LIVESTOCK FEED ADDITIVE

Abstract

A livestock feed additive that includes 1,8 cineole, and at least one of naringin or betalain, which may be used to prepare a feed product for poultry, swine or ruminates. Also, a feed product including this livestock feed additive. The livestock feed additive or feed product improves an animal's resistance to heat stress while breeding.

Claims

1-15. (canceled)

16. A livestock feed additive comprising at least 1.5% 1,8 cineole (w/w) and at least one of naringin or betalain.

17. The feed additive of claim 16, which comprises up to 6% 1,8 cineole (w/w).

18. The feed additive of claim 16, which comprises at least 3% naringin (w/w), preferably up to 6% (w/w), and/or at least 0.1% betalain (w/w), preferably up to 3% (w/w).

19. The feed additive of claim 16, which comprises a) 1,8 cineole and naringin at a ratio of at least 0.2:1 (w/w); and/or b) 1,8 cineole and betalain at a ratio of at least 4:1 (w/w).

20. The feed additive of claim 16, wherein said 1,8 cineole is comprised in an essential oil, preferably of lamiaceae plants, preferably of rosemarinus genus.

21. The feed additive of claim 16, wherein said naringin is comprised in citrus plant material, preferably of orange or grapefruit.

22. The feed additive of claim 16, wherein said betalain is comprised in plant material of plants of the Caryophyllales order, preferably of the amaranthaceae family, preferably of Beta vulgaris.

23. The feed additive of claim 16, which comprises a flowable mixture of phytogenic compounds, preferably comprising one or more of essential oils, dried herbs, spices, carbohydrates, bulking or anti-caking agents, or further excipients.

24. The feed additive of claim 16, which further comprises at least one essential oil comprising carvacrol and/or thymol.

25. The feed additive of claim 24, which comprises at least 0.05% carvacrol (w/w) and/or at least 0.05% thymol (w/w).

26. The feed additive of claim 16, which is a storage stable, pelletable dry preparation, with a stability of at least 18 months at room temperature.

27. The feed additive of claim 16, which is provided in a preparation for use in preparing a feed product for poultry, swine or ruminants.

28. A feed product comprising the feed additive composition of claim 16, wherein the feed product comprises a) 1,8 cineole at a concentration of at least 5 mg/kg; and b) naringin at a concentration of at least 10 mg/kg and/or betalain at a concentration of at least 0.3 mg/kg.

Description

FIGURES

[0130] FIG. 1: A, B, C, D, E: Trial I: Life span extension in C. elegans under heat stress by reference substances (Ascorbic acid, Betaine) and compounds “A”, “B”, and “C”.

[0131] FIG. 2: F, G, H, I, J, K: Trial I: Comparative example: Life span extension in C. elegans under heat stress by further substances, frequently recommended to lower negative consequences of heat stress (Piperine, Capsaicin, Gingerol, Eucalyptus oil, Mint oil, Eugenol).

[0132] FIG. 3: Trial II: Dose-dependent induction of heat shock response in CaCo2 cells under heat stress by A) Betaine (reference substance), B), C), D) compounds “A”, “B”, “C”; and E) the combination of compounds “B” and “C” vs. compounds “B” and “C” alone.

[0133] FIG. 4: Trial II: Effect of betalain (compound “C”) at 1.10 mg/L on TEER development of transwell CaCo2 cell cultures exposed to heat stress at 42° C.

[0134] FIG. 5: Trial IV: Effect of betaine (reference substance) and compound “A” on body weight at days 21 and 42 in broilers exposed to heat stress from day 22 to day 42 —A) absolute body weight; B) relative difference of betaine and compound A to the negative control; C) relative difference of compound A to betaine; *a,b,c: different small letters indicate significant differences between means.

[0135] FIG. 6: Trial IV: Effect of betaine (reference substance) and compound “A” on daily weight gain in the periods 0-21d, 22-42d and 0-42d in broilers exposed to heat stress from day 22 to day 42—A) absolute daily weight gain; B) relative difference of betaine and compound “A” to negative control; C) relative difference of compound “A” compared to betaine; *a,b,c: different small letters indicate significant differences between means.

