in terms of the number of the synthesis pathway in the gut epithelial cells to replen- colony-forming units per ml as well as microbial compo- ish glutathione [60, 61] as it is consumed and to maintain sition [69]. We focused on the ileum in this study, largely cellular oxidative/nitrosative homeostasis. because of the large information gaps on the microbiome Metabolites are an important link in the interac- and its host interactions compared to the large intes- tions between the host, its gut microbiota and the tine [70]. We observed histological evidence of an ongo- lumen environment. For example, metabolites such as ing inflammatory process in animals fed the CON and metabolically-derived itaconate, generated during the NFDM diets. In contrast, we observed a reduction in epi- immune response and identified in our screening can thelial cell nitrooxidative stress in BC-fed animals which regulate both the magnitude and duration of the immune may be associated with the greater population diver- response [62]. In addition, Henrick et  al. established the sity that we observed in the ileal microbiome of these cause-and-effect relationships between gut increases in animals. The decreased gut inflammation-associated Elsasser et al. Animal Microbiome (2023) 5:6 Page 14 of 22 generation of NT proteins in BC-supplemented chick- NFDM-supplemented feed compared to CON. The lev - ens is consistent with other known anti-inflammatory els of these oligosaccharides were significantly higher in effects of colostrum such as the observed reduction in BC than marginally-increased levels in NFDM. However, NFk-B-mediated proinflammatory cytokine expression they did not statistically contribute to the Ai-i per se, the in intestinal epithelial cells [71]. Those nitration reac - positive correlation largely driven by the levels found in tions can play out in the cascade of responses initiated by BC. Oligosaccharides in BC may facilitate microbiota such mediators as TNF-α cascading through increases in remodeling in the chicken by reducing the ability of intracellular NO and superoxide anion culminating in the pathogens to gain a niche in the gut microenvironment generation of nitrating reactants like ON OO and the and through promoting growth of beneficial microbiota more reactive ONOOCO , as produced when higher as has been seen in humans and mice The increased oli - pCO is present in sections of the gut with more anaero- gosaccharides in BC associate strongly with the selec- bic status with perturbed mesenteric blood flow. The ileal tive increase in ileum mucosa-associated segmented microbiome also differed from that of the duodenal and filamentous bacteria (SFB), namely Candidatus Arthro - colonic segments analyzed in that the segmented fila - mitus, in chickens in the BC-supplemented group. SFB mentous bacteria Candidatus Arthromitus was localized were sparsely detected in the ileal mucosa of both CON to the cell layer scraped from the ileal bowel (rather than or NFDM chickens. Furthermore, Candidatus Arthromi- the digesta per se). These data suggest that anatomical tus was not detected in the raw colostrum after incuba- localization of microbiota composition in the gastroin- tion on media for 48 h suggesting that some aspect of the testinal tract may be important in the regional status of BC diet promoted the increase in SFB already present in immune activation in different gut segments as well as the ileum. These bacteria, living predominantly attached the inflammation-anti-inflammation status thus suggest - to the ileal epithelial mucosa, are known to promote gut ing the need for spatially distinct analyses along the gas- maturation and gut mucosal and adaptive immune devel- trointestinal tract. opment including the development of germinal centers We hypothesized that BC-initiated changes in the in Peyer’s patches that generate potent IgA and Th17 gut microbiota may enhance the metabolic process- responses [74, 75]. By itself, SFB can induce intestinal T ing of nutrients into anti-inflammatory metabolites, a cell development similar to that induced by the complete feature we characterized with the development of the gastrointestinal microbiota [76]. SFB colonization also anti-inflammatory index, Ai-i. These observations were has been shown to improve barrier protection against reproducible over time (i.e., replicated experiments) and enteric viral infections and associated diarrheal dis- across different sources of colostrum (cow breed and eases [77] as well as serve as a coordination link between collection time after parturition). The ability of BC to metabolism and immune function [78]. Danzeisen et  al. beneficially modify the gut environment was due to the [79] demonstrated that in turkeys a fundamental deter- nutrient-derived metabolites and microbial populations minate of growth success was the gut microbial milieu, in the ileum not matched by the NFDM diet. The group particularly the presence of significantly more SFB. of metabolites identified after statistical regression align - Tryptophan, an essential amino acid, is metabolized by ment supported the hypothesis of an increased abun- gut bacteria to generate metabolites specific to indole, dance of metabolites with anti-inflammatory character kynurenine and serotonin pathways, the latter a key neu- and aligned with observed effects on gut morphology and rotransmitter in both the enteric and central nervous an improved efficiency of nutrient use for body weight systems [65]. The tryptophan metabolite, indolelactic gain in BC. acid, is elevated in NFDM, but even more so in BC- Several classes of compounds were altered by the BC supplemented diets. Indolelactate acts on gut epithelial supplementation. Compared to CON feed, BC feed cells, Paneth cells and enteroendocrine cells through increased the microbially-derived secondary bile acids, specific receptors to (1) stimulate the release of antimi - chenodeoxycholic acid, 7-ketolithocholic acid, in the crobial peptides, (2) upregulate tight junction proteins ileum. Studies in humans and mice have shown bile acids to maintain barrier function; (3) suppress inflamma - to have an anti-inflammatory effect on immune cells tory cytokine production, (4) decrease superoxide anion [72] and appear to function in their host-microbe cross- generation, and (5) decrease apoptosis. Tryptophan talk signaling capacity through the farnesoid-X receptor metabolites generated by the microbiota can bind to the (FXR) and the G-protein-coupled bile acid receptor-1 endogenous tryptophan receptor, the aryl hydrocarbon (TGR5) [73]. receptor (AHR) [64]. Indolelactate is generated only by Oligosaccharides 3’-sialyllactose (3SL) and 6’-sialyl- bacteria, particularly Lactobacillus, abundant in BC, and lactose (6SL) with prebiotic properties were markedly bifidobacteria with demonstrated benefits to gut health increased in the ileal contents of chickens fed BC- and in colostrum-fed infants [63, 80]. Elsasser  et al. Animal Microbiome (2023) 5:6 Page 15 of 22 While the methods used by Metabolon Inc. do not initiation of the BC supplement at an earlier time point allow us to measure short chain fatty acid (SCFAs), they may have an even greater effect, or to define the changes undoubtedly were present in the BC that are high in in the immune response provoked by the presence of the indigestible carbohydrates (“prebiotics”)” that selectively SFBs. enhance the growth of Bifidobacterium and Lactoba - cillus that have been shown to modulate inflammation Materials and methods and modulate a vast range of physiologic processes that Animal model include suppression of inflammatory signals and carcino - The rapidly growing commercial meat chicken (Gallus genesis [81]. The microbially-derived SCFAs share the gallus, Strain: Ross 708, Amick Farms Hatchery, Hurlock, use of receptors and signaling pathways that are used by MD) was used for the nutrition, inflammation, metabo - the host gut epithelium to detect molecules that signal lome, and microbiome objectives of the study. Genetic between the gut microbiota and the host. selection for characteristics of allometric growth result Our findings suggest that BC feeding enhances the gen - in an enhanced accretion of ingested nutrients in lean eration of several classes of anti-inflammatory metabo - muscle deposition largely associated with breast and lites. Collectively, the findings support the need to better leg tissue. The rapidity of growth (< 49  days from hatch understand not only what colostrum components other to commercial processing; [> 100  g body weight (BW) than traditionally assessed factors like immunoglobulins, gain/day]) and high rate of nutrient utilization for lean antimicrobial factors, growth factors and cytokines con- tissue accretion (< 1.7  kg feed to attain 1  kg increase in tribute to an anti-inflammatory environment in changing BW) make these animals exquisitely sensitive to pertur- the composition of the digesta in the gut lumen. Perhaps bations in environmental conditions, including those of more critical though, information gaps exist needing diet [82, 83] and require stringent controls on environ- resolution regarding what anti-inflammatory factors and ment and feed [84]. For these chickens, it is recognized metabolite precursors reach the different sections of the that the growth process dynamics and feed efficiency as gut and are processed through host-diet-microbe interac- well as changes in the gut microbiome are highly volatile tions in maintaining gut health and homeostasis. and change over time in as little as 1  week, essentially as the animals mature [85, 86]. With this awareness, we assessed what was consistently affected by the presented Conclusion dietary treatments and what metabolite and gut micro- The addition of BC to standard feed exerts profound biota attributes of the treatment diets might mitigate low effects on the immunometabolism of chickens, a model level gut inflammation as specifically addressed in terms for gut health exquisitely sensitive to metabolic perturba- of nitrooxidative stress, i.e., the abundance of tyrosine tions that reflects the impact of gut stress on feeding effi - nitrated proteins in the gut [26, 27]. ciency and growth [50]. Compared to chickens fed CON or a NFDM-supplemented diet, BC-fed chickens had an Experimental treatments: diets supplementation increased ileal absorptive surface and developed changes with colostrum extract or nonfat dry milk in the composition and structure of the gut microbi- A total of four colostrum samples (4 L each) were col- ota that generated feed- and host-derived metabolites lected from multiparous Jersey (n = 2) or Holstein (n = 2) and compounds, resulting in an anti-inflammatory gut dairy cows as first and second milkings after parturition, microenvironment. This could be attributed to significant respectively. In accordance with traditional feeding for increases in several BC-associated digesta metabolites cows at parturition and early lactation, cows were fed a with a defined anti-inflammatory character. Importantly, total mixed ration consisting of corn and grass silages only the BC-fed chickens had major increases in mucosa- supplemented with roasted soy bean meal, vitamins and associated SFBs known to promote gut maturation and minerals. As such, the colostrum from the cows would be immune mucosal development. Our study also high- naturally enriched with a spectrum of flavones and iso - lights the importance of sampling the mucosal surface in flavones, etc. derived from the plant matrix of the diet addition to the digesta to comprehensively assessing gut [87, 88]. This concept aligns well with the data in Table  1 microbiota. In addition, the results underscore the util- and Additional file  5: Table S1. Each collected colostrum ity in knowing how the total anti-inflammatory capac - was frozen at − 80  °C until needed for diet preparation. ity established in the interaction between microbes and To prepare the colostrum-containing diet, the following metabolites of a given gut region can be changed as a procedures were performed. The thawed, cold colostrum result of feeding a complex food matrix with the active from one cow at a time was thoroughly mixed, aliquoted components identified through appropriate statisti - into eight 50  ml polycarbonate tubes and centrifuged cal analysis. Future studies are required to determine if at 2  °C for 1  h at 20,000×g, centrifugation conditions Elsasser et al. Animal Microbiome (2023) 5:6 Page 16 of 22 sufficient to congeal lipids and sediment bacteria at the to individual pens where the daily feed intake for each top and bottom of the tube, respectively. In agreement animal could be accurately measured. The abrupt change with Morrill et  al. [89], the fat/lipid content of the dif- in local environment and housing as well as separation ferent colostrums was not different as a function of cow from a group is recognized as a short-term, low-level breed or day obtained. After centrifugation, the solidi- stress situation impacting the hypothalamic–pituitary– fied lipid (nonaqueous long chain fatty acid component) adrenal axis with impact on gastrointestinal function in was removed from the top of the samples and the liquid the chicken [90, 91]. The quantity of fresh diet given to underneath constituting the middle third of the tube each chicken daily was recorded. The next morning the volume poured off and collected. Higher density sedi - residual feed not eaten from the previous day was meas- mented material in the bottom third of the centrifuge ured and the difference between given and residual used tubes containing cells, bacteria and cell and membrane as the measure of feed intake. Live body weight of each debris was discarded. The fluid from each of the four chicken was obtained daily at a standardized time of day cow’s colostrum was pooled across tubes but maintained relative to feed management. Chickens and their envi- as separate pools for the generation of the respective BC- ronment were observed three times daily as regards the supplemented diets. After the pooling, the material was health and welfare of the animals per approved animal passed through glass fiber matt filters. Collected material care protocol. was sprayed onto a base diet previously used for chicken The overall experiment was conducted in 2 sepa - experiments by our laboratory [corn-soybean meal base; rate replicates of the following treatments: control diet 23 crude protein and 13  MJ metabolizable energy [27]] (CON), nonfat dry milk diet (NFDM), and colostrum to supply the equivalent of 100  ml colostrum/kg feed, diets (BC), the replications conducted 6 months apart. with continuous mixing. The damp diet was put into 4 L Colostrum was obtained from four different sources; glass high-vacuum containers, frozen at − 80  °C, lyophi- sources consisted of Jersey cow first postpartum milk - lized to dryness, and fed as such. To assess the effect of ing, Jersey cow second postpartum milking, Holstein cow the addition of this quantity of colostrum to the overall first postpartum milking, and Holstein cow second post - crude protein and energy content of the base diet, a test partum milking. Variability in colostrum composition batch of each of the BC diets was formulated and sent (day1 and day 2 of lactation postpartum and Jersey and out for nutritional analysis (Cumberland Valley Analyti- Holstein breeds) was intentionally built into the design cal Services, Mercersburg, PA). Following analysis, it was so that across the variability a set of metabolites with observed