WO2024263687A2 - Composition and methods for increasing lipid synthesis in mammary epithelial cells - Google Patents

Abstract

The present disclosure provides compositions and methods for increasing milk fat content or milk fat yield in milk produced by an animal. The compositions include a lipid enhancing bioactive compound in an amount effective to increase milk fat synthesis in mammary epithelial cells of an animal. The lipid enhancing bioactive compound includes at least one of anandamide, oleamide, 7,7-dimethylelcosadienoic acid, 10(Z)-heptadecenoic acid, or arachidonic acid, or derivatives or mixtures thereof. The methods include administering the compositions herein to an animal or group of animals. Other aspects are also provided.

Description

COMPOSITION AND METHODS FOR INCREASING LIPID SYNTHESIS IN

MAMMARY EPITHELIAL CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/509,846, filed June 23, 2023, which is incorporated by reference herein in its entirety.

FIELD

[0002] The present disclosure relates to compositions and methods for increasing lipid synthesis in mammary epithelial cells. In particular, the present disclosure relates to compositions and methods for increasing lipid synthesis in mammary epithelial cells of livestock animals to increase the milk fat in milk produced by these animals.

BACKGROUND

[0003] Milk produced by livestock animals is one of the most widely cultivated food commodities and sources of nutrition around the world. Milk consists mainly of components including water, milk fat, and skim solids, where the skim solids can include proteins, lactose, minerals, and other trace elements. Milk quality can be measured by quantifying any one of the components found in milk, and the financial and nutritive value of the milk can be positively or negatively correlated with the quantities of these components. Thus, it is of great importance to milk producers to ensure that their cows can produce milk of high quality corresponding to these components. In particular, milk fat quantity and quality are of great importance to milk producers, as these are a key drivers in the nutritive value of the milk and to the production of various foodstuffs, including ice creams and other frozen desserts, butter, creams, and cheeses.

[0004] Milk fat is the most variable component in milk, both in quantity and profile, including a broad variety of lipids in varying quantities. Numerous factors can affect milk fat quantity or composition, including dietary feed and supplement composition, frequency of feeding, health and age of the animal, timing of milking during lactation, season, and the amount of time that passes between milking, to name a few. The fatty acids in an animal’s milk lipids can arise from two sources - preformed fatty acids from an animal’s diet or mobilization of body reserves and de novo synthesis in the mammary gland. Even though various animal diets, such as the ruminant diet, contain predominantly unsaturated fatty acids, the rumen microorganisms can be capable of modifying these fatty acids through bio-hydrogenation, which can result in saturated, conjugated, and trans fatty acids that are transferred to milk. Various attempts at adding saturated fats to livestock diets, such as palm oil and its derivatives, have recently met resistance from consumers due to a variety of concerns related to sustainability or the quality of downstream food products that incorporate milk fat from milk produced by animals fed diets high in saturated fats. Thus, a need exists to target nutrigenomic approaches that can effectively increase milk fat content or milk fat yield in milk without causing additional complications to milk quality.

SUMMARY

[0005] The present disclosure provides a composition for increasing milk fat content or milk fat yield in milk. The composition can include a lipid enhancing bioactive compound in an amount effective to increase milk fat synthesis in mammary epithelial cells of an animal. The lipid enhancing bioactive compound includes at least one of anandamide, oleamide, 7,7- dimethylelcosadienoic acid, 10(Z)-heptadecenoic acid, or arachidonic acid, or derivatives or mixtures thereof.

[0006] In an aspect, the effective amount of lipid enhancing bioactive compound present in the composition can be sufficient to increase milk fat synthesis within the mammary epithelial cells as compared to mammary epithelial cells not exposed to the lipid enhancing bioactive compound.

[0007] In an aspect, the effective amount of lipid enhancing bioactive compound is sufficient to increase lipogenesis within the mammary epithelial cells as compared to mammary epithelial cells not exposed to the lipid enhancing bioactive compound.

[0008] In an aspect, the composition is encapsulated.

[0009] In an aspect, the composition can be a component of a complete feed, a premix, a concentrate, a base mix, a supplement, a top dress, or any combination thereof.

[0010] The present disclosure provides a method for increasing milk fat content or milk fat yield in milk. The method can include administering a composition including a lipid enhancing bioactive compound in an amount effective to increase milk fat synthesis in mammary epithelial cells of the animal, where the milk fat lipid enhancing bioactive compound includes at least one of anandamide, oleamide, 7,7-dimethylelcosadienoic acid, 10(Z)-heptadecenoic acid, or arachidonic acid, or derivatives or mixtures thereof.

[0011] In an aspect, the method includes where the effective amount of milk fat enhancing bioactive compound present in the composition can be sufficient to increase milk fat synthesis within the mammary epithelial cells as compared to mammary epithelial cells not exposed to the lipid enhancing bioactive compound.

[0012] In an aspect, the method includes where the effective amount of lipid enhancing bioactive compound is sufficient to increase lipogenesis within the mammary epithelial cells by as compared to mammary epithelial cells not exposed to the lipid enhancing bioactive compound. [0013] In an aspect, the method includes where the composition is administered as a feed additive or a dietary supplement.

[0014] In an aspect, the method includes where the animal is a bovine animal.

[0015] In an aspect, the method includes where the animal is a deer, elk, caribou, moose, equine, goat, sheep, or swine animal.

[0016] In an aspect, the method includes where the composition is administered to a cow or group of cows at any time during active milk production.

[0017] In an aspect, the method includes where the composition is administered to a cow or group of cows once a day during active milk production.

[0018] In an aspect, the method includes where the composition is administered to a cow or group of In an aspect, the method includes where the composition is administered to a cow or group of cows at any time immediately following the onset of active milk production.

[0019] In an aspect, the method includes where the composition is administered to a cow or group of cows once a day during active milk production, twice a day during active milk production, three times a day during active milk production, or four times a day during active milk production. [0020] In an aspect, the method includes where the composition is administered upon a showing that the milk fat content or milk fat yield are not sufficient for a milk producer’s demands. [0021] In an aspect, the method includes where the composition is administered to an animal or group of animals, such as a cow or group of cows, on a daily basis throughout milk production to sustain milk fat content or milk fat yield.