[0136] FIG. 7: Trial IV: Effect of betaine (reference substance) and compound “A” on FCR in the periods 0-21d, 22-42d and 0-42d in broilers exposed to heat stress from day 22 to day 42-A) absolute FCR; B) relative to the negative control; C) relative to the positive control betain; *a,b,c: different small letters indicate significant differences between means

[0137] FIG. 8: Trial V: Effect of betaine (reference substance) and combinations of compound “A” with different concentrations of compound “B” on body weight at days 21 and 42 in broilers exposed to heat stress from day 22 to day 42—A) absolute body weight; B) relative difference of betaine and combinations of compound “A” and “B” to the negative control; C) relative difference of combinations of compound “A” and “B” to betaine; *a,b,c: different small letters indicate significant differences between means.

[0138] FIG. 9: Trial V: Effect of betaine (reference substance) and combinations of compound “A” with different concentrations of compound “B” on daily weight gain in the periods 0-21d, 22-42d and 0-42d in broilers exposed to heat stress from day 22 to day 42—A) absolute daily weight gain; B) relative difference of betaine and combinations of compound “A” and “B” to the negative control; C) relative difference of combinations of compound “A” and “B” to betain; *a,b,c: different small letters indicate significant differences between means.

[0139] FIG. 10: Trial V: Effect of betaine (reference substance) and combinations of compound “A” with different concentrations of compound “B” on FCR in the periods 0-21d, 22-42d and 0-42d in broilers exposed to heat stress from day 22 to day 42—A) absolute FCR; B) relative difference of betaine and combinations of compound “A” and “B” to the negative control; C) relative difference of combinations of compound “A” and “B” to betaine; *a,b,c: different small letters indicate significant differences between means.

[0140] FIG. 11: Trial VI: Effect of betaine (reference substance) and combinations of compound “B” with compound “A” or “C” on body weight at days 21 and 42 in broilers exposed to heat stress from day 22 to day 42—A) absolute body weight; B) relative difference of betaine and combinations of compound “B” with compounds “A” or “C” to the negative control; C) relative difference combinations of compound “B” with compounds “A” or “C” to betaine; *a,b,c: different small letters indicate significant differences between means.

[0141] FIG. 12: Trial VI: Effect of betaine (reference substance) and combinations of compound “B” with compound “A” or “C” on daily weight gain in the periods 0-21d, 22-42d and 0-42d in broilers exposed to heat stress from day 22 to day 42—A) absolute daily weight gain; B) relative difference of betaine and combinations of compound “B” with compounds “A” or “C” to the negative control; C) relative difference combinations of compound “B” with compounds “A” or “C” to betaine; *a,b,c: different small letters indicate significant differences between means.

[0142] FIG. 13: Trial VI: Effect of betaine (reference substance) and combinations of compound “B” with compound “A” or “C” on FCR in the periods 0-21d, 22-42d and 0-42d in broilers exposed to heat stress from day 22 to day 42—A) absolute FCR; B) relative difference of betaine and combinations of compound “B” with compounds “A” or “C” to the negative control; C) relative difference combinations of compound “B” with compounds “A” or “C” to betaine; *a,b,c: different small letters indicate significant differences between means

DETAILED DESCRIPTION OF THE INVENTION

[0143] Specific terms as used throughout the specification have the following meaning.

[0144] The term “antibiotic-free” as used herein with respect to feeding an animal, a diet or a feed product, shall refer to the feeding of an animal with a feed product devoid of antibiotics. Though the animal may have been treated with antibiotics upon veterinary prescription, the regular diet would not contain additional antibiotics as growth enhancer. Thus, accumulation of such harmful substances as antibiotics and the like in persons who have consumed the meat or eggs of poultry is prevented. The compositions as described herein effectively improve the food conversion thereby substituting antibiotics in feed products. Thus, it is possible to increase the productivity of meat of good quality.

[0145] The term “biophysical characteristics” of an animal like livestock or poultry is herein understood as the biotic and abiotic function of an animal, or population, and includes particularly the factors that have an influence in their survival, development and evolution, in particular including factors improving the feed conversion efficiency in animals, e.g. as determined by in vitro or in vivo models. Such factors include e.g. intestinal membrane permeability, nutrient digestibility, (ileal) protein digestibility, nutrient transport, antimicrobial or antioxidative effects.

[0146] The “feed conversion efficiency” (FCE) as herein understood specifically refers to a measure of an animal's efficiency in converting feed mass into increased body mass (e.g. muscle or egg mass). The efficiency may be determined as the feed conversion rate (FCR), which is the mass of the food eaten divided by the body mass gain, all over a specified period. For example, an animal being fed with a feed additive designed for improved feed conversion efficiency may consume less food than an animal that received feed without such feed additive, producing a similar amount of meat. Typically, the feed conversion rate of poultry is in the range of 1.2 to 2.5, and the feed conversion rate of swine is in the range of 1.5 to 3.0, depending on the genetic breed. An improval (i.e., a reduction) in the feed conversion rate or a factor influencing the feed conversion efficiency may be determined, if the feed conversion rate is increased, or the factor is decreased, e.g., by at least 2%, preferably at least 2.5%.

