WO2018146643A1 - Nature-identical supplements to improve the health and performance of animals - Google Patents

Abstract

Compositions comprising and methods of administering high-purity nature-derived additives for inclusion in the feed, feed additives or drinking water of warm-blooded mono-gastric animals and birds to improve health, reduce the need for antibiotics, and increase the productivity and quality of farmed or companion warm-blooded mono-gastric animals and birds.

Description

Nature-identical supplements to improve the health and performance of animals Field of the Invention

[0001] The present invention relates to feeds, feed additives or drinking water supplements comprising a blend of high-purity nature-derived additives suitable for consumption by animals and birds to improve health, reduce the need for traditional medication with antibiotics and heavy metals, and to increase the productivity and quality of produce from farmed animals and birds.

Background of the invention

[0002] Resveratrol and its derivatives are phytoalexin stilbenes found in very low concentrations in grapes, peanuts, cranberries, blueberries, strawberries, Polygonum cuspidatum and some other botanical sources. The content of resveratrol and resveratrol glycoside increases in grapes in grapes increases upon infection with Botrytis cinerea (Roldan 2003; Sahin et al. 2010; Alagawany et al. 2015).

[0003] In reared animals, resveratrol has been shown to reduce the oxidative stress in heat-stressed broilers and also improve the weight gain (Liu et al. 2014), and which ameliorate the damages caused by aflatoxins in broiler feeds (Sridhar et al. 2015). Aflatoxins may contaminate crops in the field, at harvest, or during transportation and storage as a result of infestations of Aspergillus flavus and parasiticus, which are abundant in warm and humid regions. It is ingested that aflatoxins ingested in feed remain present in meat or dairy products and are passed up the food chain, including for human consumption. Resveratrol in diets for quails has shown to increase the serum vitamin E level (Sahin et al. 2010).

[0004] Furthermore, resveratrol has natural antibacterial properties, which can be used in both food preservation and medicinal use (Paulo et al. 2010), and may also be a potent antiviral molecule against DNA and RNA viruses (Campagna & Rivas 2010).

[0005] Resveratrol as a feed additive has also shown to improve pork meat quality by increasing the longissimus dorsi pH₂4 (a measurement of pH increase in the longissimus dorsi muscle over the 24 hours following slaughter), shear force, drip loss, glycolytic potential, backfat depth, lactase dehydrogenase activity and mRNA level (Zhang et al. 2015).

[0006] Trials with newly weaned piglets have shown that the inclusion of 0.2% of a resveratrol containing product in the piglets' feeds improved the efficiency of the feed conversion and the daily live weight gain compared to the negative control group (Ahmed et al. 2013). [0007] However, although there is much interest in the use of resveratrol in addressing a variety of health issues, difficulties associated with producing the compound restrict its use in feed, feed additives or drinking water supplements suitable for consumption by animals and birds. In the current market, the predominant resveratrol production processes relies on extracting resveratrol from knotweed, a plant from the polygonaceae family grown commercially in China . Unfortunately, the extraction process is labour intensive and the yield of resveratrol is low, which is especially a problem for large volumes of feed or additives required for commercial farming practices. A further problem with conventional resveratrol extraction processes from knotweed is the presence of contaminants. For example, commercially available resveratrol is often contaminated with emodin, a natural product of the knotweed plant, which can induce diarrhoea if consumed . Diarrhoea is especially a problem in mammals in the days post weaning when the animals are generally dehydrated as a consequence of their reluctant transition from milk to water. However, diarrhoea is in general a very serious problem in farmed animals, to the extent that many farmers administer antibiotics prophylactically to ensure good animal health and improved overall production through consistently higher aggregate weight gain of the group.

[0008] Other contaminants that may be found in resveratrol derived from knotweed are ground and/or air pollutants from the environment in which the knotweed plants have grown . For example, very high concentrations of poly-aromatic hydrocarbons have been detected in commercially available resveratrol samples derived from knotweed plants grown in various locations in China . Table 1 shows the results of analysis using gas chromatography coupled to mass spectrometry performed on 40 g samples of commercially available resveratrol derived from knotweed plants grown in China (sa mples 1, 2 and 3) when compared with fermentation derived resveratrol (samples 4 and 5) .

Chrysene 1.2 pg/kg 1.3 pg/kg 360 pg/kg < 0.5 pg/kg < 0.5 pg/kg

Dibenzo(a,h) < 0.5 pg/kg < 0.5 pg/kg 31 pg/kg < 0.5 pg/kg < 0.5 pg/kg anthracene

Fluoranthene 15 pg/kg 15 pg/kg 940 pg/kg < 0.5 pg/kg < 0.5 pg/kg

Fluorene 94 pg/kg 98 pg/kg 280 pg/kg < 0.5 pg/kg 1.3 pg/kg

Indeno( l,2,3- < 0.5 pg/kg < 0.5 pg/kg 190 pg/kg < 0.5 pg/kg < 0.5 pg/kg cd)pyrene

Phenanthrene 67 pg/kg 69 pg/kg 860 pg/kg 1.5 pg/kg 2.8 pg/kg

Pyrene 13 pg/kg 13 pg/kg 920 pg/kg < 0.5 pg/kg < 0.5 pg/kg

Sum PAHs (4 or 3.3 pg/kg 3.5 pg/kg 1 400 < 0.5 pg/kg < 0.5 pg/kg less rings) pg/kg

Sum of heavy" 1.8 pg/kg 1.3 pg/kg 1 300 < 0.5 pg/kg < 0.5 pg/kg PAHs (5 or pg/kg

more rings)

Sum of all 210 pg/kg 210 pg/kg 5 200 1.5 pg/kg 4.8 pg/kg positive pg/kg

identified PAHs

Table 1 : Polycyclic aromatic hydrocarbon content in resveratrol samples derived from knotweed grown in China (sa mples 1, 2 and 3) and in resveratrol derived from recombinant microbial fermentation (samples 4 and 5) .

[0009] Polycyclic aromatic hydrocarbons (PAHs), otherwise known as polynuclear aromatic hydrocarbons, are chemicals released from incomplete combustion of organic matter, typically by the burning of fossil fuels in smelting, manufacturing, power stations, vehicles and home fires, as well as burning the by-products of agriculture such as crop residues and dung . PAHs generally have low solubility in water, and are predominantly seen in solid form as particulate air pollution, soil or sediment pollution . Although cancer is the primary health risk of exposure to PAHs, they have also been linked to cardiovascular disease and poor fetal development. Hence, the administration to animals and birds of resveratrol extracts comprising environmental pollutants such as has been observed in knotweed- derived resveratrol may be undesirable not only for the animals and birds themselves, but raises concerns from the ultimate human consumers of the resulting farmed products (such as meat or eggs) .

[0010] The rising global population human population is placing increasing stress on the current means of production of food and on the use of land areas. There is an environmental need to maximize the efficient use of land already allocated to farming, whilst simultaneously reducing the impact of such modern farming practices to the surrounding natural environments.

[0011] Therefore, in light of the current challenges to the domestic and commercial rearing of animals and birds, there is a growing need for effective, sustainable, environmentally friendly compositions and methodologies to benefit the health of warm animals and birds, and to improve the quality and yield of farmed products resulting from farmed animals and birds.

Summary of the Invention

[0012] Provided herein are effective natural compositions and methods of their use in the rearing of animals and birds.

