WO2018148563A1

WO2018148563A1 - Composition comprising silica, mineral clay, glucan and mannans and its administration to mammals

Info

Publication number: WO2018148563A1
Application number: PCT/US2018/017649
Authority: WO
Inventors: Derek MCLEAN; James D. CHAPMAN; Geoffrey DAHL; David Kirk; Jimena LAPORTA
Original assignee: Mclean Derek; Chapman James D; Dahl Geoffrey; David Kirk; Laporta Jimena
Priority date: 2017-02-09
Filing date: 2018-02-09
Publication date: 2018-08-16

Abstract

Disclosed herein is a method for at least maintaining, and preferably increasing the number of alveoli, such as in the mammary tissue; for increasing the prolactin levels, changing the level of expression of prolactin receptors; and/or decreasing the number of exfoliated mammary epithelial cells (MEC), in mammals, such as dairy cattle, as well as a composition for use in the disclosed method. The composition comprises a glucan, silica, mineral clay, mannan, and optionally an endoglucanohydrolase, and may be administered to an animal to provide the desired beneficial effect.

Description

COMPOSITION COMPRISING SILICA, MINERAL CLAY, GLUCAN AND MANNANS

AND ITS ADMINISTRATION TO MAMMALS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos. 62/457,144, filed February 9, 2017; U.S. Provisional Application No. 62/461,568, filed February 21, 2017, and U.S. Provisional Application No. 62/621,452, filed on January 24, 2018, which are herein incorporated by reference in their entirety.

FIELD

This disclosure concerns a method and composition for administration to a mammal to increase the number of alveoli, increase the level of prolactin, decrease expression of prolactin receptors, and/or decrease the number of exfoliated mammary epithelial cells in milk.

BACKGROUND

In the mammary gland, the alveoli are the site of milk production and storage. Milk producing animals, such as dairy cattle, typically have a drying off period between periods of lactation, during which milk is not produced. Typically, the number of alveoli in the mammary gland is reduced after a drying off period, compared to the number of alveoli in the mammary gland before the drying off period. This reduction may affect milk yield during the later lactation period.

Milk yield may also be affected by the level of prolactin in the mammal. Prolactin is a protein that is secreted from the pituitary gland. While is it is also produced by certain non- mammals, prolactin in mammals has been associated with milk production. Prolactin interacts with a prolactin receptor. Prolactin receptors are associated with lobuloalveolar maturation of the mammary glands, typically during pregnancy.

Additionally, or alternatively, milk yield may be influenced by the number of mammary epithelial cells (MEC) in the mammary gland. It has been suggested that an increase in exfoliation of MEC from the mammary gland may influence milk yield by reducing the number of MEC in mammary tissue. SUMMARY

The object of the present disclosure is to provide a novel method for at least maintaining, and preferably increasing the number of alveoli, such as in the mammary tissue, for increasing the prolactin levels, changing the level of expression of prolactin receptors, and/or decreasing the number of exfoliated mammary epithelial cells (MEC) in milk. The method may comprises administering a composition comprising silica, mineral clay, glucan and/or mannans, to an animal, such as a mammal. Certain embodiments concern a composition comprising silica, mineral clay, glucan, and mannans for use in a method to at least maintain a number of alveoli in mammary tissue, to increase an amount of Prolactin in milk and/or blood, to decrease an amount of Prolactin receptor expression, to decrease a number of exfoliated mammary epithelial cells in milk, or a combination thereof. The method may comprise administering an effective amount of the composition to a mammal. The mammal may be a bovine, such as a dairy cow. The number of alveoli in a mammal that is fed the composition may be increased compared to a number of alveoli in a mammal that is not fed the composition. The composition may be administered substantially continuously, or it may be administered daily. The composition may be a powdered composition, a granular composition, or a combination thereof. In some embodiments, a granular composition comprises plural granules where each granule comprises a substantially homogeneous blend of silica, mineral clay, glucan, and mannans. In any embodiments, the glucan and mannans may be provided by yeast cell wall or an extract thereof. The yeast cell wall or an extract thereof may further comprises endoglucanohydrolase, and/or the composition may comprise

endoglucanohydrolase as an affirmatively added component.

The effective amount of the composition may be administered to the mammal for an effective period of time starting after beginning of a lactation period for the mammal and continuing through at least a portion of a subsequent drying off period for the mammal. The effective period of time may start from 100 days to 240 days after beginning of a lactation period for the mammal. Administration of the composition may begin before the mammal commences a pregnancy, or while the mammal is pregnant. In any embodiments, the administration of the composition may continue at least through to birth of an offspring following the drying off period. And in certain embodiments, the administration continues into a subsequent lactation period following the birth of the offspring, and optionally throughout the entire subsequent lactation period. In any embodiments, the effective amount of the composition may be from 0.01 gram to 20 grams per kilogram of live body weight per day.

The mammal may additionally be administered a treatment for stress, such as heat stress. In some embodiments, the mammal is administered a cooling treatment, such as a provision of shade, use of water sprinklers to externally administer water to the mammal, use of a fan to provide air movement to the mammal, air-conditioning, or any combination thereof.

The composition may comprise 1-40 wt% silica, 0.5-25 wt% glucan and mannans, and 40- 92 wt% mineral clay. And, the composition may further comprise a feed, a metal carbonate, a copper species, a trace mineral, a bulking agent, yeast, a carrier, a colorant, a taste enhancer, a preservative, an oil, a vitamin, yucca, quillaja, a probiotic, allicin, alliin, allinase, algae, a polyphenol or plant material comprising polyphenol, or a sorbic acid or a salt thereof. In particular embodiments the copper species is copper sulfate; the yucca is Yucca schidigera; the quillaja is Quillaja saponaria; the probiotic is or comprises Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis; or a combination thereof.

Also disclosed herein is a method to at least maintain a number of alveoli in mammary tissue of a mammal, increase an amount of Prolactin in milk, decrease an amount of Prolactin receptor expression, decrease a number of exfoliated mammary epithelial cells in milk, or a combination thereof. The method may comprise administering a composition comprising silica, mineral clay, glucan, and mannans to a mammal, thereby at least maintain the number of alveoli in mammary tissue of a mammal, increase the amount of Prolactin in milk, decrease the amount of Prolactin receptor expression, and/or decrease the number of exfoliated mammary epithelial cells in milk. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar chart illustrating on-dairy milk yield (kg) by cows that are administered the disclosed composition and those that are not, including both initial and on-dairy conditions.

FIG. 2 is a bar chart illustrating milk yield for control cows and cows that have been administered the disclosed composition during thermoneutral conditions (TN), heat stress conditions (HS), and recovery conditions (Recovery).

FIG. 3 is a graph illustrating mean milk production by day for control cows and those that have been administered the disclosed composition.

FIG. 4 is a graph illustrating feed intake by day for control cows and those that have been administered the disclosed composition.

FIG. 5 is a bar chart illustrating feed intake for control cows and cows that have been administered the disclosed composition during thermoneutral conditions, heat stress conditions, and recovery conditions.

FIG. 6 is a graph illustrating somatic cell count by day for control cows and those that have been administered the disclosed composition.

FIG. 7 is a bar chart illustrating mean serum Cortisol for control cows and cows that have been administered the disclosed composition during thermoneutral conditions (TN) and heat stress conditions (HS), measured after a certain number of days. FIG. 8 is a bar chart illustrating mean serum insulin levels for control cows and cows that have been administered the disclosed composition during thermoneutral conditions (TN) and heat stress conditions (HS), measured after a certain number of days.

FIG. 9 is a bar chart illustrating mean serum glucose levels for control cows and cows that have been administered the disclosed composition during thermoneutral conditions (TN) and heat stress conditions (HS), measured after a certain number of days.

FIG. 10 is a bar chart illustrating IL8R receptor gene expression leukocytes in lactating control cows and cows that have been administered the disclosed composition during thermoneutral conditions and heat stress conditions, measured after a certain number of days.

FIG. 11 is a bar chart illustrating Regulated on Activation, Normal T Expressed and

Secreted (RANTES) protein levels in control cows and cows that have been administered one embodiment of the disclosed composition prior to heat stress, during acute heat stress, chronic heat stress, heat stress recovery and long-term recovery.

FIG. 12 is a bar chart illustrating serum adrenocorticotrophic hormone (ACTH) levels in control cows and lactating dairy cows that have been administered the disclosed composition during thermoneutral conditions (TN) and heat stress conditions (HS), measured after a certain number of days.

FIG. 13 is a graph showing rectal temperature of newly received steers supplemented with one embodiment of the disclosed composition at a rate of 4 g/cwt (composition) during the receiving period (28 days) or no composition (Control) during a subcutaneous lipopolysaccharide challenge.

FIG. 14 is a graph showing change in rectal temperature of newly received steers supplemented with one embodiment of the disclosed composition at a rate of 4 g/cwt (composition) during the receiving period (28 days) or no composition (Control) during a subcutaneous lipopolysaccharide challenge.

FIG. 15 is a graph showing rectal temperature of newly received steers supplemented with the one embodiment of the disclosed composition at a rate of 4 g/cwt (composition) during the receiving period (28 days) or no composition (Control) during a lipopolysaccharide challenge.

FIG. 16 is a graph showing change in rectal temperature of newly received steers supplemented with the one embodiment of the disclosed composition at a rate of 4 g/cwt

(composition) during the receiving period (28 days) or no composition (Control) during a lipopolysaccharide challenge.

FIG. 17 is a bar graph showing overall Cortisol concentrations of newly received steers supplemented with the one embodiment of the disclosed composition at a rate of 4 g/cwt (composition) during the receiving period (28 days) or no composition (CON) during a lipopolysaccharide challenge.

FIG. 18 is a graph showing Cortisol concentrations of newly received steers supplemented with the one embodiment of the disclosed composition at a rate of 4 g/cwt (composition) during the receiving period (28 day) or no composition (Control) during a lipopolysaccharide challenge.

FIG. 19 is a series of bar graphs showing treatment, prior, and post lipopolysaccharide challenge concentrations of blood urea nitrogen (BUN) for newly received steers supplemented with the one embodiment of the disclosed composition at a rate of 4 g/cwt (composition) during the receiving period (28 days) or no composition (Control).

FIG. 20 is a bar graph showing treatment concentrations of glucose concentrations for newly received steers supplemented with one embodiment of the disclosed composition at a rate of 4 g/cwt (composition) during the receiving period (28 days) or no composition (Control) during a lipopolysaccharide challenge.

FIG. 21 is a graph showing glucose concentrations of newly received steers supplemented with the one embodiment of the disclosed composition at a rate of 4 g/cwt (composition) during the receiving period (28 days) or no composition (Control) during a lipopolysaccharide challenge.

FIG. 22 is a series of bar graphs showing treatment, prior, and post lipopolysaccharide challenge concentrations of non-esterified fatty acids of newly received steers supplemented with the one embodiment of the disclosed composition at a rate of 4 g/cwt (composition) during the receiving period (28 days) or no composition (Control).

FIG. 23 is a series of bar graphs showing treatment, prior, and post lipopolysaccharide challenge concentrations of interferon - γ concentrations of newly received steers supplemented with the one embodiment of the disclosed composition at a rate of 4 g/cwt (composition) during the receiving period (28 days) or no composition (Control).

FIG. 24 is two bar graphs showing treatment and post lipopolysaccharide challenge concentrations of tumor necrosis - a (TNF-a) overall and post lipopolysaccharide (LPS) challenge concentrations of newly received steers supplemented with the one embodiment of the disclosed composition at a rate of 4 g/cwt (composition) during the receiving period (28 days) or no composition (Control).

FIG. 25 is a graph showing tumor necrosis - a (TNF-a) concentrations of newly received steers supplemented with the one embodiment of the disclosed composition at a rate of 4 g/cwt (Composition) during the receiving period (28 days) or no composition (Control) during a lipopolysaccharide challenge. FIG. 26 is a graph showing interleukin-6 (IL-6) concentrations of newly received steers supplemented with the one embodiment of the disclosed composition at a rate of 4 g/cwt

FIG. 27 is a graph showing lymphocyte concentrations of newly received steers supplemented with the one embodiment of the disclosed composition at a rate of 4 g/cwt

FIG. 28 is a graph showing the neutrophil to lymphocyte ratio of newly received steers supplemented with the one embodiment of the disclosed composition at a rate of 4 g/cwt

FIG. 29 is a graph of alveoli number versus treatment, illustrating the alveoli number in mammary tissue biopsy samples from cows fed one embodiment of the disclosed composition during late lactation and dry-off compared to the alveoli number from cows that were not fed the composition. The number of alveoli were determined by microscope examination of tissue sections recovered from the mammary tissue. Alveoli were differentiated in the tissue based on their ductule-type appearance that creates a lumen with a distinct epithelium.

FIG. 30 is a graph of relative gene expression versus treatment, illustrating the gene expression of HSP70 in mammary tissue from cows fed one embodiment of the disclosed composition (composition) and cows not fed the composition (CON). The expression of HSP70 in tissue from composition-fed cows was significantly less than control cows (p=0.04).

FIG. 31 is a graph of relative gene expression versus treatment, illustrating the gene expression of Prolactin Receptor (PRLR) in mammary tissue from cows fed one embodiment of the disclosed composition (composition) and cows not fed the composition (CON). The expression of PRLR in tissue from the composition-fed cows was significantly less than control cows (p=0.10).

DETAILED DESCRIPTION

I. Definitions

The following explanations of terms and abbreviations are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, "comprising" means "including" and the singular forms "a" or "an" or "the" include plural references unless the context clearly dictates otherwise. The term "or" refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise.

Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the disclosure are apparent from the following detailed description and the claims.

Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims are to be understood as being modified by the term "about." Accordingly, unless otherwise indicated, implicitly or explicitly, the numerical parameters set forth are approximations that may depend on the desired properties sought and/or limits of detection under standard test conditions/methods. When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximates unless the word "about" is recited.

As used herein, mesh sizes refer to standard U.S. mesh sizes.

Animal: This term includes species that are produced for human consumption and/or that are domesticated animals. Exemplary species of such animals include, but are not limited to, mammals such as ruminant species, including sheep, goat, bovine (such as dairy cow, beef cow, calf, bullock, or bull), deer, bison, buffalo, or llama, and ungulate species, such as horse, donkey, or

Pig- Administering: Administration by any route to the subject. As used herein, administration typically refers to oral administration.

Feed: As used herein, the term "feed" refers to solid and liquid animal feeds (e.g. , a feed ration), supplements (e.g. , a mineral supplement, a protein supplement), a premix, water, feed additive carriers (e.g. , molasses), and combinations thereof.

Granule: A granule is a particle that has a mean diameter of greater than -100 mesh, i.e. typically larger than 0.15 mm. In some embodiments, a granule has at least one dimension, such as one dimension, two dimensions, or three dimensions, greater than -100 mesh and less than 4 mesh

(4.8 mm).

Mannans: A class of polysaccharides including the sugar mannose. The mannan family includes pure mannans (i.e. , the polymer backbone consists of mannose monomers), glucomannans (the polymer backbone comprises mannose and glucose), and galactomannans (mannans or glucomannans in which single galactose residues are linked to the polymer backbone). Mannans are found in cell walls of some plant species and yeasts.

Mineral Clay: The term "mineral clay" refers to hydrous aluminum silicates. Mineral clays usually include minor amounts of impurities, such as potassium, sodium, calcium, magnesium, and/or iron. Mineral clays typically have a two-layer sheet structure including tetrahedral silicate sheets and octahedral hydroxide sheets or a three-layer structure including a hydroxide sheet between two silicate sheets.

Polyphenols: A class of natural, synthetic, or semisynthetic organic chemicals characterized by the presence of plural phenolic ( ^x-a^0H) 'n structural units.

Therapeutic agent: An agent that is capable of providing a therapeutic effect, e.g., preventing a disorder, inhibiting a disorder, such as by arresting the development of the disorder or its clinical symptoms, or relieving a disorder by causing regression of the disorder or its clinical symptoms.

Therapeutically effective amount: A quantity or concentration of a specified compound or composition sufficient to achieve a desired effect in a subject being treated for a disorder. The therapeutically effective amount may depend at least in part on the species of animal being treated, the size of the animal, and/or the severity of the disorder.