[0147] There are direct factors influencing the feed conversion efficiency, e.g. including nutrient digestibility, (ileal) nutrient or protein digestibility, intestine membrane permeability, nutrient transport systems of the brush border membrane, or indirect factors, such as those improving the health status of the animal and reducing the energy and protein demand for immune reactions, including antimicrobial effects.

[0148] The term “intestine membrane permeability” is herein understood as a factor determining intestinal absorption of nutrients, such as by passing though a cellular membrane of the intestine. Such biophysical characteristics may be determined by the ex vivo model employing epithelial colorectal cells to test a change in permeability upon contact with specific substances. The term “nutrient transport systems” is herein understood as an active transport of nutrients, including e. g. glucose, peptides and amino acids, from the lumen of the small intestine across the brush border membrane into the enterocytes. Such nutrient transport systems are specific enzymes, including e.g. the sodium-dependent glucose transporter (SGLT1) and small peptide and amino acid transporter (PEPT1). Such biophysical characteristics may be determined by the ex vivo model employing epithelial colorectal cells to test a change in gene expression of the SGLT1 transporter enzyme upon contact with specific substances. The term “antimicrobial effects” as understood herein refers to the possible bacteriostatic effects of bacteria that are possibly pathogenic to monogastric animals, including poultry. Such biophysical characteristics may be determined by the ex vivo model employing bacterial cells to test a change in bacterial cell growth upon contact with specific substances.

[0149] The term “component” with regard to a feed additive is herein understood as a part of a composition, which may include one or more further compounds, components and excipients. The feed additive described herein is a composition specifically comprising at least an (essential) oil component comprising 1,8-cineole as a compound or an active compound, and at least one further component including the compounds (or active compounds) naringin and/or betalain, but may further include biological components, primarily phytogenic components and further excipients, including e.g., anorganic excipients.

[0150] Specifically, the 1,8-cineole component is an essential oil comprising 1,8-cineole in a specified amount. Exemplary plant materials used as a source of the 1,8-cineole compound as used herein, are any one or more of: thyme, rosemary, or eucalyptus

[0151] The oil component as described herein is specifically obtained by extracting an oil from a plant material e.g., by cold extraction or hot extraction techniques, employing aqueous and optionally an oily phase, so to obtain a w/o emulsion of the oil.

[0152] According to a specific example, the 1,8-cineole component is produced as follows: In a mixer employing spray technology, the essential oil component is sprayed on silica in any one of the ratios of 30:70 or 40:60 or 50:50 or 60:40 (w/w) and thoroughly mixed e.g., for at least 30 minutes.

[0153] Essential oils are typically extracted from plant materials through removal methods that are suited to the specific plant part containing the oils. Popular extraction methods include: steam distillation, solvent extraction, CO.sub.2 extraction, maceration, enfleurage, cold press extraction, and water distillation.

[0154] According to a specific aspect, the production method employs the preparation of an o/w emulsion including the oil, and optionally a polymer, solubilizer and/or detergent, followed by spray-drying at controlled temperature to obtain a flowable dry oil component.

[0155] Essential oils can be provided in the microencapsulated form. Typically, the microencapsulation of an essential oil provides for its isolation from its surroundings e.g., isolating an oils from deteriorating effects of further substances in the aqueous phase e.g., in the gastrointestinal environment, retarding evaporation of the volatile oil, protection against friction and evaporation due to humidity and high temperature during the feed processing (pelleting) or improving the handling properties of the sticky material. In addition, the rate may be effectively controlled at which the oil leaves the microcapsule, as in the sustained or controlled release of the oil in the gastrointestinal tract, aiming at providing an effective amount of the active compounds in the intestines. Thereby the release of the oil and other components may be achieved in a synchronized way to achieve synergistic effects in vivo.