that BC diets were approximately 2.7% higher consistent effects on inflammation and gut parameters in crude protein but otherwise (total fat, gross energy, could be ascertained. In preparing the diets for the first fiber, etc.) not different from the base diet. To correct replicate of the study, one BC diet was prepared using for the protein difference, the base diet to be used to Jersey-derived first milking colostrum and the other BC prepare the BC was modified by lowering the soybean- diet prepared from the Holstein-derived first milking. derived protein content by 2.7% and that protein dif- Similarly, in the second replication the two BC diets were ference then made up for with the inclusion of the BC. made from the Jersey and Holstein colostrum second When the colostrum component was added to this lower milkings. protein base diet, reanalysis of the diets showed them to Transfer of the chickens to the individual pens and the be equivalent in protein. A third diet was developed, ana- respective switches to the test diets were considered the lyzed, and formulated to more closely approximate the first day of the start of the experiment. Test diets were amino acid composition of the BC diets and for this pur- fed for 7 days with the animals euthanized (per American pose nonfat dry milk (NFDM, Carnation, Inc.) was added Veterinary Medical Association Guidelines for Euthana- to the lower crude protein base. sia of Animals: 2013 Edition, https:// www. avma. org/ KB/ Polic ies/ Docum ents/ eutha nasia. pdf ) on day 8 following Study design the recording of the final weight. As pooled across the All research was conducted under an animal manage- two experimental replicates, there were 11 CON diet ani- ment and welfare protocol approved by the USDA Belts- mals, 11 NFDM diet animals and a total of 22 animals fed ville Institutional Animal Care and Use Committee. One colostrum-containing diets, the data from which animals day old chicks were transported from the hatchery to was used for the metabolomic and microbiome analy- the USDA Poultry Research Facility (Beltsville, MD) and sis. Variation in the n across the various diet treatment placed in group brooder housing with the environmen- groups reflects further refinement based on the quality tal temperature set according to the age of the animal. of data primarily obtained from the metabolomic evalu- The CON feed and water were provided ad  libitum. At ations. When an animal’s metabolite data were identified 15 days of age, chickens were moved from the group pens as an outlier (> 3 S.D.) or compromised as a result of an Elsasser  et al. Animal Microbiome (2023) 5:6 Page 17 of 22 inefficient extraction, that animal was eliminated from 4  °C in a humidified chamber with anti-3′-nitrotyrosine the study. The numbers of animals per treatment group (rabbit polyclonal, Millepore-Sigma, Burlington, MA, from which the data were amassed exceeded the mini- Inc., 1:100) with subsequent antigen visualization [goat mum number for achieving statistical significance based anti-rabbit IgG Alexa 680 (1:400; 1  h, Thermofisher Sci - on statistical power analysis (Introduction to Power and entific, Grand Island, NY)]. For validation purposes, Sample Size Analysis., https:// suppo rt. sas. com/ docum an additional set of slides of the adjacent serial sections entat ion/ onlin edoc/ stat/ 131/ intro pss. pdf). Samples pre- were prepared and immunostained to identify villi api- pared for immunofluorescence were obtained from rep - cal epithelial cells using anti-cytokeratin-18 (Abcam resentative numbers of animals in each replication of the Inc., Cambridge, MA, mouse monoclonal, Clone-C04, experiment. Following euthanasia, the intestinal tract 1:200) with antigen visualization (goat anti-mouse IgG was rapidly excised and divided into segment samples Alexa 488, 1:400, Thermofisher Scientific, Grand Island, for duodenum (central loop), distal ileum, and cecum NY; green pseudocolored immunofluorescence). Nuclei (with the present work focusing on the ileum). These were stained with 4–6-diamino-2-phenylindol-dihydro- samples were further subdivided into 2 components. chloride (DAPI, Thermofisher Scientific). Morphomet - Each segment was opened longitudinally. From one the ric measurements to determine the length relationships digesta was carefully removed to eliminate intestinal tis- between the villi and crypts were performed using the sue potentially scraped from the wall. The other section DAPI blue channel using a calibrated and validated digi- was rinsed free of remaining digesta and debris with ice tal micrometer internal to the ImagePro 9.3 software cold PBS and the epithelial surface gently scraped away (Media Cybernetics, Rockville, MD). Quantitative image using a fresh, chilled glass slide. One additional sec- analysis to ascertain the pixel density of the NT antigen tion of rinsed intact gut was immersed overnight in 4% per villus epithelial cell was performed using ImagePro paraformaldehyde and transferred to ethanol for further 9.3 software (Media Cybernetics, Rockville, MD) as paraffin embedding and sectioning for microscopy as described in the Supplementary Information—Detailed subsequently described. To evaluate the live growth of Methods. the chickens we measured the average daily body weight gain and feed efficiency (the grams of ingested feed cor - Metabolome profiling and analyses responding to the grams of weight gained per day). Samples of digesta were collected from each of the ani- mals in both replicates of the study (CON, n = 10; NFDM, Fluorescence microscopy and quantitative imaging n = 10; BC, n = 20, with 4 samples failing to be extracted analyses efficiently and the results excluded) from a region of the The detailed methods for the immunofluorescent tis - ileum 3–5 cm cephalad to the ileocecal junction. For the sue antigen immunostaining as well as the exact pro- metabolomic profiling of the digesta, a homogenous sam - cess used to quantify the cellular pixel content of the ple (approximately 100 mg) was accurately weighed to the 3’nitrotyrosine antigen used to represent the intensity 0.1 mg into a polypropylene vial, with the vial capped and of nitrooxidative stress can be found in the online Sup- frozen in liquid nitrogen. Metabolite identification and plementary Information—Detailed Methods. The pro - quantification was performed under contract by Metab - tocols for tissue fixation, fluorescence immunostaining olon, Inc., Research Triangle Park, NC, and all steps for for 3′-tyrosine-nitrated proteins (NT) as the inflamma - this analysis were conducted according to the company’s tion marker and morphometric analysis of ileal samples protocols (Supplementary Information—Detailed Meth- published by our laboratory previously were applied in ods). In brief, the basic process was as follows: Metabo- the present study [27, 34]. In brief, representative tissue lon’s first preparation step was to lyophilize the weighed sections for immunofluorescence were collected from samples and then reconstitute the samples for analysis by the first and second replicates of the experiment (CON, ultrahigh performance liquid chromatography-tandem n = 6, 3/replicate; NFDM, n = 6, 3/replicate; BC, N = 10, mass spectroscopy (UPLC-MS/MS). In the lyophilization 5/replicate, randomly selected within treatment). Dual step, the low molecular weight volatile short chain fatty color immunofluorescence labeling of the target antigen acids (SCFA), acetate, propionate, and butyrate, were dif- and nuclei was performed to localize and subsequently ferentially sublimated off the samples and not available quantify epithelial cell inflammation with NT proteins for analysis. Raw data was extracted, peaks identified, and (red pseudocolored immunofluorescence) as the target QC processed using Metabolon’s hardware and software. antigen along with the respective cell nuclei blue pseu- Data were made available as E xcel files containing val - docolored immunofluorescence). Following standard ues for each identified compound representing an area- deparaffinizing, rehydration, and blocking of nonspe - under the curve which were normalized, per Metabolon, cific antibody binding, slides were incubated overnight at Inc., in terms of raw area counts and presented as the Elsasser et al. Animal Microbiome (2023) 5:6 Page 18 of 22 “Original Scale” (Additional file  9: Data S2). For a single attached to the epithelial cells. After a bead-beating step day run, this was equivalent to the raw data. For compar- on a TissueLyzer II (Qiagen Inc., Germantown, MD), ing diet treatments, each metabolite’s value in Original DNA was extracted from all samples (150 mg tissue wet Scale was mathematically processed to set the median weight) using the MagAttract PowerMicrobiome DNA/ for that metabolite equal to 1 and each animal’s value RNA kit (Qiagen Inc.) implemented on a Hamilton STAR for that metabolite proportionately scaled accordingly. robotic platform (Hamilton, Reno, NV). Amplification These scaled values were referred to “Scaled Imputed of the 16S rRNA gene V4 hypervariable region was per- Value” (SIV). The preliminary statistical ANOVA analy - formed using dual-barcoded universal primers 515F and sis of the data was performed by Metabolon, Inc. with 806R as previously described [92]. High-throughput comparisons and separated effects established using con - sequencing of the amplicons was performed on an Illu- trast statements such that an overall understanding of the mina MiSeq instrument using the 300  bp paired-end sources of variation as affected by diet, colostrum source, protocol. Raw data was demultiplexed as cited [93, 94]. and replication could be assessed. Barcode, adapter, and primer sequences were trimmed using TagCleaner (2013-10-14 vers; [95]). Quality assess- “Anti‑inflammatory index” of the metabolome ment and sequencing error correction was performed A major objective of the study was to define a set of mass using the software package DADA2 (version 1.14; [96]) spectrometry-identified metabolites in the ileal digesta and the following parameters: forward reads were trun- that could facilitate a reduction in epithelial cell inflam - cated at position 220 and the reverse reads at position mation and reflect the metabolite profiles associated with 160 based on the sequencing quality plot, no ambigu- the fed BC or NFDM diets. To accomplish this, we devel- ous bases and a maximum of 2 expected errors per- oped the term “Anti-inflammatory-index” (Ai-i) to serve read were allowed [97]. The quality-trimmed reads were as an integrative parameter intended to reflect the selec - used to infer ribosomal sequence variants and their tion and grouping of a set of specific metabolites that relative abundance in each sample after removing chi- contributed significantly to relieving the magnitude of mera. Sequencing analyses includes denoising, de novo nitration stress present in epithelial cells of the ileum. A and reference-based chimera detection conducted with detailed description of the A-i-i and rationale for devel- UCHIME v5.1 [98]. Taxonomic ranks were assigned to opment is further presented in accompanying on-line each sequence using the Ribosomal Database Project Supplementary Information—Detailed Methods. Briefly, [[99] Naïve Bayesian Classifier v.2.2 [100] trained on the the A-i-i was derived from summing the Scaled Imputed Greengene database (Aug 2013 version) [101]], using 0.8 Values for the metabolites that (a) were defined in the lit - confidence values as cutoff. The heatmap and bar plot erature (Additional file  10: References 1) as having anti- were generated using statistical package R (v3.2.1) and inflammatory character and not simply antioxidative Phyloseq packages [102]. Clustering of taxonomic com- function, (b) anti-inflammatory metabolites that were position and abundance in a sample were performed significantly greater in abundance in one diet compared using Ward linkage hierarchical clustering based on (ANOVA; Proc GLM, SAS ver. 9.4: https:// docum entat Jensen-Shannon divergence metrics. Jensen-Shannon ion. sa s . c om/ do c/ en/ p g msa s c dc/9. 4_3. 3/ st a t ug/ st a t ug_ divergence is a measurement of dissimilarity between glm_ synta x01. htm) to the other diets, and (c) repeatably probability distribution, and Jensen-Shannon metric is different between diets across the two experiment rep - the square root of the normalized Jensen-Shannon diver- lications and the four kinds of colostrum or the NFDM gence value [103, 104]. The resulting clusters were used used in the diet replications. To be included in the final to define microbiota type, which indicates the cluster - multiple regression model set, a given metabolite when ing of similar community compositional profile that is a added to the regression model needed to promote an vector of the percentage of the sequences assigned to a improvement (increase) in the adjusted R of the model phylotype in each sample. Linear Discriminant Analy- as tested for either a linear or exponential fit. sis (LDA) effect size (LEfSe) algorithm was adapted to quantitatively characterize the phylotypes that could Microbial community analyses explain the differences observed between two biological The assessment of the bacterial milieu of the ileum was conditions [105]. It identifies phylotypes in their relative made on individual samples collected from the ileum at abundance profiles through building an LDA model to a point immediately cephalad to the section obtained estimate the “effect size” of each phylotype with respect for metabolite profiling. In addition to digesta samples, to biological conditions under inspections. The alpha one additional sample of the digesta-free epithelial layer value for the non-parametric factorial Kruskal–Wallis of each specimen was carefully scraped from the tissue (KW) sum-rank test [106] is 0.05, the threshold on the segments to address what would be considered microbes logarithmic LDA model [107] score for discriminative Elsasser  et al. Animal Microbiome (2023) 5:6 Page 19 of 22 features is 2.0. We used all-against-all BLAST search in as well as diet combinations on the dependent variables. multi-class analysis that is stricter than the one-against- “Animal-within-treatment” was used as the error term. all search, and we denote both target classes and subject For microbial population inferences, in order to esti- names in the analysis. mate differences in relative abundances of different In order to ascertain if any contribution to the micro- bacterial clusters within different diet status, a Bayes - biota in chickens fed BC might have originated in the ian Poisson model was employed and the structure is as colostrum itself, especially that of the SFB, samples of follows: the BC matrix as applied to the feed was analyzed. The y ∼ Poisson( ) i i metagenomic approach was chosen to assess the micro- biota of the colostrum. Given the low biomass in colos- log ( ) = a + b + c + d trum, applying the PCR-based 16S rRNA on such a i diet(i) cluster(i) diet(i),cluster(i) specimen type may have introduced additional biases. where y is the count in the i-th cell and diet(i), cluster(i) To access the microbiota of the colostrum, the non-pro- are the diet and cluster of the i-th cell, respectively. cessed colostrum was filtered through the sterile 0.22um The model was fitted using JAGS R package [114], and syringe filter (Sterivex, Germany), and the materials left 100,000 iterations with the same number of burn-in on the filter was subjected to 45  s beating cycle using a iterations was used. The convergence of the model was FastPrep instrument (MP Biomedicals) at 5.5  m/s and assessed using Gelman and Rubin’s potential scale reduc- centrifuged at 14,000  rpm for 1  h at 4  °C. The superna - tion factor [115] and visual inspection of each coeffi - tant after beads beating was used for DNA extraction cient’s Markov chains. using the same procedure as above 16S rRNA samples. Metagenomic sequencing libraries were constructed Supplementary Information from the same DNA using the Nextera XT Flex kit (Illu- The online version contains supplementary material available at https:// doi. mina, San Diego, CA) according to the manufacturer’s org/ 10. 