EP4622471A1 - Bovine colostrum-derived nutritional supplements and uses thereof - Google Patents

Abstract

Provided herein are nutritional supplements of a plurality of metabolite compounds effective as anti-inflammatories in an animal gut for an animal feed and the animal feed and animal food products containing the same and nutritional supplements formulated as a mix¬ ture of gut-associated metabolites. Also provided are methods for increasing growth of a food animal or a farm animal, improving a gut microenvironment in an animal and increasing an anti-inflammatory response in an animal that utilize the supplemented animal feed and animal food products and a method for improving gut health in an animal that is administered or takes the nutritional supplement formulation.

Description

BOVINE COLOSTRUM-DERIVED NUTRITIONAL SUPPLEMENTS AND USES THEREOF

Cross-Reference to Related Applications

This international application claims benefit of priority under 35 U.S.C. §119(e) of provisional application U.S. Serial No. 63/427,335, filed November 22, 2022, the entirety of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to the fields of animal science and animal nutrition. More particularly, the present invention relates to a dietary and nutritional supplements deduced from the effects of consumption of a refined bovine colostrum by production animals.

Description of the Related Art

The One Health concept (1 ) recognizes the interdependence between food animal and human health and includes such issues as nutrition, appropriateness of antibiotic use, zoonotic pathogen transfer, the emergence of antimicrobial resistance and alternatives to antimicrobial drug therapies. More specifically, it recognizes the commonality of physiological systems of and interdependence between food animal health and human health (2).

For decades, antibiotic drugs (ABDs) were administered to animals in their food for the purpose of enhancing their growth (3-5). This practice has possible links to increased incidence of antimicrobial resistance, a major public health concern. Although the mechanisms by which antibiotic drugs impact growth have been debated, the diminution of the local inflammatory stress in the gut was attributed to the now recognized non-antimicrobial, antiinflammatory effects of select antibiotic drugs, particularly in the tetracycline/doxycy- cline/minocycline and macrolide families (4-8). Much of the diminution of local inflammatory stress in the gut was coincident with the improved efficiency with which feed was channeled into tissue deposition in the antibiotic drug-fed animals (6,7). Through legislative actions, the subtherapeutic level use of antibiotic drugs for growth promotion in animals has been restricted thus requiring other strategies for promoting growth via reduced gut inflammation in food animals (3-5) as highlighted by the National Research Council (5). Over the last ten years, the traditional concept of “nutrition” has evolved from feeding the body to now include feeding the microbes that inhabit microenvironments and niches throughout the digestive tract (8,9). The nuances of nutrition include not only the composition of the foods and supplements ingested and the way the digestive system processes these nutrients, but also must address the interactions of the gut microbiota with both the ingested nutrients and the host’s gastrointestinal cells. The importance of the interaction between the microbes and the host cells, and the nutrient-derived metabolites that function as signaling molecules between the two ’’compartments” is encompassed in the term “immunometabolism” (10,11 ). A regulatory model of immunometabolism suggests that it occurs within the interactive relationships among endocrine, immune, metabolic, and microbial inputs that results in the preferential partitioning of nutrients to different tissue beds during times of health or stress (11 ).

Mammals “jump-start” the immune system of their offspring at birth by providing the nutrient-rich and physiologically stabilizing first milk, the colostrum. Colostrum not only contains fat, protein, mineral, and vitamin nutrients, but also immunoglobulins, antimicrobial peptides, hormones, oligosaccharides, and a plethora of small molecules derived from the diet and physiological processes of the mother. Bovine colostrum is rich in Bifidobacterium and Lactobacillus phylotypes and this combination of compound constituents along with the beneficial bacteria promotes gut maturation, mucosal integrity and tissue repair (12) with these beneficial effects attributed to these components being transferred to the young in their initial suckling. The composition and volume of colostrum rapidly changes within days following parturition, as milk production evolves over time (13).

To date, the vast majority of studies that concern the beneficial effects of colostrum have examined either aspects of neonatal organ maturation or the ability to thrive in the ex- trauterine environment (14). Other studies have explored the potential for vaccine-induced antibodies in colostrum (through immunization of cows prior to parturition) to mitigate complications of parasitic and bacterial gut infections (15-18). Few studies have comprehensively examined the effects of colostrum supplementation on gut health with the simultaneous assessment of changes in a whole-body parameter such as feed use efficiency, impact on the gut microbiome, patterns of intestinal digesta-associated anti-inflammatory metabolites, and host gut cell responses. More specifically, few studies, if any, have assessed and defined what metabolites and conditions in the gut are favorably altered by the consumption of colostrum. There is a recognized need in the art for such an assessment.

The prior art is deficient in non-antibiotic supplements with defined molecular components for animal feeds that improve gut health in food animals. The present invention fulfills this long-standing need and desire in the art. SUMMARY OF THE INVENTION

The present invention is directed to a nutritional supplement for an animal feed. The nutritional supplement comprises a plurality of metabolite compounds effective as anti-inflammatories in an animal gut. The present invention is directed to a related nutritional supplement that further comprises a mixed tocopherol oil of gamma-tocopherol and alpha-tocopherol or eicosanoic acid or a conjugated analog thereof.

The present invention also is directed to a method for improving a gut microenvironment in an animal. In this method, food for the animal is supplemented with the nutritional supplement described herein. The animal is fed a diet comprising the supplemented food over a period of time sufficient to cause an improvement in the gut microenvironment.

The present invention is directed further to an animal feed. The animal feed comprises the nutritional supplement described herein.

The present invention is directed further still to a method for increasing growth of a food animal or a farm animal. In this method, the food animal or the farm animal is fed a diet comprising the antibiotic-free animal feed described herein.

The present invention is directed further still to an animal food product supplemented with a defatted bovine colostrum. The present invention is directed to a related animal food product that is further supplemented with at least one of gamma-tocopherol or alpha tocopherol or a mixed tocopherol oil thereof, eicosanoic acid or a conjugated analog thereof.

The present invention is directed further still to a method for increasing an anti-inflammatory response in an animal. In this method, the animal is fed the animal food product described herein, whereby the defatted bovine colostrum therein produces an increase in antiinflammatory metabolites and beneficial bacteria in the gut microbiome.

The present invention is directed further still to a nutritional supplement formulated as a mixture of gut-associated metabolites. The present invention is directed to a related nutritional supplement that further comprises gamma-tocopherol or alpha tocopherol or a mixed tocopherol oil thereof, eicosanoic acid or a conjugated analog thereof or a combination thereof.

The present invention is directed further still to a method for improving gut health in an animal. In this method, an amount of the nutritional supplement formulated as a mixture of gut-associated metabolites effective to decrease inflammation and to increase beneficial bacteria in the gut is administered to the animal.

Other and further aspects, features, benefits, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure. BRIEF DESCRIPTION OF THE FIGURES

The appended drawings have been included herein so that the above-recited features, advantages and objects of the invention will become clear and can be understood in detail. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and should not be considered to limit the scope of the invention.

FIG. 1 shows that a bovine colostrum-supplemented diet improved feed efficiency. Mean body weight gain ± SEM (grams/day) and feed efficiency (grams feed consumed per gram body weight gained) in chickens fed the base diet (Control, n=10) or diets supplemented with either nonfat dry milk (NFDM, a milk product comparison control diet matrix, n=10) or a processed bovine colostrum preparation (BC, n=20). Intestinal measurements indicate significant biological impacts of the BC diet on gut morphology.

FIGS. 2A-2D show that the bovine colostrum-supplemented diet increases the villus surface abundance and the effects of inclusion of nonfat dry milk 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 (FIG. 2A) or the calculated area of the villi as associated with the diet treatments fed to the chickens (FIG. 2B), or the ratio of the villus height to crypt depth (FIG. 2C). FIG. 2D depicts representative images of the diet-associated ileal architecture. (Control, basal diet, n=6. nonfat dry milk. n=6. bovine colostrum, n=10). For reference, “V” = villi, “C” = crypts, and “SM” = submucosa.

FIGS. 3A-3J show decreased protein tyrosine nitration (NT), equating to a decrease in inflammation, in epithelial cells of villi in samples of ileum from chickens fed a bovine colostrum-supplemented diet. FIGS. 3A-3C show colocalization of signals from anti-cytokeratin-18 (anti-cytokerati 18 epithelial cell marker) with anti-3’-nitrotyrosine. FIGS. 3D-3F show an isolated signal for anti-3’-nitrotyrosine as the marker for nitrated proteins, NT. FIGS. 3G-3I are image analysis of epithelia-specified nitrated proteins pixels outlined areas of interest (AOI) for analysis. Immunofluorescence is presented in FIGS. 3A-3F and image analysis depicted in FIGS. 3G-3L Epithelial cell presence (anti-cytokeratin 18 epithelial cell marker) is indicated by green fluorescence pixels (FIGS. 3A, 3C). Nitrated proteins, as detected using an anti- nitrotyrosine antibody marker, is presented as red (cell presence, FIGS. 3B and 3D-3F, for isolated single channel image analysis quantification) or orange (colocalized in green cy- tokeratin 18-positive cells, FIG. 3C). Representative patterns of image analysis-selected nitrotyrosine pixels (FIGS. 3G-3I, yellow pixels) were summated and processed to yield the statistical analysis summarized in the lower bar graph (FIG. 3J). Values are group means ± SEM for Control (n=6), NFDM (n=6), and BC (n=10). FIGS. 4A-4C show correlation between anti-inflammatory index (A-i-i, the mass spectroscopic metabolome estimate of the abundance of anti-inflammatory metabolites in the gut as associated with consumption of the different test diets) and epithelial cell protein nitration (NT). Multiple regression was used to assess the statistical correlative relationship between the level of nitrated proteins (Log10, pixels/cell, the regression dependent variable) present in intestinal epithelial cells and the A-i-l (the regression independent variables). Inclusion of a metabolite (class) to the model was valid if the resulting adjusted R² of the regression analysis was increased over that of the prior iterative assessments (FIG. 4A). The effects of dietary treatment on the A-i-i (the sum of the Scaled Imputed Values (i.e. , the mass spectroscopic analysis-derived data) of the individual metabolites retained in the model, FIGS. 4A-4B). Linear regression of the nitrated proteins pixel content on the A-i-i suggested a significant negative correlation where the greater the A-i-i- the lower the nitration stress (FIG. 4C).