[0013] In some aspects, resveratrol is shown herein to have very positive effects on animal performance when consumed by animals or birds, including improving the weight gain, ameliorate the negative effects of heat stress and aflatoxins in animals.

[0014] In one aspect, resveratrol may be added to the feed or water of animals and birds to increase the homogeneity of the group as a whole. As evidenced herein, the resveratrol provides gain-improving effects within a shared cage, house or pen that are strongest in the smaller animals that are stressed by their co-habitation with larger animals. Resveratrol may thus be used for example, to reduce the variance of animals and birds grown for meat (such as finisher-pigs) in a pen such that the runts are brought closer to the finishing weight of their larger siblings. Similarly, the smaller female broiler chickens present in a pen or cage in general take longer to grow to a sufficient size. According to some aspects disclosed herein, the application of resveratrol in the feed of birds (such as breeder chickens) has a greater effect of weight-gain on the smaller birds such that they may begin laying eggs sooner and thus improve the aggregate productivity of the entire group of framed birds.

[0015] In other aspects disclosed herein, resveratrol is present in the food or drink of warm animals or birds to assist in reducing stress in high density farm environments. Without being limited by theory, it is postulated that resveratrol acts on chronic, low-grade inflammation (including joint inflammation) so as to reduce chronic inflammation in the joints. This is of particular benefit to caged animals and birds with limited movement (such as in intensive modern farming practices) who are particularly susceptible to such joint inflammation in the later stages of their life cycle when they have limited room in the cage or pen. Chronic inflammation is also a factor in ageing, including cardiovascular disease, asthma, etc., so administration of resveratrol into the feed or drink of animal or bird food may influence the overall health state of the animals or birds.

[0016] In another aspect, the invention provides a composition suitable for use in the manufacture of feeds, feed additives and/or drinking water supplements capable of preventing, treating or reducing an infectious disease, or pathogenic microbes in animals and birds, when fed to said animal or bird, a stilbene or a methylated or glycosylated stilbenoid (such as resveratrol or pinosylvin). The inclusion of resveratrol has antibacterial properties, providing medicinal benefits and helping to preserve the feed or feed additive, thus increasing shelf life. Exemplar infectious diseases common in farmed animals include but are not limited to ileitis, sudden death, weaning diarrhea caused by, among others, coli infections, as well as diarrhea and respiratory diseases in calves and various infectious diseases in livestock and poultry.

[0017] In another aspect, the invention provides a feed, feed supplement or feed additive composition suitable for feeding to animals or birds. In some embodiments, the composition further comprises at least one additional active ingredient that is a natural antioxidant.

Description of drawings and figures

Figure 1 provides the chemical structure of trans-resveratrol.

Figure 2 provides the layout of the building where the pigs were housed in Example 1. Figure 3 provides the layout of the building where the pigs were housed in Example 2. Figures 4 (a-h) provides the difference in live weight of males and females at the different measurement days during Example 3.

Detailed Description of the Invention

[0018] All publications, patents and patent applications cited herein are hereby expressly incorporated by reference in their entirety for all purposes.

Definitions

[0019] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element.

[0020] The term "comprise" and "include" as used throughout the specification and the accompanying claims as well as variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. These words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows. For the purposes of describing and defining the present invention it is noted that the term "substantially" is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term "substantially" is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

[0021] As used herein, the term "about" refers to ± 10% of any particular value.

[0022] As used herein, the terms "or" and "and/or" are utilized to describe multiple components in combination or exclusive of one another. For example, "x, y, and/or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z."

[0023] It is noted that terms like "preferably," "commonly," and "typically" are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.

[0024] The term "isolating" or "separating" means any human intervention which change the relative amount of the compound compared to another selected constituent in a given matrix to a higher relative amount of the compound relative to the other constituent. In an embodiment, the compound may be isolated into a pure or substantially pure form. In this context, a substantially pure compound means that the compound preparation contains less than 10%, such as less than 8%, such as less than 6%, such as less than 5%, such as less than 4%, such as less than 3%, such as less than 2%, such as less than 1 %, such as less than 0.5% by weight of other selected constituents. In an embodiment, an isolated compound is at least 50% pure, such as at least 60% pure, such as at least 80% pure, such as at least 90% pure, such as at least 91% pure, such as at least 92% pure, such as at least 93% pure, such as at least 94% pure, such as at least 95% pure, such as at least 96% pure, such as at least 97% pure, such as at least 98% pure, such as at least 99% pure, such as at least 99.5% pure, such as 100 % pure by dry weight.

[0025] The term "synthetic" or "non-naturally occurring" means that a compound is not normally found in nature or natural biological systems. In this context, the term "found in nature or in natural biological systems" does not include the finding of a compound in nature resulting from releasing the compound to nature by deliberate or accidental human intervention. Synthetic compounds may include compounds completely or partially synthetized by human intervention and/or compounds prepared by human modification of a natural compound .

[0026] As used herein, "animals or birds" comprises farmed animals and birds, companion animals and birds, livestock, domesticated animals and birds, animals and birds bred for competition (such as racing), collection, working (such as hunting, draft, load bearing, human transportation), breeding or display. Animals include without limitation mono- gastric animals, pseudoruminants, ruminants, mammals, marsupials, amphibians, reptiles (including alligators and crocodiles) and macropods (including wallabies and kangaroos) .

[0027] As used herein, the term "ruminants" includes but is not limited to sheep, deer, antelope, goats, giraffes, yaks, buffalo, and cattle (including but not limited to cows) . As used herein, the term "pseudoruminants" includes but is not limited to camels, alpacas, llamas, guanacos, and vicunas.

[0028] As used herein, the term "mono-gastric animals" includes but is not limited to pigs, horses, sheep, rabbits, dogs, and cats.

[0029] As used herein, the term "birds" includes but is not limited to a chickens (such as a broiler, breeder or a laying hens), turkey, goose, duck, pheasant, quail, grouse, ostrich, emu or pigeon . As used herein, the term "broiler chicken" may refer to a chicken grown and slaughtered for meat, rather than a "laying chicken", which may refer to a hen chicken grown for the production of eggs.

[0030] As used herein, the term "finisher pig" may refer to commercially reared pigs with a live weight of about 75 kg or higher, typically achieved as the pigs approach the final 4 weeks prior to slaughter.

[0031] As used herein, the term "piglets" may refer to commercially reared pigs from the time they begin eating solid feed in the farrowing house until they achieve 30kg, typically at the age of about 10- 11 weeks old .

[0032] As used herein, the term "active ingredient" refers to a chemical compound or mixture of chemical compounds capable of treatment of microbial infections. As such, the active ingredient may have properties including but not limited to antimicrobial, antibacterial, and/or antiviral properties against microbes capable of infecting, growing and reproducing on or in the bodies of animals and birds. For example, the active ingredients may be effective against Gram positive and/or Gram negative bacteria .

[0033] As used herein, the term "feed conversion ratio" (FCR) otherwise known as the feed conversion rate, refers to the ratio of the efficiency with which the a nimals or birds convert mass of feed into mass of desired output. As used herein in relation to animals and birds raised for meat (such as pigs, beef cows, fish and broiler chickens), the output is meat as measured by improvement in the final mass of the animal or bird . As used herein in relation to diary animals, the output is milk. As used herein in relation to layer birds (such as layer chickens, quail, ducks or geese), the output is mass of eggs. A higher FCR may be used to raise production rates of a farmed animal or bird, or it may be used economically to permit the provision of less feed by weight to the animal or bird whilst retaining or substantially retaining the same average increase in mass.