[0156] A wide range of materials and methods may be used for encapsulation to create the degree of durability and method of release suitable to the intended use. Non-limiting polymeric exemplary materials suitable for use with the microencapsulation of oil may include natural polymers of eukaryotic or prokaryotic origin, e.g. including starch hydrolysates, like dextrins, modified starch, Gummi Arabicum, alginates, cellulose derivatives, like hydroxypropylcellulose, Na-carboxycellulose, methylcellulose, ethylcellulose, animal or plant proteins or protein hydrolysates, like gelatin, collagen, egg yolk, wheat protein, casein, milk protein, soy protein, pea protein, or mixtures thereof. Various physical and chemical methods of microencapsulation may be used, depending on the oil and the desired polymeric shell coating to be used. Conveniently, the essential oil is encapsulated by dehydrating an o/w emulsion by any suitable means, including spray drying, freeze drying, fluid bed drying, tray drying, adsorption, and combinations thereof. Preferably, the microencapsulated oil is produced by spray-drying an emulsion having an aqueous phase as defined above containing a polymeric encapsulation agent. The spray-drying parameters are dictated by the physical characteristics desired in the final microencapsulated oil. Such physical parameters include particle size, flow and water content.

[0157] The oil component typically has good flowability and can be distributed homogeneously throughout the composition. Conveniently, the oil component is a powder. Any suitable additive may be added to the oil e.g., a flow agent such as silicon dioxide, to increase the flowability of the oil.

[0158] The naringin component and/or the betalain component as described herein is specifically a plant material comprising the compounds naringin and betalain, respectively, in the specified amount. Specifically, the plant material is provided as particulate dry powder.

[0159] The term “powder” as used herein is specifically understood as a flowable material comprising a plurality of particles. The particles may have a smooth outer surface and/or a flattened morphology. In certain embodiments, the particulate plant material is a beige to dark brown powder with a characteristic smell.

[0160] The particulate plant material used herein is specifically obtained by grinding dry plant material to obtain a specific particle size, e.g. corresponding to flowable material, e.g., SiO.sub.2 (powder), a flour and/or semolina.

[0161] The particles may have an average largest dimension of 250-500 μm. The typical particulate material has a particle size of min 95% below 500 μm. Preferably, the particulate plant material has a mean particle size of 100-350 μm.

[0162] The plant powder material can be derived from various portions of the plant, specifically fibrous plant materials may be used, e.g. including bark, roots, stalks, stems, leaves, fruits, peels, flowers, seeds, or combinations thereof.

[0163] Exemplary plant materials used as a source of the naringin component as used herein, are plant materials of plants of the family Rutaceae, such as citrus fruits, e.g., citrus fruit peels or pomace. Specifically, naringin can be extracted from such plant products and used as feed additive component, or the plant products can be used as such.

[0164] Exemplary plant materials used as a source of the betalain component as used herein, are plant materials from plants of the Caryophyllales, such as fruits, leaves, stems, and roots of plants e.g., beets. Specifically, betalains can be extracted from such plant products and used as feed additive component, or the plant products can be used as such.

[0165] According to a specific example, the naringin component and/or betalain component is admixed in a feed mixer to the essential oil component on silica and optionally with other excipients.

[0166] The moisture content of the feed additive or feed product described herein is typically less than 12%, preferably less than 8%.

[0167] The term “excipients” as used herein shall refer to additive components commonly used in feed compositions, e.g. phytogenic and/or inorganic feed additive components. Specifically, the feed additive and its additive components are understood as products used in animal nutrition for purposes of improving the quality of feed, or to improve the animals' performance and health. Feed additives are typically carefully selected which have no harmful effects, on human and animal health and on the environment.

[0168] In this regard, the term “feed” typically refers to any mixture of animal feed ingredients providing energy and nutrient requirements, e.g., protein, fat, carbohydrates, minerals and micronutrients. For example, the daily intake of poultry feed is typically between 50-250 g/head and day for a broiler for fattening. According to another example, the daily intake of swine feed, according to the physiological status, is typically between 50-1500 g/head and day. According to another example, the daily intake of ruminant feed, according to the species and the physiological status, is typically between 150-35000 g/head and day.

[0169] The feed additive described herein may specifically include excipients, such as further essential oils, dried herbs, spices and further excipients, including colors, flavoring substances, preservatives, or any substance needed to formulate the composition to the desired form, such as bulking, anti-caking agents, diluents, fillers, binders, disintegrants, adsorbents, or granulating agents. Typical excipients are for example rosemary leafs, juniper berries, psyllium hulls, wheat bran, limestone, SiO.sub.2 or bentonites.