FIGS. 5A-5H show chicken intestinal microbiota analyses. FIG. 5A is a heatmap of the 50 most abundant bacterial 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 R package (23). Identified microbiota types are labeled as l-V. FIG. 5B is a distribution of different community type by cluster as shown in FIG. 5A 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 bovine colostrum and NFDM. Type II is borderline significantly higher in bovine colostrum and NFDM than control, based on statistical modeling using Bayesian Poisson model. FIGS. 5C-5H show phylotype biomarkers for digesta (FIGS. 5C, 5E, 5G) and scrapings (FIGS. 5D, 5F, 5H) for Candidates Arthromitus (FIGS. 5C-5D), Streptococcus (FIGS. 5E-5F) and Bifidobacterium (FIGS. 5G-5H) generated using program LEfSe. 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 score for discriminative features was set at 2.0

FIGS. 6A-6B show that Candidates Arthromitus (a key segmented filamentous bacteria (SFB) positively affecting gut immune function) is present in the ileal scraping and increased by bovine colostrum. Mean (± SEM) abundance of Candidates Arthromitus represented as Log10 (OTU, operational taxonomic units, i.e., an estimate of bacterial numbers) in the three different segments of the gut and further differentiated by digesta vs. scraping (FIG. 6A). Mean (± SEM) abundance of Candidates Arthromitus represented as Log10 (OTU) in the ileum as measured in digesta or cell scrapings (attached) as affected by diet. *, P<0.04 (FIG. 6B). FIGS. 7A-7B show sialyllactose (protective diet-derived oligosaccharides) content of ileal digesta is correlated with presence of segmented filamentous bacteria. 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 from chickens fed the three diets (FIG. 7A). Summated 3’-sialyllactose and 6'-sialyllactose values were regressed on the Log10 (OTU of the segmented filamentous bacteria) yielding a significant positive linear correlation (FIG. 7B).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the invention. It is contemplated that any method described herein can be implemented with respect to any other method described herein.

As used herein, the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”

As used herein, “comprise” and its variations, such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated item, element or step or group of items, elements or steps but not the exclusion of any other item, element or step or group of items, elements or steps unless the context requires otherwise. Similarly, "another" or “other” may mean at least a second or more of the same or different claim element or components thereof.

As used herein, “animal” refers to, but is not limited to, food animals, farm animals, sport animals, companion animals, service animals, or a human.

As used herein, “animal feed” and “animal food product” refer to foods and food products standard for any of the animals as defined herein.

As used herein, “nutritional supplement” is interchangeable with “dietary supplement”.

In one embodiment of the present invention, there is provided a nutritional supplement for an animal feed, comprising a plurality of metabolite compounds effective as anti-inflammatories in an animal gut. Further to this embodiment the nutritional supplement may comprise a mixed tocopherol oil of gamma-tocopherol and alpha-tocopherol or eicosanoic acid or a conjugated analog thereof. In both embodiments the plurality of metabolite compounds may be indolelactic acid, apigenin, diadzein, genestein, naringinin, chenodeoxycholic acid, 7- ketolithocholic acid, glycine, A/-acetylglycine, y-glutamylcysteine, glutathione, 3’-sialyllactose, 6’-sialyllactose, and itaconate.

In another embodiment of the present invention, there is provided a method for improving a gut microenvironment in an animal, comprising supplementing food for the animal with the nutritional supplement as described supra; and feeding the animal a diet comprising the supplemented food over a period of time sufficient to effect an improvement in the gut microenvironment.

In this embodiment, the supplemented food may be antibiotic-free. Also in this embodiment, the animal may be a food animal or a farm animal. In addition, the supplemented food may produce an increase in immune function-modifying segmented filamentous bacteria (SFB or Candidates Arthromitus), a decrease in toxicity-associated nitrated proteins or an increase in gut absorptive surface characteristics or a combination thereof.

In yet another embodiment of the present invention, there is provided an animal feed comprising the nutritional supplement, as described supra. In an aspect of this embodiment, the animal feed may be an antibiotic-free food.

In yet another embodiment of the present invention, there is provided a method for increasing growth of a food animal or a farm animal, comprising feeding the food animal or the farm animal a diet comprising the antibiotic-free food, as described supra. In this embodiment, efficiency of nutrient use may be increased in the food animal or the farm animal fed the diet.

In yet another embodiment of the present invention, there is provided an animal food product supplemented with a defatted bovine colostrum. Further to this embodiment the animal food product may be supplemented with at least one of gamma-tocopherol or alpha tocopherol or a mixed tocopherol oil thereof, eicosanoic acid or a conjugated analog thereof. In an aspect of both embodiments, the animal food product may be antibiotic-free.

In yet another embodiment of the present invention, there is provided a method for increasing an anti-inflammatory response in an animal, comprising feeding the animal the animal food product, as described supra, where the defatted bovine colostrum therein produces an increase in anti-inflammatory metabolites and beneficial bacteria in the gut microbiome.

In an aspect of this embodiment, the animal may be a food animal or a farm animal and where the animal food product is antibiotic-free. In this embodiment and aspect thereof, the beneficial bacteria may be immune function-modifying segmented filamentous bacteria. Further in this embodiment the animal may be a food animal, a farm animal, a sport animal, a companion animal, a service animal, or a human. In yet another embodiment of the present invention, there is provided a nutritional supplement formulated as a mixture of gut-associated metabolites. In this embodiment, the nutritional supplement may be indolelactic acid, apigenin, diadzein, genestein, naringinin, che- nodeoxycholic acid, 7-ketolithocholic acid, glycine, A/-acetylglycine, y-glutamylcysteine, glutathione, 3’-sialyllactose, 6’-sialyllactose, and itaconate. In this embodiment, the gut-associated metabolites further may comprise gamma-tocopherol or alpha tocopherol or a mixed tocopherol oil thereof, eicosanoic acid or a conjugated analog thereof or a combination thereof. In these embodiments, each of the gut-associated metabolites in the mixture may have a content dose based on a metabolic body weight (BW) percentage of a recipient animal of BW⁰⁷⁵.

In yet another embodiment of the present invention, there is provided a pharmaceutical composition comprising the nutritional supplement as described supra and a pharmaceutically acceptable carrier.

In yet another embodiment of the present invention, there is provided a method for improving gut health in an animal, comprising administering to the animal an amount of the nutritional supplement as described supra effective to decrease inflammation and to increase beneficial bacteria in the gut. In this embodiment the beneficial bacteria may be immune function-modifying segmented filamentous bacteria. Also in this embodiment the animal is a food animal, a farm animal, a sport animal, a companion animal, a service animal, or a human.

Provided herein is a nutritional supplement or dietary supplement that comprises a plurality, i.e., all or varying numbers, of a specific colostrum diet-derived metabolite compounds identified from a metabolomic survey of compounds generated in the gut after digestion of a defatted and lyophilized bovine colostrum that are beneficial or effective towards decreasing gut inflammation. The metabolic survey may be performed via mass spectroscopic analysis of the gut digesta and statistical regression analysis. Also provided are the animal food, animal feed or food products comprising the same. It is contemplated that the nutritional supplement or dietary supplement may be useful in the practice of veterinary medicine or may be formulated for human use.

When fed to an animal bovine colostrum produces an increase in the anti-inflammatory index in the gut digesta, an increased abundance of the immune function-modifying segmented filamentous bacteria (Candidates Arthromitus) attached to the ileum, a decreased abundance of nitrated proteins associated with toxicity, and an increase in the numbers of villi per unit of gut, i.e., improved gut absorptive surface characteristics.

Also provided is an anti-inflammatory formulation comprising a matrix of specific metabolites, such as a mixture of gut-associated metabolites. Each of the gut-associated metabolites in the mixture has a content dose or live body weight (BW) percentage based on the metabolic body weight of an animal where metabolic body weight is traditionally calculated as a mathematical exponent of the live BW, i.e., BW⁰⁷⁵ for a recipient animal. These metabolites increased consistently in the gut lumen digesta with an animal’s consumption of the fractionated bovine colostrum and correlated very well with the pattern(s) of observed mitigation of inflammation, improvement in feed efficiency for growth, and importantly a shift in microbe abundances with beneficial impact on immune function and gut health. The reduction of inflammation in the presence of nitrated proteins in gut cells is considered a biomarker of inflammation more sensitive to inflammatory stimuli than other conventional oxidation markers.

The fractionated bovine colostrum utilized herein is produced by first centrifuging the bovine colostrum to produce a top layer of lipids, a middle layer of the desired bovine colostrum and a bottom layer of pelleted solids and cellular debris. Next the middle layer of bovine colostrum is separated from the top layer and the bottom layer to defat the bovine colostrum. The retained middle layer is lyophilyzed to produce the fractionated bovine colostrum.

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.