[0034] As used herein, the term "average daily gain" (ADG) refers to the average net increase in weight per animal or bird . As used herein in relation to animals and birds raised for meat (such as pigs, beef cows, fish and broiler chickens), the ADG is a measure of how rapidly the a nimals or birds are growing in mass, which may translate to either a higher final live weight prior to slaughter, and/or to an ea rlier achievement of slaughter weight. Compounds

[0035] According to some aspects, the resveratrol for inclusion in the feed, feed additives or water, may be provided at suitable concentrations as purified extracts of Japanese knotweed, yucca, grapes, etc. According to preferred aspects, the resveratrol for inclusion in the feed, feed additives or water may be produced by recombinant microbial fermentation, including but not limited to fermentation extract, purified resveratrol, or dried fermentation cake from recombinant microbes capable of producing resveratrol. Resveratrol derived from fermentation of recombinant microbes is preferred not least because it is inherently free of emodin and hazardous levels of Polycyclic Aromatic Hydrocarbons (PAHs) .

[0036] As used herein, the terms "stilbene" and "stilbenoid" are interchangeable and refer to compounds based on the compound of formula (I) :

wherein formula (I) may be substituted at one or more suitable positions. Exemplary substituents include, but are not limited to, halogen, cyano, nitro, Ci-C₆ alkyl, d-C₆ haloalkyl, d-C₆ hydroxyalkyl, hydroxy, Ci-C₆ alkoxy, thiol, Ci-C₆ alkylthio, amino, Ci-C₆ alkyl amino, di- Ci-C₆ alkyl amino, carboxyl, Ci-C₆ alkoxycarbonyl, amido, methyl, and glycosyl. Generally, stilbenes, including resveratrol, and flavonoids are produced in plants and yeast through the phenylpropanoid pathway as illustrated by the reactions shown in Figure 1.

[0037] As used herein, the term "phenylpropanoid" refers to compounds based on a 3- phenylprop-2-enoate backbone. Examples of such compounds include, but are not limited to, cinnamic acid, coumaric acid, caffeic acid, ferulic acid, S-hydroxyferuhc acid_f sinapinic acid, cinnamoyl-CoA, p-coumaroyi-CoA, and the like.

[0038] As used herein, the term "phenylpropanoid derivative" refers to any compound derived from, synthesized from, or biosynthesized from a phenylpropanoid; i.e. a phenylpropanoid derivative includes any compound for which a phenylpropanoid compound is a precursor or intermediate. Examples of phenylpropanoid derivatives include, but are not limited to, stilbenoid compounds and chalcone compounds. Specific examples of phenylpropanoid derivatives include, but are not limited to, resveratrol, pinosylvin, pinocembrin chalcone, and pinocembrin,

[0039] As used herein, the term "dihydrophenylpropanoid" refers to compounds based on a phenylpropanoate backbone. Examples of such compounds include, but are not limited to, dihydrocinnamic acid, phloretic acid, 3,4-dihydroxyhydrocinnamic acid, hydroferulic acid, dihydrocoumaroyl-CoA, dihydrocinnamoyl-CoA, and the like.

[0040] As used herein, the term "dihydrophenylpropanoid derivative" refers to any compound derived from, synthesized from, or biosynthesized from a dihydrophenylpropanoid; i.e. a dihydrophenylpropanoid derivative includes any compound for which a dihydrophenylpropanoid compound is a precursor or intermediate. Examples of dihydrophenylpropanoid derivatives include, but are not limited to, dihydrostilbenoid compounds and dihydrochalcone compounds. Specific examples of dihydrophenylpropanoid derivatives include, but are not limited to, phloretin, phlorizin, dihydropinosylvin, dihydropinosylvincarboxylate, 3-O-methyldihydropinosylvincarboxylate, 4-isoprenyl-3-0- methyldihydropinosylvincarboxylate (amorfrutin 1), 3-O-methyldihydropinosylvin, 4- isoprenyl-3-O-methyldihydropinosylvin (amorfrutin 2), 5-hydroxy-lunularic acid, and di hydro resveratrol.

[0041] As used herein, the terms "phenylpropanoid pathway," "phenylpropanoid derivative pathway," "phenylpropanoid derivative synthesis pathway," and "phenylpropanoid derivative biosynthesis pathway" are interchangeable and refer to any biosynthesis pathway in which a phenylpropanoid is a precursor or intermediate.

[0042] The term "stilbene" or a derivative thereof can be hydroxylated derivatives of stilbene and are thus encompassed by the term "stilbene" as used herein. The term "stilbene" includes but is not limited to at least one of resveratrol, dihydroresveratrol, and pinosilvin; a glycosylated stilbene comprising piceid (3-resveratrol monoglucoside or 5- resverarol monoglucoside), resveratroloside (4'-resveratrol monoglucoside), M ulberroside E (3,4'-resveratrol diglucoside), 3,5- resveratrol diglucoside, and 3,5,4'-resveratrol triglucoside and their dihydro- reduced equivalents; a methylated stilbene comprising pterostilbene (3,5-dimethoxy-4'-hydroxy-trans-stilbene), 3,5,4'-trimethoxystilbene, pinostilbene, tetramethoxystilbene, pentamethoxystilbene, and N-Hydroxy-N- (trimethoxphenyl)-trimethoxy-benzamidine and their and their dihydro- reduced equivalents. See Figures 2 and 3 for the names and aqueous solubilities of various resveratrol molecules.

[0043] As used herein, the term "resveratrol" refers to a compound seen in Figure 1 that may be synthesized, isolated, and purified from of a mixture of products produced in a host modified to express enzymes of the resveratrol biosynthetic pathway or that can be produced from natually occurring sources, such as grapes. "Resveratrol" further refers to derivatives and analogs analogs thereof, including but not limited to forms of methylated and/or glucosylated resveratrol. For example, the resveratrol compound contemplated for use herein may be produced in vivo through expression of one or more enzymes involved in the resveratrol biosynthetic pathway in a recombinant yeast or in vitro using isolated, purified enzymes involved in the resveratrol biosynthetic pathway, such as those described in WO2006/089898, WO2008/009728, WO2009/016108, WO2009/ 124879, WO2009/ 124967, WO2011/ 147818, which are incorporated by reference in their entirety. Therefore, resveratrol as defined herein can differ chemically from other sources of resveratrol, such as extracts from plants and derivatives thereof, or may include such plant extracts and derivatives thereof.

[0044] As used herein, the terms "modified resveratrol," "resveratrol derivative," and "resveratrol analog" can be used interchangeably to refer to a compound that can be derived from resveratrol or a compound with a similar structure to resveratrol. For example, the terms "modified resveratrol," "resveratrol derivative," and "resveratrol analog" can refer to resveratrol-like molecules such as to glycosylated resveratrol molecules, methylated resveratrol molecules, or resveratrol molecules that are glycosylated and methylated .

[0045] As disclosed herein, the term "glycosylated resveratrol" refers to resveratrol glycosylated at the 3 hydroxyl group, or the 4' hydroxyl group, or the 5 hydroxyl group of resveratrol, wherein glycosylation comprises covalently attaching one or a plurality of sugar or saccharide residues at one or more of the 3, 4', or 5 hydroxyl groups of resveratrol . The saccharide moiety in each position can be independently zero, one, two, three, or multiple sugar residues, wherein all the sugar residues can be the same sugar residues or different sugar residues.