[0022] In an aspect, the method includes periodically sampling milk from a cow or group of cows to measure levels of milk fat content or milk fat yield in response to the lipid enhancing bioactive compounds; and based on the measured levels of milk fat content or milk fat yield, increasing the effective amount of lipid enhancing bioactive compound in response to a level of milk fat content or milk fat yield that does not meet producer’s needs. BRIEF DESCRIPTION OF THE FIGURES

[0023] The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed herein.

[0024] This patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and the payment of the necessary fee.

[0025] FIG. l is a graph showing the effect of various bioactive lipids on lipid accumulation in bovine mammary epithelial cells in accordance with various aspects herein.

[0026] FIG. 2 shows microscopy imaging of bovine mammary epithelial cells exposed to various bioactive lipids in accordance with various aspects herein.

[0027] FIG. 3 shows the dose response effect of anandamide on lipid droplet accumulation in bovine mammary epithelial cells in accordance with various aspects herein.

[0028] FIG. 4 shows the dose response effect of oleamide on lipid droplet accumulation in bovine mammary epithelial cells in accordance with various aspects herein.

[0029] FIG. 5 shows a pairwise comparison for each treatment for lipid droplet accumulation in accordance with various aspects herein.

[0030] FIG. 6 shows a plot of the relative expression of ACACA in bovine mammary epithelial cells for various test conditions in accordance with various aspects herein.

[0031] FIG. 7 shows a plot of the relative expression of FABP3 in bovine mammary epithelial cells for various test conditions in accordance with various aspects herein.

[0032] FIG. 8 shows a plot of the relative expression of FASN in bovine mammary epithelial cells for various test conditions in accordance with various aspects herein.

[0033] FIG. 9 shows a plot of the relative expression of SREBF1 in bovine mammary epithelial cells for various test conditions in accordance with various aspects herein.

[0034] FIG. 10 shows is a graph showing the effect of various bioactive fatty acids on lipid accumulation in bovine mammary epithelial cells in accordance with various aspects herein.

[0035] FIG. 11 shows microscopy imaging of bovine mammary epithelial cells exposed to various bioactive fatty acids in accordance with various aspects herein.

[0036] FIG. 12 shows the dose response effect of 7,7-dimethyleicosadienoic acid on lipid droplet accumulation in bovine mammary epithelial cells in accordance with various aspects herein.

[0037] FIG. 13 shows the dose response effect of 10(Z)- heptadecenoic acid on lipid droplet accumulation in bovine mammary epithelial cells in accordance with various aspects herein. [0038] FIG. 14 shows the dose response effect of arachidonic acid on lipid droplet accumulation in bovine mammary epithelial cells in accordance with various aspects herein.

[0039] FIG. 15 shows a pairwise comparison for each treatment for lipid droplet accumulation in accordance with various aspects herein.

[0040] FIG. 16 shows a plot of the relative expression of AC AC A in bovine mammary epithelial cells for various test conditions in accordance with various aspects herein.

[0041] FIG. 17 shows a plot of the relative expression of FABP3 in bovine mammary epithelial cells for various test conditions in accordance with various aspects herein.

[0042] FIG. 18 shows a plot of the relative expression of SREBF1 in bovine mammary epithelial cells for various test conditions in accordance with various aspects herein.

DETAILED DESCRIPTION

[0043] Reference will now be made in detail to certain aspects of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0044] The mammary gland of an animal produces milk components such as fat, lactose, and proteins. Milk fat is largely composed of triacylglycerols. The lipid composition in bovine milk is highly complex, due to the large number of fatty acids present therein (e.g., greater than 400 unique fatty acid types). Various bioactive compounds can influence milk production. Intermediate products of this process, such as trans-10, cis-12 conjugated linoleic acid, are thought to act as bioactive molecules to alter lipid synthesis in mammary cells through transcription factors such as sterol regulatory element-binding proteins (gene name SREBPF ) to control the expression of key lipogenic genes.

[0045] The present disclosure is directed to compositions and methods for increasing lipid synthesis (i.e., lipogenesis) in various cell types, such as mammary epithelial cells in animals. The disclosure herein shows that various lipid enhancing bioactive compounds, including molecules such as fatty acids or bioactive lipids, are able to increase mammary epithelial cell lipid production when administered to mammary epithelial cells. The lipid enhancing bioactive compounds can be included in compositions used as a feed additives or dietary supplement for use in the diet of various animals. The lipid enhancing bioactive compounds can be included in compositions used as a feed additives or dietary supplement for use in the diet of various livestock animals, including bovines, deer, elk, moose, caribou, equines, goats, sheep, or swine.

Compositions

[0046] The present disclosure provides various compositions for increasing milk fat content or milk fat yield in milk produced by an animal. As used herein, the term “milk fat content” refers to the percentage of fat in a given mass of milk. As used herein, the term “milk fat yield” refers to the grams of milk fat produced in a day. It will be appreciated that the mass of milk can be a standard unit of measure, such as the kilogram (kg).

[0047] The compositions herein can be included in the diet of an animal as a feed additive or as a dietary supplement. In an aspect, the compositions herein can be provided as an ingredient of a feed product, such as a complete feed product. A complete feed product can include a nutritionally complete and balanced daily dietary composition that is fed as the sole ration and can maintain life, promote growth and performance, and sustain reproduction without any additional substances being consumed except water. Complete bovine feed products can include mixtures containing appropriate levels of the nutrients required to sustain the life of the bovines, provided among them proteins, fats, carbohydrates, and the like. In an aspect, the feed product is not a complete feed product. The compositions herein can be administered directly to any suitable animal species or can be administered to the animal as an ingredient of a feed product or as a dietary supplement. In some aspects, the animal is a bovine animal. In other aspects, the animal is a deer, elk, moose, caribou, equine, goat, sheep, or swine.