EXAMPLE 1

Materials and methods

Animal model

The rapidly growing commercial meat chicken (Gallus gallus, Strain: Ross 708, Amick Farms Hatchery, Hurlock, MD) was used for the nutrition, inflammation, metabolome, and microbiome objectives. Genetic selection for characteristics of allometric growth result in a tremendous accretion of ingested nutrients in lean muscle deposition largely associated with breast and leg tissue. The rapidity of growth (<49 days from hatch to commercial processing; (>100 g body weight (BW) gain/day) and high rate of nutrient utilization for lean tissue accretion (< 1.7 kg feed to attain 1 kg increase in body weight) make these animals exquisitely sensitive to perturbations in environmental conditions, including those of diet (80,81 ) and require stringent controls on environment and feed (82). For these chickens, it is recognized that the growth process dynamics and feed efficiency as well as changes in the gut microbiome are highly volatile and change over time in as little as 1 week, essentially as the animals mature (83,84) With this awareness, what was consistently affected by the presented dietary treatments and what metabolite and gut microbiota attributes of the treatment diets might mitigate low level gut inflammation as specifically addressed in terms of nitrooxidative stress, i.e., the abundance of tyrosine nitrated proteins in the gut (26,27) was assessed. Experimental treatments: diets supplementation with colostrum extract or nonfat dry milk

A total of four colostrum samples (4 L each) were collected from multiparous Jersey (n=2) or Holstein (n=2) dairy cows as first and second milkings after parturition, respectively. In accordance with traditional feeding for cows at parturition and early lactation, cows were fed a total mixed ration consisting of corn and grass silages supplemented with roasted soy bean meal, vitamins and minerals. As such, the colostrum from the cows would be naturally enriched with a spectrum of flavones and isoflavones, etc. derived from the plant matrix of the diet. Each collected colostrum was frozen at -80°C until needed for diet preparation.

To prepare the colostrum-containing diet, the following procedures were performed. The thawed, cold colostrum from one cow at a time was thoroughly mixed, aliquoted into eight 50 ml polycarbonate tubes and centrifuged at 2°C for 1 hour at 20,000 x g, centrifugation conditions sufficient to congeal lipids and sediment bacteria at the top and bottom of the tube, respectively. The fat/lipid content of the different colostrums was not different as a function of cow breed or day obtained. After centrifugation, the solidified lipid (nonaqueous long chain fatty acid component) was removed from the top of the samples and the liquid underneath constituting the middle third of the tube volume poured off and collected. Higher density sedimented material in the bottom third of the centrifuge tubes containing cells, bacteria and cell and membrane debris was discarded. The fluid from each of the four cow’s colostrum was pooled across tubes but maintained as separate pools for the generation of the respective bovine colostrum-supplemented diets. After the pooling, the material was passed through glass fiber matt filters. Collected material was sprayed onto a base diet previously used for chicken experiments (corn-soybean meal base; 23 crude protein and 13 MJ metabolizable energy (26) to supply the equivalent of 100 ml colostrum/kg feed, with continuous mixing. The damp diet was put into 4 L glass high-vacuum containers, frozen at -80° C, lyophilized to dryness, and fed as such.

To assess the effect of the addition of this quantity of colostrum to the overall crude protein and energy content of the base diet, a test batch of each of the bovine colostrum diets was formulated and sent out for nutritional analysis (Cumberland Valley Analytical Services, Mercersburg, PA). Following analysis, it was observed that bovine colostrum diets were approximately 2.7% higher in crude protein but otherwise (total fat, gross energy, fiber, etc.) not different from the base diet. To correct for the protein difference, the base diet to be used to prepare the bovine colostrum was modified by lowering the soybean-derived protein content by 2.7% and that protein difference then made up for with the inclusion of the bovine colostrum. When the colostrum component was added to this lower protein base diet, reanalysis of the diets showed them to be equivalent in protein. A third diet was developed, analyzed and formulated to more closely approximate the amino acid composition of the bovine colostrum diets and for this purpose nonfat dry milk (NFDM, Carnation, Inc.) was added to the lower crude protein base.

Study design

All research was conducted under an animal management and welfare protocol approved by the USDA Beltsville Institutional Animal Care and Use Committee. One day old chicks were transported from the hatchery to the USDA Poultry Research Facility (Beltsville, MD) and placed in group brooder housing with the environmental temperature set according to the age of the animal. The CON feed and water were provided ad libitum. At 15 days of age, chickens were moved from the group pens to individual pens where the daily feed intake for each animal could be accurately measured. The abrupt change in local environment and housing as well as separation from a group is recognized as a short-term, low-level stress situation impacting the hypothalamic-pituitary-adrenal axis with impact on gastrointestinal function in the chicken (85,86). The quantity of fresh diet given to each chicken daily was recorded. The next morning the residual feed not eaten from the previous day was measured and the difference between given and residual used as the measure of feed intake. Live body weight of each chicken was obtained daily at a standardized time of day relative to feed management. Chickens and their environment were observed three times daily as regards the health and welfare of the animals per approved animal care protocol.

The overall experiment was conducted in 2 separate replicates of the following treatments: control diet (CON), nonfat dry milk diet (NFDM), and bovine colostrum diets (BC), the replications conducted six months apart. Colostrum was obtained from four different sources; sources consisted of Jersey cow first postpartum milking, Jersey cow second postpartum milking, Holstein cow first postpartum milking, and Holstein cow second postpartum milking. Variability in colostrum composition (day 1 and day 2 of lactation postpartum and Jersey and Holstein breeds) was intentionally built into the design so that across the variability a set of metabolites with consistent effects on inflammation and gut parameters could be ascertained. In preparing the diets for the first replicate of the study, one bovine colostrum diet was prepared using Jersey-derived first milking colostrum and the other bovine colostrum diet prepared from the Holstein-derived first milking. Similarly, in the second replication the two bovine colostrum diets were made from the Jersey and Holstein colostrum second milkings.

Transfer of the chickens to the individual pens and the respective switches to the test diets were considered the first day of the start of the experiment. Test diets were fed for 7 days with the animals euthanized (per American Veterinary Medical Association Guidelines for Euthanasia of Animals: 2013 Edition), on day 8 following the recording of the final weight. As pooled across the two experimental replicates, there were 11 CON diet animals, 11 NFDM diet animals and a total of 22 animals fed colostrum-containing diets, the data from which animals was used for the metabolomic and microbiome analysis. Variation in the n across the various diet treatment groups reflects further refinement based on the quality of data primarily obtained from the metabolomic evaluations. When an animal’s metabolite data were identified as an outlier (> 3 S.D.) or compromised as a result of an inefficient extraction, that animal was eliminated from the study. The numbers of animals per treatment group from which the data were amassed exceeded the minimum number for achieving statistical significance based on statistical power analysis (Introduction to Power and Sample Size Analysis (113)). Samples prepared for immunofluorescence were obtained from representative numbers of animals in each replication of the experiment.

Following euthanasia, the intestinal tract was rapidly excised and divided into segment samples for duodenum (central loop), distal ileum, and cecum (with focus on the ileum). These samples were further subdivided into 2 components. Each segment was opened longitudinally. From one, the digesta was carefully removed to eliminate intestinal tissue potentially scraped from the wall. The other section was rinsed free of remaining digesta and debris with ice cold PBS and the epithelial surface gently scraped away using a fresh, chilled glass slide. One additional section of rinsed intact gut was immersed overnight in 4% paraformaldehyde and transferred to ethanol for further paraffin embedding and sectioning for microscopy as subsequently described. To evaluate the live growth of the chickens, the average daily body weight gain and feed efficiency (the grams of ingested feed corresponding to the grams of weight gained per day) was measured.

Fluorescence microscopy and quantitative imaging analyses

The detailed methods for the immunofluorescent tissue antigen immunostaining as well as the exact process used to quantify the cellular pixel content of the 3’nitrotyrosine antigen used to represent the intensity of nitrooxidative stress are well known in the art. The protocols for tissue fixation, fluorescence immunostaining for 3’-tyrosine-nitrated proteins (NT) as the inflammation marker and morphometric analysis of ileal samples published previously were applied herein (26,33). In brief, representative tissue sections for immunofluorescence were collected from the first and second replicates of the experiment (CON, n=6, 3/replicate; NFDM, n=6, 3/replicate; BC, N=10, 5/replicate, randomly selected within treatment). Dual color immunofluorescence labeling of the target antigen and nuclei was performed to localize and subsequently quantify epithelial cell inflammation with NT proteins (red pseudocolored immunofluorescence) as the target antigen along with the respective cell nuclei blue pseudocolored immunofluorescence). Following standard deparaffinizing, rehydration, and blocking of nonspecific antibody binding, slides were incubated overnight at 4 °C in a humidified chamber with anti-3’-nitrotyrosine (rabbit polyclonal, Millepore-Sigma, Burlington, MA, Inc., 1 :100) with subsequent antigen visualization (goat anti-rabbit IgG Alexa 680 (1 :400; 1 h, Thermofisher Scientific, Grand Island, NY)). For validation purposes, an additional set of slides of the adjacent serial sections were prepared and immunostained to identify villi apical epithelial cells using anti-cytokeratin-18 (Abeam Inc., Cambridge, MA, mouse monoclonal, Clone-C04, 1 :200) with antigen visualization (goat anti-mouse IgG Alexa 488, 1 :400, Thermofisher Scientific, Grand Island, NY; green pseudocolored immunofluorescence). Nuclei were stained with 4-6-diamino-2-phenylindol-dihydrochloride (DAPI, Thermofisher Scientific). Morphometric measurements to determine the length relationships between the villi and crypts were performed using the DAPI blue channel using a calibrated and validated digital micrometer internal to the ImagePro 9.3® software (Media Cybernetics, Rockville, MD). Quantitative image analysis to ascertain the pixel density of the NT antigen per villus epithelial cell was performed using ImagePro 9.3® software (Media Cybernetics, Rockville, MD).

Metabolome profiling and analyses

Samples of digesta were collected from each of the animals in both replicates of the study (CON, n=10; NFDM, n=10; BC, n=20, with 4 samples failing to be extracted efficiently and the results excluded) from a region of the ileum 3 to 5 cm cephalad to the ileocecal junction. For the metabolomic profiling of the digesta, a homogenous sample (approximately 100 mg) was accurately weighed to the 0.1 mg into a polypropylene vial, with the vial capped and frozen in liquid nitrogen. Metabolite identification and quantification was performed under contract by Metabolon, Inc., Research Triangle Park, NC, and all steps for this analysis were conducted according to the company’s protocols. In brief, the basic process was as follows: Metabolon’s first preparation step was to lyophilize the weighed samples and then reconstitute the samples for analysis by ultrahigh performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS). In the lyophilization step, the low molecular weight volatile short chain fatty acids (SCFA), acetate, propionate, and butyrate, were differentially sublimated off the samples and not available for analysis. Raw data was extracted, peaks identified, and QC processed using Metabolon’s hardware and software. Data containing values for each identified compound representing an area-under the curve were normalized, per Metabolon, Inc., in terms of raw area counts and presented as the “Original Scale”. For a single day run, this was equivalent to the raw data. For comparing diet treatments, each metabolite’s value in Original Scale was mathematically processed to set the median for that metabolite equal to 1 and each animal’s value for that metabolite proportionately scaled accordingly. These scaled values were referred to as Scaled Imputed Value (SIV). The preliminary statistical ANOVA analysis of the data was performed by Metabolon, Inc. with comparisons and separated effects established using contrast statements such that an overall understanding of the sources of variation as affected by diet, colostrum source, and replication could be assessed.