[0046] Thymol is a natural monoterpene phenol known to have antioxidant and antimicrobial properties. Thymol is for all practical purposes found at commercially relevant concentrations in agriculture only in leaves of common thyme Thymus vulgaris) and extracts thereof. Thymol is also found at much lower concentrations in other natural plants such as Euphrasia rostkoviana, Monarda didyma, Monarda fistulosa, Trachyspermum ammi, Origanum compactum, Origanum dictamnus, Origanum onites, Origanum vulgare, Thymus glandulosus, Thymus hyemalis, Thymus vulgaris, Thymus zygis.

[0047] Hu and Coats (2008) have shown that thymol degrades rapidly (DT50 16 days in water, 5 days in soil) in the environment and has thus subsequently been recognized as low environmental risk because of rapid dissipation and low bound residues.

[0048] In one aspect the leaves and/or stems of the Thymus vulgaris plant may be used to form a concentrated component of animal feed or feed additive rich in thymol . In one aspect, this may be thyme oil, or it may be a waste product of a thyme oil production such as the remaining crushed, pressed thyme leaves and/or stems. Although thymol may be extracted from the Thymus vulgaris plant or any of the other thymol-producing plants listed above, thymol is just one of a large number of antioxidants and other molecules present in these raw materials and crude extracts derived therefrom. Additionally, the level of thymol in even the same sub-species or breed of Thymus vulgaris will vary as a consequence of the climatic conditions experienced by the plants during a particular growing season . Hence there may be some difficulty in consistently achieving the desired concentration and purity of thymol to meet the demand associated with the global farming and rearing of animals and birds.

[0049] In some embodiments, thymol for use in the methods and incorporation in the compositions disclosed herein can be produced synthetically, such as is taught for example in US 1432298 and US 1332680) .

[0050] Crude compositions comprising thymol suitable for incorporation into animal or bird feed or feed additives may therefore comprise at least one of thyme stems, thyme flowers, thyme leaves, thyme leaf marc (cake remaining from oil extraction), all preferably dried, or thyme oil. Compositions with higher concentrations of thymol suitable for incorporation into animal or bird feed or feed additives may be achieved by immersing or washing these crude compositions with solvents suitable for use in feed or feed additives and then optionally further concentrated a nd/or selectively purified for thymol by evaporation, distillation, chromatography, or equivalent techniques. The resulting compositions comprising higher concentrations of thymol suitable for incorporation into animal or bird feed or feed additives may be present in the form of water extracts or alcohol extracts or combinations and blends thereof. Alcohol extracts may be preferred, since both resveratrol and thymol have significantly better solubilities in alcohols such but not limited to ethanol, than their solubilities in water.

[0051] According to some aspects, the thymol may be present in substantially pure form or be incorporated into the feed of feed additives to an effective concentration as an element of biological material comprising thymol such as at least one of thyme stems, thyme flowers, thyme leaves, thyme leaf marc (all preferably dried 0.1-0.3 dry wt%), or thyme oil and higher thymol concentration extracts thereof.

[0052] As used herein, the term "enzyme suitable for inclusion in animal or bird feed" means any enzyme that may be incorporated into animal or bird feed, feed additives, or water supplements. Enzymes suitable for inclusion in animal or bird feed are typically included to improve the efficiency of feed utilization (i.e. the FCR) and/or to improve the health of the animal and/or to reduce the environmental impact of the animal or bird excreta. Non-limiting examples of enzymes that may be added to the animal or bird feeds, additives or supplements, or produced by the recombinant resveratrol and/or thymol- producing microorganisms to be incorporated into these compositions, includes without limitation one or more selected from the group comprising beta-glucanase, alpha- galactosidase, invertase, lactase, lipase, phospholipase, acetyltransferase, phytase, cellulase, beta-galactosidase, protease, amylase, or xylanase.

Compositions

[0053] The active ingredients contemplated herein are used in the form of compositions. The active ingredients can be applied to feed, feed additives, drinking water or drink supplements, to form a comestible composition suitable for ingestion by animals and birds. The active ingredients can be applied, if desired, together with other carriers conventionally used in the formulation art of feed, feed additives and drink supplements, surfactants or other additives which aid application. Suitable carriers and additives can be solid or liquid and are the substances expediently used in the art of formulation, for example natural or regenerated mineral materials, solvents, dispersants, wetting agents, adhesives, thickeners, binders or attractants.

[0054] In one aspect, the resveratrol and thymol-containing compositions may comprise the fermentation cake of microorganisms capable of producing resveratrol and/or thymol. The microorganism engineered to produce resveratrol may be any food or feed-approved microorganism, preferably a yeast, such as baker's or brewer's yeast {Saccharomyces cerevisiae), Kluveromyces marxianus, Candida utilis. Incorporation of fermentation cake comprising resveratrol-producing microorganisms into animal or bird feed, feed additives or water supplement compositions has the advantage of providing additional nutrients, vitamins and minerals present in the fermentation broth or produced naturally by the microbes. In a preferred embodiment of this aspect, the resveratrol-producing recombinant microorganism is saccharomyces cerevisiae free of antibiotic markers and grown in a fermentation medium not comprising antibiotics. Saccharomyces cerevisiae has been included in feed for many years, and is sometimes known as "Feed yeast". The use of feed yeast (an example of a "feed microorganism") has increased since the Bovine Spongiform Encephalopathy crisis, in consequence of the demand for "clean" protein sources which could not support the growth or reproduction of viruses or prions able to cross animal species barriers. Saccharomyces cerevisiae are now appreciated to have a high Protein Efficiency Ratio, have a high lysine content, are an excellent source of vitamin D and further comprise significant concentrations of the nutrients glutathione, inositol and choline. Yeast beta-glucans and mannans a lso help stimulate the immune system of young and infants in many animal species.

[0055] In some aspects, the resveratrol and/or thymol-producing microorganisms may also be engineered to produce enzymes desirable for incorporation into animal or bird feeds, additives or supplements, such as to improve the efficiency of feed utilization or the health of the animal or bird .

[0056] In some aspects, the resveratrol and/or thymol-producing microorganisms (such as recombinant cerevisiae) are present as whole cells in "direct-fed microbial" compositions such as active dry yeast, yeast culture (such as molasses yeast condensed solubles) .

[0057] In some aspects, the resveratrol and/or thymol-producing microorganisms (such as recombinant cerevisiae) are present as whole cells that are heat treated, irradiated, lysed (such as by sonication or by hydrolysis), pasteurized, or in another way rendered "non- fermentive" so that the viability of recombinant microorganisms in the resulting composition is significantly reduced, preferably reduced to the extent that no viable recombinant microbes may be detected . The resveratrol, optionally with thymol and recombinantly produced enzymes suitable for inclusion in animal or bird feed may be added to the animal or bird feed, feed additive or water supplement as a "yeast extract" comprising concentrated soluble materials recovered from lysed yeast cells following fermentative growth.