[0170] The term “flowable” as used herein shall specifically refer to a mixture of components in the powder form, including e.g. particulate material, which may flow. For example, a flowable mixture may flow through a funnel or hopper into another container under the influence of gravity. In the present invention, a flowable powder mixture is suitable for use with a device for mixture with feed material and pelleting. The term “flowable” is well known in the food and feed industry and has a clear meaning to the person skilled in the art.

[0171] A flowable mixture has several advantages in use, particularly on an industrial scale. The mixture may be handled, stored and transported relatively easily and energy-efficiently, as compared with, for example, solid materials that are not flowable. This advantage is particularly important in combination with the ability to avoid a liquefying step in the pelleting process.

[0172] The flowable mixture as used herein specifically comprises at least an (essential) oil component comprising 1,8-cineole, and at least one further component including naringin or betalain, and optionally other components and excipients, wherein the components are all mixed together without the inclusion of any substantial amount of liquid to form a dry mixture, which is optionally ground into a flowable, preferably pelletable powder.

[0173] Specifically, a 1,8-cineole component, fed in a mixture with at least one further component including naringin or betalain, provide for a phytogenic system that inherently improves the sustained release properties of the active compounds in vivo, to improve the biophysical characteristics as required. Preferably, any one or more or all of the 1,8-cineole component, the naringin component or the betalain component do not dissolve or dissolve only poorly in the stomach. The components of the feed additive composition can be sufficiently provided to the intestine and/or colon, so that the active compounds may concomitantly act on the biophysical characteristics.

[0174] Thus, it is possible to obtain synergistic effects in the intestines when using the feed additive or feed product described herein.

[0175] The term “poultry” as used herein shall specifically refer to domesticated fowl kept primarily for meat (broilers) and eggs (layers); including birds of the order Galliformes, e.g., the chicken, turkey, guinea fowl, pheasant, quail, and peacock; and Anserigormes (swimming birds,) e.g., the duck and goose.

[0176] The poultry may be fed with the same feed composition throughout the growth period, or at least during a period of at least 3 weeks, preferably at least 4 weeks to improve the efficiency of feed use or feeding efficiency, e.g. output per unit of feed. Significant differences may be found between control and experimental treatments in final body weight and weight gain at the entire growth periods till 28 days after hatching. While the feed intake may be almost equal between treatments, the feed efficiency, i.e. g feed/g weight gain may be significantly better for poultry and specifically chicken fed with the feed additive composition of the invention.

[0177] The term “swine” as used herein shall refer to stout-bodied short-legged omnivorous artiodactyl mammals (family Suidae) with a thick bristly skin and a long flexible snout; especially the domestic pig and the wild boar.

[0178] The feed described herein as used for poultry or pigs is specifically pelleted or mash.

[0179] Exemplary compositions of a feed additive composition described herein for use in breeding poultry or pigs are described in Example 1.

[0180] The term “ruminants” as used herein shall refer to ruminating mammals including e.g., cattle, goats, sheep, giraffes, yaks, deer, antelope, and some macropods (kangaroos).

[0181] The feed described herein as used for ruminants, such as cattle, specifically is pelleted, or a feed additive admixed to one or more of the following: grass-silage, corn-silage, soy bean meal, or bruised grain.

[0182] Exemplary compositions of a feed additive composition described herein for use in breeding ruminants, such as cattle, are described in Example 1.

[0183] Further examples provided herein are directed to the analytical methods to determine the active substances 1,8-cineole, naringin and betalain in a feed additive composition.

[0184] Further examples provided herein are directed to in vivo and ex vivo testing of the effect of the feed additive, such as [0185] i. In vivo testing the effect on life span extension in C. elegans worms which are exposed to heat stress, [0186] ii. Ex vivo testing the effect heat shock protein induction in CaCo2 cells; [0187] iii. Ex vivo intestinal barrier function by transepithelial electrical resistance (TEER) in transwell CaCo2 cells; [0188] iv. In vivo validation of beneficial effects of naringin on growth performance in broilers under heat stress and evaluation of a beneficial dose level; [0189] v. In vivo validation of beneficial effects of combinations of naringin with different 1,8-cineole concentrations on growth performance, heat shock protein response and intestinal barrier integrity in broilers under heat stress; and [0190] vi. In vivo validation of beneficial effects of the combinations of naringin with 1,8-cineole, or 1,8-cineole with betalain.

[0191] The foregoing description will be more fully understood with reference to the following examples. Such examples are, however, merely representative of methods of practicing one or more embodiments of the present invention and should not be read as limiting the scope of invention.