Anti-inflammatory Index of the metabolome

A major objective of the study was to define a set of mass spectrometry-identified metabolites in the ileal digesta that could facilitate a reduction in epithelial cell inflammation and reflect the metabolite profiles associated with the fed bovine colostrum or nonfat dry milk diets. To accomplish this, the term Anti-inflammatory-index (Ai-i) was developed to serve as an integrative parameter intended to reflect the selection and grouping of a set of specific metabolites that contributed significantly to relieving the magnitude of nitration stress present in epithelial cells of the ileum. Briefly, the anti-inflammatory-index was derived from summing the Scaled Imputed Values for the metabolites that (a) were defined in the literature as having anti-inflammatory character and not simply antioxidative function, (b) anti-inflammatory metabolites that were significantly greater in abundance in one diet compared (ANOVA; Proc GLM, SAS® ver. 9.4: documentation. sas.com/doc/en/pgmsascdc/9.4_3.3/statug/statug_glm syntaxO1.htm) to the other diets, and (c) repeatably different between diets across the two experiment replications and the four kinds of colostrum or the NFDM used in the diet replications. To be included in the final multiple regression model set, a given metabolite when added to the regression model needed to promote an improvement (increase) in the adjusted R² of the model as tested for either a linear or exponential fit.

Microbial community analysis

The assessment of the bacterial milieu of the ileum was made on individual samples collected from the ileum at a point immediately cephalad to the section obtained for metabolite profiling. In addition to digesta samples, one additional sample of the digesta-free epithelial layer of each specimen was carefully scraped from the tissue segments to address what would be considered microbes attached to the epithelial cells. After a bead-beating step on a Tis- sueLyzer II (Qiagen Inc., Germantown, MD), DNA was extracted from all samples (150 mg tissue wet weight) using the MagAttract PowerMicrobiome DNA/RNA kit (Qiagen Inc.) implemented on a Hamilton STAR robotic platform (Hamilton, Reno, NV). Amplification of the 16S rRNA gene V4 hypervariable region was performed using dual-barcoded universal primers 515F and 806R as previously described (87). High-throughput sequencing of the amplicons was performed on an Illumina MiSeq instrument using the 300 bp paired-end protocol. Raw data was demultiplexed as cited (88,89). Barcode, adapter, and primer sequences were trimmed using TagCleaner (2013-10-14 vers (90)). Quality assessment and sequencing error correction was performed using the software package DADA2 (version 1.14; (91 )) and the following parameters: forward reads were truncated at position 220 and the reverse reads at position 160 based on the sequencing quality plot, no ambiguous bases and a maximum of 2 expected errors per-read were allowed

(92). The quality-trimmed reads were used to infer ribosomal sequence variants and their relative abundance in each sample after removing chimera. Sequencing analyses includes denoising, de novo and reference-based chimera detection conducted with UCHIME v5.1

(93). Taxonomic ranks were assigned to each sequence using the Ribosomal Database Project (94) Naive Bayesian Classifier v.2.2 (94) trained on the Greengene database (Aug 2013 version) (96), using 0.8 confidence values as cutoff. The heatmap and bar plot were generated using statistical package R (v3.2.1 ) and Phyloseq packages (97). Clustering of taxonomic composition and abundance in a sample were performed using Ward linkage hierarchical clustering based on Jensen-Shannon divergence metrics. Jensen-Shannon divergence is a measurement of dissimilarity between probability distribution, and Jensen-Shannon metric is the square root of the normalized Jensen-Shannon divergence value (98,99).

The resulting clusters were used to define microbiota type, which indicates the clustering of similar community compositional profile that is a vector of the percentage of the sequences assigned to a phylotype in each sample. Linear Discriminant Analysis (LDA) effect size (LEfSe) algorithm was adapted to quantitatively characterize the phylotypes that could explain the differences observed between two biological conditions (24). It identifies phylotypes in their relative abundance profiles through building an LDA model to estimate the “effect size” of each phylotype with respect to biological conditions under inspections. The alpha value for the non-parametric factorial Kruskal-Wallis (KW) sum-rank test (100) is 0.05, the threshold on the logarithmic LDA model (101 ) score for discriminative features is 2.0. All- against-all BLAST search was used in multi-class analysis that is stricter than the one-against- all search, and both target classes and subject names were denoted in the analysis.

In order to ascertain if any contribution to the microbiota in chickens fed bovine colostrum might have originated in the colostrum itself, especially that of the SFB, samples of the bovine colostrum matrix as applied to the feed was analyzed. The metagenomic approach was chosen to assess the microbiota of the colostrum. Given the low biomass in colostrum, applying the PCR-based 16S rRNA on such a specimen type may have introduced additional biases. To access the microbiota of the colostrum, the non-processed colostrum was filtered through the sterile 0.22um syringe filter (Sterivex, Germany), and the materials left on the filter was subjected to 45 seconds beating cycle using a FastPrep instrument (MP Biomedicals) at 5.5 m/s and centrifuged at 14,000 rpm for 1 h at 4°C. The supernatant after beads beating was used for DNA extraction using the same procedure as above 16S rRNA samples. Metagenomic sequencing libraries were constructed from the same DNA using the Nextera XT Flex kit (Illumina, San Diego, CA) according to the manufacturer’s recommendations. Libraries were then pooled together in equimolar proportions and sequenced on Illumina HiSeq 4000 platform using the 150bp protocol at the Genomic Resource Center at the University of Maryland School of Medicine. Metagenomic sequence reads were removed using BMTagger v3.101 (102) using a Genome Reference Consortium Gallus gallus Build 5.0 (GCA_000002315.3, (103)). Sequence read pairs were removed even if only one of the reads matched to the genome reference. Taxonomic profiling was conducted in MetaPhlAn vers 2 (104). Reads mapping of the metagenomic reads to published genomes of Candidates arthro- mitus isolated from chicken, turkey, mouse, and rat (NZ_CP008713.1 , NC_015913.1 , NZ_AGAG01000005.1 , NZ_AGVP01000010.1 , NC_017294.1 , NC .016012.1 ,

LXFF01000001.1 ) using bowtie (v1 , parameters: “-I 25--fullref--chunkmbs 512 -best -strata - m 20”, (105)) were performed.

Metabolite formulation

The metabolites in the anti-inflammatory formulation are: Indolelactic acid, chenodeox- ycholic acid, and 3’-sialyllactose (Sigma Aldrich, Inc., St. Louis, MO), itaconate (Sigma Aldrich, Inc., St. Louis, MO; BOC Biosciences, London, England), tryptophan, apigenin, diadzein, gen- estein, naringinin, glycine, N-acetylglycine, gamma-glutamylcysteine, and glutathione (Spectrum Chem. Mfg., New Brunswick, NJ), 7-ketolithocholic acid (Alfa chem. Lipids, Inc., Ronko- koma, NY), 6;-sialyllactose (GeneChem Inc., Daejeon, Korea).

Statistical analyses:

The basic statistical analysis for the effects of diet treatment on growth, feed efficiency, morphometric measurements, NT pixel content, and the assessment of specific metabolites on an anti-inflammatory index was analysis of variance performed using the General Linear Models procedure of the Statistical Analysis System® ver. 9.4 (106). Specific contrast statements (107) were used to test hypotheses regarding the effects of the various diets as well as diet combinations on the dependent variables. “Animal-within-treatment” was used as the error term.

For microbial population inferences, in order to estimate differences in relative abundances of different bacterial clusters within different diet status, a Bayesian Poisson model was employed and the structure is as follows: where y_t is the count in the i-th cell and diet(i), cluster(i) are the diet and cluster of the i-th cell, respectively. The model was fitted using JAGS R package (108), and 100,000 iterations with the same number of burn-in iterations was used. The convergence of the model was assessed using Gelman and Rubin's potential scale reduction factor (109) and visual inspection of each coefficient’s Markov chains.

EXAMPLE 2

Results

Short-term bovine colostrum supplementation of the standard diet significantly improved feeding efficiency and consistently modified the metabolic and bacteriological milieu of the gut compared to CON and in a manner superior to that of NFDM. Diet-associated changes in epithelial anatomy, anti-inflammatory metabolites and gut microbiota of the ileum were found.

Feeding efficiency was significantly improved in short-term BC diet-fed chickens

The impact of feeding diets supplemented with bovine colostrum or the nonfat dry milksupplement (NFDM, a macronutrient balanced control to bovine colostrum) compared to CON on weight gain and feeding efficiency is summarized in FIG. 1. For chickens fed the bovine colostrum diet, the feed efficiency was improved approximately 9 percent (P<0.05) over CON and NFDM diets. Although the lowest and highest mean weight gain responses were observed in NFDM and bovine colostrum chickens, respectively, the differences between the means of these treatment groups as well as the difference from CON were only trends (P > 0.05 <0.1 ).

Intestinal measurements indicate significant biological impacts of the BC diet on gut morphol- ogy

A visual depiction of the ileum structure attributes as associated with the three diets as well as measured and estimated morphological attributes of the ileum are summarized in FIGS. 2A-2D. Where nuclei are depicted as white objects against the black/grey background, the increased number of nuclei in the lamina propria of villi of animals in the CON and NFDM groups, higher than that in observable in bovine colostrum, is consistent with a larger number of infiltrating immune cells, a hallmark of inflammation or at the least, a sensed perturbation.