Additional disclosure is found in U.S. Patent Application No. 13/566,433, U.S. Patent

Application No. 13/872,935, U.S. Patent Publication No. 2013/0017211, U.S. Patent Publication No. 2012/0156248, U.S. Patent Publication No. 2007/0253983, U.S. Patent Publication

No. 2007/0202092, U.S. Patent Publication No. 20070238120, U.S. Patent Publication

No. 2006/0239992, U.S. Patent Publication No. 2005/0220846, U.S. Patent Publication

No. 2005/0180964, and Australian Patent Application No. 2011201420, all of which are incorporated herein by reference.

II. Composition

Certain disclosed embodiments of the composition comprise glucan (e.g., β-1,3 (4)glucan), silica, mineral clay, and mannans, and may further comprise an endoglucanohydrolase, such as β- 1,3 (4)-endoglucanohydrolase, either endogenously or as an affirmatively added ingredient.

In any embodiments disclosed herein, the composition may comprise, consist essentially of, or consist of, glucan (e.g., β-1,3 (4)glucan), silica, mineral clay and mannans. In some

embodiments, the composition comprises, consists essentially of, or consists of, glucan (e.g., β-1,3 (4)glucan), silica, mineral clay, mannans and endoglucanohydrolase. In any embodiments disclosed herein, the glucan and mannans may be provided, at least in part, by yeast cell wall or an extract thereof. Thus, in some embodiments, the composition may comprise, consist essentially of, or consist of, silica, mineral clay and yeast cell wall or an extract thereof, or the composition may comprise, consist essentially of, or consist of, silica, mineral clay, yeast cell wall or an extract thereof, and endoglucanohydrolase.

Suitable sources of silica include, but are not limited to, sand, diatomaceous earth, and synthetic silica. In one embodiment, quartz may be used. In certain embodiments, the mannans comprise glucomannan.

The components of the composition are prepared by methods commonly known in the art and can be obtained from commercial sources, β-1,3 (4)-endoglucanohydrolase may be produced from submerged fermentation of a strain of Trichoderma longibrachiatum. Diatomaceous earth is available as a commercially-available product with from 70% to 95% silica (S1O2) and with its remaining components not assayed but primarily ash (minerals) as defined by the Association of Analytical Chemists (AOAC, 2002). The mineral clays (e.g., aluminosilicates) used in this composition may be any of a variety of commercially-available clays including, but not limited to, montmorillonite clay, bentonite and/or zeolite. Glucan, mannans, and/or endoglucanohydrolase can be obtained from plant cell walls, yeast or yeast cell wall or an extract thereof (e.g., Saccharomyces cerevisiae, Candida utilis), certain fungi (e.g., mushrooms), algae, and bacteria. In certain embodiments, yeast, yeast culture, and/or yeast cell wall or an extract thereof, can be administered affirmatively to provide glucan, mannans and endoglucanohydrolase endogenously.

In one embodiment, the composition comprises, consists essentially of, or consists of, 1-40 wt% silica, 0.5-25 wt% glucan and mannans, and 40-92 wt% mineral clay, in amounts relative to each other. In another embodiment, the composition comprises, consists essentially of, or consists of, 5-40 wt% silica, 0.5-15 wt% glucan and mannans, and 40-80 wt% mineral clay, in amounts relative to each other. In another embodiment, the composition comprises, consists essentially of, or consists of, 20-40 wt% silica, 0.5-10 wt% glucan and mannans, and 50-70 wt% mineral clay, in amounts relative to each other. In another embodiment, the composition comprises, consists essentially of, or consists of, 15-40 wt% silica, greater than zero to 15 wt% glucans, greater than zero to 10 wt% mannans, and 50-81 wt% mineral clay, in amounts relative to each other. In another embodiment, the composition comprises, consists essentially of, or consists of, 15-40 wt% silica, 0.5-5.0 wt% glucans, 0.5-8.0 wt% mannans, and 50-81 wt% mineral clay, in amounts relative to each other. In another embodiment, the composition comprises, consists essentially of, or consists of, 20-30 wt% silica, 0.5-3.5 wt% glucans, 0.5-6.0 wt% mannans, and 60-70 wt% mineral clay, in amounts relative to each other. In some embodiments, β-glucans and mannans are obtained from yeast or yeast cell wall or an extract thereof. The composition may comprise, consist essentially of, or consist of, 1-40 wt% silica, 1-30 wt% yeast cell wall or an extract thereof, and 40-92 wt% mineral clay, in amounts relative to each other. In one embodiment, the composition comprises, consists essentially of, or consists of, 10-40 wt% silica, 5-20 wt% yeast cell wall or an extract thereof, and 40-80 wt% mineral clay, in amounts relative to each other. In another embodiment, the composition comprises, consists essentially of, or consists of, 15-30 wt% silica, 5-15 wt% yeast cell wall or an extract thereof, and 50-70 wt% mineral clay, in amounts relative to each other.

In any of the above embodiments, the composition may further comprise an

endoglucanohydrolase, such as β-1,3 (4)-endoglucanohydrolase. The composition may include from 0.025 wt% endoglucanohydrolase to 5 wt% endoglucanohydrolase or more, such as from 0.05 wt% to 3 wt% β-1,3 (4)-endoglucanohydrolase, relative to the amounts of silica, mineral clay, glucan, mannans, and/or yeast, yeast cell wall, or yeast cell wall extract present in the composition. In one embodiment, the composition comprises, consists essentially of, or consists of, 0.1-3 wt% β- 1,3 (4)-endoglucanohydrolase, 20-40 wt% silica, 0.5-20 wt% glucan and mannans, and 50-70 wt% mineral clay, in amounts relative to each other. In another embodiment, the composition comprises, consists essentially of, or consists of, 0.1-3 wt%, β-1,3 (4)-endoglucanohydrolase, 20-40 wt% silica, 0.5-10 wt% glucan and mannans, and 50-70 wt% mineral clay, in amounts relative to each other. Alternatively, the composition may comprise, consist essentially of, or consist of, 0.1-3 wt% β-1,3 (4)-endoglucanohydrolase, 1-40 wt% silica, 5-30 wt% yeast cell wall or an extract thereof, and 40-92 wt% mineral clay, in amounts relative to each other. In one embodiment, the composition comprises, consists essentially of, or consists of, 0.1-3 wt% β-1,3 (4)- endoglucanohydrolase, 10-40 wt% silica, 5-20 wt% yeast cell wall or an extract thereof, and 40-80 wt% mineral clay, in amounts relative to each other. In another embodiment, the composition comprises, consists essentially of, or consists of, 0.1-3 wt% β-1,3 (4)-endoglucanohydrolase, 15-30 wt% silica, 5-15 wt% yeast cell wall or an extract thereof, and 50-70 wt% mineral clay, in amounts relative to each other.

In any of the above embodiments, the silica may be provided by diatomaceous earth. In any of the above embodiments, the glucans may be β glucans. In some embodiments, the β glucans can be obtained from yeast, or other materials, such as fungi, algae, bacteria, or the like. In any of the above embodiments, the mannans may comprise glucomannan. In some embodiments, the composition does not comprise a separate binder in addition to the components of the composition.

The glucan and mannans (or yeast or yeast cell wall or an extract thereof) can be prepared by a method known to a person of ordinary skill in the art and as further disclosed by the patent documents referenced herein and incorporated herein by reference. Yeast cell wall or an extract thereof may have a composition comprising 0-15% moisture and 85-100% dry matter. The dry matter may comprise 10-65 % protein, 0-25 % fats, 0-3% phosphorus, 5-30% β-glucan, 5-35% mannans, and 0-15% ash. In an independent embodiment, a commercial source of β 1,3 (4) glucan and glucomannan derived from primary inactivated yeast (Saccharomyces cerevisiae) may be used. The commercially available β 1,3 (4) glucan and glucomannan may have a chemical composition of moisture 2-5%; proteins 40-50%; fats 3-8%; phosphorus 0-2%; mannans 10-16%; β-1,3-(4) glucan 10-20%; and ash 2-12%.

In another independent embodiment, the yeast cell wall or an extract thereof comprises moisture 1-7% and dry matter 93-99%, and the dry matter may comprise proteins 18-28%, fats 10- 17%, phosphorus 0-2%, mannans 20-30%, β-1,3-(4) glucan 18-28%, and ash 2-5%.

In an independent embodiment of the composition, silica, glucan and mannans, and mineral clay are combined at 1-40%, 0.5-25% and 40-92% by weight, respectively. In an independent embodiment of the composition, β-1,3 (4)-endoglucanohydrolase, diatomaceous earth, yeast cell wall or an extract thereof, and mineral clay are combined at 0.05-3%, 1-40%, 1-20% and 40-92% by weight, respectively. In an independent composition, β-1,3 (4)-endoglucanohydrolase, diatomaceous earth, yeast cell wall or an extract thereof, and mineral clay are combined at 0.1-3%, 5-40%, 2-15% and 40-80% by weight, respectively. In another independent embodiment of the composition, β-1,3 (4)-endoglucanohydrolase, diatomaceous earth, yeast cell wall or an extract thereof, and mineral clay are combined at 0.1-3%, 30-40%, 4-15% and 50-65% by weight, respectively.

The disclosed composition may include one or more additional components. In some embodiments, the composition comprises a substantially homogenous blend of silica, mineral clay, glucan, mannans, one or more additional components, and optionally endoglucanohydrolase.

Additional components may be used for any desired purpose, such as a substantially biologically inert material added, for example, as a filler, or to provide a desired beneficial effect. For example, the composition may include a carbonate (including a metal carbonate such as calcium carbonate); a sulfate, including a metal sulfate, such as, but not limited to, copper sulfate, zinc sulfate, sodium sulfate, and/or potassium sulfate; a copper species, such as a copper species that provides a copper ion, for example, a copper salt, including, but not limited to, copper sulfate, copper fluoride, copper chloride, copper bromide, copper iodide, copper oxide, copper carbonate, or a combination thereof, typically copper sulfate or comprising copper sulfate; a trace mineral, such as, but not limited to, chloride, fluoride, iodide, chromium, copper, zinc, iron, magnesium, manganese, molybdenum, phosphorus, potassium, sodium, sulfur, selenium, or a combination thereof; a bulking agent; a micro tracer, such as iron particles coated with a dye; yeast; a carrier; a colorant; a taste enhancer; a preservative; an oil; a vitamin; yucca; quillaja; a probiotic; allicin; alliin; allinase; algae; a polyphenol or plant material comprising polyphenol; a sorbic acid or a salt thereof; or a

combination thereof. The yeast may be yeast culture, active yeast, a live yeast, a dead yeast, yeast extract, or a combination thereof. The yeast may be a baker's yeast, a brewer's yeast, a distiller's yeast, a probiotic yeast or a combination thereof. Exemplary yeasts include, but are not limited to, Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces pastorianus, Brettanomyces bruxellensis, Brettanomyces anomalus, Brettanomyces custersianus, Brettanomyces naardenensis, and Brettanomyces nanus, Candida utilis, Candida stellate, Schizosaccharomyces pombe,

Torulaspora delbrueckii, or Zygosaccharomyces bailii. The preservative may be benzoic acid or a salt thereof, e.g. sodium benzoate; lactic acid or a salt thereof, e.g. sodium lactate, potassium lactate or calcium lactate; propionic acid or a salt thereof, e.g. sodium propionate; ascorbic acid or a salt thereof, e.g. sodium ascorbate; gallic acid or a salt thereof e.g. sodium gallate; sulfur dioxide and/or sulfites; nitrites; nitrates; choline, or a salt thereof, such as an anion salt of choline, e.g. choline hydroxide or choline halide, such as choline chloride, choline bromide, choline iodide, or choline fluoride; or any combination thereof. The oil may be mineral oil, corn oil, soybean oil, or a combination thereof. The yucca may be one or more of Yucca aloijolia, Yucca angustissima, Yucca arkansana, Yucca baccata, Yucca baileyi, Yucca brevifolia, Yucca campestris, Yucca capensis, Yucca carnerosana, Yucca cernua, Yucca coahuilensis, Yucca constricta, Yucca decipiens, Yucca declinata, Yucca de-smetiana, Yucca elata, Yucca endlichiana, Yucca faxoniana, Yucca

filamentosa, Yucca filif era, Yucca flaccida, Yucca gigante an, Yucca glauca, Yucca gloriosa, Yucca grandiflora, Yucca harrimaniae, Yucca intermedia, Yucca jaliscensis, Yucca lacandonica, Yucca linearifolia, Yucca luminosa, Yucca madrensis, Yucca mixtecana, Yucca necopina, Yucca neomexicana, Yucca pallida, Yucca periculosa, Yucca potosina, Yucca queretaroensis, Yucca reverchonii, Yucca rostrata, Yucca rupicola, Yucca schidigera, Yucca schottii, Yucca sterilis,

Yucca tenuistyla, Yucca thompsoniana, Yucca treculeana, Yucca utahensis, or Yucca valida. In some embodiments, the yucca is or comprises Yucca schidigera. The quillaja may be one or more of Quillaja brasiliensis, Quillaja lanceolata, Quillaja lancifolia, Quillaja molinae, Quillaja petiolaris, Quillaja poeppigii, Quillaja saponaria, Quillaja sellowiana, or Quillaja smegmadermos. In some embodiments, the quillaja is or comprises Quillaja saponaria. The probiotic may be a Bacillus species, such as Bacillus alcalophilus, Bacillus alvei, Bacillus aminovorans, Bacillus amyloliquefaciens, Bacillus aneurinolyticus, Bacillus anthracis, Bacillus aquaemaris, Bacillus atrophaeus, Bacillus boroniphilus, Bacillus brevis, Bacillus caldolyticus, Bacillus centrosporus, Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus firmus, Bacillus flavothermus, Bacillus fusiformis, Bacillus galliciensis, Bacillus globigii, Bacillus infernus, Bacillus larvae, Bacillus laterosporus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus mesentericus, Bacillus mucilaginosus, Bacillus mycoides, Bacillus natto, Bacillus pantothenticus, Bacillus polymyxa, Bacillus pseudoanthracis, Bacillus pumilus, Bacillus schlegelii, Bacillus sphaericus, Bacillus sporothermodurans, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thermoglucosidasius, Bacillus thuringiensis, Bacillus vulgatis, or Bacillus weihenstephanensis, or a combination thereof. In some embodiments, the probiotic is, or comprises Bacillus coagulans. In some embodiments, the probiotic is, or comprises Bacillus subtilis. In some embodiments, the probiotic is, or comprises Bacillus amyloliquefaciens. In some embodiments, the probiotic is, or comprises Bacillus licheniformis. In certain embodiments, the probiotic is, or comprises, a combination of Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis. In other embodiments, the probiotic is or comprises Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus coagulans. In certain embodiments, the probiotic comprises from 25% to 75% Bacillus amyloliquefaciens, and from 75% to 25% in total of Bacillus subtilis and Bacillus licheniformis.

Allicin (diallyl thiosulfate; 2-Propene-l-sulfinothioic acid S-2-propenyl ester) is a compound found in garlic, such as raw garlic. Allicin is typically produced from alliin ((2R)-2- amino-3-[(5)-prop-2-enylsulfinyl]propanoic acid) in damaged garlic cells by the action of the enzyme alliinase. Allicin, alliin, and/or alliinase may be provided as whole garlic cloves or bulbs; crushed, mashed, or chopped garlic; a garlic extract; and/or as a synthesized or isolated compound.

The sorbic acid or salt thereof may be potassium sorbate, sodium sorbate, ammonium sorbate, or a combination thereof. In some embodiments, the sorbic acid or salt thereof is potassium sorbate. The vitamin may be vitamin A; vitamin B i, such as thiamine mononitrate; vitamin B2, such as riboflavin- 5 -phosphate; vitamin B3, such as niacin or niacinamide; vitamin B5, such as pantothenic acid or d-calcium pantothenate; vitamin B₆, such as pyridoxine or pyridoxine hydrochloride; vitamin B 12; vitamin C, such as ascorbic acid, sodium ascorbate, or calcium ascorbate; vitamin D; vitamin E; vitamin K, or a combination thereof. Vitamin D may comprise vitamin Di, vitamin D2, vitamin D3, vitamin D₄, vitamin D5, 25 -hydroxy vitamin D3, 25-dihydroxy vitamin D3, or combinations thereof.