[0048] The compositions herein can include one or more lipid enhancing bioactive compounds in an amount effective to increase milk fat synthesis in mammary epithelial cells of an animal. The lipid enhancing bioactive compounds can include one or more of a bioactive lipid or bioactive fatty acid including anandamide, oleamide, 7,7-dimethylelcosadienoic acid, 10(Z)- heptadecenoic acid, or arachidonic acid, or derivatives or mixtures thereof. It will be appreciated that anandamide and oleamide belong to the endocannabinoid class of compounds and 7,7- dimethylelcosadienoic acid, 10(Z)-heptadecenoic acid, or arachidonic acid belong to the class of unsaturated fatty acid compounds. The chemical structures of the lipid enhancing bioactive compounds can be found in Table 1. Table 1. Chemical Structures of Various Lipid Enhancing Bioactive Compounds

[0049] The effective amount of lipid enhancing bioactive compound present in the composition can be sufficient to increase milk fat synthesis within the mammary epithelial cells as compared to mammary epithelial cells not exposed to the lipid enhancing bioactive compound. The effective amount of lipid enhancing bioactive compound is sufficient to increase lipogenesis (i.e., de novo fatty acid synthesis) within the mammary epithelial cells by as compared to mammary epithelial cells not exposed to the lipid enhancing bioactive compound. It will be appreciated that the lipid bioactive compounds described herein can further be effective to increase lipid synthesis in other tissues such as in muscle tissues. The lipid enhancing bioactive compounds can be present in the compositions herein in an amount effective to increase milk fat synthesis in mammary epithelial cells of an animal. In one aspect, the animal is a bovine animals. The compositions herein further can be suitable for administration to one or more animal species from the groups including deer elk, moose, caribou, equines, goats, sheep, or swine.

[0050] The feed supplement compositions herein can be administered to animals at any stage of development and any stage of life. The compositions herein can include lipid enhancing bioactive compounds that are protected from degradation. In an aspect, the lipid enhancing bioactive compounds can be protected from degradation or fermentation in the rumen of ruminant animals or the stomach of non-ruminant animals. Protection from rumen fermentation can be accomplished by various mechanisms, including encapsulation with various compounds. In various aspects, encapsulation can include, but is not to be limited to, the use of fat coatings, polymer coatings, silica coatings, and the like. In an aspect, the lipid enhancing bioactive compounds can be formulated to withstand physiological pH found in the stomach or rumen of an animal.

[0051] The compositions herein can be formulated for use in any suitable species of cattle and at any suitable life stage. The term “cattle” as used herein refers to domestic cattle including those raised as livestock for meat production (e.g., beef or veal) and those raised for milk production. The term cattle as used herein can also include animals used for animal products such as collagen and hides. The cattle can include those at any life stage, including cows, calves, bulls, heifers, and steers, including those animals also raised for breeding.

[0052] Cattle (Bos taurus) suitable for administration of the compositions herein can include, any cattle breed suitable for meat and milk production, but are not limited to, breeds such as Anatolian Black, Andalusian Black, Angus (e.g., black or red), Aubrac, Belgian (e.g., blue or red), Belted Galloway, Brahman, Brangus, Braunvieh, Brown Swiss, Caracu, Charolais, Chianina, Corriente, Darkensberger, Dexter, Gelbvieh, Guernsey, Hereford, Holstein, Jersey, Limousin, Maine- Anjou, Mongolian, Piedmontese, Santa Gertruids, Salers, Scottish Highland, Shorthorn, Simmental, Tarentaise, Texas Longhorn, Wagyu, and Watusi.

[0053] The compositions herein can be administered to cattle, where the cattle include cows (i.e., female cattle) that are actively producing milk. In general, the lactation cycle of the cow can be divided into four phases, including early lactation, mid lactation, late lactation and the dry period. The early-, mid-, and late-lactation phases collectively amount to approximately 305 days in duration, whereas the dry period typically lasts for about 35 to 60 days. During early lactation, the cow’s body prepares its reserves for calving and milk production. A week before parturition, the cow’s body begins to actively produce milk (i.e., lactogenesis) and the cow reaches peak milk production at approximately 45 to 60 days in milk (i.e., after parturition). Following calving, the cow generally increases feed intake to support milk production. During mid to late lactation, milk production declines until the cow is dried-off and the cow is anymore.

[0054] The compositions herein further can be any suitable feed product designed for mixing with another composition, such as a base feed, to form the bovine feed. The compositions further can include a premix, a concentrate, a base mix, a supplement, a top dress, or any combination thereof.

[0055] A base feed can be a commercially available feed or other animal feed. A base feed suitable for bovines can refer to a ration that contains any of the various sources of nutrition, their by-products, and other sources of primary nutrition (e.g., fat, starch, and protein) such as hay, barley, grass, grains, corn (e.g., whole or meal), oats, alfalfa, silage, dry feeds, or any combinations thereof.

[0056] A premix can be a composition that can include vitamins, minerals, appropriate medications, carriers, and combinations thereof, and are typically less than 1 wt.% of the diet but can be higher. The carrier can increase bulk to improve distribution in compounding to prepare a more complete feed material. Such premixes can be used to formulate concentrates and complete feeds.

[0057] A concentrate can be a composition that can include high-protein feed components and can also include vitamins, minerals, appropriate medications, and combinations thereof. A concentrate is typically 5 wt.% to 40 wt.% of the diet but can be higher or lower. A concentrate can include other additives. Concentrates can be used to make complete feeds by adding available grains or other energy sources. An “other additive” can include an ingredient or a chemical preparation or combination of ingredients which is added to the base feed to fulfill a specific nutritional requirement. It can be used in micro quantities and may have no nutritional value but is added to the feed to improve its quality and efficacy. Other additives can include, but are not limited to, acidifiers, antioxidants, aromatics, deodorizing agents, flavor enhancers, mold inhibitors, pellet binders, preservatives, sweeteners, toxin binders, and the like.

[0058] A base mix can be similar to a supplement but can contain just a portion of the animal’ s protein requirements, so it can be used with high protein ingredients and grain (e.g., ground grain and protein source) to form the bovine feed. A base mix can include a mixture of one or more macro-mineral sources and one or more micro-ingredient sources such as vitamin premixes, trace mineral premixes, essential amino acids, and other additives, that when mixed with sources of protein and energy form a complete feed.

[0059] A supplement can include a feed ingredient, or a chemical preparation or combination of feed ingredients, intended to supply any deficiencies in an animal (e.g., bovine) feed and/or improve the nutritive balance or performance of the animal or bovine feed.