1186/ s42523- 023- 00225-z. recommendations. Libraries were then pooled together in equimolar proportions and sequenced on Illumina Additional file 1. Figure S1: Within-sample diversity by community type. Notched boxplot of diversity index of samples grouped in community HiSeq 4000 platform using the 150  bp protocol at the type (color indicated in legend). Within-sample diversity was estimated Genomic Resource Center at the University of Mary- using Shannon diversity index using Phyloseq R package [102]. The top land School of Medicine. Metagenomic sequence reads and bottom of the box are the lower and upper quartiles, and the band near the middle of the box represents the median. Box width is propor- were removed using BMTagger v3.101 [108] using a tional to the square root of the size of the category. Genome Reference Consortium Gallus gallus Build 5.0 Additional file 2. Figure S2: Statistical analyses of relative frequency of (GCA_000002315.3, [109]). Sequence read pairs were community types. The mean relative frequencies and their 95% credible removed even if only one of the reads matched to the intervals are shown. A Bayesian Poisson model was employed, and model fitting was performed using JAGS R package [114] and 100,000 iterations genome reference. Taxonomic profiling was conducted in with the same number of burn in iterations. Abbr: DUO: duodenum; CEC: MetaPhlAn vers 2 [110]. We specifically performed reads cecum; ILE: ileum. mapping of the metagenomic reads to published genomes Additional file 3. Figure S3: Community diversity analyses using of Candidatus Arthromitus isolated from chicken, turkey, weighted UniFrac principal coordinates analysis (PCoA) analyses using mouse, and rat (NZ_CP008713.1, NC_015913.1, NZ_ QIIME2 (v2019.10) [117]. Each symbol represents a sample colored by its different anatomical sites. The scatterplot is of principal coordinate 1 (PC1) AGAG01000005.1, NZ_AGVP01000010.1, NC_017294.1, plotting against principal coordinate 2 (PC2), and the percentage of the NC_016012.1, LXFF01000001.1) using bowtie [v1, variation described by the plotted principal coordinates in indicated on parameters: “-l 25-fullref -chunkmbs 512 -best -strata -m the axes. 20”, [111]]. Additional file 4. Figure S4: Relative abundance of phylotype biomark - ers. Analyses were performed using program LEfSe [105]. Bars represent the relative abundance of a phylotype in each sample. Dotted line repre- sents mean, solid line represents median relative abundance. The alpha Statistical analyses value for the non-parametric factorial Kruskal-Wallis (KW ) sum-rank test was 0.05 and the threshold for the logarithmic LDA model ([107]) score for The basic statistical analysis for the effects of diet treat - discriminative features was set at 2.0. Abbr: DUO: duodenum; CEC: cecum; ment on growth, feed efficiency, morphometric measure - ILE: ileum. ments, NT pixel content, and the assessment of specific Additional file 5. Table S1: Summary of the identified plant-derived metabolites on an anti-inflammatory index was analysis xenobiotic compounds in digests of the duodenum, ileum and cecum. Data illustrate the nature of the changes in presence/detention and rela- of variance performed using the General Linear Mod- ® tive levels of xenobiotic plant-derived compounds in the relevant digesta els procedure of the Statistical Analysis System ver. 9.4 as affected by diet. Digesta from chickens fed BC was more complex with [112]. Specific contrast statements [113] were used to more compounds present in the ileal digesta. test hypotheses regarding the effects of the various diets Additional file 6. Table S2: Taxonomic profiles of the intraluminal stool specimens collected in this study. Intraluminal stool specimens collected Elsasser et al. Animal Microbiome (2023) 5:6 Page 20 of 22 5. NRC, Council NR, Panel on Animal Health FS, Public H, Committee from A) different anatomical sites of ileum, duodenum and cecum for a on Drug Use in Food A, Institute of M, et al. The use of drugs in food total of 156 samples; B) only ileum. 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Short-term feeding of defatted bovine colostrum mitigates inflammation in the gut via changes in metabolites and microbiota in a chicken animal model

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Springer Journals
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Copyright © The Author(s) 2023
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10.1186/s42523-023-00225-z
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Abstract

Background Nondrug supplement strategies to improve gut health have largely focused on the effects of individual compounds to improve one aspect of gut homeostasis. However, there is no comprehensive assessment of the repro- ducible effects of oral, short-term, low-level colostrum supplementation on gut inflammation status that are specific to the ileum. Herein, a chicken animal model highly responsive to even mild gut inflammatory stimuli was employed to compare the outcomes of feeding a standard diet (CON) to those of CON supplemented with a centrifuge-defatted bovine colostrum (BC) or a nonfat dried milk (NFDM) control on the efficiency of nutrient use, ileal morphology, gut nitro-oxidative inflammation status, metabolites, and the composition of the microbiota. Results A repeated design, iterative multiple regression model was developed to analyze how BC affected ileal digesta-associated anti-inflammatory metabolite abundance coincident with observed changes in the ileal micro - biome, mitigation of epithelial inflammation, and ileal surface morphology. An improved whole body nutrient use efficiency in the BC group (v CON and NFDM) coincided with the observed increased ileum absorptive surface and reduced epithelial cell content of tyrosine-nitrated protein (NT, biomarker of nitro-oxidative inflammatory stress). Metabolome analysis revealed that anti-inflammatory metabolites were significantly greater in abundance in BC-fed animals. BC also had a beneficial BC impact on microbiota, particularly in promoting the presence of the bacterial types associated with eubiosis and the segmented filamentous bacteria, Candidatus Arthromitus. Conclusion The data suggest that an anti-inflammatory environment in the ileum was more evident in BC than in the other feeding groups and associated with an increased content of statistically definable groups of anti-inflam- matory metabolites that appear to functionally link the observed interactions between the host’s improved gut health with an observed increase in whole body nutrient use efficiency, beneficial changes in the microbiome and immunometabolism. Keywords Anti-inflammation, Colostrum, Ileum, Immunometabolism, Metabolome, Gut microbiome, Protein tyrosine nitration *Correspondence: Institute for Genome Sciences, University of Maryland School Alan Cross of Medicine, Baltimore, MD 21201, USA across@som.umaryland.edu Center for Vaccine Development and Global Health, University Animal Biosciences and Biotechnology Laboratory, USA Department of Maryland School of Medicine, Baltimore, MD 21201, USA of Agriculture (USDA), Agricultural Research Service (ARS), Beltsville, MD 20705, USA © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Elsasser et al. Animal Microbiome (2023) 5:6 Page 2 of 22 Mammals “jump-start” the immune system of their off - Background spring at birth by providing the nutrient-rich and physi- The One Health concept [1] recognizes the interde - ologically stabilizing first milk, the colostrum. Colostrum pendence between food animal and human health and not only contains fat, protein, mineral, and vitamin nutri- includes such issues as nutrition, appropriateness of ents, but also immunoglobulins, antimicrobial peptides, antibiotic use, zoonotic pathogen transfer, the emer- hormones, oligosaccharides, and a plethora of small mol- gence of antimicrobial resistance and alternatives to ecules derived from the diet and physiological processes antimicrobial drug therapies. More specifically, it rec - of the mother. Bovine colostrum is rich in Bifidobacte - ognizes the commonality of physiological systems of rium and Lactobacillus phylotypes and this combination and interdependence between food animal health and of compound constituents along with the beneficial bac - human health [2]. For decades, antibiotic drugs (ABDs) teria promotes gut maturation, mucosal integrity and tis- were administered to animals in their food for the pur- sue repair [12] with these beneficial effects attributed to pose of enhancing their growth [3–5]. This practice has these components being transferred to the young in their possible links to the increased incidence of antimicrobial initial suckling. The composition and volume of colos - resistance, a major public health concern. Although the trum rapidly changes within days following parturition, mechanisms by which ABD impact growth have been as milk production evolves over time [13]. To date, the debated, the diminution of the local inflammatory stress vast majority of studies that concern the beneficial effects in the gut was attributed to the now recognized non- of colostrum have examined either aspects of neonatal antimicrobial, anti-inflammatory effects of select ABDs, organ maturation or the ability to thrive in the extrau- particularly in the tetracycline/doxycycline/minocycline terine environment [14]. Still other studies have explored and macrolide families (4–8). Much of the diminution the potential for vaccine-induced antibodies in colos- of local inflammatory stress in the gut was coincident trum (through immunization of cows prior to parturi- with the improved efficiency with which feed was chan - tion) to mitigate complications of parasitic and bacterial neled into tissue deposition in the ABD-fed animals [6, gut infections [15–18]. Few studies have comprehensively 7]. Through legislative actions, the subtherapeutic level examined the effects of colostrum supplementation on use of ABDs for growth promotion in animals has now gut health with the simultaneous assessment of changes been restricted; consequently, other strategies for pro- in a whole-body parameter such as feed use efficiency, moting growth via reduced gut inflammation in food ani - impact on the gut microbiome, the patterns of intestinal mals were deemed necessary [3, 5] and was highlighted digesta-associated anti-inflammatory metabolites, and by the National Research Council [5]. Our current study host gut cell responses. In the present study, we hypoth- addresses one such alternative with a specific focus on esized that the actions of some metabolite constituents mitigating low-level inflammation. of the ileal digesta as modified by bovine colostrum Over the last 10 years the traditional concept of “nutri- intake can combine in an additive manner or synergize tion” has evolved from feeding the body to now include to mitigate inflammation resulting more efficiently in feeding the microbes that inhabit microenvironments improved nutrient use. We used mass spectroscopic and niches throughout the digestive tract [8, 9]. The metabolomics and S16 rRNA microbial analysis on the nuances of nutrition now include not only the composi- same luminal digesta samples along with quantitative tion of the foods and supplements ingested and the way measurement of intestinal morphometrics and a defined the digestive system processes these nutrients, but also epithelial cell nitro-oxidative stress inflammation marker must address the interactions of the gut microbiota with (3′-tyrosine-nitrated proteins, NT) to study the interac- both the ingested nutrients and the host’s gastrointesti- tions between gut-modifying metabolites of the bovine nal cells. The importance of the interaction between the colostrum diet and the mitigation of low-level inflamma - microbes and the host cells, and the nutrient-derived tion in the ileum. A positive association between a BC- metabolites that function as signaling molecules between supplemented diet and reduced gut inflammation would the two” compartments” is now encompassed in the term be consistent with the outcomes on growth and nutrient “immunometabolism” [10, 11]. The model developed in use reported with ABDs but devoid of the issues of drug our laboratory which was aimed at integrating multiple residues and antimicrobial resistance [19]. To explore this physiological systems [11] suggests that the regulation of further, we examined the effect of short-term feeding of a immunometabolism occurs within the interactive rela- diet containing semi-purified, centrifuge-defatted bovine tionships between endocrine, immune, metabolic, and colostrum (BC) to a commercial line of meat chickens microbial inputs to result in the preferential partitioning (broilers). While these animals are characterized as rap- of nutrients to different tissue beds in times of health or idly growing with high nutrient use efficiency under ideal stress. management conditions, they also are highly sensitive Elsasser  et al. Animal Microbiome (2023) 5:6 Page 3 of 22 to even mild stresses that negatively impact nutrient use efficiency as associated with increased gastrointes - tinal perturbation. Though less studied than oxidative stress (cell stress responses that mainly generate post- translational carbonyl modifications to proteins), nitro- oxidative stress (i.e., cell responses that result in aberrant intracellular post-translational protein tyrosine nitration) is a significant additional component of such gut distur - bances [20, 21]), indicative of sustained gut inflammation Fig. 1 BC-supplemented diet improved feed efficiency. Mean body [22–24]). We report that the refined colostrum supple - weight gain ± SEM (grams/day) and feed efficiency (grams feed ment improved feeding efficiency consistent with the consumed per gram body weight gained) in chickens fed the base observed increase in the absorptive surface area of the diet (Control, n = 10) or diets supplemented with either nonfat dry ileum and alterations in the gut microbiota and digesta milk (NFDM, n = 10) or a processed bovine colostrum preparation (BC, n = 20) metabolites that reflect an overall increase in the anti- inflammatory capacity of the luminal environment high - lighted by a significant decrease in gut epithelial protein NT content. or at the least, a sensed perturbation. Presented as an expression of the density of villi, the number of villi per Results linear unit of ileum was significantly increased in animals Short-term BC supplementation of the standard diet sig- fed the BC diet in contrast to CON (P < 0.03) and NFDM nificantly improved feeding efficiency and consistently (P < 0.004, Panel A). When the relative villus area (RVA, modified the metabolic and bacteriological milieu of the mid-villus width x the villus length x number of villi per gut compared to CON and in a manner superior to that unit villus