The algae may be a blue-green algae (cyanobacteria), a diatom (bacillariophyta), a stone wort algae (charophyta), a green algae (chlorophyta), a golden algae (chrysophyta), a dinoflagellate (dinophyta), a brown algae (phaeophyta) or a red algae (rhodophyta). In some embodiments, the algae is a chlorophyta, and may be an algae from the genus Chlorella, including, but not limited to, Chlorella vulgaris, Chlorella angustoellipsoidea, Chlorella botryoides, Chlorella capsulata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella homosphaera, Chlorella luteo-v iridis, Chlorella marina, Chlorella miniata, Chlorella minutissima, Chlorella mirabilis, Chlorella ovalis, Chlorella parasitica, Chlorella peruviana, Chlorella rugosa, Chlorella saccharophila, Chlorella salina, Chlorella spaerckii, Chlorella sphaerica, Chlorella stigmatophora, Chlorella subsphaerica, Chlorella trebouxioides, or a combination thereof. In other embodiments, the algae is a cyanobacteria, such as Arthrospira platensis or Arthrospira maxima (spirulina). Other algae include, but are not limited to, algae of the genus Pediastrum, such as Pediastrum dupl, Pediastrum boryanum, or a combination thereof; algae of the genus

Botryococcus, such as Botryococcus braunii; algae of the genus Porphyra, such as Porphyra dioica, Porphyra linearis, Porphyra lucasii, Porphyra mumfordii, Porphyra purpurea, Porphyra umbilicalis, or a combination thereof.

Additionally, or alternatively, the additional components may comprise corn, soybean meal, wheat, wheat fiber, barley, rye, rice hulls, canola, limestone, salt, distillers dried grains with solubles (DDGS), dicalcium phosphate, sodium sesquicarbonate, methionine source, lysine source, L-threonine, biotin, folic acid, kelp including dried kelp, menadione dimethylpyrimidinol bisulfite, silicon dioxide, calcium aluminosilicate, or any combination thereof.

Additional information concerning additional components can be found in PCT application No. PCT/US2015/053439, and U.S. application Nos. 15/359,342, 14/699,740, and 14/606,862, each of which is incorporated herein by reference in its entirety.

In some embodiments, the composition does not comprise additional components. In other embodiments, the composition comprises from greater than zero to 40% or more by weight additional components, such as from 0.1% to 40% by weight, or from 0.2% to 35% by weight additional components. In certain embodiments, the composition comprises from 0.1% to 5% by weight additional components, such as from 0.2% to 3% by weight. In other embodiments, the composition comprises from 5% to 20% by weight additional components, such as from 10% to 15% by weight. And in further embodiments, the composition comprises from 20% to 40% by weight additional components, such as from 30% to 35% by weight additional components.

In some embodiments, the composition comprises, consists essentially of, or consists of, silica, mineral clay, glucan, mannans, and endoglucanohydrolase; silica, mineral clay, glucan, mannans, endoglucanohydrolase, micro tracers and mineral oil; silica, mineral clay, glucan, mannans, endoglucanohydrolase, micro tracers, mineral oil, and vitamins; silica, mineral clay, glucan, mannans, endoglucanohydrolase, micro tracers, mineral oil, vitamins, and potassium sorbate; silica, mineral clay, glucan, mannans, endoglucanohydrolase, vitamins, and active yeast; silica, mineral clay, glucan, mannans, endoglucanohydrolase, micro tracers, mineral oil, and active yeast; silica, mineral clay, glucan, mannans, endoglucanohydrolase, and mineral oil; silica, mineral clay, glucan, mannans, endoglucanohydrolase, vitamins, and calcium carbonate; silica, mineral clay, glucan, mannans, endoglucanohydrolase, micro tracers, and wheat fiber; silica, mineral clay, glucan, mannans, endoglucanohydrolase and Bacillus coagulans; silica, mineral clay, glucan, mannans, endoglucanohydrolase, micro tracers, mineral oil and Bacillus coagulans; silica, mineral clay, glucan, mannans, endoglucanohydrolase, micro tracers, mineral oil, vitamins and Bacillus coagulans; silica, mineral clay, glucan, mannans, endoglucanohydrolase, micro tracers, mineral oil, vitamins, potassium sorbate and Bacillus coagulans; silica, mineral clay, glucan, mannans, endoglucanohydrolase, vitamins, active yeast and Bacillus coagulans; silica, mineral clay, glucan, mannans, endoglucanohydrolase, micro tracers, mineral oil, active yeast and Bacillus coagulans; silica, mineral clay, glucan, mannans, endoglucanohydrolase, mineral oil and Bacillus coagulans; silica, mineral clay, glucan, mannans, endoglucanohydrolase, vitamins, calcium carbonate and Bacillus coagulans; or silica, mineral clay, glucan, mannans, endoglucanohydrolase, micro tracers, wheat fiber and Bacillus coagulans. In any of these embodiments, the glucan and mannans may be provided by yeast, yeast cell wall, or yeast cell wall extract.

In some embodiments, the composition does not comprise an affirmatively added binder, such as starch.

In some embodiments, the composition does not comprise water as an affirmatively added ingredient.

In some embodiments, the composition does not comprise a peroxide compound.

In some embodiments, the composition does not comprise hydrogen peroxide.

In some embodiments, the composition does not comprise carbamide peroxide.

In some embodiments, the composition does not comprise urea.

In some embodiments, the composition does not comprise hydrogen peroxide and urea. In some embodiments, the composition does not comprise a feed.

The composition may be formulated in any suitable form, including a powder, a granule, a pellet, a solution, or a suspension. In one embodiment, the composition can be a dry, free-flowing powder suitable for direct inclusion into a commercially-available feed, food product or as a supplement to a total mixed ration or diet. The powder may be mixed with either solid or liquid feed. In another embodiment, the composition can be formed into pellets.

/. Granular formulation

In particular embodiments, the composition is formed into granules to form a granular feed supplement. In some embodiments, substantially every particle in the granular feed supplement includes each component of the composition, including silica, mineral clay, glucan and mannans optionally provided by yeast cell wall or an extract thereof, endoglucanohydrolase, and any additional components, and may comprise a substantially homogeneous blend of the components. And in any of the above embodiments, the relative amount of each component in each granule is substantially the same as the relative amount of the same component in the granular feed supplement as a whole. The coefficient of variation (C.V.) of the granular feed supplement is a measure of the variation in composition between different particles, such as between particles of different sizes. The C.V. provides a measurement of the segregation of the components across the different sizes of particles. The lower the C.V. value, the more homogenized the granular feed supplement is between different- sized particles. The C.V. may be measured for different components of the composition. In some embodiments, the C.V. for mineral content in the disclosed composition, optionally measured by measuring the calcium content, is 10% or less. That is, the mineral content of the particles varies by 10% or less in a sample of the granular feed supplement. The proximate content is a measure of the amounts of moisture, crude protein, ether extract, crude fiber, crude ash and nitrogen free extracts in the composition. In some embodiments, the C.V. for proximate content, optionally measured by measuring the protein content, is 20% or less, such as 15% or less, for particles in a sample of the granular feed supplement. For comparison, the C.V. for mineral content in a powdered composition comprising silica, mineral clay, glucan and mannans typically is about 15%, and the C.V. for proximate content in such a powdered composition is about 56%.

The granules in the granular feed supplement may have an average particle size selected to be suitable for direct inclusion into a feed, such as a commercially-available feed, food product, or as a supplement to a total mixed ration or diet. The average particle size may be selected to be compatible with the feed to which the granular feed supplement may be admixed. The term "compatible" as used herein means that the particle size is sufficiently similar to reduce or eliminate particle size segregation when the granules are admixed with the feed. For example, if the granular composition is admixed with a feed having an average particle size of 20 x 60 mesh (0.84 mm to 0.25 mm), the granules in the granular composition may have a similar average particle size, e.g., from 80%-120% of the feed particle size with which the granules are admixed. Exemplary feed material sizes include, but are not limited to, corn silage 30%-40% by weight minus 12 mesh (i.e., less than 1.7 mm), haylage 40%-50% by weight minus 12 mesh, total mixed ration (TMR) 40%-60% by weight minus 12 mesh. In some embodiments, the average particle size of the granular feed supplement is selected to overlap with the minus 12 mesh-sized feed material, thereby minimizing segregation with such feed. The granules may be mixed with either solid or liquid feed. In some embodiments, the granules in the granular composition do not comprise a feed.

The particles in the granular feed supplement may be selected to a size of less than 4.8 mm (-4 mesh), such as less than 2 mm (-10 mesh), and may have a size of less than 1.7 mm (-12 mesh). That is, the particles can pass through a sieve of 4.8 mm (4 mesh), 2 mm (10 mesh) or 1.7 mm (12 mesh), respectively. In some embodiments, at least one dimension, and optionally two or three dimensions, of the particles are less than the mesh size. In some embodiments, the particles are selected such that the amount of dust in the composition, as measured by the amount of the composition that can pass through a 100 mesh (0.15 mm) sieve, is less than 60% by weight, such as less than as less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 3% or less than 2% by weight. In some embodiments, less than 20% by weight of the particles, such as less than 15%, less than 10%, less than 7%, less than 5%, less than 3%, less than 2%, or less than 1% by weight have at least one dimension, and optionally two or three dimensions, less than 100 mesh (less than 0.15 mm size). In some embodiments, the composition is formulated as granules having a size of from smaller than 4 mesh to larger than 100 mesh size, such as 10 x 100 mesh, 10 x 80 mesh, 12 x 100 mesh, 12 x 80 mesh, 12 x 60 mesh, 12 x 40 mesh, or 12 x 30 mesh. In other embodiments, the granules have a size of from greater than 0.15 mm to less than 4.8 mm, such as from 0.15 mm to 2 mm, 0.18 mm to 2 mm, from 0.15 mm to 1.7 mm, from 0.18 mm to 1.7 mm, from 0.25 mm to 1.7 mm, from 0.42 mm to 1.7 mm, or from 0.6 mm to 1.7 mm. In any of the above embodiments, at least 40% by weight, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% by weight of the granules have a size within the stated size range. In any embodiments disclosed herein, at least one dimension, and optionally two or three dimensions of the granule is within the stated range, and in some embodiments, two or the dimensions of the granule or particle are within the stated range.

The bulk density difference of the granular feed supplement is the difference in bulk density between a loose-packed samples, and a sample that is agitated, such as tapped or shaken, to release some of the trapped air and/or provide closer packing of the particles in the sample. In some embodiments, the bulk density difference is less than 15 lb/ft³, such as less than 10 lb/ft³, or less than 8 lb/ft³.

Dispersion is a measure of the particle's ability to return to its original, non-compacted form on contact with liquid. It is measured by spraying the particles with water using a mechanical spray head positioned over a 500 mesh sieve and measuring the percentage of material that remains on the sieve after a predetermined length of time. Dispersion typically is measured at certain time points, such as 2 minutes, 5 minutes and/or 10 minutes. In some embodiments, the granular feed supplement has a dispersion value of 20% or less, such as 15% or less, at 2 minutes; a dispersion value of 15% or less, such as 12% or less, or 10% or less, at 5 minutes; and/or a dispersion value of 10% or less at 10 minutes.

Additional information concerning the granulated composition can be found in U.S.

application Nos. 62/449,959, filed January 24, 2017, and 15/878,761, filed on January 24, 2018, both of which are incorporated herein by reference in their entirety.

//. Administration

The composition may be administered alone or in combination with a feed. In some embodiments, the composition is mixed with the feed to form a feed composition. The feed may comprise a carbonate (including a metal carbonate such as calcium carbonate); a sulfate, including a metal sulfate, such as, but not limited to, copper sulfate, zinc sulfate, sodium sulfate, and/or potassium sulfate; a copper species, such as a copper species that provides a copper ion, for example, a copper salt, including, but not limited to, copper sulfate, copper fluoride, copper chloride, copper bromide, copper iodide, copper oxide, copper carbonate, or a combination thereof, typically copper sulfate or comprising copper sulfate; a trace mineral, such as, but not limited to, chloride, fluoride, iodide, chromium, copper, zinc, iron, magnesium, manganese, molybdenum, phosphorus, potassium, sodium, sulfur, selenium, or a combination thereof; a bulking agent; a micro tracer, such as iron particles coated with a dye; yeast; a carrier; a colorant; a taste enhancer; a preservative; an oil; a vitamin; yucca; quillaja; a probiotic; allicin; alliin; allinase; algae; a polyphenol or plant material comprising polyphenol; a sorbic acid or a salt thereof; or a

combination thereof, as previously described. Additionally, or alternatively, the feed may comprise corn, soybean meal, wheat, wheat fiber, barley, rye, rice hulls, canola, limestone, salt, distillers dried grains with solubles (DDGS), dicalcium phosphate, sodium sesquicarbonate, methionine source, lysine source, L-threonine, biotin, folic acid, kelp including dried kelp, menadione dimethylpyrimidinol bisulfite, silicon dioxide, calcium aluminosilicate, or any combination thereof.

In one embodiment, when incorporated directly into a feed, such as to form a feed composition, the composition may be added in amounts ranging from 0.1 kg to 100 kg per ton, such as from 0.1 kg to 20 kg per ton (2000 pounds) of feed. In some embodiments, the composition can be added to animal feeds or to food in amounts from 0.1 kg to 50 kg per ton, from 0.1 kg to 20 kg per ton, or from 0.5 kg to 10 kg per ton of feed. In certain embodiments, the composition may be added to feeds in amounts ranging from 1 kg to 5 kg per ton of feed. When expressed as a percentage of dry matter of feed, the composition may be added to animal feeds or to foods in amounts ranging from 0.01% to 2.5% by weight, such as from 0.0125% to 2% by weight. In one embodiment, the composition can be added to animal feeds or to food in amounts from 0.05% to 1.5% by weight, such as from 0.06% to 1% by weight. In another embodiment, the composition can be added in amounts from 0.1% to 0.7% by weight, such as from 0.125% to 0.5% by weight of feed.

Alternatively, the composition may be fed directly to the animal as a supplement in amounts of from 0.01 gram to 20 gram per kilogram of live body weight per day, such as from 0.01 gram to 10 gram per kilogram, 0.01 gram to 5 gram, 0.01 gram to 1 gram, 0.015 gram to 1 gram, or 0.02 gram to 0.4 gram per kilogram of live body weight per day. In some embodiments, the composition may be provided for use with many species in amounts of from 0.05 grams to 0.20 grams per kilogram of live body weight per day.

Additionally, the composition may be fed to mammalian animals species as a supplement (in combination with feed, or alone) in amounts ranging from 10 grams per head per day to 70 grams per head per day, such as from 40 grams per head per day to 70 grams per head per day, from 45 grams per head per day to 70 grams per head per day, or from 50 grams per head per day to 70 grams per head per day. In exemplary embodiments, the composition can be provided to bovines in an amount ranging from 50 grams per head per day to 60 grams per head per day, such as 56 grams per head per day.

Alternatively, the composition contained in the present composition may be fed directly to mammalian species as a supplement in amounts 0.016 grams/kg to 0.37 grams/kg of live body weight per day. In an independent embodiment, the composition may be provided to mammalian species in amounts of 0.10 grams/kg to 0.20 grams/kg of body weight per day.

In particular disclosed embodiments, the composition may be administered to the mammal species using a carrier. The carrier may be any carrier known to a person of ordinary skill in the art as being suitable for combining with a feed composition, such as molasses. One of ordinary skill in the art can appreciate that the amount of the claimed composition fed can vary depending upon the animal species, size of the animal and type of the feed to which the claimed composition is added. III. Effects of Administration

Disclosed herein is a method of administering the disclosed composition to an animal. Administration may provide a beneficial result to the animal, compared to an animal that is not administered the composition. The beneficial effects of administration include, but are not limited to, increasing the number of alveoli, increasing the level of Prolactin, changing, such as decreasing, Prolactin receptor expression, and/or decreasing the number of exfoliated mammary epithelial cells in milk, in any mammalian (including human) species that is administered the composition. The animal can be an animal raised for human consumption and/or a domesticated animal. Exemplary animals include, but are not limited to, mammals, such as ruminant species, such as a sheep, goat, cow, deer, bison, buffalo, or llama, and ungulate species, such as a horse, donkey, or pig.

A. Increasing or maintaining alveoli in mammals

Typically, mammals lactate after giving birth for a period of time before drying off. The period of time varies by species. For example, a cow usually will lactate after giving birth for approximately 10 to 11 months before drying off. During the dry-off period between lactation, typically 45-60 days, the animal, such as a cow, may lose mammary tissue (i.e., udder tissue in a cow). Such reduction in mammary tissue typically includes a reduction in the number of alveoli that are present in the mammary tissue. During a subsequent lactation period, the amount of mammary tissue typically does not return to its previous level, and accordingly the number of alveoli, and associated milk yield, is reduced compared to the number of alveoli and/or milk yield before the dry off period.