[0060] A top dress can include a solid or liquid supplement that can be added at specific time intervals to the animal’s ration to provide a specific supplement or supplements over a period of time that makes it inconvenient or difficult to include in a complete feed.

Methods for Increasing Lipogenesis

[0061] The present disclosure provides methods for administering to an animal the compositions described herein. The methods can include feeding the animals the compositions herein to increase milk fat synthesis in mammary epithelial cells. The methods can include feeding the animals the compositions herein to increase milk fat content or milk fat yield in milk produced by an animal. The methods for feeding an animal can include feeding an animal or group of animals the composition including a lipid enhancing bioactive compound in an amount effective to increase milk fat synthesis in mammary epithelial cells of the animal. The lipid enhancing bioactive compound can include at least one of anandamide, oleamide, 7,7-dimethylelcosadienoic acid, 10(Z)-heptadecenoic acid, or arachidonic acid, or derivatives or mixtures thereof. The animal suitable for administration of the compositions herein can include a bovine animal. In an aspect, the animal suitable for administration of the compositions herein can include a deer, elk, caribou, moose, equine, goat, sheep, or swine animal.

[0062] In an aspect, the effective amount of lipid enhancing bioactive compound present in the composition can be sufficient to increase lipogenesis within the mammary epithelial cells as compared to mammary epithelial cells not exposed to the lipid enhancing bioactive compound. [0063] In another aspect, the effective amount of lipid enhancing bioactive compound is sufficient to increase lipogenesis within the mammary epithelial cells by as compared to mammary epithelial cells not exposed to the lipid enhancing bioactive compound.

[0064] When administered to an animal, the compositions herein can be administered as a feed additive or a dietary supplement. As used herein, the terms “administered/administering” and “feeding” both refer to providing an animal with the compositions herein for consumption in the diet or as a dietary supplement.

[0065] As discussed above, compositions herein can be administered to cattle, where the cattle include cows (i.e., female cattle) that are actively producing milk. In general, the lactation cycle of the cow can be divided into four phases, including early lactation, mid lactation, late lactation and the dry period. The early-, mid-, and late-lactation phases collectively amount to approximately 305 days in duration, whereas the dry period typically lasts for about 35 to 60 days. During early lactation, the cow’s body prepares its reserves for calving and milk production. A week before parturition, the cow’s body begins to actively produce milk (i.e., lactogenesis) and the cow reaches peak milk production at approximately 45 to 60 days in milk (i.e., after parturition). Following calving, the cow generally increases feed intake to support milk production. During mid to late lactation, milk production declines until the cow is dried-off and is not milked anymore.

[0066] The compositions herein can be administered at any life stage or any developmental stage of the animal. In an aspect, the compositions are administered to an animal following the birth of their young and upon commencement of active milk production. The composition can be administered to a cow or group of cows at any time following the birth of a calf and upon commencement of active milk production. In an aspect, the composition can be administered to a cow or group of cows once a day during active milk production, twice a day during active milk production, three times a day during active milk production, or four times a day during active milk production. In an aspect, the composition can be administered to a cow or group of cows every other day during active milk production, or every three days during active milk production.

[0067] The compositions herein can be administered to a cow or group of cows at any frequency designed to increase lipid synthesis as needed to meet the demands of milk producers. It will be appreciated that a milk producer’s demands and the desired levels of milk fat content or milk fat yield can include a predetermined amount of milk fat content or milk fat yield in a predetermined period of time, such as a given day, a given week, a given month, or throughout all active milk production, and the like. By way of example, the milk from cows actively producing milk can be sampled periodically to determine if the milk fat content or milk fat yield are sufficient to meet the demands of milk producers. The compositions can be administered upon a showing that the milk fat content or milk fat yield are not sufficient for a milk producer’s demands. For example, upon a showing of insufficient milk fat content or milk fat yield, the compositions can be administered for a period of time configured to increase milk fat content or milk fat yield up to desired levels. The administration of the compositions can be sustained throughout the duration of the active milk production, or alternatively the administration of the compositions can be configured to last for a predetermined period of time sufficient to increase milk fat content or milk fat yield up to desired levels. The method can further include periodically sampling milk from a cow or group of cows to measure levels of milk fat content or milk fat yield in response to the lipid enhancing bioactive compounds. The method can further include, based on the measured levels of milk fat content or milk fat yield, increasing the effective amount of lipid enhancing bioactive compound in response to a level of milk fat content or milk fat yield that does not meet producer’s needs.

[0068] The compositions herein can be administered to an animal or group of animals, such as a cow or group of cows, at any time during active milk production. The compositions herein can be administered to an animal or group of animals, such as a cow or group of cows, at any time within a one week (i.e., 7 day) time period immediately following the onset of active milk production. The compositions herein can be administered to an animal or group of animals, such as a cow or group of cows, at any time within a two week (i.e., 14 day) time period immediately following calving. The compositions herein can be administered to an animal or group of animals, such as a cow or group of cows, at any time within a three week (i.e., 21 day) time period immediately following calving. The compositions herein can be administered to an animal or group of animals, such as a cow or group of cows, on a daily basis throughout milk production to sustain milk fat content or milk fat yield.

[0069] The compositions can be administered at any frequency to an animal or group of animals, such as a cow or group of cows, at any time during active milk production. In an aspect, the composition can be administered to a cow or group of cows on a daily basis during active milk production. In an aspect, the composition can be administered to a cow or group of cows on a weekly basis during active milk production. In an aspect, the composition can be administered to a cow or group of cows on a bi-weekly basis during active milk production. EXAMPLES

[0070] Various aspects of the present disclosure can be better understood by reference to the following Examples, which are offered by way of illustration. The present disclosure is not limited to the Examples given herein.

Example 1: Isolation and Differentiation of Bovine Mammary Epithelial Cells

[0071] The Examples herein use a cell culture model to screen various lipid enhancing bioactive compounds for their ability to promote milk fat synthesis in bovine mammary epithelial cells.