[0058] In other embodiments of the invention, compositions contemplated herein ca n contain a ca rrier and at least about 0.0001%, or at least about 0.001%, or at least about 0.01%, or at least about 0.1%, or at least about 1%, or at least about 2%, or at least about 5%, or at least about 7.5%, or at least about 10%, or greater than about 10%, or greater than about 15%, or greater than about 20%, or greater than about 25%, or greater than about 50% by weight stilbene, such as resveratrol . In some applications, stilbene can be present in an amount that is greater tha n about 60%, about 70%, about 80%, about 90%, about 95% or about 99% by weight of the composition . In one example, the provided compositions contain stilbene in an amount at or about 0.0001% to at or about 2%, or about 0.001% to at or about 5%, or about 0.01% to at or about 75% by weight of the composition. In another example, a composition may contain stilbene in an amount of from at or about 1% to at or about 50% by weight of the composition. In another example, a composition may contain stilbene in an amount of from at or about 5% to at or about 40% by weight of the composition . In another example, a composition may contain stilbene in an amount of from at or about 10% to at or about 30% by weight of the composition . In another example, a composition may contain stilbene in an amount of from at or about 15% to at or about 25% by weight of the composition. In another example, a composition may contain stilbene in an amount of from at or about 1% to at or about 90% by weight of the composition. In another example, a composition may contain stilbene in an amount of about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 50% by weight of the composition. In another example, a composition may contain stilbene in an amount of up to about 99% or more by weight of the composition.

[0059] In one particular embodiment, a contemplated stilbene-containing composition, such as a resveratrol-containing composition is provided as a concentrate. For example, a stilbene-containing composition may be provided as a 20X, or a 10X, or a 5X, or a 3X concentrate that can be diluted by an end user with an appropriate solvent to achieve a IX working concentration. Alternatively, a stilbene-containing composition may be provided to an end user at a IX working concentration. However, any concentration is contemplated for use herein . For example, compositions provided as concentrates can be used without dilution at all or may be diluted from a highly concentrated concentrate (e.g., about 20X to about 100X) to some multiple of concentration higher than IX, such as 2X, 2.5X, 3X, etc. or can be used at a more dilute concentration, such as 1/2X, 1/4X, 1/ lOX, etc. While concentrates are more preferred as commercially available goods, the industry and end consumers typically apply dilute compositions to animal and bird feeds or drinking water.

[0060] In another embodiment, a contemplated composition may be seen in Table 1, where ingredients can be measured in percent volume per volume, percent weight per volume, or percent by weight.

[0061] Table 1. Contemplated composition formulation.

[0062] In some aspects, the resveratrol and thymol are present in or contacted with the composition as a blend comprising 50wt% resveratrol to 50wt% thymol, or 30wt% resveratrol to 70wt% thymol, or 20wt% resveratrol to 80wt% thymol, or 15wt% resveratrol to 85wt% thymol, or 10wt% resveratrol to 90wt% thymol.

[0063] Preferred inclusion rates of the blend of stilbene (such as resveratrol or pinosylvin) and thymol include aggregate concentrations of less than 30ppm, 5 to 25ppm, 5 to 20ppm, 5 to 15ppm, or 5 to lOppm.

[0064] In preferred aspects the resveratrol is produced by fermentation of recombinant microbes, which is more cost effective. Preferred production organisms include Saccharomyces cerevisiae or other yeasts.

[0065] In some aspects the resveratrol employed in the compositions and methods described herein may be resveratrol derivatives or other forms of resveratrol, particularly more water-soluble derivatives including but not limited to acetyl-resveratrol or pinosylvin.

[0066] Further examples of additional active ingredients include plant essential oil compounds or derivatives thereof. Examples include aldehyde C16 (pure), a-terpineol, amyl cinnamic aldehyde, amyl salicylate, anisic aldehyde, benzyl alcohol, benzyl acetate, cinnamaldehyde, cinnamic alcohol, carvacrol, carveol, citral, citronellal, citronellol, p- cymene, diethyl phthalate, dimethyl salicylate, dipropylene glycol, eucalyptol (cineole) eugenol, is-eugenol, galaxolide, geraniol, guaiacol, ionone, menthol, methyl salicylate, methyl anthranilate, methyl ionone, methyl salicylate, nootkatone, a-pheliandrene, pennyroyal oil perillaldehyde, 1- or 2-phenyl ethyl alcohol, 1- or 2-phenyl ethyl propionate, piperonal, piperonyl acetate, piperonyl alcohol, D-pulegone, terpinen-4-ol, terpinyl acetate, 4-tert butylcyclohexyl acetate, thyme oil, thymol, metabolites of trans-anethole, vanillin, and ethyl vanillin.

[0067] In another embodiment, a contemplated composition may include a stilbene, such as resveratrol, to additional active ingredient ratio of about 1 : 10, or about 1 : 8, or about 1 : 6, or about 1 :4, or about 1 : 2, or about 1 : 1, or about 2: 1, or about 4: 1, or about 6: 1, or about 8 : 1, or about 10: 1.

[0068] In other embodiments, compositions contemplated herein can include a stilbene in combination with thymol and optionally one or more additives, such as a fragrance, a preservative, or an attractant.

[0069] Carriers may be added to a composition in an amount of about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 50% by weight of the composition. In some applications, a carrier can be present in an amount that is at or greater than about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% by weight of the composition.

[0070] According to some aspects, carriers may be included as part of the feed or feed additive composition. Suitable carriers may be mineral carriers or vegetable carriers. Non-limiting examples of mineral carriers include sepiolite, zeolite (such as clinoptilolite), bentonite, calcium carbonate, dicalcium phosphate, monocalcium phosphate, fine dried salt and diatomaceous earth . Although not inert, clay minerals may be suitable mineral carriers in some circumstances. Non-limiting examples of vegetable carriers include soy bean meal, maize flour, wheat flour and wheat middlings.

[0071] In other aspects, resveratrol and thymol-containing compositions can include a carrier, such as an aqueous liquid carrier, water, a saline, a gel, an inert powder, a zeolite, a cellulosic material, a microcapsule, an alcohol such as ethanol, a hydrocarbon, a polymer, a wax, a fat, an oil, and the like. Some carriers include time release materials where a stilbene-containing composition may be released over a period of hours, or days, or weeks. Suitable paniculate adsorptive carriers for granules are porous types, for example pumice, brick grit, sepiolite or bentonite, and suitable non-sorptive carrier materials are, for example, calcite or sand . Moreover, a large number of pregranulated materials of inorganic or organic nature can be used, such as, in pa rticular, dolomite or comminuted plant residues.

[0072] Solvents suitable for incorporation into compositions according to some aspects of the current invention include but are not limited to alcohols and glycols and also their ethers and esters, such as ethanol, ethylene glycol, strongly polar solvents, or free vegetable oils, such as coconut oil or soya oil; water or solvents derived from natural products. In some aspects, alcohol solvents are preferred for resveratrol and/or thymol .

[0073] Depending on the nature of the active ingredients to be formulated, suitable surface-active compounds are non-ionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties. Surfactants are also to be understood as meaning mixtures of surfactants.

[0074] Additives which aid application of compositions according to some aspects of the invention include natural or synthetic phospholipids from the series of the cephalins and lecithins, for example phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerine, or lysolecithin.

[0075] In some aspects, feed-acceptable acids may be incorporated into the feed, feed additives or drinking water supplements. For example, to enhance shelf life and stability of some compositions described herein, it is advantageous to add up to approximately 5 wt% total of a feed acceptable acid such as citric acid, ta rtaric acid, propionic acid, sorbic acid or lactic acid, or a mixture thereof. In some aspects the feed-acceptable acids also have a positive influence on the efficacy of the product. Other appropriate preservatives may include calcium propionate, potassium sorbate or sodium benzoate.