Stress events, such as heat stress, transportation, nutritional stress, environmental stress, poor bedding, etc., may exacerbate the mammary tissue loss. Without wishing to be bound by a particular theory of operation, one mechanism for the tissue loss is apoptosis, also known as programmed cell death. Mammals that are exposed to stress, including heat stress, may experience increased apoptosis of mammary tissue that may lead to reduced milk production.

Disclosed herein is the surprising result that administering the disclosed composition to animals that are not stressed, experiencing stress, or who have experienced a stress event, reduces or substantially prevents mammary tissue regression, such as udder tissue regression in animals, such as cows, during the dry off period, compared to animals that are not administered the composition. The reduction may be attributed to one or more of increased retention of mammary tissue, including alveoli, and a reduction in the apoptosis index of the mammary tissue. The reduction or substantial prevention of regression of the mammary tissue leads to substantially the same or an increased amount of mammary tissue in a subsequent lactation period compared to the amount of mammary tissue present before the dry off period, and/or compared to the amount of mammary tissue present in an animal that is not fed the composition and that is at approximately the same stage of lactation. Additionally, the number of alveoli is maintained or increased in the subsequent lactation period, compared to the number of alveoli present before the dry off period, and/or the number of alveoli present in an animal that is not fed the composition and is at approximately the same stage of lactation. This results in maintaining or increasing the milk yield in the subsequent lactation period, compared to the lactation period before the dry off.

B. Prolactin Level

Additionally, or alternatively, administration of the disclosed composition may affect the levels of Prolactin protein in the milk and blood. Prolactin is a protein that is implicated in milk production. Typically, increased Prolactin levels results in increased milk production. It is expected that administration of the disclosed composition will increase Prolactin levels in animals, such as cows, compared to animals that are not fed the composition but that are at substantially the same stage of lactation. Accordingly, milk yields in animals, such as cows, that are fed the composition will also increase, compared to animals that are not fed the composition.

C. Prolactin Receptor Expression

Administration of the disclosed composition may change the level of expression of Prolactin receptor genes in the animal. Typically, as Prolactin levels increase in an animal, the expression level of the Prolactin receptors decreases. It has been hypothesized that this might be at least partially a feedback mechanisms or nature check to prevent overstimulation of mammary gland tissue. Surprisingly, administration of the disclosed composition resulted in a decrease in Prolactin receptor expression, compared to Prolactin receptor expression in an animal that was not administered the composition, but that was at substantially the same stage of lactation. Without being bound to a particular theory, this might indicate that administration of the disclosed composition results in an increase in the Prolactin protein level, and therefore a concomitant reduction in Prolactin receptor expression occurs, to regulate the milk production that results from the Prolactin protein increase, for example, to prevent any unwanted effects.

D. Mammary Epithelial Cells (MEC)

Additionally, or alternatively, administration of the disclosed composition may affect the levels of exfoliated MEC in the milk. MEC are present in the lactating mammary gland and are, at least in part, responsible for milk production. Milk production typically is affected by the number of MEC in the gland. Exfoliation of MEC occurs during lactation, and the amount of exfoliation may be linked to the amount of apoptosis of MEC in the gland. It is expected that administration of the disclosed composition will result in a decrease in the number of exfoliated MEC detected in milk, compared to the number of MEC detected in milk from an animal that is not administered the composition, but that is at substantially the same stage of lactation. This suggests that there may be a lower apoptosis rate among MEC in the mammary gland of the animal that is administered the composition than in animals that are not fed the composition, and are at the same stage of lactation.

E. Milk Yield

The milk yield also may be maintained or increased (as compared to yields from animals that are not administered the composition) by administering the composition. For example, the milk yield may increase by 1 kg, 2 kg, 3 kg, 4 kg, up to 10 kg using the disclosed composition. In particular disclosed embodiments, animals that are provided the composition will produce milk having different milk fat and/or milk protein (in terms of percentage). For example, milk fat may be changed from 0.2% to 1%, or from 0.2% to 0.8%, or from 0.2% to 0.6%, with exemplary embodiments including a 0.4% change. In one embodiment, a dairy cow fed the composition produces a milk fat percentage of 3.8%, whereas dairy cattle not fed the composition have a milk fat percentage of 4.2% or the opposite change. F. Administration

With respect to maintaining or increasing alveoli numbers, decreasing exfoliated MEC, increasing Prolactin protein levels, and/or decreasing Prolactin receptor expression, administration of the composition may start during a lactation period, such as during the late lactation period, during the dry-off period, or be substantially continuous throughout the lactation and dry-off periods. In some embodiments, administration of the composition starts during the late lactation period. For example, in cows the late lactation period may be during the last 120 days of the lactation period. Additionally, or alternatively, the late lactation period for cows may be from 100 days from the start of lactation to the end of lactation and the start of the dry off period, such as from 150 days from the start of lactation to the end of lactation, from 200 days from the start of lactation to the end of lactation, from 220 days from the start of lactation to the end of lactation and the start of the dry off period, or from 240 days from the start of lactation to the end of lactation and the start of the dry off period. Without being bound to a particular theory, the number of alveoli may be increased or maintained in a subsequent lactation period because administration of the composition during this time period may reduce or substantially prevent the udder regression.

In some embodiments, the animal, such as a cow, will become pregnant again during the lactation period, and the administration may start before the pregnancy commences, or while the animal is pregnant, and continue through to at least birth of the offspring, such as a calf, and optionally, into the next lactation period. In some embodiments, a method of increasing or maintaining alveoli in an animal, such as a cow, comprises administering a disclosed amount of a composition disclosed herein to an animal. The animal may be lactating and may be in a late lactation period of lactating. The animal may be pregnant or may be expected to become pregnant. The administration of the composition of the animal may continue throughout the remainder of the late lactation period and through the dry off period and may further continue through birthing and into the subsequent early lactation period due to that birth. In some embodiments, the administration of the composition is maintained throughout the entire subsequent lactation period, including early, mid and late lactation, and into the next dry off period. In other embodiments, the combination of administering the composition and is stopped once the subsequent early lactation period is reached. The administration may be restarted during a subsequent late lactation period.

Administration of the disclosed composition may start during the dry-off period, or it may start during the lactation period before the dry-off period, such as during late lactation. In either case, administration typically, continues through the dry-off period and into the subsequent lactation period. In some embodiments, administration is continuous, but in other embodiments, administration may be stopped during the lactation period and restarted during late lactation or the subsequent drying off period.

IV. Reducing Stress

Additionally, or alternatively, the composition is administered to animals experiencing, or at risk of experiencing a stress event. The stress event may be, but is not limited to, heat,

environmental, bad or improper nutrition, poor bedding, and/or transportation. In some

embodiments, the composition prevents and/or promotes reduction of stress in the animal due to the stress event. Stress can impair production, reproduction, and animal health. Stress may lead to increased respiratory rate, increased body temperature, increased fluid intake, decreased feed intake, decreased weight gain, decreased milk yield, decreased reproduction, respiratory alkalosis, ruminal alkalosis, metabolic acidosis, increased oxidative stress, decreased immune function, and/or increased cell damage and death. Accordingly, any of the foregoing factors may be assessed as stress indicators. In some embodiments, the stress indicator is feed intake, water consumption, respiration rate, rectal temperature, milk yield, milk fat, milk protein, an immune biomarker, or any combination thereof. In some embodiments, animals experiencing a stress event may have a reduced number of alveoli, and administering the composition may alleviate this symptom. The composition may be administered in combination with a treatment and/or preventative measure associated with stress. For example, animals suffering, or at risk of suffering, from heat stress may be cooled, such as, by misting, fans, air conditioning, or a combination thereof.

For example, in particular disclosed embodiments, the composition disclosed herein may be used to prevent and/or promote reduction of heat stress in animals. Heat stress can impair production, reproduction, and animal health. For example, heat stress can lead to increased respiratory rate, increased body temperature, increased fluid intake, decreased feed intake, decreased weight gain, decreased milk yield, decreased reproduction, respiratory alkalosis, ruminal alkalosis, metabolic acidosis, increased oxidative stress, decreased immune function, and/or increased cell damage and death. Accordingly, any of the foregoing factors may be assessed as heat stress indicators. In some embodiments, the heat stress indicator is feed intake, water consumption, respiration rate, rectal temperature, milk yield, milk fat, milk protein, an immune biomarker, or any combination thereof. In certain embodiments, the heat stress indicator is feed intake, water consumption, respiration rate, rectal temperature, milk yield, milk fat, milk protein, or any combination thereof.

In some embodiments, heat stress conditions occur when a temperature humidity index (THI) is greater than 68, such as greater than 75, greater than 79 (regarded as a dangerous level), or greater than 84 (regarding as an emergency level). In another embodiment, heat stress conditions occur when the heat index, commonly reported by the media for humans, is above 100, such as above 110, above 115, or above 120.

The composition may be administered to an animal that is susceptible to heat stress or that suffers from heat stress. In one embodiment, the composition is administered to the animal when the temperature humidity index is, or is expected to be, greater than 68. The composition may be administered to the animal at set intervals. For example, the composition may be administered to the animal on a daily basis. In some embodiments, a daily amount of the composition is divided and administered to the animal at two or more feedings.

In some embodiments, the animal can be an animal raised for human consumption and/or a domesticated animal. Exemplary animals include, but are not limited to, mammals, such as livestock (e.g., feed or dairy cattle) or pigs. In some embodiments, the animal can be a ruminant species, such as a sheep, goat, dairy cow, beef cow, deer, bison, buffalo, or llama. In yet other embodiments, the animal can be an ungulate, such as a horse, donkey, or pig.

The composition disclosed herein may be administered in an amount effective to promote reduction of heat stress in animals, such as dairy cows, particularly lactating dairy cows. The composition may be administered pre- or post-calving for a suitable number of days. The composition also may be administered prior to lactation or after lactation. For example, the composition may be administered to the animal for a period of from 1 day prior to lactation onset to 100 days prior to lactation onset. In other disclosed embodiments, the composition may be administered to the animal for 40 days to 100 days post calving, or for 45 days to 95 days post calving, or for 50 days to 90 days post calving.

In some embodiments, a dairy cow is fed the composition for a period of time to increase milk production relative to dairy cattle not fed the composition. In particular embodiments, the dairy cow has lower milk fat relative to dairy cattle not fed the composition. In one embodiment, the dairy cow had reduced water intake relative to dairy cattle not fed the composition. In another embodiment, the dairy cow had a reduced respiration rate relative to dairy cattle not fed the composition. In yet another embodiment, the dairy cow had a reduced rectal temperature relative to dairy cattle not fed the composition. The dairy cow may have increased food intake relative to dairy cattle not fed the composition. In particular disclosed embodiments, the dairy cow may have decreased respiratory alkalosis, rumen acidosis, metabolic acidosis, and any and all combinations thereof, relative to dairy cattle not fed the composition. In another embodiment, the dairy cow has decreased serum Cortisol levels relative to dairy cattle not fed the composition.

In additional disclosed embodiments, the composition may be administered

prophylactically. In certain embodiments, the composition may be administered prophylactically and continuously. For example, the animal may be administered the composition every day for a certain period of time prior to and during lactation.

The disclosed composition may be formulated for administration to animals in order to prevent and/or promote reduction of heat stress. In particular disclosed embodiments, the effective amount of the composition administered can be affirmatively chosen based on the ability of that effective amount to promote heat stress reduction in an animal based on particular factors disclosed herein. In particular disclosed embodiments, the effective amount may be determined by administering a particular first dose to the animal, monitoring the animal during heat stress, and then adjusting the dose in order to determine an effective amount of a second dose that will ameliorate, or further ameliorate the heat stress of the animal.

In one embodiment, the composition is administered to a group of animals that have or are at risk of developing heat stress. The composition may be administered to the group of animals in a first amount for a first period of time. Subsequently a heat stress indicator of at least one animal in the group of animals is measured. Based at least in part on the heat stress indicator measurement, the amount of composition administered to the group of animals for a subsequent period of time may be adjusted. For example, the amount may be increased if the heat stress indicator measurement indicates that the animals are stressed. Alternatively, the amount may be decreased if the heat stress indicator measurement indicates that the animals are not stressed or have relatively little heat stress.

In another embodiment, individual animals in a group of animals are evaluated for heat stress. Heat stress can be evaluated, for example, by measuring a level of one or more heat stress indicators. Based at least on the measurement, a determination is made whether one or more of the individual animals is experiencing heat stress. Individual animals in the group that are

experiencing heat stress may be selected to receive the composition for a period of time effective to promote reduction of heat stress.

In particular disclosed embodiments, the composition may be administered with a molasses carrier once or multiple times a day (e.g., from two to five times per day). The composition may be mixed into the total mixed ration of feed that can be provided to the mammal species, such as in the top one-fourth, top one-third, or top one -half of the total mixed ration.

In some embodiments, the animal is further administered a therapeutic process and/or a therapeutic agent suitable for treating stress. Exemplary therapeutic processes and agents include, but are not limited to, provision of shade to the animal, use of water sprinklers to externally administer water to the animal, use of a fan to provide air movement, addition of high-fat feeds or bypass fats, meloxicam, corticosteroids (isoflupredone, fludrocortisone, triamcinolone, dexamethasone, betamethasone, flumethasone, methylprednisolone acetate, methylprednisolone sodium succinate), oral electrolytes (e.g., sodium, glucose, glycine, potassium, chloride) alkalinizing agents (e.g., bicarbonate, acetate, and/or citrate salts), direct-fed microbials, and combinations thereof.

The ability of the composition to reduce and/or prevent heat stress may be determined by comparing heat stress indicators with animals that are not administered the composition. In particular disclosed embodiments, heat stress indicators may be used to determine the effect of the composition on heat stress. Suitable heat stress indicators/factors include, but are not limited to, feed intake, milk yield, milk fat, milk protein, water consumption, respiration rates, rectal temperatures, and combinations thereof.

In particular disclosed embodiments, animals that are administered the composition will have a higher feed intake during heat stress compared to animals that are not administered the composition. The feed intake during heat stress may increase from 2 kg to 10 kg (or from 2 kg to 8 kg, or from 2 kg to 6 kg, or 2 kg to 4 kg) compared to the feed intake of an animal of the same species has not been administered the composition. In exemplary embodiments, a bovine that is administered the composition will have feed intake of 3 kg higher than a bovine who has not been administered the composition. The milk yield also may be maintained or increased (as compared to yields from animals that are not administered the composition) during heat stress by administering the composition. For example, the milk yield may increase by 1 kg, 2 kg, 3 kg, 4 kg, up to 10 kg using the disclosed composition. In particular disclosed embodiments, animals that are provided the composition will produce milk having lower milk fat and/or milk protein (in terms of percentage). For example, milk fat may be reduced from 0.2% to 1%, or from 0.2% to 0.8%, or from 0.2% to 0.6%, with exemplary embodiments including a 0.4% reduction. In one embodiment, a dairy cow fed the composition produces a milk fat percentage of 3.8%, whereas dairy cattle not fed the composition have a milk fat percentage of 4.2%. The increased milk yield may be due to increasing or maintaining the number of alveoli in the animal that is administered the composition, increasing the prolactin level, changing the amount of prolactin expression, and/or decreasing the exfoliation of MEC, ameliorating stress effects, or a combination thereof.

Water consumption, respiration rates, and serum Cortisol levels also may be lowered by administering the disclosed composition, with some embodiments exhibiting from 4 to 10 fewer respirations per minute. Another factor indicating that the disclosed composition is capable of reducing heat stress is rectal temperature, which typically may be lowered by 0.10 °C to 0.3 °C, in comparison to the rectal temperatures taken from animals that are not administered the

composition. Additionally, animals that have been administered the disclosed composition may have decreased respiratory alkalosis, rumen acidosis, metabolic acidosis, and combinations thereof in comparison to animals that were not administered the composition.