length) was calculated for each animal and the of NFDM. To examine these effects in greater detail, we mean group RVAs compared, the calculated area for BC particularly focused on the ileum. We found diet-asso- was 42 percent (P < 0.05) and 24 percent (P < 0.06) greater ciated changes in epithelial anatomy, anti-inflammatory than the mean areas calculated for CON and NFDM, metabolites and gut microbiota of the ileum. respectively (Panel B). Mean villus lengths were 265, 280 and 250  µm for CON, NFDM and BC, respectively Feeding efficiency was significantly improved in short‑term (P = NS, data not shown). By comparison, corresponding BC diet‑fed chickens mean crypt depths, associated with enterocyte cell pro- The impact of feeding diets supplemented with BC or liferation and digestion capacity of the small intestine, of the nonfat dry milk-supplement (NFDM, a macronutri- 28, 31 and 37  µm were observed for CON, NFDM and ent balanced control to BC) compared to CON on weight BC, respectively, yielding the resulting villus-to-crypt gain and feeding efficiency is summarized in Fig.  1. ratios (Fig.  2, Panel C). Though the mean values calcu - For chickens fed the BC diet, the feed efficiency was lated for villus length and crypt depths alone across the improved approximately 9 percent (P < 0.05) over CON dietary treatments did not attain statistical significance, and NFDM diets. Although the lowest and highest mean the mean villus:crypt ratio for chickens in BC was 27 per- weight gain responses were observed in NFDM and BC cent lower (P < 0.03) and 24 percent lower (P < 0.05) than chickens, respectively, the differences between the means that calculated for CON and NFDM, respectively (Panel of these treatment groups as well as the difference from B). The value for the calculated estimate of villus area per CON were only trends (P > 0.05 < 0.1). unit length was significantly increased in BC compared to the other fed diets (Panel C). Collectively, the increase Intestinal measurements indicate significant biological in estimated villus surface area with BC is consistent with impacts of the BC diet on gut morphology an effect of BC to stimulate a more efficient use of nutri - A visual depiction of the ileum structure attributes as ents and the observed improved feed efficiency in BC. associated with the three diets as well as measured and estimated morphological attributes of the ileum are sum- marized in Fig.  2. Where nuclei are depicted as white BC‑ and to a lesser extent nonfat dry milk objects against the black/grey background, the increased (NFDM)‑supplemented diets are associated with less ileal number of nuclei in the lamina propria of villi of ani- epithelial cell nitro‑oxidative stress mals in the CON and NFDM groups, higher than that We measured the cellular pixel density associated with in observable in BC, is consistent with a larger number immunofluorescence localization of NT, on the exte - of infiltrating immune cells, a hallmark of inflammation rior, epithelial layer of the villi, thereby excluding lamina Elsasser et al. Animal Microbiome (2023) 5:6 Page 4 of 22 Fig. 2 The BC-supplemented diet increases the villus surface abundance. The effects of inclusion of nonfat dry milk (NFDM) or the bovine colostrum fraction (BC) to the standard feed (Control) on the morphology of the ileum. Data represent the means ± SEM of the number of villi counted per standardized length of tissue (Panel A) or the calculated area of the villi as associated with the diet treatments fed to the chickens (Panel B), or the ratio of the villus height to crypt depth (Panel C). Panel D depicts representative images of the diet-associated ileal architecture. (Control, basal diet, n = 6. NFDM. n = 6. BC, n = 10). For reference, “V” = villi, “C” = crypts, and “SM” = submucosa propria and interior layers of the villi that contain infil - measure of the number of cells specified in the demar - trating immune inflammatory cells. Though nitrated cated area of interest, was significantly decreased by 37% proteins were observed in the gut epithelial cells of in BC-fed chickens compared to that present in chicken all chickens, the highest levels were detected in those tissues from either the CON (P < 0.05) or the NFDM chickens fed the CON and NFDM diets (Fig.  3G, H, I). (P < 0.04) diets (Fig. 3J). The overall decrease in NT pixel The epithelial cell NT protein content, characterized density per epithelial cell was attributable to “clusters” of as pixel density as normalized by nuclear counting as a nitration elements that were both fewer in number and Elsasser  et al. Animal Microbiome (2023) 5:6 Page 5 of 22 Fig. 3 Decreased protein tyrosine nitration (NT ) in epithelial cells of villi in samples of ileum from chickens fed a BC-supplemented diet. Immunofluorescence is presented in Panels A–F and Image analysis depicted in Panels G–I. Epithelial cell (anti-cytokeratin 18 epithelial cell marker) presence is indicated by green fluorescence pixels (A, C). NT, as detected using an anti-nitrotyrosine antibody marker, is presented as red (cell presence, B and D–F, for isolated single channel image analysis quantification) or orange (colocalized in green cytokeratin 18-positive cells, C Representative patterns of image analysis-selected nitrotyrosine pixels (G–I, yellow pixels) were summated and processed to yield the statistical analysis summarized in the lower panel bar graph(J). Values are group means ± SEM for Control (n = 6), NFDM (n = 6), and BC (n = 10) smaller in the contained number of pixels. The lower a literature-documented capacity to mitigate tissue level of nitrated proteins in the ileal epithelial cells sug- inflammation. Thus, the goal was to identify those anti- gests that these cells were less subject to nitro-oxidative inflammatory compounds derived from NFDM or BC stress in the presence of BC-derived diet than were the present in amounts over and above those contributed ileal cells of chickens fed the Con and NFDM diets. through consumption of the basal CON diet that had a potential to lower the NT proteins and then test them Short‑term feeding of BC‑ and NFDM‑supplemented in a statistical model for association with any impact on diets alters the anti‑inflammatory metabolite profile ileum inflammation status. Across dietary treatments, of the ileum metabolomic profiling of the ileal digesta revealed 649 We focused on the identified metabolite digesta com - named and 300 unnamed compounds (Additional file  8: pounds derived from the animal treatment diets Data S1). Quantified levels of the identified compounds themselves or as further derived from host and/ or in the metabolite data base in our third-party analyti- microbial enzymatic action on these compounds with cal contract (Metabolon, Inc., Morrisville, NC) were Elsasser et al. Animal Microbiome (2023) 5:6 Page 6 of 22 reported to us as “scaled imputed values (SIV, see “Meth- The effects of the NFDM and BC diets on the changes ods” and Additional file  9: Data S2 for full definition and in these compounds as well as those identified in the derivation). other relevant seven pathway metabolite groups are pre- The digesta of BC was significantly more complex in sented in Table  1. We identified numerous compounds metabolite richness (number of detected compounds) with potential anti-inflammatory activity (as cited in the than that of CON or NFDM. Plant-derived compounds literature) in the ileal digesta. Comparing the levels of the are well-known for having anti-inflammatory and anti- identified metabolites in the ileum digesta between the nitro-oxidative properties and as such were of significant diet treatments, data revealed, perhaps not unexpect- interest here. Of seventy-seven compounds in the ileal edly, that the mean levels of many individual metabolites digesta classified as xenobiotics of plant origin identified varied numerically, sometimes greatly, among the three at or higher than levels stated as the threshold limits of diet treatments and the two replications of the experi- detection (indicated as “DETECTED” in the accompa- ment. Further analysis revealed that fewer individual nying table), 40 compounds were in CON, 46 in NFDM metabolites consistently differed in the two experimental and all 77 in BC (Additional file  5: Table  S1). Interest- replications between CON and BC or CON and NFDM. ingly, though beyond the scope of the present paper, This became apparent with respect to the animal-to-ani - data in Additional file  5: Table  S1 are also presented for mal variation and replication variation for many of the the sake of relative comparison of the composition of metabolites. However, when multiple related metabo- digesta in the ileum compared to that in the duodenum lites for a given common pathway and mechanism of and cecum. These data clearly demonstrate that the avail - anti-inflammatory action were grouped and concentra - ability of metabolites changes with the host-microbiome tion levels considered additively as a singular entity (for interactions that process the digesta as it flows down the example, if several anti-inflammatory compounds like alimentary canal. This finding is consistent with the con - flavonoids/isoflavones or active metabolites like glu - cept that anti-inflammatory compounds available in one tathione and its major precursors), the abundance of a gut segment might not be available in other gut segments select set of compounds/groups were significantly greater to provide their beneficial effects and that bacterial popu - for BC than those measured in CON or NFDM. The liter - lations differ between gut segments. Our considerations ature-supported anti-inflammatory pathway metabolite for inclusion of a metabolite compound in a list of com- categories included tryptophan indole-conjugates, toco- pounds with diet-derived anti-inflammatory character pherols (α-, γ-, δ-tocopherols), flavonoids/isoflavones stemmed from a PubMed (https:// pubmed. ncbi. nlm. (apigenin, daidzein, genistein, and their conjugates), pri- nih. gov/) search for literature citations of studies inves- mary and secondary bile acids (chenodeoxycholic acid, tigating such biochemical modes of action of qualifying 7-ketolithocholic acid), glutathione (N-acetylglycine, compounds with specific reference to compounds in the γ-glutamylcysteine, cysteine,), polyunsaturated fatty acid digesta identified by the metabolomic separation and (docosahexaenoic acid, arachidonic and eicosapentaenoic analysis (Additional file  10: Online References S1). The acids), oligosaccharides (3′- and 6′-sialyllactose), and output from a Boolean search using the terms “polyphe- mitochondrial TCA cycle compounds (itaconate). (see nol, flavonoids, bile acids, amino acids, fatty acids, or oli - Additional file  10: Anti-inflammatory Metabolite Refer - gosaccharides along with small intestine, metabolite, and ences in the online material), inflammation or anti-inflammation” yielded 1968 cita - We also observed some significant effects of diet on tions and formed the basis of our assessments. the potent tripeptide anti-inflammatory compound glu - With focus on the ileum, ten xenobiotic compounds tathione and its precursors in the luminal digesta which with or without anti-inflammatory character were found are critical to the anti-nitro-oxidative capacity of the gut. to be significantly different in abundance between CON The metabolomic report contained data on the reduced and BC and reproducibly across both treatment trials. form of glutathione, GSHr. Overall, the mean level of Apigenin, 2,3-dihydroxyisovalerate, carotenediol, daid- GSHr in BC was half that present in CON (P < 0.04) and zein, daidzein sulfate, genistein, gluconate, naringenin, 70 percent of that present in the digesta of NFDM (NS). and 2-piperidinone were higher in BC than CON and However, the digesta from chickens in BC had a sum- panose (a purported prebiotic) lower, respectively. Simi- mated glutathione precursor pool of compounds sig- larly, digesta from NFDM chickens contained six more nificantly increased over that measured in CON. This is compounds than were present in the digesta from CON consistent with the need for an abundance of precursors with 2,8-quinolinediol sulfate, 2-oxindiole-3-acetate, (cystine, glycine, glutamate, and associated dipeptides) 1-kestose, 1,1-kestotetraose higher and homostachydrine being available to the synthesis pathway in the gut epi- lower. thelial cells to replenish glutathione as it is consumed in countering nitro-oxidative processes. When considered Elsasser  et al. Animal Microbiome (2023) 5:6 Page 7 of 22 Table 1 The effects of inclusion of nonfat dry milk (NFDM) or the bovine colostrum fraction (BC) on ileal digesta components with literature-supported anti-inflammatory effects on tissues varied according to the involved biochemical pathway components Data represent the means ± SEM of the Scaled Imputed Values of metabolites that were increased by either BC or NFDM identified by the metabolomic analysis to be consistently changes across the two experimental replicates and the source(s) of the colostrum. Control, basal diet, n = 11. NFDM. N = 11. BC, n = 20. General Linear Model (GLM) contrast statement coding and weighting indicated in line 2 under GLM Specific Treatment Contrasts. The statistical comparisons of Scaled Imputed Values between control (#1), NFDM (#2) and BC (#3) treatments are shown in the far-right columns with the shades of red colors indicating increases or shades of green colors indicating decreases in significance (or trends). Comparisons highlighted in grey are not significant. as a precursor group, the summated mean of glycine, For some pathways, for example tryptophan or toco- cystine, N-acetylglycine, and γ-glutamylcysteine digesta pherols, only a single pathway compound with pur- abundances averaged 158 percent greater levels in BC ported anti-inflammatory potential was significantly than in CON (3.46 ± 0.46 v 1.34 ± 0.22, P < 0.001). Both different between one or among all diets. Only indole - the difference in summated abundance (P < 0.001) as lactic acid in the tryptophan pathway or γ-tocopherol well as the ratio (P < 0.002) of GSH -to-GSHr may in the tocopherol pathway, respectively, were affected precursors reflected the potential for BC to increase the anti-nitro- by inclusion of nonfat dry milk or the colostrum matrix oxidative capacity of the gut over that present in CON into the basal diet. Surprisingly, the effect amounted by contributing to the metabolites the gut needs to make to a reduction in relative mean ileal SIV. The overall glutathione. impact of this reduction in ileal gamma-tocopherol Statistical evaluation of the pathway components in content was sufficient to account for the majority of the Table  1 showed that polyunsaturated fatty acid (PUFA) pathway change in total tocopherols. compounds, and especially metabolites of eicosapen- The inclusion of BC to the basal diet consistently taenoic and arachidonic acids, were not significantly increased the ileal digesta content of four anti-inflam - affected by diet (all grey). This lack of change with BC matory flavonoid compounds: apigenin, daidzein, feeding may relate to the defatting process used to genistein, and naringin. In contrast, none of the com- generate the BC matrix and the NFDM supplemented pounds was affected by adding NFDM to the basal feeds. However, seven other identified pathways were diet. The effect of adding BC to the diet resulted in a significantly affected by diet either as single compounds doubling of the amount of flavonoid present in