Presented as an expression of the density of villi, the number of villi per linear unit of ileum was significantly increased in animals fed the bovine colostrum diet in contrast to CON (P<0.03) and NFDM (P<0.004, FIG. 2A). When the relative villus area (RVA, mid-villus width x the villus length x number of villi per unit villus length) was calculated for each animal and the mean group RVAs compared, the calculated area for bovine colostrum was 42 percent (P<0.05) and 24 percent (P<0.06) greater than the mean areas calculated for CON and NFDM, respectively (FIG. 2B). Mean villus lengths were 265, 280 and 250 pm for CON, NFDM and bovine colostrum, respectively (P=NS, data not shown).

By comparison, corresponding mean crypt depths, associated with enterocyte cell proliferation and digestion capacity of the small intestine, of 28, 31 and 37 pm were observed for CON, NFDM and bovine colostrum, respectively, yielding the resulting villus-to-crypt ratios (FIG. 2C). Though the mean values calculated for villus length and crypt depths alone across the dietary treatments did not attain statistical significance, the villus:crypt ratio for chickens in bovine colostrum was 27 percent lower (P<0.03) and 24 percent lower (P<0.05) than that calculated for CON and NFDM, respectively (FIG. 2B). The value for the calculated estimate of villus area per unit length was significantly increased in bovine colostrum compared to the other fed diets (FIG. 2C). Collectively, the increase in estimated villus surface area (i.e., improved nutrient absorptive surface) with bovine colostrum is consistent with an effect of bovine colostrum to stimulate a more efficient use of nutrients and the observed improved feed efficiency in bovine colostrum.

BC- and to a lesser extent nonfat dry milk (NFDM)-supplemented diets are associated with less ileal epithelial cell nitro-oxidative stress

The cellular pixel density associated with immunofluorescence localization of NT, on the exterior, epithelial layer of the villi, thereby excluding lamina propria and interior layers of the villi that contain infiltrating immune inflammatory cells was measured. FIGS. 3A-3C illustrate the colocalization of the NT with epithelial cells with the red-green color shift to orange. Though nitrated proteins were observed in the gut epithelial cells of all chickens, the highest levels were detected in those chickens fed the CON and nonfat dry milk diets (FIGS. 3G-3I). The epithelial cell NT protein content, characterized as pixel density as normalized by nuclear counting as a measure of the number of cells specified in the demarcated area of interest, was significantly decreased by 37% in bovine colostrum-fed chickens compared to that present in chicken tissues from either the CON (P<0.05) or the nonfat dry milk (P<0.04) diets (FIG. 3J). The overall decrease in NT pixel density per epithelial cell was attributable to “clusters” of nitration elements that were both fewer in number and smaller in the contained number of pixels. The lower level of nitrated proteins in the ileal epithelial cells suggests that these cells were less subject to nitrooxidative stress (indicator of inflammation) in the presence of bovine colostrum-derived diet than were the ileal cells of chickens fed the Con and nonfat dry milk diets. Short-term feeding of BC- and NFDM-supplemented diets alters the anti-inflammatory metabolite profile of the ileum

The identified metabolite digesta compounds derived from the animal treatment diets themselves or as further derived from host and/ or microbial enzymatic action on these compounds with a literature-documented capacity to mitigate tissue inflammation was examined. The goal was to identify those anti-inflammatory compounds derived from NFDM or bovine colostrum present in amounts over and above those contributed through consumption of the basal CON diet that had a potential to lower the NT proteins and then test them in a statistical model for association with any impact on ileum inflammation status. Across dietary treatments, metabolomic profiling of the ileal digesta revealed 649 named and 300 unnamed compounds. Quantified levels of the identified compounds in the metabolite data base in a third-party analytical contract (Metabolon, Inc., Morrisville, NC) were reported as “scaled imputed values (SIV).

The digesta of bovine colostrum was significantly more complex in metabolite richness (number of detected compounds) than that of CON or NFDM. Plant-derived compounds are well-known for having anti-inflammatory and anti-oxidative properties and as such were of significant interest here. Of seventy-seven compounds in the ileal digesta classified as xeno- biotics of plant origin identified at or higher than levels stated as the threshold limits of detection (indicated as “DETECTED”), 40 compounds were in control (CON), 46 in non-fat dry milk (NFDM) and all 77 in bovine colostrum (Table 1 ).

TABLE 1 Table 1 shows that the effects of inclusion of nonfat dry milk or the bovine colostrum fraction on ileal digesta components with literature-supported anti-inflammatory effects on tissues varied according to the involved biochemical pathway components. The com- pounds/metabolites were identified by analyzing the gut digesta mass spectroscopy. These data clearly demonstrate that the availability of metabolites changes with the host-microbiome interactions that process the digesta as it flows down the alimentary canal. This finding is consistent with the concept that anti-inflammatory compounds available in one gut segment might not be available in other gut segments to provide their beneficial effects and that bacterial populations differ between gut segments. Considerations for inclusion of a metabolite compound in a list of compounds with diet-derived anti-inflammatory character stemmed from a PubMed search.

With focus on the ileum, ten xenobiotic compounds with or without anti-inflammatory character were found to be significantly different in abundance between CON and bovine colostrum and reproducibly across both treatment trials. Apigenin, 2,3-dihydroxyisovalerate, carotenediol, daidzein, daidzein sulfate, genistein, gluconate, naringenin, and 2-piperidinone were higher in bovine colostrum than CON and panose (a purported prebiotic) lower, respectively. Similarly, digesta from NFDM chickens contained six more compounds than were present in the digesta from CON with 2,8-quinolinediol sulfate, 2-oxindiole-3-acetate, 1-kestose, 1 ,1-kestotetraose higher and homostachydrine lower.

The effects of the NFDM and bovine colostrum diets on the changes in these compounds as well as those identified in the other relevant seven pathway metabolite groups are presented in Table 2. Data represent the means ± SEM of the Scaled Imputed Values of metabolites that were increased by either bovine colostrum or NFDM identified by the metab- olomic 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 . bovine colostrum, 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 bovine colostrum (#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. TABLE 2 The metabolite data in Table 2 were the basis on which the Anti-inflammatory index (A-i-i) was derived leading to the determination of which compounds were to be included in the supplement as provided herein. Numerous compounds with potential anti-inflammatory activity in the ileal digesta were identified. Comparing the levels of the identified metabolites in the ileum digesta between the diet treatments, data revealed, perhaps not unexpectedly, that the mean levels of many individual metabolites varied numerically, sometimes greatly, among the three diet treatments and the two replications of the experiment. Further analysis revealed that fewer individual metabolites consistently differed in the two experimental replications between CON and bovine colostrum or CON and NFDM. This became apparent with respect to the animal-to-animal variation and replication variation for many of the metabolites. However, when multiple related metabolites for a given common pathway and mechanism of anti-inflammatory action were grouped and concentration levels considered additively as a singular entity (for example, if several anti-inflammatory compounds like flavonoids/ isoflavones or active metabolites like glutathione and its major precursors), the abundance of a select set of compounds/groups were significantly greater for bovine colostrum than those measured in CON or NFDM. The literature-supported anti-inflammatory pathway metabolite categories included tryptophan indole-conjugates, tocopherols (a-, y-, 6-tocopherols), flavonoids/ isoflavones (apigenin, daidzein, genistein, and their conjugates), primary and secondary bile acids (chenodeoxycholic acid, 7-ketolithocholic acid ), glutathione (N-acetylglycine, y- glutamylcysteine, cysteine), polyunsaturated fatty acid (docosahexaenoic acid, arachidonic and eicosapentaenoic acids), oligosaccharides (3’- and 6’-sialyllactose), and mitochondrial TCA cycle compounds (itaconate),

There were significant effects of diet by the potent tripeptide anti-inflammatory compound glutathione and its precursors in the luminal digesta which are critical to the anti-oxidative capacity of the gut. The metabolomic report contained data on the reduced form of glutathione, GSHr. Overall, the mean level of GSHr in bovine colostrum was half that present in CON (P<0.04) and 70 percent of that present in the digesta of NFDM (NS). However, the digesta from chickens in bovine colostrum had a summated glutathione precursor pool of compounds significantly increased over that measured in CON. This is consistent with the need for an abundance of precursors (cystine, glycine, glutamate, and associated dipeptides) being available to the synthesis pathway in the gut epithelial cells to replenish glutathione as it is consumed in countering oxidative processes. When considered as a precursor group, the summated mean of glycine, cystine, N-acetylglycine, and y-glutamylcysteine digesta abundances averaged 158 percent greater levels in bovine colostrum than in CON (3.46 ±0.46 v 1 ,34±0.22, P<0.001 ). Both the difference in summated abundance (P<0.001 ) as well as the ratio (P<0.002) of GSHprecursors-to-GSHr may reflected the potential for bovine colostrum to increase the antioxidative capacity of the gut over that present in CON by contributing to the metabolites the gut needs to make glutathione.

Statistical evaluation of the pathway components in Table 2 showed that polyunsaturated fatty acid (PUFA) compounds, and especially metabolites of eicosapentaenoic and arachidonic acids, were not significantly affected by diet (all grey). This lack of change with bovine colostrum feeding may relate to the defatting process used to generate the bovine colostrum matrix and the NFDM supplemented feeds. However, seven other identified pathways were significantly affected by diet either as single compounds or, as summed components of an individual pathway. For some pathways, for example tryptophan or tocopherols, only a single pathway compound with purported anti-inflammatory potential was significantly different between one or among all diets. Only indolelactic acid in the tryptophan pathway or y- tocopherol in the tocopherol pathway, respectively, were affected by inclusion of nonfat dry milk or the colostrum matrix into the basal diet. Surprisingly, the effect amounted to a reduction in relative mean ileal SIV. The overall impact of this reduction in ileal gamma-tocopherol content was sufficient to account for the majority of the pathway change in total tocopherols.