V. Examples

Example 1

A total of 60 cows on a commercial dairy in Arizona were balanced for DIM , parity and milk production and assigned to 1 of 2 treatment groups fed one embodiment of the disclosed composition (composition comprising between 15% and 40% silica, between 50% and 81% mineral clay, between 1.0% and 5.0% β-glucans, between 0.05% and 3.0% β-1,3 (4)-endoglucanohydrolase and between 1% and 8.0% mannan, 30 cows) or control (CON, 30 cows) diets for 52 days post calving. At 52 days of lactation cows were randomly selected (n = 12) from both groups (6 composition and 6 CON) and housed in environmentally controlled modules for 21 days. The composition was top-dressed 2x/day with molasses as the carrier and the CON cows received the molasses carrier 2x/day. Both were mixed into the top one-third of the TMR (total mixed ration). During the environmental room phase of the study cows fed the composition had higher feed intake than CON during heat stress (HS) (46.8 kg vs. 42.9 kg, P < 0.0001) and no difference during thermoneutral (TN). A temperature-humidity index (THI) threshold of 68 or greater was used to achieve HS. Feeding the composition maintained a numerical 1 kg milk yield advantage compared with CON (30.3 kg vs. 31.4 kg, P = 0.26) during HS but not during TN. Cows fed the composition had lower milk fat (%) (4.2% vs. 3.8%, P = 0.02) and milk protein (%) (P = 0.04). There was no difference in 3.5% FCM between treatments. Water consumption was lower (12.4 1/day in composition treated cows, P < 0.01) than control cows. Respiration rates were lower in treated cows at 1400 hours and 1700 hours (4.7 and 8.4 less respirations/min, P = 0.05, < 0.001) and rectal temperatures were also lower (0.15°C and 0.25 °C lower that CON, P = 0.05, < 0.001) in treated cows. Feeding the composition reduced physiological responses to heat stress in lactating dairy cows.

Example 2

A total of 30 cows on a commercial dairy in Arizona were balanced for DIM, parity and milk production and assigned to 1 of 2 treatment groups fed one embodiment of the disclosed composition (composition comprising between 15% and 40% silica, between 50% and 81% mineral clay, between 1.0% and 5.0% β-glucans, between 0.05% and 3.0% β-1,3 (4)-endoglucanohydrolase and between 1% and 8.0% mannan, 15 cows) or control (CON, 15 cows) diets for 90 days post calving. At 90 days of lactation, cows were randomly selected (n = 12) from both groups (6 composition and 6 CON) and housed in environmentally controlled modules for 21 days. The composition was top-dressed 2x/day with molasses as the carrier. The CON cows received the molasses carrier 2x/day. Both were mixed into the top one-third of the TMR. During the environmental room phase of the study, cows fed the composition had higher feed intake than CON during heat stress (HS) (46.8 kg vs. 42.9 kg, P < 0.0001) and no difference during thermoneutral (TN). A temperature-humidity index (THI) threshold of 68 or greater was used to achieve HS. Feeding the composition maintained a numerical 1 kg milk yield advantage compared with CON (30.3 kg vs. 31.4 kg, P = 0.26) during HS but not during TN. Cows fed the composition had lower milk fat (%) (4.2% vs. 3.8%, P = 0.02) and milk protein (%) (P = 0.04). There was no difference in 3.5% FCM between treatments. Water consumption was lower (12.4 1/day in composition-treated cows, P < 0.01) than control cows. Respiration rates were lower in treated cows at 1400 hours and 1700 hours (4.7 and 8.4 less respirations/min, P = 0.05, < 0.001) and rectal temperatures were also lower (0.15 °C and 0.25 °C lower that CON, P = 0.05, < 0.001) in treated cows. Feeding the composition reduced physiological responses to heat stress in lactating dairy cows. Experimental Design: The study consisted of two phases; 1) the commercial dairy, and 2) the controlled environmental chambers. During the commercial dairy phase, multiparous lactating Holstein cows (n=30) were balanced by DIM, milk production and parity (91 + 5.9 DIM, 36.2 + 2.5 kg/d, and 3.1 + 1.4). Cows were separated into one of two groups. The control group received the base TMR with no supplement. The treatment group was fed the base diet plus 56 g/ head/ day of the composition mixed into the TMR. Daily milk production was measured. The dairy phase lasted for 45 days. The dairy portion was used to meet the manufacture's recommended 45 days feeding for the composition to function.

After the on-dairy portion was complete, 12 cows (6 control and 6 treatment) were housed in the environmentally controlled rooms at the Agricultural Research Center (ARC). Cows continued the ARC portion in the same treatment groups from the on-dairy portion.

The ARC portion lasted for 21 days. Cows were subjected to 7 days of TN conditions, 10 days of HS, and 4 days of recovery (TN). The diurnal cycle during thermoneutral (TN) and recovery maintained a temperature humidity index (THI) < 68. During HS, the THI was greater than 68 for 16 hours/day. Temperatures mimicked ambient temperatures at a southwest United

States dairy during summer heat and TN conditions. Fresh feed was provided twice daily and cows were individually fed. Control animals received base TMR, and the treatment cows received 56 g/head of the composition per day, split between two meals. Feed intake, milk production, and milk composition were measured daily. Rectal temperatures and respiration rates were recorded 3x /day (600, 1400, and 1800 hours). Blood samples were taken by venipuncture from the tail

(coccygeal) vein on days 7 (TN), 8 (HS), 10 (HS), 17 (HS) and 18 (TN) during the ARC segment. Samples were collected 6 times per day (0400, 0800, 1200, 1600, 2000, and 2400 hours) on days 7, 8, 17, and 18, and once per day on day 14 (0800 hours). Blood was collected in Vacutainer (BD Vacutainer, Franklin Lakes, NJ) tubes containing sodium heparin for plasma and in sterile blank tubes for serum.

Statistical analyses were performed using the PROC MIXED procedure (version 9.3, SAS Institute, Cary, NC). Cow was the experimental unit (ARC portion). Data is presented in least square means with significance declared with a P- value < 0.05. (See Table 1, below).

There were no initial differences in milk yield (control = 38.6 kg/day and treatment = 38.6 kg/day) at the start of the on-dairy phase of the study. There was a numerical advantage to feeding the composition (FIG. 1) of 1.5 kg of milk/day, but this was not significant (control = 36.8 kg/day and treatment = 38.3 kg/day).

There was a period effect on milk yield (P < 0.01) during the environmental room (ARC) phase associated with a decline in milk yield in both groups during HS. Milk yield at the ARC (P < 0.23) did not differ between control and composition-fed groups (FIGS. 2 and 3), however, there was a numerical advantage (1.1 kg/day) for cows fed the composition during HS (P < 0.26) which was similar to the pattern in milk yield noted during the on-farm phase

Feeding the disclosed composition to heat stressed dairy cows maintained feed intake during heat stress. Feed intakes in the two groups did not differ during TN but was higher during HS in composition-fed cows (46.8 kg/d and 42.9 kg/d, P < 0.01, FIGS. 4, 5; Table 1).

Milk protein (%) and fat (%) were lower in composition-fed cows (Table 1) during HS but not during TN. There was no difference in FCM or protein yield between treatments. Cows fed the composition displayed decreased somatic cell count (SCC) compared to control cows (59.4 and 26.3 x 1000, P < 0.03; Table 1) with the greatest difference during the recovery period (FIG. 6). There was a spike in SCC around day 5 (TN) and during recovery around day 17 (FIG. 6).

Table 1. Effects of feed supplementation with one embodiment of the disclosed composition to heat-stressed lactating dairy cows

Respiration rate and rectal temperatures did not differ between treatments during TN;

however, during HS, administration of the composition reduced respiration rate, (Table 2, P < 0.01) in both environments at 1400 hours and in HS animals at 1800 hours when environmental heat load was greatest. Rectal temperatures were lower in cows fed the composition at 1400 and 1800 hours compared to controls when environmental heat loads were maximal. Table 2. Effects of feed supplementation with one embodiment of the disclosed composition and environment on respiration rate and rectal temperature in lactating dairy cows

Hormones in plasma are important as potential indicators of the physiological status of a cow and reflect the physiological compensations a cow undergoes at various stages of lactation and exposure to HS. Serum Cortisol levels were highest on day 8 (first day of HS, FIG. 7). This is in agreement with prior reports that acute but not chronic HS is associated with increases in circulating Cortisol concentrations (Christian and Johnson, 1972, Wise et al., 1988). Composition- treated cows had significantly lower serum Cortisol on day 8 (0.8372 vs. 0.4838 μg/dL for control and composition respectively, P < 0.006) and did not differ on other days. This suggests that the composition may reduce impact of acute stress on the Cortisol response in lactating dairy cows.

Serum insulin and plasma glucose levels (FIGS. 8 and 9) were not different between groups (P = 0.8248 and 0.945). Serum insulin concentrations in both groups rose during the latter part of the HS period and during the recovery period. The reason for this pattern is unknown.

The immune function of cattle on this study was evaluated by looking at the expression of the interleukin-8 receptor (FIG. 10) and expression of Regulated on Activation, Normal T

Expressed and Secreted (RANTES) protein (FIG. 11) which is a member of the interleukin-8 family of cytokines.

Heat stress exposure was mild to moderate in this study. The threshold for heat stress in lactating dairy cows is a THI > 68, respiration rates > 60 bpm, and rectal temperatures > 38.5°C (Zimbleman et al., 2009). The composition reduced impact of thermal stress on stress of lactating dairy cows. Cows fed the composition had reduced rectal temperatures and respiration rates during periods of peak thermal load. Respiration rates in treated cows did not exceed 60 bpm and mean rectal temperatures were 0.2 to 0.3 °C cooler. Cows fed the composition displayed higher feed intakes during HS as well. Cows fed the composition also displayed a lower Cortisol spike on the first day of heat stress.

Milk yield decreased with heat stress in both control animals and the composition-fed animals. However, feed intake was unchanged in cows fed the composition and milk yields were numerically higher. Changes in SCC were consistent between groups. Cows fed the composition displayed decreased SCC compared to control cows with the greatest difference during the recovery period.

Serum Cortisol levels were similar to previous findings (Christison and Johnson, 1972) and increased within the first day of heat exposure. The animals in the ARC had higher Cortisol levels compared to published levels, but the confinement and changes in surrounding from the dairy to the ARC may account for some of the changes.

Cytokine (RANTES) gene expression was higher in cows fed the composition during the HS portion of the study but not during recovery. The elevated cytokine gene expression may be associated with improved immune function in cows fed the composition.

Example 3

Embodiments of the disclosed composition, such as compositions comprising between 15% and 40% silica, between 50% and 81% mineral clay, between 1.0% and 5.0% β-glucans, between 0.05% and 3.0% β-1,3 (4)-endoglucanohydrolase and between 1% and 8.0% mannan

(composition), have been demonstrated to reduce physiological measures of heat stress (e.g., body temperature, respiration rate, and water intake) in cattle subjected to temperature and humidity conditions above their thermoneutral zone. Concomitantly, in composition-fed animals, feed intake was significantly increased and milk yield was numerically increased during periods of thermal stress. Furthermore, measures of immune function were improved along with reduced Cortisol concentrations during acute heat stress. Paradoxically, adrenocorticotrophic hormone (ACTH) which regulates Cortisol secretion from the adrenal cortex was significantly increased in animals fed the composition (FIG. 12). This suggests that either the adrenal is less responsive to ACTH or that corticoid binding globulin is increased in animals fed the composition. Both conditions would result in increased ACTH secretion from the pituitary since negative feedback in both cases would be reduced. The objective of this study is to determine if feeding one embodiment of the disclosed composition to lactating dairy cows decreases adrenal responsiveness to ACTH or increases corticosteroid binding globulin in either thermoneutral or heat stress conditions. Heat stress in dairy cows may result in reductions in dry matter intakes and milk yields and elevated somatic cell counts, respiration rates, body temperature and plasma heat shock protein and serum Cortisol. Feeding the composition may reduce the impact of heat stress on these measures of stress response. Feeding the composition to non-heat stressed lactating dairy cows for 52 days prior to a heat stress bout may increase the expression of immune markers of neutrophil function (L- selectin, IL8-R and IL-1B) associated with altered secretion of Cortisol from the adrenal gland. This may be associated with reduced Cortisol secretion in response to ACTH infusion in cows fed the composition. Heat stress may reduce the expression of immune markers of neutrophils even in cows supplemented with the composition. However, the reduced Cortisol secretion response may also be present in heat stressed dairy cows fed the composition.

Experimental Design:

Animal Selection and Treatment Assignment: Thirty lactating dairy cows will be assigned to one of two treatments (15 head/treatment). Treatment 1 cows will be fed a control diet without the composition, and Treatment 2 cows will be fed the control diet plus the composition. The composition will be pre -blended in a grain mix to provide 56 gram/hour/day. Each cow will be housed in an individual tie-stall where individual feed and water intake can be controlled and recorded prior to moving to the Agricultural Research Center (ARC). After 45 days on study, all cows will be given a low dose ACTH challenge (20 mg) via tail vein infusion, and blood samples will be taken at time zero, 1 hour, 4 hours and 8 hours after ACTH challenge. Subsequently, a subgroup of 12 cows, which have been on their respective diets on a commercial dairy for 55 days prior to the moving to the ARC environmental controlled rooms, will be added. From the 30 original cows assigned to treatments, 6 cows from each treatment will be selected for the trial and will continue on their respective treatment diets while in the ARC. Cow will be the experimental unit.

Blood Sample Schedule: Blood samples will be collected on all treatment cows while at the commercial dairy at days 1, 21 and 42 after initial treatment assignment. On days 45- 50 ten cows each day will be subjected to low dose ACTH challenge. On each sampling day, approximately 2 hours after the morning feeding, cows will be restrained in headlocks, taking care to avoid stressing the animals; tail vein blood samples will be obtained from each animal and then infused via tail vein or tail artery with 20 μg (=2 IU) of a synthetic analogue of ACTH (ACTHl-24, Synacthen^® - Novartis Pharma AG - Stein, CH). Blood samples will also be taken 30 and 60 minutes after ACTH injection, leaving cows restrained in the head locks. All the blood samples will be collected in vacuum Li-heparin tubes, and immediately stored in iced water. In the blood samples, packed cell volume (PCV) will be determined and, after centrifugation (3500g for 16min. at 6°C), plasma Cortisol will be measured by the RIA method (Coat-A-Count; DPC, Los Angeles, CA, USA). The integrated response of Cortisol over 60 min will be evaluated as area under the curve. For the statistical evaluation, data will be subjected to ANOVA using GLM procedure (SAS Inst. Inc., Cary, NC, release 9.1) including in the model cow, dietary treatment (control or composition) stage of lactation as main factors. A repeated measures analysis will also be conducted on the challenge data.

At arrival to the ARC, cows will be weighed, fitted with halters and blood samples collected. Cows will remain in the ARC chambers for 21 days, the first 7 days at thermal neutral (TN) conditions followed by 10 days of heat stress (HS) and then 4 days in TN conditions. On day 7 of TN, day 8 (HS), day 17 (HS), day 18 (TN) and day 21 (TN) cows will be bled in 4 hour intervals at 0400, 0800. 1200, 1600, 2000, and 2400 hours following a low dose (20mg) of ACTH infused via the tail vein.

Physiological/Behavior Metrics: To assess the effectiveness of the imposed heat stress model, known physiological and behavioral responses will be measured and recorded daily. These will include feed intake, water consumption, milk yield, milk somatic cell concentrations (cells/ml), respiration rate, rectal temperature and skin temperature measurements (3x/day at 0600, 1400 and 1800 hours). Milk samples will be collected and stored in vials containing bronopol tablets for preservation and stored at 4 °C until analysis. Analysis will be done by infrared analysis. All treatment and health events will be recorded daily.