the gut or, as summed components of an individual pathway. digesta based on summated SIVs. Data from the larger Elsasser et al. Animal Microbiome (2023) 5:6 Page 8 of 22 metabolite profiling indicated that the levels of the fla -adjusted R (terms shaded in green) they were dropped vonoids in their conjugated forms (glucoside, glucuro- from the model (terms shaded in pink). Consequently, we nide) did not contribute to the gut levels of the parent excluded gamma-tocopherol and the two sialyloligosac- compound’s anti-inflammatory status suggesting that charides from the cumulative Ai-i, based on the fact that the parent compounds alone were to be considered the the adjusted R resolved with these metabolites included major components of interest. in the model was lower than that resolved before either The summated values of the primary and secondary metabolite was added to the model. Therefore, in the final bile acids with anti-inflammatory effects, chenodeoxy - assessment, the combination of chenodeoxycholic acid, cholic acid and 7-ketolithocholic acid content, respec- total flavonoids, glutathione precursors, 7-ketolithocho - tively, were significantly elevated in digesta from BC and late, itaconate and indolelactate accounted for approxi- NFDM compared to CON diets. However, digesta from mately 76 percent of the observed decline of cellular NT BC-fed chickens tended to have higher levels of chenode- protein content. Second, BC feeding yielded mean Ai-i oxycholic acid than that from NFDM (Table 1). values (derived from data in Panel A) significantly dif - Only two oligosaccharides were identified by the mass ferent from and higher than the average Ai-i’s from both spectroscopic analysis, 3′-sialyllactose and 6′-sialyllac- NFDM and Con groups (Panel B). Third, the variability tose. Levels of total oligosaccharides in BC digesta were in the magnitude of the derived NT protein pixel quan- 5.7-fold higher (P < 0.002) than those measured in CON tification data among treatment groups could be better digesta and 2.8-fold (P < 0.003) higher than those in analyzed with respect to a linear model after perform- digesta from NFDM. ing a logarithmic transformation of the pixel data. When The cellular energetics generated by subcellular mito - this was done, the regression analysis showed a signifi - chondrial processes is a fundamental requisite of cell cant (P < 0.005) negative linear correlation (R = 0.694) stability, immune system function, and the efficiency of wherein the higher the Ai-i, the lower the NT protein in nutrient use. Of the eleven mitochondrial compounds villus epithelial cells (Panel C). associated with energy production identified in the digesta, only one, itaconate, derived from the decar- BC and NFDM added to a standard diet altered boxylation of cis-aconitate in the mitochondrial matrix, specific populations of bacteria in the ileum rapidly was an anti-inflammatory metabolite and found to be and specifically. increased in digesta in NFDM and BC relative to CON Using 16S rRNA gene amplicon sequencing, we per- (P < 0.03). formed a community survey of the gut microbiota of 41 chickens under 3 different dietary conditions (stand - Short‑term colostrum extract supplementation (BC) ard or CON feeding, and CON feeding supplemented is associated with a significantly higher Anti‑inflammatory by NFDM or BC). We collected intraluminal digesta index (A‑i‑i) than found in CON or NFDM diets. from the proximal and distal intestine including jeju- The relative contribution of each of these metabo - num, duodenum, and cecum for a total of 156 samples lites to our model was assessed according to whether as well as scrapings from ileal epithelial cells washed free the inclusion of a particular metabolite or metabolite of digesta. We obtained a total of 25,838,078 high-qual- group improved the adjusted R in the multiple regres- ity 16S rRNA V4 amplicon sequences corresponding to sion model, and not all did. To facilitate our analysis of 51,165 (± 2,267, μ ± s.e.m.) sequences per sample (Addi- the impact of dietary-derived metabolites on chicken tional file  6: Table  S2). Overall, we found distinct differ - gut inflammation, we devised an anti-inflammatory ences in the digesta and mucosal-associated microbiota index and found associations arising from the gut pro- among animals fed the three different diets. Across diets, cessing of the ingested feed components of the spe- five distinct types or groupings of intestinal microbiota, cific diets that correlated with protein nitration (Fig.  4). Type I–Type V, were observed and summarized in the First, the data used to construct Panel A of Fig.  4 were heat map and bar graph (Fig.  5, Panels A and B); Type I derived from the SIV values in the metabolomic data- is enriched in Lactobacillus spp., Type II is enriched in base. As indicated by the terms “Yes” and “No” in the Lachnospiraceae spp., Type III is enriched in both Lach- table line “Model Inclusion”, as each successive variable nospiraceae spp. and Bacteroides fragilis, Type IV is was added to the regression model, that variable was only characteristic of Candidatus Arthromitus, and Type V is retained in the model if it added a positive incremen- enriched in Bifidobacterium, Coriobacteriaceae spp. and tal increase to the adjusted R . While some metabolites Lactobacillus spp. Type I and IV microbiota had the low- present in the digesta of chickens may have had a pur- est community diversity (Additional file  1: Figure S1) and ported anti-inflammatory character, if they did not have were highly enriched with Lactobacillus and Candidatus added a benefit in the regression model to increase the Arthromitus, respectively (Fig.  5 Panel B). In particular, Elsasser  et al. Animal Microbiome (2023) 5:6 Page 9 of 22 Fig. 4 Correlation between anti-inflammatory index (A-i-i) and epithelial cell protein nitration (NT ). Multiple regression was used to assess the relationship between the level of NT content (Log10, pixels/cell) present in intestinal epithelial cells and the independent metabolite variables. Inclusion of a metabolite (class) to the model was valid if the resulting adjusted R was increased over that of the prior iterative assessments (Panel A). The effects of dietary treatment on the A-i-i (the sum of the Scaled Imputed Values of the individual metabolites retained in the model, Panel B). Linear regression of the NT pixel content on the A-i-i suggested a significant negative correlation where the greater the A-i-i- the lower the nitration stress (Panel C) the different physiological sections of the intestine dem - consistent with the nature of the cell attachment mecha- onstrated distinct microbiota (Additional file  2: Figure nism this organism employs to persevere in its growing S2), and the Type IV microbiota was enriched in the microenvironmental niche largely specific to the ileum. ileal microbiota. The other types of microbiota were sig - Consolidating across all data comprising Fig. 5 and Addi- nificantly more diverse and had relatively more abun - tional file  2: Figure S2 and Additional file  4: Figure S4, dant Lachnospiraceae spp., Bacteroides fragilis and a the patterns, distribution, and abundance of microbes wide array of strict and facultative anaerobic bacterial is consistent with BC having greater microbial diversity species (Additional file  7: Table  S3). Statistical mod- than that in the digesta from animals in the other two eling using Bayesian Poisson model (details in Methods diets. Increases in this microorganism in the colon might and (Additional file  3: Figure S3) showed the intestinal reflect remaining pass-through organisms originating in microbiota in the digesta of BC-fed chickens was signifi - the ileum and perhaps simply shed into the digesta going cantly enriched in Type I and IV microbiota. We further into the large intestine in association with the naturally- performed LDA effect size (LEfSe) analyses to quantita - occurring programmed epithelial cell losses from the tips tively characterize the phylotypes that could explain the of the villi associated with apoptosis. In addition, Candi- differences observed under different conditions (Fig.  5C, datus Arthromitus was minimally detected in the cecum (Additional file  1: Figure S4). Candidatus Arthromi- or duodenum digesta or scrapings (Fig. 5C, left side). tus was shown to be the most differentially abundant In order to determine whether the presence of Can- phylotype in ileal mucosal scrapping samples of BC-fed didatus Arthromitus might have arisen in the BC animals but not in ileal digesta (Fig.  6A). This finding is chickens as a result of feeding the actual processed Elsasser et al. Animal Microbiome (2023) 5:6 Page 10 of 22 Fig. 5 Chicken intestinal microbiota analyses. A Heatmap of the 50 most abundant phylotypes of 156 samples collected. Five types of intestinal microbiota were revealed based on clustering patterns. Ward linkage clustering is used to cluster samples based on their Jensen-Shannon distance calculated in vegan package in R [116]. Identified microbiota types are labeled as I–V. B Distribution of different community type by cluster as shown in A in diet treatment groups of Control, colostrum and NFDM. Type I is significantly lowered in control than in other two groups while type IV is significantly higher in BC and NFDM. Type II is borderline significantly higher in BC and NFDM than control, based on statistical modeling using Bayesian Poisson model. C Phylotype biomarkers for digesta and scrapings generated using program LEfSe [105]. Vertical bars represent the relative abundance of Bacteriodes in each sample. Dotted line represents mean, solid line represents median relative abundance. The alpha value for the non-parametric factorial Kruskal–Wallis (KW ) sum-rank test was 0.05 and the threshold for the logarithmic LDA model (43) score for discriminative features was set at 2.0 colostrum fraction that contained a live Candidatus Arthromitus genomes or using marker gene-based Arthromitus, we characterized the composition of approach (Additional file  7: Table  S3). This result indi - the microbiota associated with various samples of the cated that the Candidatus Arthromitus was not likely bovine colostrum used in the diets, and showed that derived from the bovine colostrum diet. these samples contained very low abundance of bacte- Other than Candidatus Arthromitus, Streptococcus ria in general and revealed no Lactobacillus nor Candi- was another phylotype that was shown to be enriched datus Arthromitus. While fresh colostrum is known to in BC-fed animals, irrespective of the sub-anatomi- contain many microorganisms beneficial to the nursing cal location (digesta vs. mucosa-associated) (Fig.  5C). infant, the lack of such microbial abundance in our BC This result again emphasizes the importance of sub- diet preparations is consistent with the effects of the anatomical location, anatomic site, and dietary condi- 1–h centrifugation of the preparations at 20,000×g to tions for specific groups of bacteria in stark contrast sediment bacteria into the bottom of the tube. Further to data more commonly generated from fecal collec- analyses confirmed no detected Candidatus Arthro - tion sampling. Other phylotypes that demonstrated mitus via reads mapping to published Candidatus varied enrichment under different feeding conditions Elsasser  et al. Animal Microbiome (2023) 5:6 Page 11 of 22 Fig. 7 Sialyllactose content of ileal digesta is correlated with presence of SFBs. Ileal digesta content of the combined mean (± SEM) levels of the 3′-sialyllactose and 6’-sialyllactose represented as the metabolomic Scaled Imputed Values as measured in samples Fig. 6 Candidatus Arthromitus is present in the ileal scraping from chickens fed the three diets (Panel A). Summated 3′-sialyllactose and increased by BC. Mean (± SEM) abundance of Candidatus and 6′-sialyllactose values were regressed on the Log10 [OTU of the Arthromitus represented as Log10 [OTU] in the three different SFB] yielding a significant positive linear correlation. (Panel B) segments of the gut and further differentiated by digesta vs. scraping (Panel A). Mean (± SEM) abundance of Candidatus Arthromitus represented as Log10 [OTU] in the ileum as measured in digesta or cell scrapings (attached) as affected by diet. *P < 0.04 (Panel B) the combined oligosaccharides versus the lo g of the abundance of total Candidatus Arthromitus indicate a significant positive correlation wherein higher levels of the oligosaccharides in the digesta were associated with were also included (Fig.  5C). For example, Bifidobac - increased numbers of the Candidatus Arthromitus with terium were more enriched with the NFDM diet, and its potential impact on the immune capacity of the gut. Bacteroides fragilis, Bacteroides ovatus, and E. coli, was particularly enriched in cecal samples. Together, we observed that Candidatus Arthromitus was only Discussion in the mucosa-associated microbiota of the ileum in Significant increases in growth rate of domestic food ani - these chickens and its presence was amplified signifi - mals have been linked to some of the nonantibiotic, anti- cantly when BC was incorporated into the basal diet inflammatory effects of ABDs [5, 6], with more recent (Fig. 6A, B). The higher relative abundance of Candida - trends towards increased popularity of the use of sup- tus Arthromitus is consistent with a localized prolifera- plements with purported anti-inflammatory effects [25]. tion of the organism, most likely due to factors in the Therefore, we addressed whether the anti-inflammatory environment of the organism rather than being sourced gut benefits of ABDs could be mimicked by the feeding from the fed colostrum itself. of defatted colostrum and further, whether a model could While the oligosaccharide content of the various diets be developed to better define what aspects of colos - did not affect the anti-inflammatory index, the 3′- and trum consumption fostered these beneficial impacts. 