[0072] Briefly, bovine mammary epithelial cells were isolated by enzymatic digestion of the mammary glands from Holstein cows were isolated. The bovine mammary epithelial cells were seeded into collagen-coated 96-well plates designed for high-throughput screening. The cells were allowed to grow to confluence in a humidified, 5% CO2 incubator at 37 °C. To induce lactogenic differentiation, the cells were incubated in medium containing 1 pg/mL lactogenic hormones including insulin, prolactin and glucocorticoids, and precursors for fatty acid synthesis including acetate and butyrate, at physiological concentrations for 4 days.

Example 2: High-Throughput Screening of Various Lipid Enhancing Bioactive Compounds on Milk Fat Synthesis in Bovine Mammary Epithelial Cells

[0073] This example describes an analysis of multiple lipid enhancing bioactive compounds (also referred to herein as “test compounds”) that were screened for their ability to exhibit an increase in lipid synthesis or fatty acid synthesis (i.e., milk fat synthesis) in bovine mammary epithelial cells in vitro.

[0074] To screen various test compounds that promote lipid synthesis, two commercially available screening libraries - SCREEN-WELL® Bioactive Lipid Library and SCREEN-WELL® Fatty Acid (both of Enzo Life Sciences, Farmingdale, NY, USA) - that contain 190 bioactive lipids and 64 fatty acids, respectively, were used. The screening libraries were supplied with each test compound separately dissolved at 10 mM and 1 mM, respectively, in dimethyl sulfoxide (DMSO) and aliquoted into their own well of a 96-well plate. A vehicle control containing just DMSO was used to control for solvent effects, and rapamycin (a drug that acts as a mTORCl inhibitor) and pioglitazone (a drug that activates peroxisome proliferator-activated receptor) were used as positive controls. [0075] The test compounds from the screening libraries were transferred independently into unique wells containing lactogenic differentiated mammary epithelial cells to a final concentration of 10 pM and incubated during the final 16 hours of the 4 day differentiation period described in Example 1. Following incubation with the test compounds, the cells were fixed in 4% w/v paraformaldehyde, permeabilized using 0.1% v/v Triton X-100 solution and stained using BODIPY™ neutral lipid stain 493/503 (i.e., 4,4-Difluoro-l,3,5,7,8-Pentamethyl-4-Bora-3a,4a- Diaza-s-Indacene; excitation maximum: 493 nm and emission maximum: 503 nm), DAPI nuclear stain (i.e., 4',6-diamidino-2-phenylindole; excitation maximum: 358 nm and emission maximum: 461 nm), and/or phalloidin-TRITC cytoskeletal stain (i.e., phalloidin-tetramethylrhodamine; excitation maximum: 557 nm and emission maximum: 576 nm). Thus, microscopy imaging reveals the nucleus in blue (DAPI stain), the cytoskeleton in orange (phalloidin-TRITC), and lipid droplets in green (BODIPY 496/503). The DAPI signal was used to count cells within images and account for potential differences in cell number between wells. The phalloidin-TRITC was used to define the cell contour in images. The BODIPY 493/503 stain was used as readouts for the accumulation of neutral lipids in mammary epithelial cells, because it is understood that unpolarized mammary epithelial cells in adherent culture cannot secrete milk fat. Thus, the lipophilic stains can be used as markers of lipid droplets. Fluorescence intensity was measured using a BIOTEK Cytation 5 high-content imaging microscope (Agilent Technologies, Inc. Santa Clara, CA, USA) and results were reported to reflect an accumulation of intracellular lipids in the cells of the 96 well plates at day 4 after incubation with the test compounds.

[0076] Data from the high-throughput screening were analyzed using HiTSeekR software to determine positive hits based on a threshold (± k median absolute deviation). A k factor of 1 was used for the hit detection. Raw data were collected from the 254 bioactive compounds of the screening libraries and were analyzed for their effect on lipid droplet accumulation in bovine mammary epithelial cells.

Example 3: Characterization of Bioactive Lipid Compounds That Increase Lipid Accumulation in Bovine Mammary Epithelial Cells

[0077] Two bioactive lipids were identified from the high-throughput screening of test compounds in Example 2 and were further characterized on their ability to increase lipid synthesis by bovine mammary epithelial cells.

[0078] Bovine mammary epithelial cells were isolated and differentiated according to the method described in Example 1. During the last 16 h of the lactogenic differentiation step, the bovine mammary epithelial cells were treated with 10 pM of each of the identified bioactive lipids, anandamide and oleamide, that were conjugated independently to bovine serum albumin (BSA). Cells treated with the vehicle control used to dissolve lipids before conjugation to BSA, ethanol, were also included. For the microscopy assays, the treated bovine mammary epithelial cells were fixed, permeabilized, and stained with BODIPY 493/503, DAPI, or phalloidin-TRITC as described above. The BMEC were imaged using the BIOTEK Cytation high-content imaging microscope, where each plate contained an ethanol vehicle control, anandamide, and oleamide in triplicate. Data were analyzed by ANOVA with PROC MIXED in SAS. Means were separated using Scheffe’s test. P-values were adjusted for multiple comparisons using Bonferroni’s correction.

[0079] Image cellular analyses were used to identify lipid droplet accumulation per cell when exposed to the test compounds as reported by high-content cellular microscopy imaging. FIG. 1 shows the lipid droplet accumulation (in counts per cell) in bovine mammary epithelial cells for each condition tested, including an ethanol vehicle control (VEH), 10 pM anandamide conjugated BSA (A-BSA), or 10 pM oleamide conjugated BSA (O-BSA) following the 16-hour incubation, where a indicates P <0.05. Data are presented in triplicate with the mean ± SEM. FIG. 2 shows the fluorescence microscopy imaging for the conditions described in reference to FIG. 1 for each of the VEH, A-BSA, and O-BSA, where the scale bar is equivalent to 100 pm. These assays showed an increase in lipid droplet accumulation of 95% for anandamide and 92% for oleamide (P < 0.05) compared with vehicle control.

[0080] Dose response analyses were performed for each test compound on the effects of concentration on lipogenesis in bovine mammary epithelial cells. Referring now to FIG. 3 and FIG. 4, plots of lipid droplet accumulation (in count per cell) against a range of treatment doses from 0 pM to 125 pM for A-BSA or O-BSA, respectively, are shown. Data are reported as means ± SEM, where n = 3 replicates for each test condition. In dose-response experiments, lipid droplet accumulation was maximal at 60 pM for A-BSA whereas lipid droplet accumulation increased linearly with O-BSA up to 125 pM treatment.