Blood collected for immune biomarkers will be collected and preserved according to protocol (Wang, et al., 2003). The immune biomarkers neutrophil L-selectin, IL8-R and IL-IB will be evaluated on each sample. In addition, on days 17 and 18 from the 0800 and 1600 hours samples, neutrophils will be purified and assessed for RANTES (regulated on activation, normal T cell expressed and secreted), phagocytosis ability or ROS (reactive oxygen species) generation. Ionized serum calcium will also be determined on samples collected on days 1, 7, 17, 18 and 21. Blood samples collected on day 1 of arrival to the ARC and days 7, 8, 12, 17, 18 and 21 will be assayed for Heat Shock Protein (HSP) and Cortisol. These samples will be stored on ice until centrifugation (15,000 x g) for 15 minutes at 4 °C. Plasma will be removed and stored at -20 °C and the buffy coat fraction will be removed and stored in Trizol at -80 °C. On days of blood sampling, additional milk samples will be collected at both the AM and PM milking and neutrophils isolated. An additional possibility is the sampling of fecal Cortisol as a noninvasive measure of Cortisol output. Another consideration is to measure corticoid binding globuli (CBG). An increase in CBG would reduce effective concentrations of Cortisol and cause increases in ACTH. Example 4

Procedures: Calf-fed steers (306 head; BW 263.5 + 18.6 kg; 36 pens) were utilized in a randomized block design experiment. Steers were received over a two-day span at the feedlot. Upon arrival, steers were allowed access to water and were processed, weighed, and allocated to treatment within 12 hours. During processing, steers were identified with an individual ear tag, individually weighed, vaccinated with Bovi-Shield Gold One Shot™ (IBR, BVD), Dectomax injectable^® (internal and external parasiticide), and Somubac^® (Haemophilus somnus disease complex). Shrunk BW was a single weight collected at time of processing following arrival. Steers were blocked based on arrival date resulting in two blocks. Within blocks, steers were assigned randomly to one of three treatments by gate sorting every two steers to a pen; pen was then randomly assigned to treatment. Treatments included: a control group (Control) with no supplementation; a group that was supplemented with one embodiment of the disclosed

composition (composition comprising between 15% and 40% silica, between 50% and 81% mineral clay, between 1.0% and 5.0% β-glucans, between 0.05% and 3.0% β-1,3 (4)-endoglucanohydrolase and between 1% and 8.0% mannan) at 4 g/cwt of BW for 28 days (receiving period); and a group that was supplemented with the composition at 4 g/cwt of BW for 215 days (the entire feeding period). Supplementation with the composition for both the 28 days and 215 days treatment groups was formulated and added to the daily delivery of the diet prior to feeding. A ninth steer was randomly added to nine pens of the control group and the treatment group that only received the composition for 28 days during processing of the 2^nd block. These 18 steers (9 hd/treatment group) would allow for the selection of one steer from each of these 18 pens to be utilized for the intravenous lipopolysaccharide (LPS) challenge portion of the trial.

At the conclusion of the 28 day receiving period, steers were limit fed for a period of 4 days at 2% BW to obtain an end of receiving period BW. At the conclusion of the 28 day receiving period, the composition was no longer supplemented to the group of steers within the treatment group that only received the composition for the 28 day of the receiving period; steers within the other composition treatment group (fed for 215 days) continued to receive the composition.

Composition supplementation for the remaining treatment group was recalculated every 30 days to supply 4 g/cwt of BW on average. All steers were implanted with Revalor^® XS at the conclusion of the receiving period. During the last 28 days of the finishing period, all cattle were supplemented Optaflexx^® ractopamine composition at a rate of 300 mg/hour/day. At the end of the trial, steers were transported 51.5 km to a commercial abattoir and held over-night. The following morning steers were harvested; at which time hot carcass weights (HCW) were recorded.

Following a 48-hour chill, fat thickness, LM area, and USDA marbling score were determined. Final BW, ADG, and F:G were calculated using HCW adjusted to a standard (63%) dressing percentage.

To evaluate the immune response, two LPS challenges were conducted; a subcutaneous LPS challenge (steers from the Control group and the composition-fed for 215 day group) and an intravenous LPS challenge (steers from the Control group and the composition-fed for 28 day group). For the subcutaneous LPS, three pens from the Control and the composition-fed 215 day treatment group were randomly selected. From the selected pens, 6 steers/pen were randomly selected and challenged with LPS via subcutaneous injection on day 21. On day 21, steers from selected pens were removed from the pen and moved to the processing barn. As each steer was processed, BW was determined and an indwelling rectal probe was inserted. After determining BW, each steer was challenged with a subcutaneous injection of LPS at a rate of O^g/kg of BW and then returned to the feedlot pen. For the intravenous LPS challenge, on day 25 of the receiving period, 18 steers (nine steers from Control and composition- fed for 28 day treatment groups) were randomly selected from the 9 Control and 9 composition- fed for 28 day pens that contained 9 steers/pen. Steers were identified and moved into a tie stall barn. After a 3 day adjustment period, steers were fitted with indwelling jugular vein catheters for serial blood collection and indwelling rectal temperature (RT) recording devices, set to record RT at 1-min intervals continuously throughout the immune challenge study. After insertion of the jugular catheter and RT probe, steers were returned to the individual tie stalls and allowed to rest for the remainder of the day.

On the following day, from 0800 to 1600 hours, blood samples were collected at 30-min interval; two hours prior to the challenge (0800 - 1000 hours) and six hours after the challenge (1000 - 1600 hours). At 1000 (0 hour), following the collection of the blood sample, steers were administered an i.v. dose of lipopolysaccharide (LPS, 0.5 μg/kg BW; purified from E. coli 0111 :B4; Sigma- Aldrich, St. Louis, MO). A final blood sample was collected 24 hours post LPS challenge. At each collection point, 9 mL of blood was collected via monovette tubes containing no additive for serum. After collection, blood samples were allowed to clot for 30 min at room temperature and then centrifuged at 2,000 x g for 30 min (39.2 °F). Serum was collected and transferred into 1.5 mL microcentrifuge tubes and stored at -112 °F until analyzed. Serum was analyzed for Cortisol and pro-inflammatory cytokines (tumor necrosis factor - a, TNF-a; interferon - γ, IFN-γ; and interleukin - 6, IL-6). Feedlot performance data were analyzed as a randomized block design using MIXED procedures of SAS (SAS Institute, Inc., Cary, NC). Steers were blocked by arrival date and pen was the experimental unit; model included the fixed effect of treatment and block was a random effect. Immune response data were analyzed as a completely randomized design with repeated measures using the MIXED procedures of SAS; model included fixed effects of treatment and time, treatment x time was used as the error term to test whole plot effect. For both feedlot and immune data, when results of F-test were significant (P < 0.05), group means were compared by use of least significant difference. Pair wise differences among least squares means at various sample times were evaluated with the PDIFF option of SAS. Distribution of USDA Quality Grade data were analyzed as a randomized block design using the Glimmix procedure of SAS.

Results: For the receiving portion of the trial, there was no difference in initial BW (P = 0.82), ending BW (P = 0.43), ADG (P = 0.32), DMI (P = 0.76), and F:G (P = 0.35) between the three treatments (Table 2). In terms of morbidity related to respiratory diseases, there was no difference (P = 0.21) in the % of steers treated (Table 3). There was also no difference in overall feedlot performance; DMI (P = 0.89), ADG (P = 0.66) and F:G (P = 0.90) were similar across the three treatments (Table 2). There was no difference in final BW (P = 0.59), HCW (P = 0.60), LM area (P = 0.31), marbling score (P = 0.96), 12^th rib fat thickness (P = 0.86), or calculated yield grade (P = 0.52) between the three treatment groups. The distribution of USDA Quality Grade was analyzed across all three treatments. There was no difference in the amount of USDA Prime, USDA Choice, or USDA Select Quality Grades for all three treatments (Table 3). While there was no statistical difference in terms of the percentage of carcasses grading USDA Choice or greater, the economic significance is still of major importance. Steers supplemented with the composition for 215 days had a rate of USDA Choice that was 81.91%, compared to 76.40 for Control steers. With a $13.58 Choice / Select spread (11 Dec 14), this difference in Choice between the two treatment groups is the equivalent of about $850 difference in carcass value.

Table 3. Receiving period and overall feedlot performance and carcass merit for steers not fed the composition (CON), one embodiment of the disclosed composition fed during the receiving period (comp-28), or the composition for 215 days (comp-215)

Treatment groups¹

Item Con comp-28 comp-215 SEM P-value

Receiving Performance

Initial BW (kg) 259 256 262 3.2 0.82

Ending BW (kg)² 300 304 306 3.6 0.43 DMI (kg/d) 7.5 7.7 7.6 0.4 0.76

ADG (kg/d)³ 1.44 1.54 1.58 0.15 0.32

G:F⁴ 0.9 0.9 0.09 - 0.35

Morbidity (%)⁵ 6.9 2.0 3.0 3 0.12

Feedlot Performance

Initial BW (kg) 261 572 572 6 0.87

Final BW (kg)⁶ 649 642 643 11 0.59

DMI (kg/d) 9.80 9.80 9.88 0.3 0.89

ADG (kg)⁷ 1.81 1.79 1.79 0.04 0.66

G:F 0.18 0.18 0.18 - 0.90 rcass Merit

HCW (lbs) 409 405 405 3.2 0.96

LM area (in²) 94.78 91.36 94.06 1.61 0.31

Calculated YG 3.09 3.28 3.09 0.14 0.52

12^th rib fat (cm) 1.37 1.42 1.40 0.08 0.86

Marbling⁸ 503 498 508 24 0.96

Prime (%) 4.49 2.13 5.32 2.32 0.53

Choice (%) 76.40 76.60 81.91 4.50 0.59

Select (%) 19.10 21.28 13.83 4.22 0.41

¹ CON - No composition; comp-28 - composition during receiving; comp-215 - composition for 215 days.

²Limit fed at 2% of BW for 4 days prior to single BW to determine ending BW of receiving period

³Calculated from ending BW of receiving period

4Analyzed as G:F, the reciprocal of F:G.

⁵Overall percentage of steers treated for bovine respiratory disease

6Calculated from carcass weight, adjusted to 63% common dressing percent.

7ADG for the entire feeding period (including receiving period)

8Marbling Score: 400 = Small, 500 = Modest, etc. For the subcutaneous LPS challenge, there was a treatment (P = <0.001) and time (P = <0.001) effect for RT (FIG. 13). Both treatment groups responded to the subcutaneous LPS challenge, with both treatment groups achieving a maximum RT approximately 5 hours post challenge (Control = 40.10 °C vs. composition = 39.92 °C; FIG. 13). Furthermore, both maintained similar patterns (P = 1.00) throughout the subcutaneous LPS challenge. Steers in the Control treatment group had an average RT that was greater than the composition treatment group (39.22 °C and 39.07 °C, respectively). There was no treatment effect (P = 0.75) or treatment x time interaction (P = 0.99) for change in RT from baseline (FIG. 14). Again, there was a treatment effect for RT (P = <0.001), but this treatment difference was not observed when comparing the change in RT from baseline between the treatment groups (average change from baseline was 0.03 °C for Control and 0.03 °C for the composition).

For the intravenous LPS challenge portion of the trial, there was a treatment (P = 0.002) and time effect (P = <0.001), however there was no treatment x time interaction (P = 0.99) for RT (FIG. 15). Steers supplemented with the composition had a greater RT when compared to Control (39.26 °C vs. 39.17 °C, respectively) during the intravenous challenge. This average RT is very similar to the RT that was observed for the steers that were utilized in the subcutaneous LPS challenge. In terms of response to the intravenous LPS challenge, both treatment groups responded to the LPS challenge similarly (P= 0.99). For both groups of steers, maximum RT was observed 2.5 hours post intravenous LPS administration, and within 6 hours, RT had returned to baseline temperatures (-21 to 0 hours prior to the challenge: FIG. 15). There was also no treatment effect (P = 0.49) or treatment x time interaction (P = 0.99) for change in RT from baseline (-21 to 0 h: FIG. 16).

Baseline line temperatures were different (P < 0.001) between the two treatment groups, but the change from baseline was similar (P = 0.49) between the Control and composition-supplemented steers (FIG. 16).

For serum concentrations of Cortisol, there was a treatment (P = 0.005) and time effect (P =

<0.001); though there was no treatment x time interaction (P = 0.99). Steers supplemented with the composition had decreased concentrations of Cortisol, when compared to the CON steers (25.5 vs 29.2 ng/ml, respectively; FIG. 17). In terms of response to the intravenous LPS challenge, both treatment groups responded similarly (P = 0.99). Prior to the LPS challenge, both treatment groups had Cortisol concentrations below 10 μg/mL. Thirty minutes after the LPS challenge, Cortisol concentrations had increased (P = < 0.001) to above 35 μg/mL; Cortisol concentrations for both treatment groups did not return to baseline concentrations until 24 hours post LPS challenge (FIG. 18). Blood urea nitrogen (BUN), non-esterified fatty acids, and glucose were analyzed to evaluate metabolic alterations during the intravenous LPS challenge. For BUN' s, there was a treatment (P = < 0 .001) and time (P = < 0.001) effect, but there was no treatment x time interaction (P = 0.99). Control steers had a greater (P = < 0.001) concentration of BUNs when compared to the composition-supplemented steers (FIG. 19). This difference in BUNs was observed prior (P =

0.01) and post LPS challenge (P = < 0001). Regardless of the LPS challenge, Control steers' BUN concentrations were on average 12.4 mg/mL while composition-supplemented steers' BUN concentrations were on average 11.5 mg/mL (FIG. 19.) There was a treatment (P = 0.009) and time (P = < 0.001) effect for glucose concentrations; no treatment x time interaction (P = 0.19). Serum glucose concentrations were greater (P = < 0.001) for composition-supplemented steers, when compared to Control steers (76.4 mg/mL vs. 72.4 mg/mL, respectively; FIG. 20). This difference in glucose was not apparent prior to the LPS challenge, as baseline concentrations (-2 to 0 hours) were similar (0.62; FIG. 21). However, following the LPS challenge, while both treatment groups had a decrease in serum glucose, this reduction in glucose was greater (P = 0.009) in the Control steers, when compared to the composition-supplemented steers (FIG. 21). Prior to the LPS challenge, both treatment groups maintained serum glucose concentrations greater than 75 mg/mL. However, following the LPS challenge (1 hour post LPS challenge) serum glucose concentrations started to decrease, and continued to decrease until 3.5 hours after administrations of the LPS challenge (FIG. 21). During this decrease in glucose concentrations, concentrations in the Control steers decreased to a greater extent than the composition-supplemented steers. While this decrease of glucose concentrations did not result in a treatment x time interaction, this decrease did result in an overall treatment difference between the two treatment groups (FIG. 21). There was a treatment (P = < 0.001) and time (P = < 0.001) effect, and a tendency for a treatment x time interaction (P = 0.007) for serum NEFA concentrations. Steers within the Control treatment group had greater (P = < 0.001) serum NEFA concentrations, when compared to composition-supplemented steers (FIG. 22). This difference in NEFA concentrations was present prior (P = 0.006) to the intravenous LPS challenge. However, following the LPS challenge these differences were even greater (P = < 0.001 : FIG. 22). Prior to the LPS challenge, NEFA concentrations for Control steers were 0.10 mmol/ml while composition-supplemented steers were 0.07. However, following the LPS challenge, NEFA concentrations in the Control steers increased to 0.23 mmol/mL while the composition-supplemented steers only increased to 0.11 mmol/mL.

There was a treatment (P = < 0.001) and a time effect (P = <0.001) for the proinflammatory cytokine IFN - γ; there was no treatment x time interaction (P = 0.77). composition- supplemented steers had a greater production of IFN - γ, when compared to the Control steers (FIG. 23). Prior (P = 0.05) and post (P = < 0.001) LPS challenge, the concentrations of IFN - γ were greater in the composition-supplemented steers, when compared to the Control steers (FIG. 23). There was also a treatment (P = 0.03) and time (P = < 0.001) effect for TNF - a; there was no treatment x time interaction (P = 0.42). Overall serum concentrations for TNF - a were greater (P = 0.03) in the composition-supplemented steers, when compared to the Control steers (FIG. 24). For both treatment groups, prior to the LPS challenge, no detectable concentrations of TNF - a were present, and this no detectable concentrations continued until 0.5 hours post challenge (FIG. 25). At 0.5 hours, concentrations of TNF - a dramatically increased (P = < 0.001) for both treatment groups. Concentrations of TNF - a returned to near baseline (-2 to 0 h) around 4 hours after the LPS challenge (FIG. 25). There was only a time effect (P = < 0.001) for the proinflammatory cytokine IL- 6 (FIG. 26). As with TNF - a, prior to the LPS challenge,

concentrations of IL - 6 were not detectable. However, 1 hour after the LPS challenge, concentrations of IL - 6 dramatically increased (P = < 0.001); reaching maximum concentrations at 2 hours post LPS challenge. From 1 to 8 hours post LPS challenge, IL - 6 concentrations remained elevated above baseline concentrations, and did not return to baseline concentrations until 24 hours post LPS challenge (FIG. 26).

The mean complete blood cell count (CBC) analysis is reported in Table 4.