The inclusion of bovine colostrum to the basal diet consistently increased the ileal digesta content of four anti-inflammatory flavonoid compounds: apigenin, daidzein, genistein, and naringin. In contrast, none of the compounds was affected by adding NFDM to the basal diet. The effect of adding bovine colostrum to the diet resulted in a doubling of the amount of flavonoid present in the gut digesta based on summated SIVs. Data from the larger metabolite profiling indicated that the levels of the flavonoids in their conjugated forms (glucoside, glucuronide) did not contribute to the gut levels of the parent compound’s anti-inflammatory status suggesting that the parent compounds alone were to be considered the major components of interest.

The summated values of the primary and secondary bile acids with anti-inflammatory effects, chenodeoxycholic acid and 7-ketolithocholic acid content, respectively, were significantly elevated in digesta from bovine colostrum and NFDM compared to CON diets. However, digesta from bovine colostrum-fed chickens tended to have higher levels of chenodeoxycholic acid than that from NFDM (Table 2). Only two oligosaccharides were identified by the mass spectroscopic analysis, 3’-sialyllactose and 6'-sialyllactose. Levels of total oligosaccharides in bovine colostrum digesta were 5.7-fold higher (P<0.002) than those measured in CON digesta and 2.8-fold (P<0.003) higher than those in digesta from NFDM.

The cellular energetics generated by subcellular mitochondrial processes is a fundamental requisite of cell stability, immune system function, and the efficiency of nutrient use. Of the eleven mitochondrial compounds associated with energy production identified in the digesta, only one, itaconate, derived from the decarboxylation of cis-aconitate in the mitochondrial matrix, was an anti-inflammatory metabolite and found to be increased in di- gesta in NFDM and bovine colostrum relative to CON (P<0.03).

Short-term colostrum extract supplementation is associated with a significantly higher antiinflammatory index (A-i-i) than found in CON or NFDM diets

The relative contribution of each of these metabolites to the model was assessed according to whether the inclusion of a particular metabolite or metabolite group improved the adjusted R² in the multiple regression model, and not all did. To facilitate analysis of the impact of dietary-derived metabolites on chicken gut inflammation, an anti-inflammatory index was devised and associations arising from the gut processing of the ingested feed components of the specific diets that correlated with protein nitration (FIGS. 4A-4C) were found.

Firstly, the data used to construct FIG. 4A were derived from the SIV values in the metabolomic database. As indicated by the terms “Yes” and “No” in the table line “Model Inclusion”, as each successive variable was added to the regression model, that variable was only retained in the model if it added a positive incremental increase to the adjusted R². While some metabolites present in the digesta of chickens may have had a purported anti-inflammatory character, if they did not have added a benefit in the regression model to increase the adjusted R² (terms shaded in green) they were dropped from the model (terms shaded in pink). Consequently, gamma-tocopherol and the two sialyloligosaccharides were excluded from the cumulative Ai-i. Therefore, in the final assessment, the combination of chenodeoxy- cholic acid, total flavonoids, glutathione precursors, 7-ketolithocholate, itaconate and indole- lactate accounted for approximately 76 percent of the observed decline of cellular NT protein content.

Secondly, bovine colostrum feeding yielded mean Ai-i values (derived from data in FIG. 4A) significantly different from and higher than the average Ai-i’s from both NFDM and Con groups (FIG. 4B).

Thirdly, the variability in the magnitude of the derived NT protein pixel quantification data among treatment groups could be better analyzed with respect to a linear model after performing a logarithmic transformation of the pixel data. When this was done, the regression analysis showed a significant (P<0.005) negative linear correlation (R²=0.694) wherein the higher the Ai-i, the lower the NT protein in villus epithelial cells (FIG. 4C).

BC and NFDM added to a standard diet altered specific populations of bacteria in the ileum rapidly and specifically

Using 16S rRNA gene amplicon sequencing, a community survey was performed of the gut microbiota of 41 chickens under 3 different dietary conditions (standard or CON feeding, and CON feeding supplemented by NFDM or bovine colostrum). Intraluminal digesta was collected from the proximal and distal intestine including jejunum, duodenum, and cecum for a total of 156 samples as well as scrapings from ileal epithelial cells washed free of digesta. A total of 25,838,078 high-quality 16S rRNAV4 amplicon sequences corresponding to 51 ,165 (±2,267, p ± s.e.m.) sequences per sample were obtained. Overall, distinct differences were found in the digesta and mucosal-associated microbiota among animals fed the three different diets. Across diets, five distinct types of intestinal microbiota l-V were observed and summarized in a heat map and bar graph (FIGS. 5A-5B). Type I is enriched in Lactobacillus spp., Type II is enriched in Lachnospiraceae spp., Type III is enriched in both Lachnospiraceae spp. and Bacteroides fragilis, Type IV is characteristic of Candidates Arthromitus (SFB), and Type V is enriched in Bifidobacterium, Coriobacteriaceae spp. and Lactobacillus spp. Type I and IV microbiota had the lowest community diversity and were highly enriched with Lactobacillus and Candidates Arthromitus, respectively (FIG. 5B). In particular, the different physiological sections of the intestine demonstrated distinct microbiota, and the type IV microbiota was enriched in the ileal microbiota. The other types of microbiota were significantly more diverse and had relatively more abundant Lachnospiraceae spp., Bacteroides fragilis and a wide array of strict and facultative anaerobic bacterial species. Statistical modeling using Bayesian Poisson model showed the intestinal microbiota in the digesta of BC-fed chickens was significantly enriched in Type I and IV microbiota. LDA effect size (LEfSe) analyses was performed to quantitatively characterize the phylotypes that could explain the differences observed under different conditions (FIGS. 5C-5H).

Candidates Arthromitus was shown to be the most differentially abundant phylotype in ileal mucosal scraping samples of bovine colostrum-fed animals but not in ileal digesta (FIG. 6A). This finding is consistent with the nature of the cell attachment mechanism this organism employs to persevere in its growing microenvironmental niche largely specific to the ileum. Consolidating across all data in FIGS. 5A-5H, the patterns, distribution, and abundance of microbes is consistent with bovine colostrum having greater microbial diversity than that in the digesta from animals in the other two diets. Increases in this microorganism in the colon might reflect remaining pass-through organisms originating in the ileum and perhaps simply shed into the digesta going into the large intestine in association with the naturally-occurring programmed epithelial cell losses from the tips of the villi associated with apoptosis. In addition, Candidates Arthromitus (SFB) was minimally detected in the cecum or duodenum digesta or scrapings (FIGS. 5C-5D).

In order to determine whether the presence of Candidates Arthromitus might have arisen in the bovine colostrum chickens as a result of feeding the actual processed colostrum fraction that contained a live Candidates Arthromitus, the composition of the microbiota associated with various samples of the bovine colostrum used in the diets was characterized, and showed that these samples contained very low abundance of bacteria in general and revealed no Lactobacillus nor Candidates Arthromitus. While fresh colostrum is known to contain many microorganisms beneficial to the nursing infant, the lack of such microbial abundance in the bovine colostrum diet preparations is consistent with the effects of the one-hour centrifugation of the preparations at 20,000 x g to sediment bacteria into the bottom of the tube. Further analyses confirmed no detected Candidates Arthromitus via reads mapping to published Candidates Arthromitus genomes or using marker gene-based approach. This result indicated that the Candidates Arthromitus was not likely derived from the bovine colostrum diet.

Other than Candidates Arthromitus, Streptococcus was another phylotype that was shown to be enriched in bovine colostrum-fed animals, irrespective of the sub-anatomical location (digesta vs. mucosa-associated) (FIGS. 5E-5F). This result again emphasizes the importance of sub-anatomical location, anatomic site, and dietary conditions for specific groups of bacteria in stark contrast to data more commonly generated from fecal collection sampling. Other phylotypes that demonstrated varied enrichment under different feeding conditions were also included. For example, Bifidobacterium (FIGS. 5G-5H were more enriched with the NFDM diet, and Bacteroides fragilis, Bacteroides ovatus, and E. coli, was particularly enriched in cecal samples. Candidates Arthromitus was only in the mucosa-associated microbiota of the ileum in these chickens and its presence was amplified significantly when bovine colostrum was incorporated into the basal diet (FIGS. 6A-6B). The higher relative abundance of Candidates Arthromitus is consistent with a localized proliferation of the organism, most likely due to factors in the environment of the organism rather than being sourced from the fed colostrum itself.

While the oligosaccharide content of the various diets did not affect the anti-inflammatory index, the 3’- and 6’-sialyl-oligosaccharides did lead to a dramatic site-specific increase in Candidates Arthromitus. The oligosaccharide content of the ileal digesta was increased approximately 4- and 2.5-fold in bovine colostrum compared to that measured in digesta from CON and NFDM, respectively (FIGS. 7A-7B). Further regression analysis between the SIVs for the combined oligosaccharides versus the logw of the abundance of total Candidates Arthromitus indicate a significant positive correlation wherein higher levels of the oligosaccharides in the digesta were associated with increased numbers of the Candidates Arthromitus with its potential impact on the immune capacity of the gut. EXAMPLE 3

Metabolite matrix for the mitigation of gut inflammation

Table 3 summarizes the metabolites in a fundamental base anti-inflammatory formulation. The formulation may contain other metabolites or natural compounds with anti-inflam- matory properties and/or an ability to prevent pathogen attachment in the gut such as tocopherols and polyunsaturated anti-inflammatory compounds. Non-limiting examples are gammatocopherol or alpha-tocopherol or a soy-derived mixed tocopherol oil (>60% gamma-tocopherol), and eicosanoic acid or a conjugated analog thereof (110-112). TABLE 3

Metabolite formulation as an anti-inflammatory supplement Supplement content and percent of composition was calculated on the basis of the weight of each component consumed per unit metabolic body weight, body weight in kg⁰⁷⁵. The use of metabolic body size is used rather than straight body weight because it better compensated for body differences between species. For example, for a 70 kg man or woman, the amounts of each component in the mixture would be adjusted by multiplying the basic amount of the compound by 24.2 which is the body weight of 7O⁰⁷⁵ kg.