6′-sialyl-oligosaccharides did lead to a dramatic site- Our results were based on the simultaneous assessment specific increase in Candidatus Arthromitus. The oli - of how a diet enriched in a partially refined BC prepa - gosaccharide content of the ileal digesta was increased ration differed from other diets in their impact on the approximately 4- and 2.5-fold in BC compared to that host gut morphology, the gut metabolite milieu, and the measured in digesta from CON and NFDM, respectively gut microbiota with particular emphasis on the ileum. (Fig.  7). Further regression analysis between the SIVs for The terminal ileum segment of the gut was focused on Elsasser et al. Animal Microbiome (2023) 5:6 Page 12 of 22 because of its role as the transition segment between enzymes such as JAK-2 [34, 35] or mitochondrial pro- the small and large intestine, the unique presence of the teins thereby causing dysfunction and ATP shortage in immune-modulating segmented filamentous bacteria, cells [28, 36, 37]. Additionally, epithelial cells can have Candidatus Arthromitus, and further in light of the rela- levels of NT proteins with cell function deficits that tive paucity of research compared to that of the cecum vary by diet in the absence of detectable infectious dis- and colon on integrated host, metabolome, and microbi- ease [26, 27]. Oxidative/nitration protein and lipid dam- ome interactions. age has been generated in chickens simply by low levels Research from our laboratory has demonstrated that of partially oxidized corn oil or polyunsaturated fatty nitro-oxidative stress occurs in the intestine as observed acids being in the diet [38]; similarly, that oxidized oil- with the increased tissue content of nitrated proteins in induced inflammation was relieved with the addition of conjunction with parasitic infection and correlated with the flavonoid antioxidant quercetin to the chicken diet the severity of infection [26, 27]. In comparison to exten- [39]. Mitigation of gut dysfunction by reducing inflam - sive research done on oxidative stress in the gut, there is mation is a goal of both human and veterinary medicine. less research on nitration stresses in the gut, even though In the present study the short-term feeding of the refined nitration stress and oxidative stress share a pool of cell BC matrix was significantly associated with the observed response-induced common oxygen- and nitrogen-based reduction in ileal epithelia-specific NT protein content. reactants (nitric oxide and superoxide anion [20, 24]). Gut metabolism in health can account for up to 20 per- With peroxynitrite (ONOO ) substantively being the cent of maintenance energy expenditure [40–42]. Gut principal effector relating to this form of inflammation, inflammation increases energy demands by as much the term nitro-oxidative stress therefore encompasses as 10 percent of the body’s metabolic energy need [43, the result of nitric oxide (the “nitro” component) and 44] with a significant portion of the caloric substrates superoxide anion (the “oxidative” component) react- diverted from growth and muscle accretion to support ing to generate the ONOO insult [22, 23, 28, 29]. The immune function particularly in young animals [22, 24]. present study demonstrates that the fractionated prepa- Microbial populations in the gut, and more specifically ration of BC developed in this study, when added to a changes in such populations, significantly impact host standard feed and consumed by animals for 8  days, is immune and metabolic gene expression [45] and there- associated with a significant reduction of the amount of fore the energetic needs of the host’s physiological sys- nitro-oxidative inflammation in the epithelial cells of the tems. Signals from the gut microbiome can prioritize the ileum, denoted as a reduction in the content of intracel- flux of nutrients between the nervous, endocrine, and lular NT proteins. In concert with this effect, feeding BC immune systems and shape their interactions [22, 24, was strongly associated with an increase in the absorp- 46, 47]. Since oxidative and nitration stress impacts the tive surface area of the ileum. The rapidity with which energy utilization of affected cells, even marginal changes this occurred is consistent with an increased capacity to in gut health result in energetic deficiencies [48, 49] that generate epithelial cells [30] as reflected in the calculated rapidly translate into growth and nutrient use efficiency crypt-to-villus ratio. These observations on the increased deficits but improved significantly in BC. These observa - ileal surface area are also consistent with the observed tions are consistent with the regulatory model proposed improvement in nutrient absorption as reflected in feed for the controlling inputs to immunometabolism [50] and utilization efficiency. The presence of naturally-occur - further impacts on nutrient use in the young growing ring, low-level gut stressors such as changes in environ- animal. ment and social structure, present in our select, high The enhanced genetic lines of commercial production growth rate animal model, has been identified previously chickens with a high growth rate used in our chicken as having the capability of significantly altering the archi - model required no direct disease or chemical challenge tecture of the villus absorptive area as well as the mor- to trigger gut nitrooxidative stress. Further, low levels of phogenesis of the crypts [31–33]. nitrated proteins were shown to be generated by entero- Elevated levels of intracellular tyrosine-nitrated cytes in response to non-infectious perturbations such (3’-nitrotyrosine-) NT proteins are a cause of protein as changes in feed composition, allergens, endogenous dysfunction in non-immune cells, as documented in bacterial endo- and exotoxins, mold toxins, and even many tissue inflammation pathologies [23, 24, 29]. Epi- metabolic ketosis [51–53]. In the past, this stress sensi- thelial cells generate highly reactive nitrogen interme- tivity was managed in part through the extensive use of diates (i.e., peroxynitrite, ONOO ) from nitric oxide antibiotics added to the diet [3, 5]. However, herein we and superoxide anion [23, 24, 28], which can react with report that consumption of components of BC can ame- critical tyrosine(s) in phosphorylation sites of many criti- liorate inflammation in this gastrointestinal situation and cal intracellular proteins including signal transduction improve nutrient use. Elsasser  et al. Animal Microbiome (2023) 5:6 Page 13 of 22 Dietary factors are potent modulators of the micro- the tryptophan pathway metabolite, indolelactate, also biota composition and its interaction with the host [54]. identified in our metabolomic screening, concurrent with Reciprocally, microbes metabolizing ingested nutrients the enhanced presence of bifidobacteria, and their ben - and generating nutrient-derived metabolites play a criti- eficial effects on the balance between inflammatory and cal role in regulating the host immune response and gut anti-inflammatory states [63]. The capacity for microbe- cell function, as documented in the newly emerging area derived indolelactate to function as an anti-inflammatory of immunometabolism [55]. Active microbial modifica - metabolite [64, 65] is consistent with our findings of the tion of feed-derived nutrients was evident (Additional reduction in nitrated proteins, the increased nutrient use file  8: Data S1) as observed in the presence of microbe- efficiency, and favorable microbial populations in BC- derived compounds like p-cresol sulfate and hippurate as compared to CON- or NFDM-fed chickens. These previ - well as deconjugation of flavonoid compounds differen - ously published data lend support to the validity of our tially affecting their bioavailability [56–59] and levels of regression analysis that identified indolelactate among the various secondary bile acids consistent with litera- the many metabolites in the digesta. Collectively, the pre- ture reports [57]. From the metabolomic analysis of the sent data demonstrate the potential for diet composition ileal digesta of test animals fed the various dietary treat- to significantly impact gut health through changes in the ments, we were able to identify and statistically model a microbiota, the gut metabolite milieu and host responses. defined set of nutrient molecules and microbially-derived Specific metabolites generated by the localized bacte - metabolites that correlated with the reduction in level rial populations acting on ingested dietary components of nitrooxidative stress present in the ileal epithelial facilitate processes needed for healthy eubiotic regula- cells. The positive effects of the colostrum feeding were tion of the gut epithelia. In studies of the murine colonic reflected in the increased portion of bacteria with anti- microbiota, Tiffany and Baumler [66] proposed that dur - inflammatory/ immunomodulatory properties in the ing gut homeostasis, bacteria of the phyla Firmicutes and microbiota profiles, and the generation of anti-inflamma - Bacteroidetes, obligate anaerobic bacteria, fermented tory metabolites bathing the cells of the lumen. dietary fiber and maintained a hypoxic environment We determined that classes of related microbially- that could reduce colonization with Enterobacteriaceae derived metabolites should be considered for their (phylum Proteobacteria). In the absence of hypoxia, Pro- anti-inflammatory effect in addition to the more com - teobacteria gained a foothold. This deviation from an monly literature-cited approach of identifying single obligate anaerobic microbiota was defined as gut “dysbio - compounds. For example, the polyphenol flavonoids sis”. Dysbiosis in the ileal microbiome stems from lower identified in the mass spectroscopic analysis revealed levels of microbial diversity with imbalances between that as a class they were significantly increased in BC both beneficial and pathogenic organisms as well as dis - and NFDM feeding in comparison to CON. If, however, proportionate imbalances in commensal bacteria that only one flavonoid, apigenin, of the identified group was can change the local microenvironments downstream assessed, the larger group effect would have been missed. thus facilitating a bacterial overgrowth syndrome or Similarly, while the effect of adding NFDM or BC to the pathogen emergence [67, 68]. Thus, in the present study, basal diet on the individual components identified in the the increased diversity and favorable ratios in microbial anti-inflammatory glutathione pathway may have been populations in the BC animals would be consistent with a insignificant (i.e., P > 0.1), collectively the sum of the pre- greater opportunity for eubiosis and sound gut health to cursor pool and the glutathione components improved be maintained. the regression model (Table  1). This is consistent with The microbiome of the small intestine, the primary the need for an abundance of precursors (cystine, glycine, site for the absorption of nutrients, differs substantially glutamate, and associated dipeptides) being available to from that of the colon both in terms of the number of the synthesis pathway in the gut epithelial cells to replen- colony-forming units per ml as well as microbial compo- ish glutathione [60, 61] as it is consumed and to maintain sition [69]. We focused on the ileum in this study, largely cellular oxidative/nitrosative homeostasis. because of the large information gaps on the microbiome Metabolites are an important link in the interac- and its host interactions compared to the large intes- tions between the host, its gut microbiota and the tine [70]. We observed histological evidence of an ongo- lumen environment. For example, metabolites such as ing inflammatory process in animals fed the CON and metabolically-derived itaconate, generated during the NFDM diets. In contrast, we observed a reduction in epi- immune response and identified in our screening can thelial cell nitrooxidative stress in BC-fed animals which regulate both the magnitude and duration of the immune may be associated with the greater population diver- response [62]. In addition, Henrick et  al. established the sity that we observed in the ileal microbiome of these cause-and-effect relationships between gut increases in animals. The decreased gut inflammation-associated Elsasser et al. Animal Microbiome (2023) 5:6 Page 14 of 22 generation of NT proteins in BC-supplemented chick- NFDM-supplemented feed compared to CON. The lev - ens is consistent with other known anti-inflammatory els of these oligosaccharides were significantly higher in effects of colostrum such as the observed reduction in BC than marginally-increased levels in NFDM. However, NFk-B-mediated proinflammatory cytokine expression they did not statistically contribute to the Ai-i per se, the in intestinal epithelial cells [71]. Those nitration reac - positive correlation largely driven by the levels found in tions can play out in the cascade of responses initiated by BC. Oligosaccharides in BC may facilitate microbiota such mediators as TNF-α cascading through increases in remodeling in the chicken by reducing the ability of intracellular NO and superoxide anion culminating in the pathogens to gain a niche in the gut microenvironment generation of nitrating reactants like ON OO and the and through promoting growth of beneficial microbiota more reactive ONOOCO , as produced when higher as has been seen in humans and mice The increased oli - pCO is present in sections of the gut with more anaero- gosaccharides in BC associate strongly with the selec- bic status with perturbed mesenteric blood flow. The ileal tive increase in ileum mucosa-associated segmented microbiome also differed from that of the duodenal and filamentous bacteria (SFB), namely Candidatus Arthro - colonic segments analyzed in that the segmented fila - mitus, in chickens in the BC-supplemented group. SFB mentous bacteria Candidatus Arthromitus was localized were sparsely detected in the ileal mucosa of both CON to the cell layer scraped from the ileal bowel (rather than or NFDM chickens. Furthermore, Candidatus Arthromi- the digesta per se). These data suggest that anatomical tus was not detected in the raw colostrum after incuba- localization of microbiota composition in the gastroin- tion on media for 48 h suggesting that some aspect of the testinal tract may be important in the regional status of BC diet promoted the increase in SFB already present in immune activation in different gut segments as well as the ileum. These bacteria, living predominantly attached the inflammation-anti-inflammation status thus suggest - to the ileal epithelial mucosa, are known to promote gut ing the need for spatially distinct analyses along the gas- maturation and gut mucosal and adaptive immune devel- trointestinal tract. opment including the development of germinal centers We hypothesized that BC-initiated changes in the in Peyer’s patches that generate potent IgA and Th17 gut microbiota may enhance the metabolic process- responses [74, 75]. By itself, SFB can induce intestinal T ing of nutrients into anti-inflammatory metabolites, a cell development similar to that induced by the complete feature we characterized with the development of the gastrointestinal microbiota [76]. SFB colonization also anti-inflammatory index, Ai-i. These observations were has been shown to improve barrier protection against reproducible over time (i.e., replicated experiments) and enteric viral infections and associated diarrheal dis- across different sources of colostrum (cow breed and eases [77] as well as serve as a coordination link between collection time after parturition). The ability of BC to metabolism and immune function [78]. Danzeisen et  al. beneficially modify the gut environment was due to the [79] demonstrated that in turkeys a fundamental deter- nutrient-derived metabolites and microbial populations minate of growth success was the gut microbial milieu, in the ileum not matched by the NFDM diet. The group particularly the presence of significantly more SFB. of metabolites identified after statistical regression align - Tryptophan, an essential amino acid, is metabolized by ment supported the hypothesis of an increased abun- gut bacteria to generate metabolites specific to indole, dance of metabolites with anti-inflammatory character kynurenine and serotonin pathways, the latter a key neu- and aligned with observed effects on gut morphology and rotransmitter in both the enteric and central nervous an improved efficiency of nutrient use for body weight systems [65]. The tryptophan metabolite, indolelactic gain in BC. acid, is elevated in NFDM, but even more so in BC- Several classes of compounds were altered by the BC supplemented diets. Indolelactate acts on gut epithelial supplementation. Compared to CON feed, BC feed cells, Paneth cells and enteroendocrine cells through increased the microbially-derived secondary bile acids, specific receptors to (1) stimulate the release of antimi - chenodeoxycholic acid, 7-ketolithocholic acid, in the crobial