Supplementation with the composition did not impact red blood cell counts (P = 0.45) or the monocyte percentage (P = 0.26). Hemoglobin concentrations (P = 0.008), Hematocrit % (P = 0.03), and white blood cell concentration (P = 0.001) were greater (P = 0.008) in Control steers, when compared to composition- supplemented steers. Neutrophil % (P = 0.04) and eosinophils concentration (P = 0.02) were greater in composition-supplemented steers, when compared to Control steers. For the % of lymphocytes, there was a treatment x time interaction (P = 0.006). Prior to the LPS challenge (- 2 to 0 h), Control steers had a greater (P = < 0.001) concentration of lymphocytes compared to the composition-supplemented steers (FIG. 27). One hour after the challenge, there was no difference (P = 0.35) between the treatment groups, and from 1 hour to 8 hours, there was no difference (P = 0.66). However, 24 hours after the LPS challenge, Control steers had a greater (P = 0.005) concentration of lymphocytes (FIG. 27). There was also a treatment x time interaction (P = 0.006) for the neutrophil: lymphocyte ratio. Prior to the LPS challenge, the neutrophil: lymphocyte ration was greater (P = < 0.001) for steers fed the

composition, when compared to Control steers (FIG. 28). Table 4. Mean Complete Blood Cell Count values newly received steers supplemented with one embodiment of the composition at a rate of 4 mg/cwt during the receiving period (composition) or no composition (CON) during a lipopolysaccharide challenge

P - value

Variables composition Control SEM TRT TIME TxT

Red Blood Cells (mil/μΐ,) 8.46 8.47 .30 0.45 0.41 0.99

Hemoglobin (g/dL) 10.61 10.97 0.09 0.008 0.18 0.99

Hematocrit (%) 31.06 32.29 0.38 0.03 0.46 0.99

White Blood Cells (Κ/μ]_) 4.47 5.42 0.12 0.001 <0.001 0.88

Neutrophils (%) 0.43 0.26 0.05 0.04 <0.001 0.34

Lymphocytes (%) 3.02 3.63 0.09 <0.001 <0.001 0.006

Monocytes (%) 1.06 1.14 0.20 0.26 <0.001 0.23

Eosinophils (K/uL) 0.07 0.04 0.005 0.02 0.76 0.76

Neutrophils:Lymphocytes 0.14 0.09 0.016 0.01 <0.001 0.006

Conclusions: In this study, newly received calf-fed steers supplemented with the composition for a period of 28 days (receiving period) or for 215 days (entire feeding period) did not impact feedlot performance or carcass merit. While there was no statistical difference in terms of the percentage of carcasses grading USDA Choice or greater, the economic significance should outweigh the lack of a statistical difference. With a $13.58 Choice / Select spread (11 Dec 2014), this difference in Choice between the two treatment groups is the equivalent of about $850 in carcass value. Furthermore, supplementation with the composition alters the immune response. When challenged with a lipopolysaccharide, composition supplementation appears to prime the pro-inflammatory response (as evident by increased concentrations of IFN - γ and TNF - a). In addition, there was the difference in metabolism that was observed. The increased concentrations of BUN's and NEFA's within the Control steers indicate a possible greater energy need to initiate and sustain an immune response, when compared to the composition-supplemented steers. This increase in greater energy demand is further indicated as the composition-supplemented steers had greater concentrations of serum glucose, when compared to the Control steers. Overall, these results suggest that supplementation with embodiments of the composition may enhance the immune response of calf-fed steers upon feedlot entry.

Example 5

A study was conducted to identify genes expressed by circulating immune cells that are regulated by one embodiment of the disclosed composition (Composition). Rats (n=6 per group) were randomly assigned to Composition and control groups. One embodiment of the disclosed composition (comprising between 15% and 40% silica, between 50% and 81% mineral clay, between 1.0% and 5.0% β-glucans, between 0.05% and 3.0% β-1,3 (4)-endoglucanohydrolase and between 1% and 8.0% mannan) was supplemented in the diet at 0.5% in the Composition group. Total RNA was purified from whole blood and gene expression was analyzed with the use of the Rat Innate and Adaptive Immune Responses RT2 Profiler Polymerase Chain Reaction (PCR) Array (SABiosciences, Qiagen). A total of 84 target genes were present on the array. Gene expression of circulating immune cells was analyzed at seven, fourteen, twenty-one and twenty-eight days of Composition supplementation. The expression of 67 genes changed following Composition supplementation across the time points. Table 5 lists the genes with altered gene expression following Composition supplementation and includes information indicating stimulation (+) or repression (-) of gene expression.

Table 5

Ccl3 - Tlr7 +

Ccr6 - Irf7 +

Cd40 - Rorc +

Ddx58 - Cd401g +

1118 - Tbx21 +

Jun - Casp8 +

Tnf - 1123a +

Traf6 - Cdl4 +

Statl - Cd8a +

Cxcr3 +

Foxp3 +

Lbp +

Mapkl +

Myd88 +

Stat6 +

Agrin +

IL33 +

Example 6

Feeding one embodiment of the disclosed composition at 0.5% of the diet supports immune function in ruminant livestock. Targeted profiling of immune-associated genes in whole blood is an established methodology to evaluate the efficacy of feed additives with immune- altering properties. We hypothesized that higher daily inclusion rate of the composition than 0.5% may be required to optimize immune function. The objective of this study was to evaluate the effect of dietary composition inclusion rate (1% vs. 0.5%) on the expression profile of immune- associated genes. Male CD rats (5/treatment) weighing 180-200 grams had ad libitum access to a diet with 0 (control), 0.5 (lx), or 1% (2x) of the composition for 28 days. At the end of the feeding period, whole blood was collected. RNA was purified from whole blood samples and used to generate cDNA that acted as template in the Rat Innate and Adaptive Immune Responses RT² Profiler PCR array (SABiosciences). Using PROC GLM, we compared cDNA abundance of immune-associated genes between control and supplemented groups (0.5 or 1%) with a P < 0.05 cut-off value for significance. Of the 79 immune-associated genes that were expressed above the detection limit in all samples, 16 (7 up-regulated) and 13 genes (8 up-regulated) were altered by 0.5% and 1% composition supplementation, most of which (11 with 6 up-regulated) were altered at both composition inclusion rates. Genes that were up-regulated at both rates include IL13 (0.5%: +3.16, 1%: +3.70 fold-change), IL5 (0.5%: +2.64, 1%: +2.62), Iraki (0.5%: +2.50, 1%: +1.98), Nod2 (0.5%: +1.83, 1%: +2.02), IFNal (0.5%: +1.81, 1%: +2.10), and Cd80 (0.5%: +1.77, 1%: +2.47). Genes that were down-regulated at both inclusion rates include TLR3 (0.5%: -2.22, 1%: -2.39), CxCLlO (0.5%: -2.19, 1%: -2.26), STAT1 (0.5%: -2.07, 1%: -1.99), STAT3 (0.5%: -2.05, 1%: - 1.92), and NFKM (0.5%: -1.84, 1%: -1.75). In conclusion, our results suggest that supplementation inclusion rate independently promotes immune function through various pathways including pathogen recognition, adaptive immune cell activation, and various transcription factors. Example 7

Embodiments of the disclosed composition have been shown to augment immune function in ruminants and other species. Targeted profiling of immune-associated genes in whole blood is an effective platform for identification of multiple immune response markers to feed additives with immune-altering properties. The objective of this study was to identify multiple immune response markers that were increased by administration of one embodiment of the disclosed composition (comprising between 15% and 40% silica, between 50% and 81% mineral clay, between 1.0% and 5.0% β-glucans, between 0.05% and 3.0% β-1,3 (4)-endoglucanohydrolase and between 1% and 8.0% mannan) throughout a 28-day supplementation period. It was hypothesized that several immune-associated genes in whole blood are consistently up-regulated during a 28-day

supplementation period. Fourteen male CD rats weighing 180-200 grams had ad libitum access to a diet containing 0 (control; n=5, only 28 days) or 0.5% composition for 7 (n=4) or 28 days (n=5). Whole blood was collected at the end of the feeding period. RNA was purified from whole blood samples and used to generate cDNA that acted as template in the Rat Innate and Adaptive Immune Responses RT² Profiler PCR array (SABiosciences). Using PROC GLM, cDNA abundance of immune-associated genes was compared between control and supplemented groups (7 or 28 days) with a P < 0.05 cut-off value for significance. Of the 77 immune-associated genes that were expressed above the detection limit in all samples, 6 genes were up-regulated after 7 days of composition supplementation and 4 genes were up-regulated after 28 days of composition supplementation. Three genes were up-regulated after 7 days (Cd80: +2.40; Iraki: +2.25; Nod2: +2.08 fold-change) as well as after 28 days of supplementation (Cd80: +1.77; Iraki : +2.50; Nod2: +1.83 fold-change). In conclusion, the results suggested Cd80, Iraki, and Nod2 as immune response markers that were increased by administration of the composition throughout a 28-day supplementation period. Example 8

This study was designed to determine the effect of supplementing feedlot steers with one embodiment of the disclosed composition (comprising between 15% and 40% silica, between 50% and 81% mineral clay, between 1.0% and 5.0% β-glucans, between 0.05% and 3.0% β-1,3 (4)- endoglucanohydrolase and between 1% and 8.0% mannan) on the acute phase response to a lipopolysaccharide (LPS) challenge. Steers (n=18; 270+5 kg BW) were separated into two treatment groups (n=9/treatment): one group was fed a standard receiving diet (Control, Cont) and the other group was fed the same receiving diet supplemented with the composition at 4 g/45.4 kg BW for 29d (composition). On day 27 steers were fitted with indwelling jugular cannulas and rectal temperature (RT) monitoring devices and placed in individual stalls. On day 28, steers were challenged i.v. with LPS (0.5 μg/kg BW at 0 hour). Sickness behavior scores (SBS) and two whole blood samples were collected at 30-minute intervals from -2 to 8 hours relative to the challenge at 0 hours. One vacutainer containing EDTA was collected for complete blood cell count (CBC) analysis, and the second was collected in 9-mL monovette serum tube; after collection serum was isolated and stored at -80 °C until analyzed for Cortisol and cytokine concentrations. Rectal temperature, SBS, and Cortisol were affected by time (P<0.001). Prior to the challenge, RT was greater (P<0.001) in Cont steers (39.31+0.03 °C) than composition-fed steers (39.14+0.03 °C). Therefore, post-challenge RT was analyzed as the change in response from baseline values. The change in RT relative to baseline values increased (P<0.001) in both groups in response to LPS challenge, but was not affected by treatment (P=0.49). Sickness behavior scores increased

(P<0.001) after LPS challenge and tended (P=0.09) to be greater in Control (1.57+0.02) than composition- fed steers (1.51+0.02). Cortisol concentrations were affected by treatment (P=0.005) and time (P<0.001). For both groups, Cortisol increased (P<0.001) in response to LPS challenge. Cortisol was greater in Cont (25.2+0.9 ng/niL) than composition-fed steers (25.5+0.9 ng/mL). White blood cell and lymphocyte concentrations were greater (P<0.004) in Cont than composition- fed steers throughout the study. Neutrophils were decreased (P = 0.04) in Cont steers (0.7+0.2 Κ/μί) compared composition-fed steers (1.3+0.2 Κ/μί) prior to the LPS challenge. There was a treatment (P<0.02) and time (P<0.001) effect for tumor necrosis factor-a (TNFa) and interferon-γ (IFNy). Specifically, TNFa and IFNy concentrations increased (P<0.001) in response to LPS challenge. Furthermore, concentrations of TNFa and IFNy were decreased in (P<0.02) in Cont steers compared to composition-fed steers. These data suggest that supplementation with the composition served to prime the immune system prior to the LPS challenge, allowing for an enhanced response to LPS challenge. Example 9

The use of probiotic feed supplements to enhance animal health and growth are of great interest to the beef industry. Studies have demonstrated that some probiotic supplements may affect metabolism, and therefore influence an animal's response to an immune challenge. This study was designed to determine the effect of supplementing feedlot steers with one embodiment of the disclosed composition (comprising between 15% and 40% silica, between 50% and 81% mineral clay, between 1.0% and 5.0% β-glucans, between 0.05% and 3.0% β-1,3 (4)-endoglucanohydrolase and between 1% and 8.0% mannan) during the receiving period on the metabolic response to a lipopolysaccharide (LPS) challenge. Steers (n = 18; 270 ± 5 kg BW) were obtained and transported to the feedlot. Upon arrival steers were processed and separated into 2 treatment groups (n =

9/treatment): one group was fed a standard receiving diet (Control; Cont) and the other group was fed the same receiving diet supplemented with the composition at 4 g/45.4 kg BW/d for 29 days (composition). On day 27 steers were fitted with indwelling jugular cannulas and placed in individual stalls. On day 28, steers were challenged i.v. with LPS (0.5 μg/kg BW at 0 hour) and blood samples were collected at 30-minute intervals from -2 to 8 hours and at 24 hours post- challenge. Serum was isolated and stored at -80 °C until analyzed for glucose, non-esterified fatty acids (NEFA) and blood urea nitrogen (BUN) concentrations. Glucose concentrations were affected by treatment (P = 0.009) and time (P < 0.001). Glucose was greater in composition-fed steers compared to Cont steers (76.4 + 1.1 mg/dL vs. 72.4 + 1.0 mg/dL). For NEFA concentrations, there was a treatment (P < 0.001) and time (P < 0.001) effect. Specifically, Cont (0.210 + 0.007 mmol/L) steers had greater NEFA concentrations than composition-fed steers (0.101 + 0.010 mmol/L). There was a tendency (P = 0.07) for a treatment x time interaction such that NEFA concentrations were greater (P < 0.03) in Cont steers than composition-fed steers from 3 to 8 hours after LPS challenge. For BUN, there was a treatment (P < 0.001) effect such that concentrations were greater in Cont steers (12.4 + 0.1 mg/dL) than composition-supplemented steers (11.5 + 0.1 mg/dL) throughout the study, and were not affected by time (P = 0.28). These data suggest that supplementation with the composition modulates the metabolic response to a LPS challenge and provides an indication that supplementation of feedlot steers with the composition may prevent the breakdown of other substrates (e.g., protein and fat) for energy during an immune challenge. Example 10

The disclosed composition ameliorates the negative impact of heat stress on the immune status of cows during the dry period

Heat stress (HT) of cows in the dry period decreases immune function and lowers milk yield in the next lactation compared with cooled dry cows. The objective of this study was to evaluate the effects of the disclosed composition fed to heat stressed cows before, during and after the dry period on immune function, hematology and immune related gene expression. Sixty days before dry-off, cows were cooled (i.e. shade, fans and/or soakers) and divided into two groups: control (fed 56 g/d of an anti-caking agent; CON) and treatment (fed 56 g/d of one embodiment of the disclosed composition (comprising between 15% and 40% silica, between 50% and 81% mineral clay, between 1.0% and 5.0% β-glucans, between 0.05% and 3.0% β-1,3 (4)- endoglucanohydrolase and between 1% and 8.0% mannan); DC). Cows were dried-off 45 days before parturition and further split into cooling (shade, fans and soakers; CL) or HT (only shade) pens, which resulted in 4 treatments: HT (n=17), CL (n=16), HT + DC (HTDC, n=19) and CL + DC (CLDC, n=14). In the dry period, rectal temperature (RT; °C), respiration rate (RR; breaths per minute) and temperature humidity index (THI) were recorded to evaluate heat strain. Blood samples were collected before dry-off, during the dry period and lactation from a subset of cows (HT, n=12; CL, n=12; HTDC, n=l l and CLDC, n=9) to evaluate L-selectin (CD62L, copies per ng of total mRNA) and CXCR2 mRNA (a.k.a. IL8-R) gene expression in immune cells. Other samples were used before dry-off and in the dry period to evaluate neutrophil function and blood hematology (HT, n=8; CL, n=7; HTDC, n=8 and CLDC, n=6). HT increased RR (45.2 vs.