[0081] A pairwise comparison of the lipid droplet count per cell for various treatment conditions including an ethanol vehicle control (VEH), 10 pM anandamide conjugated BSA (A- BSA), 10 pM oleamide conjugated BSA (O-BSA), or both 10 pM anandamide conjugated BSA (A-BSA) and 10 pM oleamide conjugated BSA (O-BSA) is shown in FIG. 5. Data are reported as means ± SEM, where n = 3 replicates for each test condition. Results of the pairwise comparison indicate that no synergistic effect was observed when anandamide and oleamide were combined. [0082] Cell viability was assayed using an MTT assay, a colorimetric assay that measures cellular proliferation using the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Briefly, bovine mammalian epithelial cells were incubated with each test compound and treated with 100 pL of SDS-HCL solution and incubated for four hours. The MTT cytotoxicity assay measured the concentration of bioactive lipid required to kill 50% of the cells (CC50) after treatment. Absorbance was measured at 570 nm using a microplate spectrophotometer. Cell viability and CC50 data for each bioactive lipid are reported in Table 2.

Table 2. Bioactive Lipid Cytotoxicity Against Bovine Mammalian Epithelial Cells

[0083] Fatty acid (FA) profile of total lipid extracts from bovine mammary epithelial cells exposed ethanol vehicle control (VEH), 10 pM anandamide conjugated BSA (A-BSA), or 10 pM oleamide conjugated BSA (O-BSA) were characterized using gas chromatography/mass spectroscopy (GC/MS). GC/MS analyses were conducted using an Agilent 5975C series gas chromatography machine with a single quadrupole MS detector equipped with a GC column DB- 23 30m, 0.25mm, and 0.15pm (Agilent Technologies, Inc. Santa Clara, CA, USA). FA were grouped as sum of de novo fatty acids (De novo FA), sum of saturated FA content, and sum of unsaturated FA content are summarized as a percent of the total in Table 3 for each test condition. De novo FAs were characterized as all fatty acids below or including those fatty acid being 14- carbons in length. Data shown are means ± SEM; n = 3 replicates. Profiling of fatty acids (FA) from total lipid BMEC extracts showed that oleamide numerically increased the proportion of de novo FA (<14 carbons) by 32% compared with non-treated BMEC.

Table 3. Lipid Fraction Characterization for BMEC Exposed the A-BSA or O-BSA

[0084] The expression of lipogenic genes in differentiated bovine mammary epithelial cells exposed to the test compounds was investigated. Quantitative polymerase chain reaction (qPCR) was performed on the cells using the S so Advanced Universal SYBR green in a CFX96 Touch qPCR System (BioRad Laboratories, Inc., Hercules, CA, USA). Various lipogenesis genes were investigated for gene expression, including acetyl-CoA carboxylase alpha (ACACA), fatty acid binding protein 3 (FABP3), and fatty acid synthase (FASN). The lipogenic transcription factor sterol regulatory element binding-protein 1 (SREBF1) was also investigated.

[0085] Results of the qPCR showing the expression of fatty acid synthesis markers relative to the geometric mean of three reference genes (referred to as relative expression) are reported in FIG. 6 (ACACA), FIG. 7 (FABP3), FIG. 8 (FASN), and FIG. 9 (SREBF1). Data are presented as the mean ± SEM; n = 3 replicates. *P < 0.05, **P < 0.01 vs. VEH. Anandamide exposure resulted in a statistically significant increase in the expression of the ACACA and FASN mRNA abundance in bovine mammary epithelial cells but had limited effect on the expression of FABP3 and SREBF1. Oleamide exposure resulted in a statistically significant increase in the expression of ACACA, FABP3, FASN and SREBF1. Thus, anandamide and oleamide exposure in bovine mammary epithelial cells resulted in an increased expression of markers of lipogenic enzymes. Oleamide showed a 2-fold increase and anandamide showed a 2.7-fold increase (both /^J < 0.05) in the mRNA abundance of ACACA. The test compounds did not influence gene expression for other markers of fatty acid synthesis, including diacylglycerol O-acyltransferase 1 (DGAT1) and stearoyl-CoA desaturase 1 (SCD1). (Data not shown).

[0086] Conclusions: The MTT assay showed a 50% cytotoxic concentration at 49 pM for oleamide and 68 pM for anandamide. In dose-response experiments, lipid droplet accumulation was maximal at 60 pM for anandamide whereas lipid droplet increased linearly with oleamide up to 125 pM treatment. The combination of the compounds did not show a synergistic or additive effect. Profiling of fatty acids (FA) from total lipid BMEC extracts showed that oleamide increased the proportion of de novo FA (< 14 carbons) by 32% compared with non-treated BMEC. To identify the potential mechanism of action influenced by bioactive lipids, relative mRNA abundance of genes involved in lipogenesis by RT-qPCR was measured. Anandamide showed a 2-fold increase and oleamide a 2.7-fold increase (both P < 0.05) in the mRNA abundance of ACACA. The compounds did not affect the expression of other lipogenic genes tested (DGAT1 and SCD1). Example 4: Characterization of Bioactive Fatty Acid Compounds That Increase Lipid Accumulation in Bovine Mammary Epithelial Cells

[0087] Three bioactive fatty acids were identified from the high-throughput screening of test compounds in Example 2 and were further characterized on their ability to increase milk fat synthesis by bovine mammary epithelial cells.

[0088] Bovine mammary epithelial cells were isolated and differentiated according to the method described in Example 1. During the last 16 h of the lactogenic differentiation step, the bovine mammary epithelial cells were treated with 10 pM of each of the identified bioactive fatty acids: 7,7-dimethylelcosadienoic acid, 10(Z)- heptadecenoic acid, and arachidonic acid, that were conjugated independently to BSA. Cells treated with the vehicle control used to dissolve lipids before conjugation to BSA, ethanol, were also included. For the microscopy assays, the treated bovine mammary epithelial cells were fixed, permeabilized, and stained with BODIPY 493/503, DAPI, or phalloidin-TRITC as described above. The BMEC were imaged using the BIOTEK Cytation 5 high-content imaging microscope, where each plate contained an ethanol vehicle control, 7,7-dimethylelcosadienoic acid, 10(Z)- heptadecenoic acid, and arachidonic acid in triplicate. Data were analyzed by ANOVA with PROC MIXED in SAS. Means were separated using Scheffe’s test. P-values were adjusted for multiple comparisons using Bonferroni’s correction.