peptides, (2) upregulate tight junction proteins ileum. Studies in humans and mice have shown bile acids to maintain barrier function; (3) suppress inflamma - to have an anti-inflammatory effect on immune cells tory cytokine production, (4) decrease superoxide anion [72] and appear to function in their host-microbe cross- generation, and (5) decrease apoptosis. Tryptophan talk signaling capacity through the farnesoid-X receptor metabolites generated by the microbiota can bind to the (FXR) and the G-protein-coupled bile acid receptor-1 endogenous tryptophan receptor, the aryl hydrocarbon (TGR5) [73]. receptor (AHR) [64]. Indolelactate is generated only by Oligosaccharides 3’-sialyllactose (3SL) and 6’-sialyl- bacteria, particularly Lactobacillus, abundant in BC, and lactose (6SL) with prebiotic properties were markedly bifidobacteria with demonstrated benefits to gut health increased in the ileal contents of chickens fed BC- and in colostrum-fed infants [63, 80]. Elsasser  et al. Animal Microbiome (2023) 5:6 Page 15 of 22 While the methods used by Metabolon Inc. do not initiation of the BC supplement at an earlier time point allow us to measure short chain fatty acid (SCFAs), they may have an even greater effect, or to define the changes undoubtedly were present in the BC that are high in in the immune response provoked by the presence of the indigestible carbohydrates (“prebiotics”)” that selectively SFBs. enhance the growth of Bifidobacterium and Lactoba - cillus that have been shown to modulate inflammation Materials and methods and modulate a vast range of physiologic processes that Animal model include suppression of inflammatory signals and carcino - The rapidly growing commercial meat chicken (Gallus genesis [81]. The microbially-derived SCFAs share the gallus, Strain: Ross 708, Amick Farms Hatchery, Hurlock, use of receptors and signaling pathways that are used by MD) was used for the nutrition, inflammation, metabo - the host gut epithelium to detect molecules that signal lome, and microbiome objectives of the study. Genetic between the gut microbiota and the host. selection for characteristics of allometric growth result Our findings suggest that BC feeding enhances the gen - in an enhanced accretion of ingested nutrients in lean eration of several classes of anti-inflammatory metabo - muscle deposition largely associated with breast and lites. Collectively, the findings support the need to better leg tissue. The rapidity of growth (< 49  days from hatch understand not only what colostrum components other to commercial processing; [> 100  g body weight (BW) than traditionally assessed factors like immunoglobulins, gain/day]) and high rate of nutrient utilization for lean antimicrobial factors, growth factors and cytokines con- tissue accretion (< 1.7  kg feed to attain 1  kg increase in tribute to an anti-inflammatory environment in changing BW) make these animals exquisitely sensitive to pertur- the composition of the digesta in the gut lumen. Perhaps bations in environmental conditions, including those of more critical though, information gaps exist needing diet [82, 83] and require stringent controls on environ- resolution regarding what anti-inflammatory factors and ment and feed [84]. For these chickens, it is recognized metabolite precursors reach the different sections of the that the growth process dynamics and feed efficiency as gut and are processed through host-diet-microbe interac- well as changes in the gut microbiome are highly volatile tions in maintaining gut health and homeostasis. and change over time in as little as 1  week, essentially as the animals mature [85, 86]. With this awareness, we assessed what was consistently affected by the presented Conclusion dietary treatments and what metabolite and gut micro- The addition of BC to standard feed exerts profound biota attributes of the treatment diets might mitigate low effects on the immunometabolism of chickens, a model level gut inflammation as specifically addressed in terms for gut health exquisitely sensitive to metabolic perturba- of nitrooxidative stress, i.e., the abundance of tyrosine tions that reflects the impact of gut stress on feeding effi - nitrated proteins in the gut [26, 27]. ciency and growth [50]. Compared to chickens fed CON or a NFDM-supplemented diet, BC-fed chickens had an Experimental treatments: diets supplementation increased ileal absorptive surface and developed changes with colostrum extract or nonfat dry milk in the composition and structure of the gut microbi- A total of four colostrum samples (4 L each) were col- ota that generated feed- and host-derived metabolites lected from multiparous Jersey (n = 2) or Holstein (n = 2) and compounds, resulting in an anti-inflammatory gut dairy cows as first and second milkings after parturition, microenvironment. This could be attributed to significant respectively. In accordance with traditional feeding for increases in several BC-associated digesta metabolites cows at parturition and early lactation, cows were fed a with a defined anti-inflammatory character. Importantly, total mixed ration consisting of corn and grass silages only the BC-fed chickens had major increases in mucosa- supplemented with roasted soy bean meal, vitamins and associated SFBs known to promote gut maturation and minerals. As such, the colostrum from the cows would be immune mucosal development. Our study also high- naturally enriched with a spectrum of flavones and iso - lights the importance of sampling the mucosal surface in flavones, etc. derived from the plant matrix of the diet addition to the digesta to comprehensively assessing gut [87, 88]. This concept aligns well with the data in Table  1 microbiota. In addition, the results underscore the util- and Additional file  5: Table S1. Each collected colostrum ity in knowing how the total anti-inflammatory capac - was frozen at − 80  °C until needed for diet preparation. ity established in the interaction between microbes and To prepare the colostrum-containing diet, the following metabolites of a given gut region can be changed as a procedures were performed. The thawed, cold colostrum result of feeding a complex food matrix with the active from one cow at a time was thoroughly mixed, aliquoted components identified through appropriate statisti - into eight 50  ml polycarbonate tubes and centrifuged cal analysis. Future studies are required to determine if at 2  °C for 1  h at 20,000×g, centrifugation conditions Elsasser et al. Animal Microbiome (2023) 5:6 Page 16 of 22 sufficient to congeal lipids and sediment bacteria at the to individual pens where the daily feed intake for each top and bottom of the tube, respectively. In agreement animal could be accurately measured. The abrupt change with Morrill et  al. [89], the fat/lipid content of the dif- in local environment and housing as well as separation ferent colostrums was not different as a function of cow from a group is recognized as a short-term, low-level breed or day obtained. After centrifugation, the solidi- stress situation impacting the hypothalamic–pituitary– fied lipid (nonaqueous long chain fatty acid component) adrenal axis with impact on gastrointestinal function in was removed from the top of the samples and the liquid the chicken [90, 91]. The quantity of fresh diet given to underneath constituting the middle third of the tube each chicken daily was recorded. The next morning the volume poured off and collected. Higher density sedi - residual feed not eaten from the previous day was meas- mented material in the bottom third of the centrifuge ured and the difference between given and residual used tubes containing cells, bacteria and cell and membrane as the measure of feed intake. Live body weight of each debris was discarded. The fluid from each of the four chicken was obtained daily at a standardized time of day cow’s colostrum was pooled across tubes but maintained relative to feed management. Chickens and their envi- as separate pools for the generation of the respective BC- ronment were observed three times daily as regards the supplemented diets. After the pooling, the material was health and welfare of the animals per approved animal passed through glass fiber matt filters. Collected material care protocol. was sprayed onto a base diet previously used for chicken The overall experiment was conducted in 2 sepa - experiments by our laboratory [corn-soybean meal base; rate replicates of the following treatments: control diet 23 crude protein and 13  MJ metabolizable energy [27]] (CON), nonfat dry milk diet (NFDM), and colostrum to supply the equivalent of 100  ml colostrum/kg feed, diets (BC), the replications conducted 6 months apart. with continuous mixing. The damp diet was put into 4 L Colostrum was obtained from four different sources; glass high-vacuum containers, frozen at − 80  °C, lyophi- sources consisted of Jersey cow first postpartum milk - lized to dryness, and fed as such. To assess the effect of ing, Jersey cow second postpartum milking, Holstein cow the addition of this quantity of colostrum to the overall first postpartum milking, and Holstein cow second post - crude protein and energy content of the base diet, a test partum milking. Variability in colostrum composition batch of each of the BC diets was formulated and sent (day1 and day 2 of lactation postpartum and Jersey and out for nutritional analysis (Cumberland Valley Analyti- Holstein breeds) was intentionally built into the design cal Services, Mercersburg, PA). Following analysis, it was so that across the variability a set of metabolites with observed that BC diets were approximately 2.7% higher consistent effects on inflammation and gut parameters in crude protein but otherwise (total fat, gross energy, could be ascertained. In preparing the diets for the first fiber, etc.) not different from the base diet. To correct replicate of the study, one BC diet was prepared using for the protein difference, the base diet to be used to Jersey-derived first milking colostrum and the other BC prepare the BC was modified by lowering the soybean- diet prepared from the Holstein-derived first milking. derived protein content by 2.7% and that protein dif- Similarly, in the second replication the two BC diets were ference then made up for with the inclusion of the BC. made from the Jersey and Holstein colostrum second When the colostrum component was added to this lower milkings. protein base diet, reanalysis of the diets showed them to Transfer of the chickens to the individual pens and the be equivalent in protein. A third diet was developed, ana- respective switches to the test diets were considered the lyzed, and formulated to more closely approximate the first day of the start of the experiment. Test diets were amino acid composition of the BC diets and for this pur- fed for 7 days with the animals euthanized (per American pose nonfat dry milk (NFDM, Carnation, Inc.) was added Veterinary Medical Association Guidelines for Euthana- to the lower crude protein base. sia of Animals: 2013 Edition, https:// www. avma. org/ KB/ Polic ies/ Docum ents/ eutha nasia. pdf ) on day 8 following Study design the recording of the final weight. As pooled across the All research was conducted under an animal manage- two experimental replicates, there were 11 CON diet ani- ment and welfare protocol approved by the USDA Belts- mals, 11 NFDM diet animals and a total of 22 animals fed ville Institutional Animal Care and Use Committee. One colostrum-containing diets, the data from which animals day old chicks were transported from the hatchery to was used for the metabolomic and microbiome analy- the USDA Poultry Research Facility (Beltsville, MD) and sis. Variation in the n across the various diet treatment placed in group brooder housing with the environmen- groups reflects further refinement based on the quality tal temperature set according to the age of the animal. of data primarily obtained from the metabolomic evalu- The CON feed and water were provided ad  libitum. At ations. When an animal’s metabolite data were identified 15 days of age, chickens were moved from the group pens as an outlier (> 3 S.D.) or compromised as a result of an Elsasser  et al. Animal Microbiome (2023) 5:6 Page 17 of 22 inefficient extraction, that animal was eliminated from 4  °C in a humidified chamber with anti-3′-nitrotyrosine the study. The numbers of animals per treatment group (rabbit polyclonal, Millepore-Sigma, Burlington, MA, from which the data were amassed exceeded the mini- Inc., 1:100) with subsequent antigen visualization [goat mum number for achieving statistical significance based anti-rabbit IgG Alexa 680 (1:400; 1  h, Thermofisher Sci - on statistical power analysis (Introduction to Power and entific, Grand Island, NY)]. For validation purposes, Sample Size Analysis., https:// suppo rt. sas. com/ docum an additional set of slides of the adjacent serial sections entat ion/ onlin edoc/ stat/ 131/ intro pss. pdf). Samples pre- were prepared and immunostained to identify villi api- pared for immunofluorescence were obtained from rep - cal epithelial cells using anti-cytokeratin-18 (Abcam resentative numbers of animals in each replication of the Inc., Cambridge, MA, mouse monoclonal, Clone-C04, experiment. Following euthanasia, the intestinal tract 1:200) with antigen visualization (goat anti-mouse IgG was rapidly excised and divided into segment samples Alexa 488, 1:400, Thermofisher Scientific, Grand Island, for duodenum (central loop), distal ileum, and cecum NY; green pseudocolored immunofluorescence). Nuclei (with the present work focusing on the ileum). These were stained with 4–6-diamino-2-phenylindol-dihydro- samples were further subdivided into 2 components. chloride (DAPI, Thermofisher Scientific). Morphomet - Each segment was opened longitudinally. From one the ric measurements to determine the length relationships digesta was carefully removed to eliminate intestinal tis- between the villi and crypts were performed using the sue potentially scraped from the wall. The other section DAPI blue channel using a calibrated and validated digi- was rinsed free of remaining digesta and debris with ice tal micrometer internal to the ImagePro 9.3 software cold PBS and the epithelial surface gently scraped away (Media Cybernetics, Rockville, MD). Quantitative image using a fresh, chilled glass slide. One additional sec- analysis to ascertain the pixel density of the NT antigen tion of rinsed intact gut was immersed overnight in 4% per villus epithelial cell was performed using ImagePro paraformaldehyde and transferred to ethanol for further 9.3 software (Media Cybernetics, Rockville, MD) as paraffin embedding and sectioning for microscopy as described in the Supplementary Information—Detailed subsequently described. To evaluate the live growth of Methods. the chickens we measured the average daily body weight gain and feed efficiency (the grams of ingested feed cor - Metabolome profiling and analyses responding to the grams of weight gained per day). Samples of digesta were collected from each of the ani- mals in both replicates of the study (CON, n = 10; NFDM, Fluorescence microscopy and quantitative imaging n = 10; BC, n = 20, with 4 samples failing to be extracted analyses efficiently and the results excluded) from a region of the The detailed methods for the immunofluorescent tis - ileum 3–5 cm cephalad to the ileocecal junction. For the sue antigen immunostaining as well as the exact pro- metabolomic profiling of the digesta, a homogenous sam - cess used to quantify the cellular