Formulation examples

[0076] The feed-additives are, when manufactured in their concentrated form on the basis of two or more elements, and both as regards insoluble and soluble variants be blended and packed at facilities using equipment approved of for also blending of vitamin and mineral premixes for animals. Non-limiting examples of feed additives include :

a . blend comprising 7.5 grams resveratrol and 7.5 grams thymol (or thymol-rich extracts), which is included in the feed at 15 grams per ton of feed .

b. premix blend comprising 7.5 grams resveratrol, 7.5 grams thymol (or thymol-rich extracts)and 485 grams clay mineral, which is added to the feed at 500 grams per ton of feed .

c. soluble product containing 7.5 grams resveratrol, 7.5 grams thymol (or thymol-rich extracts)and 135 grams of sorbitol, which is added to the drinking water via a 10% stock solution and added to the drinking water at 1% .

[0077] The following feed-additives, manufactured in their concentrated form on the basis of two or more elements (which may comprise insoluble and soluble variants) were blended and packed at facilities using approved equipment. The resulting compositions have also been blended with vitamins and minerals to produce premixes for animals and birds.

[0078] Non-limiting examples of feed additives include :

a . 10-30 grams 98% pure resveratrol and 5-30 grams thymol per tons of feed was included in standard bird or animal feed at 15-30 grams per ton of feed . b. 5-20 grams 98% pure resveratrol and 5-30 grams thymol was included in the feed at 15 grams per ton of feed .

c. 5-20 grams 98% pure resveratrol and 5-20 grams thymol were blended, and 15g of the blend mixed in to 485 grams clay mineral to provide a premix which is added to the feed at 500 grams per ton of feed .

d . Soluble product containing about 7.5 grams 98% pure resveratrol and 7.5 grams thymol and 135 grams of sorbitol were blended and made up to a 10% stock solution in water, suita ble for addition to the drinking water of birds or animals at 1% .

[0079] In some aspects, the resveratrol and thymol are present in or contacted with the composition as a blend comprising 50wt% resveratrol to 50wt% thymol, or 30wt% resveratrol to 70wt% thymol, or 20wt% resveratrol to 80wt% thymol, or 15wt% resveratrol to 85wt% thymol, or 10wt% resveratrol to 90wt% thymol.

[0080] Preferred inclusion rates of the blend of stilbene (such as resveratrol or a derivative thereof) and thymol include aggregate concentrations of less than 30ppm, 5 to 25ppm, 5 to 20ppm, 5 to 15ppm, or 5 to lOppm.

Methods

[0081] According to some embodiments of the current invention, compositions comprising active ingredients may be applied once per day, once per week, twice per week, once per two weeks, once per month, once per two months, once per three months, or once per lifecycle of the object to which the composition is being applied . Compositions according to aspects of the current invention may be employed as pure active ingredients or, preferably, together with the auxiliaries conventionally used in the art of formulation and are therefore processed in a known manner to give, for example, emulsion concentrates, spreadable pastes, ready-to-spray or ready-to-dilute solutions, dilute emulsions, wettable powders, soluble powders, dusts, granules, or encapsulations, for example in polymeric materials. The methods of application to feed, feed additives, may include spraying, atomizing, dusting, scattering, brushing on, submerging, coating, pouring or rubbing, and the type of composition are selected to suit the intended aims and prevailing circumstances.

[0082] A preferred method of applying a mixture of active ingredients comprising in each case at least one of the active ingredients is to blend the active ingredients into animal feed pellets or kibbles, or to coat the surfaces of pre-formed feed pellets or kibbles.

[0083] Compositions as described herein may be introduced to warm blooded mono- gastric animals and birds at any time of the life cycle (i.e. to subjects of all ages), including but not limited to piglets, lactating sows, grower pigs and finishing pigs. In some aspects, compositions as described herein are preferably used in the finishing of commercially reared animals or birds.

[0084] Generally and without limitation, compositions contemplated herein can be in the form of an aqueous liquid, an oil-based liquid, a concentrated liquid, a gel, a foam, an emulsion, a slurry, a paint, a clear coat, a wax, a block, a pellet, a puck, a granule, a powder, a capsule, a vesicle, an effervescent tablet, slow release tablet, an impregnated dissolvable sheet or film, an impregnated material, and combinations thereof.

[0085] In some embodiments, compositions comprising stilbene (such as resveratrol) or a derivative thereof, and thymol, may be administered directly to the feed, food or drinking water of piglets to be treated from the time they are freshly weened to until they achieve at least 30kg in weight. In other embodiments compositions comprising stilbene (such as resveratrol) and thymol, may be administered directly to the feed, food or drinking water of finisher pigs to be treated from the time they have achieved at least 75kg in weight (typically approximately 4 weeks before slaughter) until they are slaughtered .

[0086] In some embodiments, compositions comprising stilbene (such as resveratrol) and thymol, may be administered directly to the feed, food or drinking water of starter, grower, or finisher broiler chickens. Manufacture, Packaging and Finish

[0087] The compositions disclosed herein may be included in dry or wet feeds, and in the animals' drinking water, respectively.

[0088] In some aspects the resveratrol and thymol may be added separately or together to the drinking water, feed, premixes, supplements or concentrates described herein.

[0089] In some aspects, the thymol present in the resveratrol and thymol containing compositions described herein has natural antimicrobial properties, so may be added with or without sanitisation, pasteurisation, or sterilisation (such as microfiltration or heat treatment). In some aspects, feed suitable for use as described herein may be undergo a sufficient heat treatment during the pelletisation or extrusion process that a subsequent sterilisation step is optional.

[0090] In the aspects relating to administration via the feeds, the additives may in their concentrated forms be included via vitamin and mineral-premixes or concentrates suitable for addition to feeds. Less concentrated formulations comprising the blend of active ingredients and optional additives may be included via base-mixes, or directly into the feeds at a feed-mill, or at a home-mixing farm.

Application and use

[0091] In some aspects, feeding broiler chickens with feed compositions comprising 15 ppm of compositions rich in resveratrol and thymol results in the broilers reaching a higher live-weight.

[0092] In some aspects, feeding broiler chickens with feed compositions in which compositions rich in resveratrol and thymol are included at a combined rate of about 15ppm. This blend was found to result in the broilers reaching a live-weight of 100 grams more than broilers receiving standard feed by day 35 after hatching. Typically, broilers that are slaughtered at 35 days of age will have consumed the following percentage of total feed at each stage of their growth :

Day 0 - 10: 10%

Day 11 - 21 : 28%

Day 22 - 35 : 62

[0093] In some aspects, feeding broiler chickens with feed compositions comprising compositions comprising 15 ppm of extracts rich in resveratrol and thymol results in the broilers reaching a live-weight of 150 grams more than broilers receiving standard feed by day 42 after hatching. Typically, broilers that are slaughtered at 42 days of age will have consumed the following percentage of total feed during each stage of their growth :

Day 0 - 10: 6%

Day 11 - 21 : 19%

Day 22 - 35 : 45% Day 36 - 42 : 30%

[0094] In some aspects, feeding newly weaned piglets with a feed composition comprising 15 ppm of extracts rich in resveratrol and thymol results in the piglets by day 14 after weaning reaching a live weight of 800 grams more than piglets receiving standard feed .