The formulation contains individual purified metabolite compounds in the stated percentages and amounts determined by 75% of the body weight of the animal receiving the formulation. The formulation may be administered as a capsule, a powder, a liquid, or may be microencapsulated for specific administration for specific delivery to a given gut section or to preserve integrity of bioactivity. The formulation may be administered in a pharmaceutical composition with a pharmaceutically acceptable carrier as is known and standard in the art.

Discussion

Protein nitration stress occurs in the intestine in conjunction with parasitic infection and correlated with the severity of infection (25,26). In comparison to extensive research done on oxidative stress in the gut, there is less research on nitration stresses in the gut, even though nitration stress and oxidative stress share a pool of cell response-induced common oxygen- and nitrogen-based reactants (22). The present invention demonstrates that the fractionated preparation of bovine colostrum developed herein, when added to a standard feed and consumed by animals for 8 days, is associated with a significant reduction of the amount of nitrooxidative inflammation in the epithelial cells of the ileum, denoted as a reduction in the content of intracellular NT proteins. In concert with this effect, feeding bovine colostrum was strongly associated with an increase in the absorptive surface area of the ileum. The rapidity with which this occurred is consistent with an increased capacity to generate epithelial cells (27) as reflected in the calculated crypt-to-villus ratio. These observations on the increased ileal surface area are also consistent with the observed improvement in nutrient absorption as reflected in feed utilization efficiency. The presence of naturally-occurring, low-level gut stressors such as changes in environment and social structure, present in our select, high growth rate animal model, has been identified previously as having the capability of significantly altering the architecture of the villus absorptive area as well as the morphogenesis of the crypts (28-30).

Elevated levels of intracellular tyrosine-nitrated (3’-nitrotyrosine-) NT proteins are a cause of protein dysfunction in non-immune cells, as documented in many tissue inflammation pathologies (21 ,22,31 ). Epithelial cells generate highly reactive nitrogen intermediates (i.e., peroxynitrite, ONOO ) from nitric oxide and superoxide anion (21 ,22,32), which can react with critical tyrosine(s) in phosphorylation sites of signal transduction enzymes such as JAK-2 (33,34) or mitochondrial proteins thereby causing dysfunction and ATP shortage in cells (32,35,36). Additionally, epithelial cells can have levels of NT proteins with cell function deficits that vary by diet in the absence of detectable infectious disease (25,26). Oxidative/nitra- tion protein and lipid damage has been generated in chickens by low levels of partially oxidized corn oil or polyunsaturated fatty acids being in the diet (37) and oxidized oil-induced inflammation was relieved with the addition of the flavonoid antioxidant quercetin to the chicken diet (38). Mitigation of gut dysfunction by reducing inflammation is a goal of both human and veterinary medicine. In the present invention, the short-term feeding of the refined bovine colostrum matrix was significantly associated with the observed reduction in ileal epi- thelia-specific NT protein content.

Gut metabolism in health can account for up to 20 percent of maintenance energy expenditure (39-41 ). Gut inflammation increases energy demands by as much as 10 percent of the body’s metabolic energy need (42,43) with a significant portion of the caloric substrates diverted from growth and muscle accretion to support immune function particularly in young animals (20,22). Microbial populations in the gut, and more specifically changes in such populations, significantly impact host immune and metabolic gene expression (44) and therefore the energetic needs of the host’s physiological systems. Signals from the gut microbiome can prioritize the flux of nutrients between the nervous, endocrine, and immune systems and shape their interactions (20,22,45,46). Since oxidative and nitration stress impacts the energy utilization of affected cells, even marginal changes in gut health result in energetic deficiencies (47,48) that rapidly translate into growth and nutrient use efficiency deficits but improved significantly in bovine colostrum. These observations are consistent with the regulatory model proposed for the controlling inputs, i.e., endocrine, immune, metabolic, and microbial inputs, to immunometabolism (11 ) and further impacts on the nutrient use in the young growing animal.

The enhanced genetic lines of commercial production chickens with a high growth rate used in the present chicken model required no direct disease or chemical challenge to trigger gut nitrooxidative stress. Further, low levels of nitrated proteins were shown to be generated by enterocytes in response to non-infectious perturbations such as changes in feed composition, allergens, endogenous bacterial endo- and exotoxins, mold toxins, and even metabolic ketosis (49-51 ). In the past, this stress sensitivity was managed in part through the extensive use of antibiotics added to the diet (3,5). However, the present invention demonstrates that consumption of components of bovine colostrum can ameliorate gastrointestinal inflammation and improve nutrient use without the use of antibiotics. Dietary factors are potent modulators of the microbiota composition and its interaction with the host (52). Reciprocally, microbes metabolizing ingested nutrients and generating nutrient-derived metabolites play a critical role in regulating the host immune response and gut cell function, as documented in the newly emerging area of immunometabolism (53). Active microbial modification of feed-derived nutrients was evident as observed in the presence of microbe-derived compounds like p-cresol sulfate and hippurate as well as deconjugation of flavonoid compounds differentially affecting their bioavailability (54-57) and levels of the various secondary bile acids consistent with literature reports (55). From the metabolomic analysis of the ileal digesta of test animals fed the various dietary treatments, a defined set of nutrient molecules and microbially-derived metabolites that correlated with the reduction in level of nitrooxidative stress present in the ileal epithelial cells were identified and statistically modeled. The positive effects of the colostrum feeding were reflected in the increased portion of bacteria with anti-inflammatory/ immunomodulatory properties in the microbiota profiles, and the generation of anti-inflammatory metabolites bathing the cells of the lumen.

The present invention shows that classes of related microbially-derived metabolites should be considered for their anti-inflammatory effect in addition to the more commonly literature-cited approach of identifying single compounds. For example, the polyphenol flavonoids identified in the mass spectroscopic analysis revealed that as a class they were significantly increased in bovine colostrum and NFDM feeding in comparison to CON. If, however, only one flavonoid, apigenin, of the identified group was assessed, the larger group effect would have been missed. Similarly, while the effect of adding NFDM or bovine colostrum to the basal diet on the individual components identified in the anti-inflammatory glutathione pathway may have been insignificant (i.e., P>0.1 ), collectively the sum of the precursor pool and the glutathione components improved the regression model (Table 2). This is consistent with the need for an abundance of precursors (cystine, glycine, glutamate, and associated dipeptides) being available to the synthesis pathway in the gut epithelial cells to replenish glutathione (58,59) as it is consumed and to maintain cellular oxidative/nitrosative homeostasis.

Metabolites are an important link in the interactions between the host, its gut microbiota and the lumen environment. For example, metabolites such as metabolically-derived ita- conate, generated during the immune response and identified in our screening can regulate both the magnitude and duration of the immune response (60). In addition, Henrick et al. established the cause-and-effect relationships between gut increases in the tryptophan pathway metabolite, indolelactate, also identified in our metabolomic screening, concurrent with the enhanced presence of bifidobacteria, and their beneficial effects on the balance between inflammatory and anti-inflammatory states (61 ). The capacity for microbe-derived indolelactate to function as an anti-inflammatory metabolite (62,63) is consistent with the findings of the reduction in nitrated proteins, the increased nutrient use efficiency, and favorable microbial populations in BC- compared to CON- or NFDM-fed chickens. These published data support the validity of the regression analysis that identified indolelactate among the many metabolites in the digesta. Collectively, the present data demonstrate the potential for diet composition to significantly impact gut health through changes in the microbiota, the gut metabolite milieu and host responses.

Specific metabolites generated by the localized bacterial populations acting on ingested dietary components facilitate processes needed for healthy eubiotic regulation of the gut epithelia. In studies of the murine colonic microbiota, Tiffany and Baumler (64) proposed that during gut homeostasis, bacteria of the phyla Firmicutes and Bacteroidetes, obligate anaerobic bacteria, fermented dietary fiber and maintained a hypoxic environment that could reduce colonization with Enterobacteriaceae (phylum Proteobacteria). In the absence of hypoxia, Proteobacteria gained a foothold. This deviation from an obligate anaerobic microbiota was defined as gut “dysbiosis”. Dysbiosis in the ileal microbiome stems from lower levels of microbial diversity with imbalances between both beneficial and pathogenic organisms as well as disproportionate imbalances in commensal bacteria that can change the local microenvironments downstream thus facilitating a bacterial overgrowth syndrome or pathogen emergence (65,66).

The microbiome of the small intestine, the primary site for the absorption of nutrients, differs substantially from that of the colon both in terms of the number of colony-forming units per ml as well as microbial composition (67). The ileum was examined herein largely because of the large information gaps on the microbiome and its host interactions compared to the large intestine (68). The present invention showed a reduction in epithelial cell nitrooxidative stress in BC-fed animals which may be associated with the greater population diversity that observed in the ileal microbiome of these animals. The decreased gut inflammation-associated generation of NT proteins in bovine colostrum-supplemented chickens is consistent with other known anti-inflammatory effects of colostrum such as the observed reduction in NFk-B- mediated proinflammatory cytokine expression in intestinal epithelial cells (69). Those nitration reactions can play out in the cascade of responses initiated by such mediators as TNF-a cascading through increases in intracellular NO and superoxide anion culminating in the generation of nitrating reactants like ONOO' and the more reactive ONOOCO?', as produced when higher pCO? is present in sections of the gut with more anaerobic status with perturbed mesenteric blood flow. The ileal microbiome also differed from that of the duodenal and colonic segments analyzed in that the segmented filamentous bacteria Candidates Arthromitus was localized to the cell layer scraped from the ileal bowel (rather than the digesta per se). These data suggest that anatomical localization of microbiota composition in the gastrointestinal tract may be important in the regional status of immune activation in different gut segments as well as the inflammation-anti-inflammation status thus suggesting the need for spatially distinct analyses along the gastrointestinal tract.