pixel content of the ple (approximately 100 mg) was accurately weighed to the 3’nitrotyrosine antigen used to represent the intensity 0.1 mg into a polypropylene vial, with the vial capped and of nitrooxidative stress can be found in the online Sup- frozen in liquid nitrogen. Metabolite identification and plementary Information—Detailed Methods. The pro - quantification was performed under contract by Metab - tocols for tissue fixation, fluorescence immunostaining olon, Inc., Research Triangle Park, NC, and all steps for for 3′-tyrosine-nitrated proteins (NT) as the inflamma - this analysis were conducted according to the company’s tion marker and morphometric analysis of ileal samples protocols (Supplementary Information—Detailed Meth- published by our laboratory previously were applied in ods). In brief, the basic process was as follows: Metabo- the present study [27, 34]. In brief, representative tissue lon’s first preparation step was to lyophilize the weighed sections for immunofluorescence were collected from samples and then reconstitute the samples for analysis by the first and second replicates of the experiment (CON, ultrahigh performance liquid chromatography-tandem n = 6, 3/replicate; NFDM, n = 6, 3/replicate; BC, N = 10, mass spectroscopy (UPLC-MS/MS). In the lyophilization 5/replicate, randomly selected within treatment). Dual step, the low molecular weight volatile short chain fatty color immunofluorescence labeling of the target antigen acids (SCFA), acetate, propionate, and butyrate, were dif- and nuclei was performed to localize and subsequently ferentially sublimated off the samples and not available quantify epithelial cell inflammation with NT proteins for analysis. Raw data was extracted, peaks identified, and (red pseudocolored immunofluorescence) as the target QC processed using Metabolon’s hardware and software. antigen along with the respective cell nuclei blue pseu- Data were made available as E xcel files containing val - docolored immunofluorescence). Following standard ues for each identified compound representing an area- deparaffinizing, rehydration, and blocking of nonspe - under the curve which were normalized, per Metabolon, cific antibody binding, slides were incubated overnight at Inc., in terms of raw area counts and presented as the Elsasser et al. Animal Microbiome (2023) 5:6 Page 18 of 22 “Original Scale” (Additional file  9: Data S2). For a single attached to the epithelial cells. After a bead-beating step day run, this was equivalent to the raw data. For compar- on a TissueLyzer II (Qiagen Inc., Germantown, MD), ing diet treatments, each metabolite’s value in Original DNA was extracted from all samples (150 mg tissue wet Scale was mathematically processed to set the median weight) using the MagAttract PowerMicrobiome DNA/ for that metabolite equal to 1 and each animal’s value RNA kit (Qiagen Inc.) implemented on a Hamilton STAR for that metabolite proportionately scaled accordingly. robotic platform (Hamilton, Reno, NV). Amplification These scaled values were referred to “Scaled Imputed of the 16S rRNA gene V4 hypervariable region was per- Value” (SIV). The preliminary statistical ANOVA analy - formed using dual-barcoded universal primers 515F and sis of the data was performed by Metabolon, Inc. with 806R as previously described [92]. High-throughput comparisons and separated effects established using con - sequencing of the amplicons was performed on an Illu- trast statements such that an overall understanding of the mina MiSeq instrument using the 300  bp paired-end sources of variation as affected by diet, colostrum source, protocol. Raw data was demultiplexed as cited [93, 94]. and replication could be assessed. Barcode, adapter, and primer sequences were trimmed using TagCleaner (2013-10-14 vers; [95]). Quality assess- “Anti‑inflammatory index” of the metabolome ment and sequencing error correction was performed A major objective of the study was to define a set of mass using the software package DADA2 (version 1.14; [96]) spectrometry-identified metabolites in the ileal digesta and the following parameters: forward reads were trun- that could facilitate a reduction in epithelial cell inflam - cated at position 220 and the reverse reads at position mation and reflect the metabolite profiles associated with 160 based on the sequencing quality plot, no ambigu- the fed BC or NFDM diets. To accomplish this, we devel- ous bases and a maximum of 2 expected errors per- oped the term “Anti-inflammatory-index” (Ai-i) to serve read were allowed [97]. The quality-trimmed reads were as an integrative parameter intended to reflect the selec - used to infer ribosomal sequence variants and their tion and grouping of a set of specific metabolites that relative abundance in each sample after removing chi- contributed significantly to relieving the magnitude of mera. Sequencing analyses includes denoising, de novo nitration stress present in epithelial cells of the ileum. A and reference-based chimera detection conducted with detailed description of the A-i-i and rationale for devel- UCHIME v5.1 [98]. Taxonomic ranks were assigned to opment is further presented in accompanying on-line each sequence using the Ribosomal Database Project Supplementary Information—Detailed Methods. Briefly, [[99] Naïve Bayesian Classifier v.2.2 [100] trained on the the A-i-i was derived from summing the Scaled Imputed Greengene database (Aug 2013 version) [101]], using 0.8 Values for the metabolites that (a) were defined in the lit - confidence values as cutoff. The heatmap and bar plot erature (Additional file  10: References 1) as having anti- were generated using statistical package R (v3.2.1) and inflammatory character and not simply antioxidative Phyloseq packages [102]. Clustering of taxonomic com- function, (b) anti-inflammatory metabolites that were position and abundance in a sample were performed significantly greater in abundance in one diet compared using Ward linkage hierarchical clustering based on (ANOVA; Proc GLM, SAS ver. 9.4: https:// docum entat Jensen-Shannon divergence metrics. Jensen-Shannon ion. sa s . c om/ do c/ en/ p g msa s c dc/9. 4_3. 3/ st a t ug/ st a t ug_ divergence is a measurement of dissimilarity between glm_ synta x01. htm) to the other diets, and (c) repeatably probability distribution, and Jensen-Shannon metric is different between diets across the two experiment rep - the square root of the normalized Jensen-Shannon diver- lications and the four kinds of colostrum or the NFDM gence value [103, 104]. The resulting clusters were used used in the diet replications. To be included in the final to define microbiota type, which indicates the cluster - multiple regression model set, a given metabolite when ing of similar community compositional profile that is a added to the regression model needed to promote an vector of the percentage of the sequences assigned to a improvement (increase) in the adjusted R of the model phylotype in each sample. Linear Discriminant Analy- as tested for either a linear or exponential fit. sis (LDA) effect size (LEfSe) algorithm was adapted to quantitatively characterize the phylotypes that could Microbial community analyses explain the differences observed between two biological The assessment of the bacterial milieu of the ileum was conditions [105]. It identifies phylotypes in their relative made on individual samples collected from the ileum at abundance profiles through building an LDA model to a point immediately cephalad to the section obtained estimate the “effect size” of each phylotype with respect for metabolite profiling. In addition to digesta samples, to biological conditions under inspections. The alpha one additional sample of the digesta-free epithelial layer value for the non-parametric factorial Kruskal–Wallis of each specimen was carefully scraped from the tissue (KW) sum-rank test [106] is 0.05, the threshold on the segments to address what would be considered microbes logarithmic LDA model [107] score for discriminative Elsasser  et al. Animal Microbiome (2023) 5:6 Page 19 of 22 features is 2.0. We used all-against-all BLAST search in as well as diet combinations on the dependent variables. multi-class analysis that is stricter than the one-against- “Animal-within-treatment” was used as the error term. all search, and we denote both target classes and subject For microbial population inferences, in order to esti- names in the analysis. mate differences in relative abundances of different In order to ascertain if any contribution to the micro- bacterial clusters within different diet status, a Bayes - biota in chickens fed BC might have originated in the ian Poisson model was employed and the structure is as colostrum itself, especially that of the SFB, samples of follows: the BC matrix as applied to the feed was analyzed. The y ∼ Poisson( ) i i metagenomic approach was chosen to assess the micro- biota of the colostrum. Given the low biomass in colos- log ( ) = a + b + c + d trum, applying the PCR-based 16S rRNA on such a i diet(i) cluster(i) diet(i),cluster(i) specimen type may have introduced additional biases. where y is the count in the i-th cell and diet(i), cluster(i) To access the microbiota of the colostrum, the non-pro- are the diet and cluster of the i-th cell, respectively. cessed colostrum was filtered through the sterile 0.22um The model was fitted using JAGS R package [114], and syringe filter (Sterivex, Germany), and the materials left 100,000 iterations with the same number of burn-in on the filter was subjected to 45  s beating cycle using a iterations was used. The convergence of the model was FastPrep instrument (MP Biomedicals) at 5.5  m/s and assessed using Gelman and Rubin’s potential scale reduc- centrifuged at 14,000  rpm for 1  h at 4  °C. The superna - tion factor [115] and visual inspection of each coeffi - tant after beads beating was used for DNA extraction cient’s Markov chains. using the same procedure as above 16S rRNA samples. Metagenomic sequencing libraries were constructed Supplementary Information from the same DNA using the Nextera XT Flex kit (Illu- The online version contains supplementary material available at https:// doi. mina, San Diego, CA) according to the manufacturer’s org/ 10. 1186/ s42523- 023- 00225-z. recommendations. Libraries were then pooled together in equimolar proportions and sequenced on Illumina Additional file 1. Figure S1: Within-sample diversity by community type. Notched boxplot of diversity index of samples grouped in community HiSeq 4000 platform using the 150  bp protocol at the type (color indicated in legend). Within-sample diversity was estimated Genomic Resource Center at the University of Mary- using Shannon diversity index using Phyloseq R package [102]. The top land School of Medicine. Metagenomic sequence reads and bottom of the box are the lower and upper quartiles, and the band near the middle of the box represents the median. Box width is propor- were removed using BMTagger v3.101 [108] using a tional to the square root of the size of the category. Genome Reference Consortium Gallus gallus Build 5.0 Additional file 2. Figure S2: Statistical analyses of relative frequency of (GCA_000002315.3, [109]). Sequence read pairs were community types. The mean relative frequencies and their 95% credible removed even if only one of the reads matched to the intervals are shown. A Bayesian Poisson model was employed, and model fitting was performed using JAGS R package [114] and 100,000 iterations genome reference. Taxonomic profiling was conducted in with the same number of burn in iterations. Abbr: DUO: duodenum; CEC: MetaPhlAn vers 2 [110]. We specifically performed reads cecum; ILE: ileum. mapping of the metagenomic reads to published genomes Additional file 3. Figure S3: Community diversity analyses using of Candidatus Arthromitus isolated from chicken, turkey, weighted UniFrac principal coordinates analysis (PCoA) analyses using mouse, and rat (NZ_CP008713.1, NC_015913.1, NZ_ QIIME2 (v2019.10) [117]. Each symbol represents a sample colored by its different anatomical sites. The scatterplot is of principal coordinate 1 (PC1) AGAG01000005.1, NZ_AGVP01000010.1, NC_017294.1, plotting against principal coordinate 2 (PC2), and the percentage of the NC_016012.1, LXFF01000001.1) using bowtie [v1, variation described by the plotted principal coordinates in indicated on parameters: “-l 25-fullref -chunkmbs 512 -best -strata -m the axes. 20”, [111]]. Additional file 4. Figure S4: Relative abundance of phylotype biomark - ers. Analyses were performed using program LEfSe [105]. Bars represent the relative abundance of a phylotype in each sample. Dotted line repre- sents mean, solid line represents median relative abundance. The alpha Statistical analyses value for the non-parametric factorial Kruskal-Wallis (KW ) sum-rank test was 0.05 and the threshold for the logarithmic LDA model ([107]) score for The basic statistical analysis for the effects of diet treat - discriminative features was set at 2.0. Abbr: DUO: duodenum; CEC: cecum; ment on growth, feed efficiency, morphometric measure - ILE: ileum. ments, NT pixel content, and the assessment of specific Additional file 5. Table S1: Summary of the identified plant-derived metabolites on an anti-inflammatory index was analysis xenobiotic compounds in digests of the duodenum, ileum and cecum. Data illustrate the nature of the changes in presence/detention and rela- of variance performed using the General Linear Mod- ® tive levels of xenobiotic plant-derived compounds in the relevant digesta els procedure of the Statistical Analysis System ver. 9.4 as affected by diet. Digesta from chickens fed BC was more complex with [112]. Specific contrast statements [113] were used to more compounds present in the ileal digesta. test hypotheses regarding the effects of the various diets Additional file 6. Table S2: Taxonomic profiles of the intraluminal stool specimens collected in this study. Intraluminal stool specimens collected Elsasser et al. Animal Microbiome (2023) 5:6 Page 20 of 22 5. NRC, Council NR, Panel on Animal Health FS, Public H, Committee from A) different anatomical sites of ileum, duodenum and cecum for a on Drug Use in Food A, Institute of M, et al. The use of drugs in food total of 156 samples; B) only ileum. The taxonomic composition of the animals: benefits and risks. Washington: National Academy Press; 1999. microbiomes was established using 16S rRNA gene V4 hypervariable 6. Culic O, Erakovic V, Parnham MJ. Anti-inflammatory effects of macrolide region and dual-barcoded universal primers 515F and 806R as previously antibiotics. Eur J Pharmacol. 2001;429(1–3):209–29. described [92]. 7. Krakauer T, Buckley M. Doxycycline is anti-inflammatory and inhibits staphylococcal exotoxin-induced cytokines and chemokines. Antimi- Additional file 7. Table S3: Taxonomic profiles of the colostrum crob Agents Chemother. 2003;47(11):3630–3. specimens. Two non-processed colostrum was included in this study. 8. Conlon MA, Bird AR. The impact of diet and lifestyle on gut microbiota The taxonomic composition of the microbiomes was established using and human health. Nutrients. 2014;7(1):17–44. MetaPhlAn version 2 [110]. 9. Ercolini D, Fogliano V. Food design to feed the human gut microbiota. 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Journal

Animal MicrobiomeSpringer Journals

Published: Jan 26, 2023

Keywords: Anti-inflammation; Colostrum; Ileum; Immunometabolism; Metabolome; Gut microbiome; Protein tyrosine nitration

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