Examples

EXAMPLE 1: Effects of the combination of Resveratrol and Thymol in the growth of porcine livestock.

[0095] 167 newly weaned piglets were divided by weight and sex into two equivalent groups. Each group was divided into two pens with a shared feed dispenser (as shown in Figure 2) . The study was carried out in a farm in Norre Asmindrup, Denmark.

[0096] Five days after arrival, the piglets were vaccinated with Porcilis PCV (Intervet) against porcine circovirus. All the piglets were fed ad libitum (meaning, providing an excess of feed such as the piglets may consume as much as they wish) with the same standard feed and free access to water. The two groups in the trial were :

• Control group (C) : No inclusion of additive

• Trial group (RT) : Inclusion of 15 ppm R (resveratrol) and 15 ppm T (thymol) [0097] The results, after 14 days, are shown in Table 1.

Table 1: Results after 14 days

ADG = Average daily gain, ADFI = Average daily feed intake, FCR = Feed conversion ratio

[0098] No pigs died or were removed during the trial, nor was there any need for medication; all the pigs remained healthy during the trial period .

[0099] The RT group showed a big increase on the ADG (about 28%), leading to a lower FCR (about 17.5%) . The combination of Resveratrol and Thymol in the RT group seems to have beneficial effects on the daily weight gain and the feed conversion ratio. EXAMPLE 2: Effects of the combination of Resveratrol and Thymol compared to Resveratrol alone in the growth of porcine livestock.

[00100] 156 newly weaned piglets were divided by weight and sex into four equivalent groups. Each group was divided into two pens with a shared feed dispenser (Figure 3) . No prescription level of zinc oxide (ZnO) was given to either control nor tria l pigs in this trial setup. The study was carried out in a farm in Ruds Vedby, Denmark.

[00101] All the piglets were fed ad libitum with the same standard feed and free access to water. The three groups in the trial were :

• Control group (C) : No inclusion of additive.

· Trial group (R) : Inclusion of 15 ppm R (resveratrol) .

• Trial group (T) : Inclusion of 15 ppm T (thymol) .

• Trial group (RT) : Inclusion of 15 ppm R (resveratrol) and 15 ppm T (thymol) . The results are shown in Table 2.

Table 2: Results after 14 days

Pigs removed/dead 0 0 0 - -

ADG = Average daily gain, ADFI = Average daily feed intake, FCR = Feed conversion ratio

[00102] No pigs died or were removed during the trial . The R group showed a moderate increase on the ADG (about 4%), leading to a lower FCR (about 4%) . Resveratrol alone did not appear to affect ADFI. The RT group showed a moderate increase on the ADG (about 5%), leading to a lower FCR (about 11%), due to a reduction in ADFI (about 10%) . The combination of Resveratrol and Thymol in the RT group has beneficial effects on the daily weight gain and the feed conversion ratio beyond the effects of Resveratrol alone.

EXAMPLE 3: Effects of the combination of Resveratrol and Thymol compared to Resveratrol alone in the growth of avian livestock.

[00103] A total of 600 Ross 308 broiler newly hatched chicks were divided by weight and sex into three groups with 200 broilers in each group. The birds were allocated to 15 floor pens with 40 birds in each. The size of the pens was 2m², giving a stocking density of 19.3 broilers/m². On day 21, five female broilers from each pen were removed, so the stocking density thereafter was 16.9 broilers/m². The study was carried out in a farm in Llstrasice, Czech Republic.

[00104] All the chicks were fed ad libitum with the same standard feed (see Table 3) and free access to water. The diets were fed in meal form for the first 10 days, and in pelleted form during the rest of the trial . In all experiments included in Example 3, the different trial groups received :

• Control group (C) : No inclusion of additive

· Trial group (R) : Inclusion of 15 ppm R (resveratrol)

• Trial group (RT) : Inclusion of 7.5 ppm R (resveratrol) and 7.5 ppm T (thymol)

[00105] The chicks were vaccinated with IB Primer (a lyophilisate for active immunization against infections bronchitis) on day 1 and day 12.

[00106] The trial was divided into four periods :

Period 1 (from day 0 to day 10) also known as the "Starter" phase of growth.

Period 2 (from day 11 to day 21) also known as the "Grower" phase of growth.

Period 3 (from day 22 to day 35)

Period 4 (from day 36 to day 42)

Periods 3 and 4 together are also known as the "Finisher" phase of growth.

[00107] The temperature was initially set to 32°C on day 1 and was decreased by 0.5°C per day until the temperature reached 21°C. The light cycle was 23 hours of light and 1 hour of dark for the first 7 days, then 18 hours of light and 6 hours of dark from day 8 to day 39, and finally 23 hours of light and 1 hour of dark from day 40 to day 42.

Table 3. Feed composition of basal diets :

[00108] The data was analysed using Rstudio (http ://www. rstudio.com/) . Analysis of Variance (ANOVA) was applied to test the level of significance. A significant difference was defined as P ≤ 0.05, and a trend towards significance was defined as P ≤ 0.1 (Δ) . The different treatments were compared individually against the control group.

[00109] The results are shown in Tables 4 to 9.

[00110] Table 4 shows the average individual feed inta ke of broilers in the different groups in the different periods (days 0- 10, 11-21, 22-35, 36-42 and 0-42) .

Table 4. Average total feed intake (F7) per bird for the groups in different periods from day 0 to 42. Ross broilers¹ Control R RT

FT for % Of FT for % of FT for % of FI for % of

Period period total period total period total period total

(g) FI (g) FI (g) FI (g) FI

Days 0-10 295 6.2 301 6.6 306 6.7 315 7.0

Days 11-21 887 18.6 892 19.5 903 19.6 942 20.8

Days 22-35 2149 45.2 2099 45.8 2065 44.9 2026 44.7

Days 36-42 1426 30.0 1291 28.2 1325 28.8 1248 27.5

Day 0-42 4757 4583 4599 4531

^} The expected average performance for Ross 308 female and male broilers (Aviagen, 2012. Ross 308 Broiler Performance Objectives.). [00111] The birds' feed intake was not significantly different between the treatments in the period from 0 to 42 days or in any of the intermediate periods -although the RT group seems to promote a slightly higher FI that the C and the R groups during the first 21 days while it seems lower during the last 21 days.

[00112] Table 5 shows the average weight gain of broilers in the different groups in the different periods (days 0-10, 11-21, 22-35, 36-42 and 0-42).

Table 5. Average weight gain per bird for the groups in different periods from day 0 to 42.

^} The expected average performance for Ross 308 female and male broilers (Aviagen, 2012. Ross 308 Broiler Performance Objectives).

[00113] For all periods, both R and RT groups displayed a higher average weight gain when compared to the C group. The weight of the chicks was recorded on day 10, day 21, day 35, and day 42. The weights are shown in Table 6.

Table 6. Average live weight of broilers on measurement days. Average Ross Resveratrol Resveratrol + Thymol live standard C R Index Sign. RT Index Sign.²

1

weight 2

R/C RT/C

(kg)

Day 0 0.042 0.036 0.036 100.0 NS 0.036 100.0 NS

Day 10 0.289 0.198 0.213 107.8 _*** 0.220 111.1 _***

Day 21 0.916 0.727 0.774 106.5 _*** 0.765 105.4 _**

Day 35 2.113 1.778 1.978 111.2 _*** 1.898 106.7 _***

Day 42 2.768 2.306 2.486 107.8 _*** 2.452 106.3 _***

^} The expected average performance for Ross 308 female and male broilers (Aviagen, 2012. Ross 308 Broiler Performance Objectives.) .