[0089] Image cellular analyses were used to identify lipid droplet accumulation per cell when exposed to the test compounds as reported by high-content cellular microscopy imaging. FIG. 10 shows the lipid droplet accumulation in bovine mammary epithelial cells for each condition tested, including an ethanol vehicle control (VEH), 10 pM 7,7-dimethylelcosadienoic acid conjugated BSA (7,7D-BSA), 10 pM 10(Z)-heptadecenoic acid conjugated BSA (lO(Z)HA-BSA), or 10 pM arachidonic acid conjugated BSA (AA-BSA) following the 16-hour incubation, where a indicates P <0.05 vs. the vehicle control. Data are presented in triplicate with the mean ± SEM. FIG. 11 shows the fluorescence microscopy imaging for the conditions described in reference to FIG. 10 for each of the VEH, 7,7DA-BSA, 10(Z)HA-BSA, and AA-BSA, where the scale bar is equivalent to 100 pm. These assays showed an increase in LD of 75% for FA1 and FA2 and an increase of 66% for FA3 (P < 0.05) compared with vehicle control.

[0090] Dose response analyses were performed for each test compound on the effects of concentration on lipogenesis in bovine mammary epithelial cells. Referring now to FIGS. 12-14, plots of lipid droplet accumulation (in count per cell) against a range of treatment doses from 0 pM to 125 pM for 7,7DA-BSA, 10(Z)HA-BSA, and AA-BSA, respectively, are shown. Data are reported as means ± SEM, where n = 3 replicates for each test condition. In dose-response experiments, dose-response experiments, all treatments showed a linear increase in LD accumulation up to 125 pM treatment.

[0091] A pairwise comparison of the lipid droplet count per cell for various treatment conditions including an ethanol vehicle control (VEH), 10 pM 7,7-dimethylelcosadienoic acid conjugated BSA (7,7D-BSA), 10 pM 10(Z)-heptadecenoic acid conjugated BSA (lO(Z)HA-BSA), or both 10 pM 7,7-dimethylelcosadienoic acid conjugated BSA (7,7D-BSA) and 10 pM 10(Z)- heptadecenoic acid conjugated BSA (lO(Z)HA-BSA), is shown in FIG. 15. Data are reported as means ± SEM, where n = 3 replicates for each test condition. Results of the pairwise comparison did not show a synergistic or additive effect between compounds. Profiling of fatty acids from total lipid BMEC extracts showed an increase of 36% and 21% in de novo (<14 carbons) fatty acid fraction for 7,7D-BSA and AA-BSA, respectively. 10(Z)HA-BSA did not impact the de novo fatty acid fraction.

[0092] Cell viability was assayed using an MTT assay as described previously. Briefly, bovine mammalian epithelial cells were incubated with each test compound and treated with 100 pL of SDS-HCL solution and incubated for four hours. The MTT cytotoxicity assay measured the concentration of bioactive fatty acid required to kill 50% of the cells (CC50) after treatment. Absorbance was measured at 570 nm using a microplate spectrophotometer. Cell viability and CC50 data for each bioactive fatty acid are reported in Table 4.

Table 4. Bioactive Fatty Acid Cytotoxicity Against Bovine Mammalian Epithelial Cells

[0093] The lipid fractions produced by the bovine mammary epithelial cells exposed ethanol vehicle control (VEH), 10 pM 7,7-dimethylelcosadienoic acid conjugated BSA (7,7D-BSA), 10 pM 10(Z)-heptadecenoic acid conjugated BSA (lO(Z)HA-BSA), or 10 pM arachidonic acid conjugated BSA (AA-BSA) were characterized using gas chromatography/mass spectroscopy (GC/MS). GC/MS analyses were conducted using an Agilent 5975C series gas chromatography machine with a single quadrupole MS detector equipped with a GC column DB-23 30m, 0.25mm, and 0.15pm (Agilent Technologies, Inc. Santa Clara, CA, USA). The GC/MS data, including de novo fatty acid content (De novo FA), sum of saturated fatty acid (FA) content, and sum of unsaturated fatty acid (FA) content are summarized as a percent of the total in Tables 5 and 6 for each test condition. The de novo fatty acids were characterized as all fatty acids below or including those fatty acid being 14-carbons in length. Data shown are means ± SEM; n = 3 replicates. Profiling of fatty acids (FA) from total lipid BMEC extracts showed that 7,7DA-BSA increased the proportion of de novo FA (<14 carbons) by 32% compared with non-treated BMEC.

Table 5. Lipid Fraction Characterization for BMEC Exposed the 7,7DA-BSA or 10(Z)HA-BSA

Table 6. Lipid Fraction Characterization for BMEC Exposed the AA-BSA

[0094] Expression of markers of fatty acid synthesis in differentiated bovine mammary epithelial cells exposed to the test compounds was investigated. Quantitative polymerase chain reaction (qPCR) was performed on the cells using the SsoAdvanced Universal SYBR green in a CFX96 Touch qPCR System (BioRad Laboratories, Inc., Hercules, CA, USA). Various lipogenesis genes were investigated for gene expression, including acetyl-CoA carboxylase alpha (ACACA) and fatty acid binding protein 3 (FABP3). The lipogenic transcription factor sterol regulatory element binding-protein 1 (SREBF1) was also investigated.

[0095] Results of the qPCR showing the relative expression of the markers of fatty acid synthesis are reported in FIG. 16 (ACACA), FIG. 17 (FABP3), and FIG. 18 (SREBF1). Data are presented as the relative expression mean ± SEM; n (replicate) = 3. *P < 0.05, **P < 0.01 vs. VEH. All three of the bioactive fatty acid test compounds increased the relative abundance of mRNA expression for ACACA and FA BP 3^ however, the bioactive fatty acids did not affect the expression of the lipogenic transcription factor SREBF1 or other genes tested ( A N JA TL and SCDi (Data not shown).