The present invention showed that bovine colostrum-initiated changes in the gut microbiota enhanced the metabolic processing of nutrients into anti-inflammatory metabolites, a feature characterized with the development of the anti-inflammatory index. These observations were reproducible over time and across different sources of colostrum (cow breed and collection time after parturition). The ability of bovine colostrum to beneficially modify the gut environment was due to the nutrient-derived metabolites and microbial populations in the ileum not matched by the NFDM diet. The group of metabolites identified after statistical regression alignment supported the hypothesis of an increased abundance of metabolites with anti-inflammatory character and aligned with observed effects on gut morphology and an improved efficiency of nutrient use for body weight gain in bovine colostrum.

Several classes of compounds were altered by the bovine colostrum supplementation. Compared to CON feed, bovine colostrum feed increased the microbially-derived secondary bile acids, chenodeoxycholic acid, 7-ketolithocholic acid, in the ileum. Studies in humans and mice have shown bile acids to have an anti-inflammatory effect on immune cells (70) and appear to function in their host-microbe cross-talk signaling capacity through the farnesoid-X receptor (FXR) and the G-protein-coupled bile acid receptor-1 (TGR5) (71 ).

Oligosaccharides 3’-sialyllactose (3SL) and 6'-sialyllactose (6SL) with prebiotic properties were markedly increased in the ileal contents of chickens fed BC- and NFDM- supplemented feed compared to CON. The levels of these oligosaccharides were significantly higher in bovine colostrum than marginally-increased levels in NFDM. However, they did not statistically contribute to the Ai-i per se, the positive correlation largely driven by the levels found in bovine colostrum. Oligosaccharides in bovine colostrum may facilitate microbiota remodeling the chicken by reducing the ability of pathogens to gain a niche in the gut microenvironment and through promoting growth of beneficial microbiota as has been seen in humans and mice. The increased oligosaccharides in bovine colostrum associate strongly with the selective increase in ileum mucosa-associated segmented filamentous bacteria (SFB), namely Candidates Arthromitus, in chickens in the bovine colostrum- supplemented group. Segmented filamentous bacteria were sparsely detected in the ileal mucosa of both CON or NFDM chickens. Furthermore, Candidates Arthromitus was not detected in the raw colostrum after incubation on media for 48 hrs suggesting that some aspect of the bovine colostrum diet promoted the increase in segmented filamentous bacteria already present in the ileum. These bacteria, living predominantly attached to the ileal epithelial mucosa, are known to promote gut maturation and gut mucosal and adaptive immune development including the development of germinal centers in Peyer’s patches that generate potent IgA and Th 17 responses (72,73). By itself, segmented filamentous bacteria can induce intestinal T cell development similar to that induced by the complete gastrointestinal microbiota (74). Segmented filamentous bacteria colonization also has been shown to improve barrier protection against enteric viral infections and associated diarrheal diseases (75) as well as serve as a coordination link between metabolism and immune function (76). Danzeisen et al., demonstrated that in turkeys a fundamental determinate of growth success was the gut microbial milieu, particularly the presence of significantly more segmented filamentous bacteria (77).

Tryptophan, an essential amino acid, is metabolized by gut bacteria to generate metabolites specific to indole, kynurenine and serotonin pathways, the latter a key neurotransmitter in both the enteric and central nervous systems (63). The tryptophan metabolite, in- dolelactic acid, is elevated in NFDM, but even more so in BC-supplemented diets. Indolelac- tate acts on gut epithelial cells, Paneth cells and enteroendocrine cells through specific receptors to (1 ) stimulate the release of antimicrobial peptides, (2) upregulate tight junction proteins to maintain barrier function; (3) suppress inflammatory cytokine production, (4) decrease superoxide anion generation, and (5) decrease apoptosis. Tryptophan metabolites generated by the microbiota can bind to the endogenous tryptophan receptor, the aryl hydrocarbon receptor (AHR) (62). Indolelactate is generated only by bacteria, particularly Lactobacillus, that are abundant in BC, and bifidobacteria with demonstrated benefits to gut health in colostrum- fed infants (61 ,78).

While the methods used by Metabolon Inc. do not allow measurement short chain fatty acid, they undoubtedly were present in the BC that are high in indigestible carbohydrates (“prebiotics”) that selectively enhance the growth of Bifidobacterium and Lactobacillus that have been shown to modulate inflammation and modulate a vast range of physiologic processes that include suppression of inflammatory signals and carcinogenesis (79). The micro- bially-derived short chain fatty acid share the use of receptors and signaling pathways that are used by the host gut epithelium to detect molecules that signal between the gut microbiota and the host.

The present invention shows that BC feeding enhances the generation of several classes of anti-inflammatory metabolites. Collectively, the findings support the need to better understand not only what colostrum components other than traditionally assessed factors like immunoglobulins, antimicrobial factors, growth factors and cytokines contribute to an antiinflammatory environment in changing the composition of the digesta in the gut lumen. Perhaps more critical though, information gaps exist needing resolution regarding what anti- inflammatory factors and metabolite precursors reach the different sections of the gut and are processed through host-diet-microbe interactions in maintaining gut health and homeostasis.

The addition of BC to standard feed exerts profound effects on the immunometabolism of chickens, a model for gut health exquisitely sensitive to metabolic perturbations that reflects the impact of gut stress on feeding efficiency and growth (11 ). Compared to chickens fed CON or a NFDM-supplemented diet, BC-fed chickens had an increased ileal absorptive surface and developed changes in the composition and structure of the gut microbiota that generated feed- and host-derived metabolites and compounds, resulting in an anti-inflammatory gut microenvironment. This could be attributed to significant increases in several BC-associ- ated digesta metabolites with defined anti-inflammatory character. Importantly, only the BC- fed chickens had major increases in mucosa-associated SFBs known to promote gut maturation and immune mucosal development. The present invention also highlights the importance of sampling the mucosal surface in addition to the digesta to comprehensively assessing gut microbiota.

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Claims

WHAT IS CLAIMED:

1 . A nutritional supplement for an animal feed, comprising: a plurality of metabolite compounds effective as anti-inflammatories in an animal gut.

2. The nutritional supplement of claim 1 , wherein the plurality of metabolite compounds are indolelactic acid, apigenin, diadzein, genestein, naringinin, chenodeoxycholic acid, 7-ketolithocholic acid, glycine, A/-acetylglycine, y-glutamylcysteine, glutathione, 3’-sialyl- lactose, 6’-sialyllactose, and itaconate.

3. The nutritional supplement of claim 1 , further comprising: a mixed tocopherol oil of gamma-tocopherol and alpha-tocopherol or eicosanoic acid or a conjugated analog thereof.

4. A method for improving a gut microenvironment in an animal, comprising: supplementing food for the animal with the nutritional supplement of claim 1 ; and feeding the animal a diet comprising the supplemented food over a period of time sufficient to effect an improvement in the gut microenvironment.

5. The method of claim 4, wherein the supplemented food is antibiotic-free.

6. The method of claim 4, wherein the animal is a food animal or a farm animal.

7. The method of claim 4, wherein the supplemented food produces an increase in immune function-modifying segmented filamentous bacteria, a decrease in toxicity-associated nitrated proteins or an increase in gut absorptive surface characteristics or a combination thereof.

8. An animal feed comprising the nutritional supplement of claim 1 .

9. The animal feed of claim 8, wherein said animal feed is an antibiotic-free food.

10. A method for increasing growth of a food animal or a farm animal, comprising: feeding the food animal or the farm animal a diet comprising the antibiotic-free food of claim 9.

11 . The method of claim 10, wherein efficiency of nutrient use is increased in the food animal or the farm animal fed the diet.

12. An animal food product supplemented with a defatted bovine colostrum.

13. The animal food product of claim 12, further supplemented with at least one of gamma-tocopherol or alpha tocopherol or a mixed tocopherol oil thereof, eicosanoic acid or a conjugated analog thereof.

14. The animal food product of claim 12, wherein said animal food product is antibiotic-free.

15. A method for increasing an anti-inflammatory response in an animal, comprising: feeding the animal the animal food product of claim 12, said defatted bovine colostrum therein producing an increase in anti-inflammatory metabolites and beneficial bacteria in the gut microbiome.

16. The method of claim 15, wherein the animal is a food animal or a farm animal and wherein said animal food product is antibiotic-free.

17. The method of claim 15, wherein the beneficial bacteria are immune functionmodifying segmented filamentous bacteria.

18. The method of claim 15, wherein the animal is a food animal, a farm animal, a sport animal, a companion animal, a service animal, or a human.

19. A nutritional supplement formulated as a mixture of gut-associated metabolites.

20. The nutritional supplement of claim 19, wherein the gut associated metabolites are indolelactic acid, apigenin, diadzein, genestein, naringinin, chenodeoxycholic acid, 7-ke- tolithocholic acid, glycine, A/-acetylglycine, y-glutamylcysteine, glutathione, 3’-sialyllactose, 6’- sialyllactose, and itaconate.

21 . The nutritional supplement of claim 20, further comprising gamma-tocopherol or alpha tocopherol or a mixed tocopherol oil thereof, eicosanoic acid or a conjugated analog thereof or a combination thereof.

22. The nutritional supplement of claim 19, wherein each of the gut-associated metabolites in the mixture has a content dose based on a metabolic body weight (BW) percentage of a recipient animal of BW⁰⁷⁵.

23. A pharmaceutical composition comprising the nutritional supplement of claim 19 and a pharmaceutically acceptable carrier.

24. A method for improving gut health in an animal, comprising: administering to the animal an amount of the nutritional supplement of claim 19 effective to decrease inflammation and to increase beneficial bacteria in the gut.

25. The method of claim 24, wherein the beneficial bacteria are immune functionmodifying segmented filamentous bacteria.

26. The method of claim 24, wherein the animal is a food animal, a farm animal, a sport animal, a companion animal, a service animal, or a human.