^} The level of significance of each treatment compared to the control : Δ P≤ 0. 1, * P≤0.05, **P≤0.01, ***P≤0.001, NS - not significant.

[00114] In a ll periods measured, both R ( 15 ppm Resveratrol) and RT (7.5 ppm Resveratrol plus 7.5 ppm Thymol) resulted in a statistically significant increase of the live weight of the broilers.

[00115] Table 7 shows the performance of the broilers in the different trial periods.

Table 7. Growth performance traits in the different periods : 0-10, 11-21, 0 - 21, 22-35, 0 - 35, 36-42 and 0-42.

Day 22-35

ADG (g) 85.6 74.2 85.2 114.8 _** 80.0 107.8 _*

*

ADFI (g) 153.5 149.9 147.5 98.4 NS 144.7 96.5 NS

FCR³ 1.80 2.02 1.73 85.6 _** 1.81 89.6 _*

Day 0-35

ADG (g) 60.4 48.1 53.6 111.4 _** 50.8 105.6 _*

*

ADFI (g) 95.2 91.1 90.7 99.6 NS 91.1 100.0 NS

FCR 1.58 1.90 1.69 88.9 _** 1.80 94.7 NS

Day 36-42

ADG (g) 93.6 74.5 68.0 91.3 NS 79.2 106.3 NS

ADFI (g) 203.7 184.5 189.3 102.6 NS 178.3 96.6 NS

FCR 2.18 2.48 2.78 112.1 NS 2.25 90.7 NS

Day 0-42

ADG (g) 64.9 52.2 55.8 106.8 _* 55.2 10578 _*

ADFI (g) 113.3 105.4 105.8 100.4 NS 104.6 99.2 NS

FCR 1.74 2.03 1.90 93.6 Δ 1.90 93.6 Δ

^} The expected average performance for Ross 308 female and male broilers (Aviagen, 2012. Ross 308 Broiler Performance Objectives) .

²⁾ The level of significance of each treatment compared to the control : Δ P≤ 0.1, * P≤0.05, **P≤0.01, ***P≤0.001, NS - not significant.

³⁾ The FCR va lue for the Ross 308 standard birds does not account for mortality.

ADG = Average daily gain, ADFI = Average daily feed intake, FCR = Feed conversion ratio

[00116] In some of the periods measured, both R ( 15 ppm Resveratrol) and RT (7.5 ppm Resveratrol plus 7.5 ppm Thymol) resulted in a significant increase of the ADG and a decrease on the FCR. This difference was also measurable on the overall 0-42 period .

[00117] In order to analyze the effect of the treatments by sex, the average live weight of the female broilers is shown in Table 8 while the average live weight of the male broilers is shown in Table 9.

Table 8. Live weight of females at the measurement days.

age weight for Ross 308 female broilers (Aviagen, 2012. Ross 308 Broiler Performance Objectives.) .

²⁾ The level of significance of each treatment compared to the control : Δ P≤ 0. 1, * P≤0.05, **P≤0.01, ***P≤0.001, NS - not significant.

[00118] The females in the two treatment groups (R and RT) reached a significantly higher live weight after 10, 21, 35 and 42 days compa red to the control group. After 21 days, 25 females per treatment (5 per pen) were removed from the trial in order to reduce the stocking density.

Table 9. Live weight of males at the measurement days.

^} The expected average weight for Ross 308 male broilers (Aviagen, 2012. Ross 308 Broiler Performance Objectives.) .

²⁾ The level of significance of each treatment compared to the control : Δ P≤ 0. 1, * P≤0.05, **P≤0.01, ***P≤0.001, NS - not significant.

[00119] The ma les in the two treatment groups (R and RT) reached a significantly higher live weight after 10, 35 and 42 days compared to the control group. Additionally, the live weight of the R group was also higher at day 21. However, the effect is more marked when looking at the overall data for both R and RT groups effect on increasing the weight in female broilers than in male broilers. [00120] Without being bound by theory, it is considered by the inventors that this effect, rather than being influenced by the sex of the broilers, is actually the result of the treatments described herein actually being of higher benefit to the smaller animals -and, in the case of broilers, the females are frequently smaller than the males.

[00121] The difference on live weight of males and females at the different measurement days can be seen in the Figure 4.

[00122] Overall, the mixture of 7.5 ppm of Thymol with 7.5 ppm of Resveratrol, performs as well as 15 ppm of Resveratrol in increasing the weight gain and reducing the FCR of broilers. This effect is specially marked in the female or smaller animals, so has the effect of encouraging more homogeneous production i.e. to more consistently high finishing weights regardless of the starting weight. These trials also evidence that 7.5 ppm of Thymol can surprisingly replace 7.5 ppm resveratrol and still retain the full functionality of a higher resveratrol content feed.

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Claims

Claims

1. A composition comprising between 5 and 30ppm of a blend comprising thymol and at least one stilbene, suitable for inclusion within or addition to a feed, feed additives, drinking water or drink supplements of a farmed animal or bird, a companion animal or bird, livestock, a domesticated animal or bird, or an animal or bird bred for competition.

2. A composition according to claim 1 wherein the stilbene is part of a recombinant microorganism capable of producing the stilbene, or as a crude fermentation cake produced during the fermentation of said recombinant microorganism, or as lysed cellular extracts thereof, or as stilbene purified therefrom to 98% purity.

3. A composition according to claim 2, wherein the recombinant microorganism capable of producing the stilbene is Saccharomyces cerevisiae.

4. A composition according to any of the preceding claims, wherein the composition further comprises solvents, surfactants, antioxidants or enzymes, wherein the enzymes are capable of increasing the Feed Conversion Ratio.

5. A method of increasing the Average Daily Gain whilst reducing the Feed Conversion Ratio of animals of birds by:

i. contacting any of the compositions of claims 1 to 4 with the feed, feed additives, drinking water or drink supplements, to form a comestible composition comprising resveratrol and thymol suitable for ingestion by said animal or bird, and

ii. administering the comestible composition of step (i) to the animal or bird at least once per day for at least 10 days.

6. Use of a composition comprising a stilbene and thymol to increase the Average

Daily Gain and/or increase the final live weight of a farmed animal or bird, a companion animal or bird, livestock, a domesticated animal or bird, or an animal or bird bred for competition.

7. A method or use according to any of the preceding claims, wherein the animal or bird is a monogastric animal or bird.

8. A method or use according to any of the preceding claims, wherein the monogastric animal or bird is a chicken, turkey, goose, duck, pigeon, pig, horse, sheep, rabbit, dog or cat.

9. A method or use according to any claims 1 to 8, wherein the monogastric animal is a farmed piglet, a finisher pig, a broiler chicken, or a laying chicken.

10. A method or use according to claim 9 wherein the composition comprising thymol and a stilbene increases the homogeneity of final live weight of a group of piglets, finisher pigs, or broiler chickens compared to a group not having received the composition.

11. A composition, method or use according to any of the preceding claims, wherein the stilbene is resveratrol, pinosylvin, or a methylated or glycosylated derivative thereof.