[0096] Conclusions: The MTT assay showed a 50% cytotoxic concentration at 190 pM, 395 pM, and 230 pM for 7,7DA-BSA, 10(Z)HA-BSA, and AA-BSA, respectively. In dose-response experiments, all treatments showed a linear increase in lipid droplet accumulation up to 125 pM treatment. Pairwise combination did not show a synergistic or additive effect between compounds. Profiling of fatty acids from total lipid BMEC extracts showed an increase of 36% and 21% in de novo (<14 carbons) fatty acid fraction for 7,7DA-BSA and AA-BSA, respectively. 10(Z)HA-BSA did not impact the de novo fatty acid fraction. To identify the potential mechanism of action influenced by our tested bioactive fatty acids, relative mRNA abundance of genes involved in lipogenesis was measured by RT-qPCR for 7,7DA-BSA and AA-BSA treated BMEC. 7,7DA-BSA showed a 2.7-fold increase and AA-BSA showed a 1.8-fold increase (both E < 0.05) in ACACA mRNA abundance. 7,7DA-BSA and AA-BSA also increase FABP3 mRNA abundance by 1.9- and 1.7-fold (both E < 0.05) respectively. The tested bioactive fatty acids did not affect the expression of SREBF1 or other lipogenic genes tested (FASN, DGAT1, and SCD1).

[0097] It should be understood that the definitions described herein apply to all aspects as described unless otherwise stated.

[0098] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference is to be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0099] Values expressed in a range format are to be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range were explicitly recited. For example, a range of “about 0.1 % to about 5 %” or “about 0.1 % to 5 %” is to be interpreted to include not just about 0.1 % to about 5 %, but also the individual values (e.g., 1 %, 2 %, 3 %, and 4 %) and the sub-ranges (e.g., 0.1 % to 0.5 %, 1.1 % to 2.2 %, 3.3 % to 4.4 %) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise. [0100] Unless expressly stated, ppm (parts per million), percentage, and ratios are on a by weight basis. Percentage on a by weight basis (% w/w or w/w %) is also referred to as weight percent (wt. %) or percent by weight (% wt.) herein.

Claims

CLAIMS What is claimed is:

1. A composition for increasing milk fat content or milk fat yield in milk comprising: a lipid enhancing bioactive compound in an amount effective to increase milk fat synthesis in mammary epithelial cells of an animal; wherein the lipid enhancing bioactive compound comprises at least one of anandamide, oleamide, 7,7-dimethylelcosadienoic acid, 10(Z)-heptadecenoic acid, or arachidonic acid, or derivatives or mixtures thereof.

2. The composition of claim 1, wherein the effective amount of lipid enhancing bioactive compound present in the composition can be sufficient to increase milk fat synthesis within the mammary epithelial cells as compared to mammary epithelial cells not exposed to the lipid enhancing bioactive compound.

3. The composition of claim 1, wherein the effective amount of lipid enhancing bioactive compound is sufficient to increase lipogenesis within the mammary epithelial cells as compared to mammary epithelial cells not exposed to the lipid enhancing bioactive compound.

4. The composition of any of claims 1-3, wherein the composition comprises a feed additive or a dietary supplement.

5. The composition of any of claims 1-4, wherein the composition is encapsulated.

6. The composition of any of claims 1-5, wherein the composition comprises a component of a complete feed, a premix, a concentrate, a base mix, a supplement, a top dress, or any combination thereof.

7. A method for increasing milk fat content or milk fat yield in milk comprising: administering a composition comprising a lipid enhancing bioactive compound in an amount effective to increase milk fat synthesis in mammary epithelial cells of the animal; wherein the milk fat lipid enhancing bioactive compound comprises at least one of anandamide, oleamide, 7,7-dimethylelcosadienoic acid, 10(Z)-heptadecenoic acid, or arachidonic acid, or derivatives or mixtures thereof.

8. The method of claim 7, wherein the effective amount of milk fat enhancing bioactive compound present in the composition can be sufficient to increase milk fat synthesis within the mammary epithelial cells as compared to mammary epithelial cells not exposed to the lipid enhancing bioactive compound.

9. The method of claim 7, wherein the effective amount of lipid enhancing bioactive compound is sufficient to increase lipogenesis within the mammary epithelial cells by as compared to mammary epithelial cells not exposed to the lipid enhancing bioactive compound.

10. The method of any of claims 7-9, wherein the composition is administered as a feed additive or a dietary supplement.

11. The method of any of claims 7-10, wherein the animal is a bovine animal.

12. The method of any of claims 7-11, wherein the animal is a deer, elk, caribou, moose, equine, goat, sheep, or swine animal.

13. The method of any of claims 7-12, wherein the composition is administered to a cow or group of cows at any time during active milk production.

14. The method of any of claims 7-12, wherein the composition is administered to a cow or group of cows once a day during active milk production.

15. The method of any of claims 7-12, wherein the composition is administered to a cow or group of cows twice a day during active milk production.

16. The method of any of claims 7-12, wherein the composition is administered to a cow or group of cows at any time immediately following the onset of active milk production.

17. The method of any of claims 7-12, wherein the composition is administered to a cow or group of cows once a day during active milk production, twice a day during active milk production, three times a day during active milk production, or four times a day during active milk production.

18. The method of any of claims 7-17, wherein the composition is administered upon a showing that the milk fat content or milk fat yield are not sufficient for a milk producer’s demands.

19. The method of any of claims 7-17, wherein the composition is administered to an animal or group of animals, such as a cow or group of cows, on a daily basis throughout milk production to sustain milk fat content or milk fat yield.

20. The method of any of claims 7-17, further comprising periodically sampling milk from a cow or group of cows to measure levels of milk fat content or milk fat yield in response to the lipid enhancing bioactive compounds; and based on the measured levels of milk fat content or milk fat yield, increasing the effective amount of lipid enhancing bioactive compound in response to a level of milk fat content or milk fat yield that does not meet producer’s needs.