77.2+1.6 bpm) and RT (38.9 vs. 39.3+0.05 °C) versus CL (P<0.01). The disclosed composition increased L-selectin expression versus CON prior to dry-off (10229 vs. 5893+2353; P=0.09). L- selectin expression did not differ during the dry period, but after calving there was an interaction of dry period heat stress and dietary treatment (P=0.05); CLDC cows had increased L-selectin expression versus CL cows (24951 vs. 7198+5061). Expression of CXCR2 and neutrophil function did not differ among groups. The disclosed composition tended to increase neutrophil (10³ /μΐ) count versus CON (3.6 vs. 3.3+0.17; P=0.13) and HT cows had lower hematocrit % versus CL (29.4 vs. 31.6+0.6; P=0.12). Supplementation with the composition increased L-selectin expression before dry-off, and that may be related to improved immune status of cows during the dry period and in the next lactation. Example 11

Administration of the disclosed composition in combination with cooling alters mammary involution and development of heat stressed dry cows

A dry period is necessary for cows to attain maximal milk yield in the next lactation, and heat stress during this phase compromises mammary gland involution and redevelopment. The objective of this study was to evaluate the effects of nutritional and housing strategies to overcome the effects of heat stress on mammary gland involution and redevelopment of cows during the dry period. Before dry-off, all cows were kept in the same environment and exposed to cooling systems, i.e. shade, fans and soakers. For 60 days before dry-off, cows were divided into two groups: control (fed 56 g/d of an anti-caking agent CON) and treatment (fed 56 g/d of one embodiment of the disclosed composition (comprising between 15% and 40% silica, between 50% and 81% mineral clay, between 1.0% and 5.0% β-glucans, between 0.05% and 3.0% β-1,3 (4)- endoglucanohydrolase and between 1% and 8.0% mannan); DC). Cows were dried off 45 days before expected calving and, within nutritional treatment, assigned to cooling (shade, fans and soakers; CL) or heat stress (only shade; HT) pens, which resulted in 4 treatment groups: HT

(n=17), CL (n=16), HT + DC (HTDC, n=19) and CL + DC (CLDC, n=14). Mammary biopsies were collected on days 3, 7, 14, and 25 during the dry period from a subset of cows (HT, n=6; CL, n=7; HTDC, n=6 and CLDC, n=5) for histological evaluation of cell apoptosis and alveolar structures. Mammary tissue was placed in 4% paraformaldehyde overnight at 4 °C, dehydrated, paraffin embedded, and sectioned at 5 μιη. Mammary alveoli and apoptotic cells were visualized by hematoxylin and eosin staining and TUNEL assay, respectively. Alveoli number and positive apoptotic cells were counted using Image J software. Data were analyzed by mixed models using the MIXED procedure of Statistical Analysis Software (SAS). There was an interaction of heat stress and dietary treatment (P=0.08), where the apoptotic rate of CLDC cows was higher versus CL, HT, and HTDC cows (2.2; 1.46; 1.52; 1.47+0.2%, P<0.05, respectively). Relative to cooling, alveolar number was reduced when cows were exposed to HT (176 vs. 144+12; P=0.06) and increased when animals received the disclosed composition versus CON (179 vs. 141+12; P=0.02). Thus, DC supplementation with CL increased mammary cell apoptotic rate; DC supplementation increased alveoli number and CL increased alveoli number during the dry period. The disclosed composition might improve the capacity of the mammary gland for milk yield after calving.

Example 12

The focus of this study was to investigate how feeding one embodiment of the disclosed composition (comprising between 15% and 40% silica, between 50% and 81% mineral clay, between 1.0% and 5.0% β-glucans, between 0.05% and 3.0% β-1,3 (4)-endoglucanohydrolase and between 1% and 8.0% mannan) regulates mammary cell function when cows are exposed to heat stress conditions. Cows experiencing heat stress during dry-off, the time when lactation has stopped and a cow is preparing to have a calf, produce less milk in their next lactation. This is due to heat exposure induced mammary tissue damage during dry-off. This tissue damage prevents necessary cellular reorganization that supports the full potential of the mammary tissue to produce milk in the subsequent lactation. The objective of this study was to determine if inclusion of the composition in the feed during the exposure to heat stress would support tissue reorganization similar to the reorganization that occurs when cows are not exposed to heat stress. To investigate the cellular response, tissue biopsies were obtained from mammary tissue when lactation ended in cows that were fed the composition and in cows that were not fed the composition during the preceding 60 days. The level of tissue reorganization in these samples was evaluated with the use of microscopic analysis of the tissue.

As can be seen in FIG. 29, the number of alveoli in the mammary tissue from cows fed the composition was greater (p = 0.02) than the number of alveoli in cows that did not have the composition in their feed. This means that more alveoli were present in the mammary tissue of cows fed the composition due to a physiological or cellular regulatory mechanism controlling tissue survival or proliferation. Alveoli are the structural functional tissue that includes mammary epithelial cells in the mammary gland. Thus, the alveoli include the cells organized to produce milk. The mammary epithelial cells respond to hormones, such as prolactin, to produce the proteins, fatty acids and other molecules present in milk. These cells secrete the milk into the lumen of ductules for eventual excretion from the mammary tissue. Thus, more alveoli means there are more mammary epithelial cells that can respond to the hormones and produce milk.

The finding that more alveoli were present in the mammary tissue suggests that unique cellular reorganization mechanisms in the mammary gland were regulated by feeding the disclosed composition. To understand the regulatory changes in the mammary cells, total RNA was purified from tissue biopsy samples removed from the mammary glands of cows from each group. Real time quantitative PCR was used for analysis of Heat Shock Protein 70 (HSP70) gene expression in mammary tissue from composition-fed cows and control cows.

As can be seen in FIG. 30, the expression of HSP70 was significantly less (p= 0.04) in the mammary tissue from cows fed the composition compared to control cows. This means that less HSP70 was expressed in composition-fed cows during periods of time the cows were exposed to heat stress conditions. HSP70 is a ubiquitously expressed protein that functions in cells to facilitate amino acid folding so proteins can exist in a functional conformation. The expression of HSP70 increases in cells following exposure to stress such as heat stress. The increase in expression is needed because heat stress induces conformational changes in proteins rendering the proteins nonfunctional. Thus, increased expression of HSP70 is a signature of cellular damage and tissue that is metabolically and functionally strained. Less expression of HSP70 in mammary tissue from composition- fed cows indicated the normal tissue reorganization that occurs during dry-off was functioning at a normal level and will have more functional cells when lactation resumed. Thus, this finding indicated that cells were protected from metabolic strain during dry-off, and thus more likely to survive and produce milk in subsequent lactation periods when cows are fed the composition.

In addition to HSP70, the expression of Prolactin Receptor was measured in total RNA purified from mammary tissue in cows fed the composition and from control cows not fed the composition. As can be seen in FIG. 31, the expression of Prolactin Receptor was lower in the mammary tissue collected from cows fed the composition. This suggests that the serum

concentration of Prolactin was higher in composition- fed cows because there is an inverse relationship between serum Prolactin concentration and the expression of Prolactin Receptor in mammary tissue. These data support the conclusion that the disclosed composition supports more milk production by regulating the reorganization and survival of mammary tissue in cows during dry-off and the subsequent lactation.

Conclusion

These data demonstrate that feeding embodiments of the disclose composition to cows during late lactation and dry-off resulted in the mammary tissue maintaining more alveoli that is linked to more milk production during subsequent lactations. The mode of action of maintaining alveoli cells is linked to cellular mechanisms preventing the induction of heat stress proteins induced during stress. Lower expression of heat shock proteins indicated mammary cells in cows fed the composition underwent less metabolic strain and protein degradation during period of physiological and cellular stress. In addition, lower expression of Prolactin Receptor indicated that the composition regulated the mammary cell survival and production in cows.

Example 13

Cows on a commercial dairy are selected based on DIM, parity and milk production and assigned to 1 of 2 treatment groups of from about 15 to about 20 cows per group. The number of cows in each treatment group is balanced based on DIM, parity and milk production. The groups are fed either (1) an embodiment of the composition comprising between 15% and 40% silica, between 50% and 81% mineral clay, between 1.0% and 5.0% β-glucans, between 0.05% and 3.0% β-1,3 (4)-endoglucanohydrolase and between 1% and 8.0% mannans (EX), or (2) control (CON) diets. The trial typically is performed for at least 90 days. The administration of the granular composition may start during the dry-off period and continue for at least 90 days, with milk production being measured for at least 30-45 days once lactation has started. The composition is top-dressed 2x/day and is mixed into the top one-third of the TMR.

Milk samples are collected once daily on days 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 60 days after calving. A total of 10 mL of milk is sampled on each day, a sodium azide

preservative tablet added and archived at -20 °F. The milk samples are analyzed for mammary epithelial cell (MEC) content due to exfoliated MEC that are present in the milk. MEC content is determined by techniques known to persons of ordinary skill in the art, such as light microscopy, flow cytometry, and/or immune-magnetic methods. Typically, the milk is de-fatted before analysis. Thawed samples centrifuged at 1,000 x g to de-fat the samples. After centrifugation, fat particles are removed from the supernatant and discarded, and the remaining cells may be pelleted. For light microscopy, calls are typically stained such that different cellular components can be observed, and to recognize the different cellular types. For flow cytometry, calls may be incubated with a selective set of monoclonal antibodies that identify each cell type, or with an anti-cytokeratin antibody selected to specifically mark MEC in the milk. For the immune-magnetic method, a total milk suspension is incubated with magnetic beads coated with a specific anti-MEC antibody, such as an antibody that detects cytokeratin. The bound cells are collected using a magnetic particle concentrator and by aspiration of the supernatant containing leukocytes. Purified milk MEC can be directly counted using a hematocytometer or with a cell counter. It is expected that the MEC count in samples from cows in the EX group will be lower that the MEC count in samples from the CON group. Example 14

Cows on a commercial dairy are selected based on DIM, parity and milk production and assigned to 1 of 2 treatment groups fed (1) an embodiment of a composition comprising between 15% and 40% silica, between 50% and 81% mineral clay, between 1.0% and 5.0% β-glucans, between 0.05% and 3.0% β-1,3 (4)-endoglucanohydrolase and between 1% and 8.0% mannans (EX) or (2) control (CON) diets for about 60 days post calving. The number of cows in each treatment group is balanced based on DIM, parity and milk production. A total of 20 cows is in each treatment group. The composition is fed at dry-off, 45 days before calving and fed until 60 DIM. The granular composition is top-dressed 2x/day and is mixed into the top one-third of the TMR. It is predicted that the cows in the EX group will have a higher milk yield than the CON cows, during the period of administration of the composition.

Milk samples are collected once daily on days 1, 2, 3, 4, 5., 10, 15, 20, 25, 30, 35, 40, 45 and 60 days after calving. A total of 10 mL of milk is sampled on each day, a sodium azide preservative tablet added and archived at -20 °F. Thawed samples centrifuged at 1 ,000 x g to de-fat the samples. After centrifugation, fat particles are removed from the supernatant, discarded and the remaining sample analyzed for Prolactin by ELISA. A 100 microliter sample assayed with a colorimetric analysis at 450 nm with the use of a standard curve from standards provided in the ELISA kit. The expected range of Prolactin in the samples is 10-250 ng/mL, and it is expected that Prolactin levels in the milk samples from cows in the EX group will be higher than Prolactin levels in samples from cows in the CON group.

In view of the many possible embodiments to which the principles of the disclosed technology may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the technology and should not be taken as limiting the scope of the technology. Rather, the scope of the technology is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

CLAIMS We claim:

1. A composition comprising silica, mineral clay, glucan, and mannans for use in a method to at least maintain a number of alveoli in mammary tissue, to increase an amount of Prolactin in milk and/or blood, decrease an amount of Prolactin receptor expression, decrease a number of exfoliated mammary epithelial cells in milk, or a combination thereof, wherein the method comprises administering an effective amount of the composition to a mammal.

2. The composition for the use of claim 1, wherein the method comprises at least maintaining the number of alveoli in mammary tissue of the mammal.

3. The composition for the use of claim 1 or claim 2, wherein the method comprises increasing the amount of Prolactin in milk and/or blood.

4. The composition for the use of any one of claims 1-3, wherein the method comprises decreasing the amount of Prolactin receptor expression.

5. The composition for the use of any one of claims 1-4, wherein the method comprises decreasing the number of exfoliated mammary epithelial cells in milk.

6. The composition for the use of any one of claims 1-5, wherein the composition is administered daily.

7. The composition for the use of any one of claims 1-5, wherein the composition is administered substantially continuously.

8. The composition for the use of any one of claims 1-5, where the effective amount of the composition is administered to the mammal for an effective period of time starting after beginning of a lactation period for the mammal and continuing through at least a portion of a subsequent drying off period for the mammal.

9. The composition for the use of any one of claims 1-8, wherein the composition is a powdered composition.

10. The composition for the use of any one of claims 1-8, wherein the composition is a granular composition.

11. The composition for the use of claim 10, wherein the granular composition comprises plural granules where each granule comprises a substantially homogeneous blend of silica, mineral clay, glucan, and mannans.

12. The composition for the use of any one of claims 1-11, wherein the glucan and mannans are provided by yeast cell wall or an extract thereof.

13. The composition for the use of claim 12, wherein the yeast cell wall or an extract thereof further comprises endoglucanohydrolase.

14. The composition for the use of any one of claims 1-13, further comprising an affirmatively added endoglucanohydrolase.

15. The composition for the use of any one of claims 1-14, where the effective period of time starts from 100 days to 240 days after beginning of a lactation period for the mammal.

16. The composition for the use of any one of claims 1-15, where administration of the composition to the mammal increases a number of alveoli in the mammal, compared to a number of alveoli in a mammal that is not administered the composition.

17. The composition for the use of any one of claims 1-16, where the mammal is further administered a cooling treatment.

18. The composition for the use of claim 17 where the cooling treatment comprises provision of shade to the mammal, use of water sprinklers to externally administer water to the mammal, use of a fan to provide air movement to the mammal, air-conditioning, or any

combination thereof.

19. The composition for the use of any one of claims 1-18, where administration of the composition begins before the mammal commences a pregnancy.

20. The composition for the use of any one of claims 1-18 where administration of the composition begins while the mammal is pregnant.

21. The composition for the use of any one of claims 1-20 where administration of the composition continues through to at least birth of an offspring following the drying off period.

22. The composition for the use of claim 21 where administration of the composition continues into a subsequent lactation period following the birth of the offspring

23. The composition for the use of claim 22, where administration of the composition continues throughout the entire subsequent lactation period.

24. The composition for the use of any one of claims 1-23, where the mammal is a dairy cow.

25. The composition for the use of any one of claims 1-24 where the composition comprises 1-40 wt% silica, 0.5-25 wt% glucan and mannans, and 40-92 wt% mineral clay.

26. The composition for the use of any one of claims 1-25 where the effective amount of the composition is from 0.01 gram to 20 grams per kilogram of live body weight per day.

27. The composition for the use of claim 1 where the method comprises administering to the mammal from 0.01 gram to 20 grams of the composition per kilogram of live body weight per day throughout at least a portion of a lactation period for the mammal and continuing through at least a portion of a subsequent drying off period for the mammal, wherein the composition comprises 1-40 wt% silica, 0.5-25 wt% glucan and mannans, and 40-92 wt% mineral clay.

28. The composition for the use of claim 27, further comprising administering a cooling treatment to the mammal, wherein administering the composition and the cooling treatment to the mammal maintains or increases the number of alveoli in the mammary tissue of the mammal compared to an animal that is not administered the composition or cooled.

29. The composition for the use of any one of claims 1-28, wherein the composition further comprises a feed.

30. The composition for the use of any one of claims 1-29, wherein the composition further comprises a metal carbonate, a copper species, a trace mineral, a bulking agent, yeast, a carrier, a colorant, a taste enhancer, a preservative, an oil, a vitamin, yucca, quillaja, a probiotic, allicin, alliin, allinase, algae, a polyphenol or plant material comprising polyphenol, or a sorbic acid or a salt thereof.

31. The composition for the use of claim 30, wherein the copper species is copper sulfate.

32. The composition for the use of claim 30 or claim 31 , wherein the yucca is Yucca schidigera.

33. The composition for the use of any one of claims 30-32, wherein the quillaja is Quillaja saponaria.

34. The composition for the use of any one of claims 30-33, wherein the probiotic comprises Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis.

35. A method to at least maintain a number of alveoli in mammary tissue of a mammal, to increase an amount of Prolactin in milk, decrease an amount of Prolactin receptor expression, decrease a number of exfoliated mammary epithelial cells in milk, or a combination thereof, the method comprising administering a composition comprising silica, mineral clay, glucan, and mannans to a mammal, thereby at least maintain the number of alveoli in mammary tissue of a mammal, increase the amount of Prolactin in milk, decrease the amount of Prolactin receptor expression, and/or decrease the number of exfoliated mammary epithelial cells in milk.

36. The method of claim 35, comprising at least maintaining the number of alveoli in mammary tissue of the mammal.

37. The method of claim 35 or claim 36, comprising increasing the amount of Prolactin in milk.

38. The method of any one of claims 35-37, comprising decreasing the amount of Prolactin receptor expression.

39. The method of any one of claims 35-38, comprising decreasing the number of exfoliated mammary epithelial cells in milk.