EXAMPLES

Example 1: Exemplary Feed Additives Named “New Feed Additive Supporting Animal Welfare Under Heat Stress Conditions”

[0192] Exemplary compositions contain the following ingredients, admixed to a dry flowable mixture. Whereas the oil component is a specific lamiaceae oil or a mixture of lamiaceae oils (e.g., of Oregano, and/or Rosemary, and/or Thyme), standardized to the concentration of 1,8 cineole, the flavonoid component derives from citrusflavonoids, with standardized Naringin concentration, and the betalain component derives from Beetroot powder, with standardized betalain concentration.

TABLE-US-00001 TABLE 1 Formula I; Composition of a new feed additive formulation for Poultry (Broilers, Layers, Turkey) and Pigs Ingredients Content (%, w/w) Lamiaceae oil mixture, in total ≥4.00 (4.00-16.0) 1,8-cineole ≥1.50 (1.50-6.00) Citrus flavonoids, in total ≥5.50 (5.50-11.0) Naringin ≥3.00 (3.00-6.00) Excipients To 100%

[0193] The exemplary feed additive formula I contains 1.50% 1,8-cineole (w/w); and an effective ratio of 0.65:1 (w/w 1,8-cineol per naringin).

TABLE-US-00002 TABLE 2 Formula II; Composition of a new feed additive formu- lation for Poultry (Broilers, Layers, Turkey) and Pigs Ingredients Content (%, w/w) Lamiaceae oil mixture, in total ≥4.00 (4.00-16.0) 1,8-cineole ≥1.50 (1.50-6.00) Beetroot powder, in total ≥10.0 Betalain ≥0.125 (0.125-0.25) Excipients To 100%

[0194] The exemplary feed additive formula II contains 1.50% 1,8-cineole (w/w); and an effective ratio of 22:1(w/w 1,8-cineol component per betalain).

TABLE-US-00003 TABLE 3 Formula III; Composition of a new feed additive formulation for Ruminants Ingredients Content (%, w/w) Lamiaceae oil mixture, in total ≥1.00 (1.00-4.00) 1,8-cineole ≥0.375 (0.375-1.50) Citrus flavonoids, in total ≥1.30 (1.30-2.60) Naringin ≥0.75 (0.75-3.00) Glycerol (1,2,3-Propanetriol) ≥50.0 Excipients To 100%

[0195] The exemplary feed additive formula III contains 0,375% 1,8-cineole (w/w); and an effective ratio of 0.5:1 (w/w 1,8-cineol per naringin).

TABLE-US-00004 TABLE 4 Formula IV; Composition of a new feed additive formulation for Ruminants Ingredients Content (%, w/w) Lamiaceae oil mixture, in total ≥1.00 (1.00-4.00) 1,8-cineole ≥0.375 (0.375-1.50) Beetroot powder, in total ≥5.00 Betalain ≥0.033 (0.033-0.066) Glycerol (1,2,3-Propanetriol) ≥50.0 Excipients To 100%

[0196] The exemplary feed additive formula IV contains 0,375% 1,8-cineole (w/w); and an effective ratio of 12:1 (w/w 1,8-cineol per betalain)

[0197] The feed additive called “New Feed Additive Supporting Animal Welfare Under Heat Stress Conditions” contains

[0198] a) naringin at an effective ratio within the range of 0.2:1-1.5:1, in particular 0.65:1 (w/w, 1,8-cineole per naringin); and/or

[0199] b) betalain at an effective ratio within the range of 4:1-64:1, in particular 22:1 (w/w, 1,8-cineole per betalain).

[0200] The formula is tested in the test systems described herein to show a synergistic effect of combinations of the individual compounds.

[0201] For example, the synergistic effect is defined as higher growth performing efficacy compared to a standard reference additive, when used at a recommended dose level.

[0202] According to the example, the dosage of the “New Feed Additive Supporting Animal Welfare Under Heat Stress Conditions” is described in a range of 200 to 400 mg/kg or in a range of 200 to 400 mg/kg of complete feed.

[0203] Moreover, in the in vivo test systems (animal trials) a usually recommended dose of betaine (500 mg/kg of complete feed) is carried along as a positive control group. This approach serves to prove the higher efficacy of the “New Feed Additive Supporting Animal Welfare Under Heat Stress Conditions” compared to a frequently applied commercial solution.

Example 2: Description of Analytical Methods and Trial Methodologies

[0204] Description of the Analytical Active Substance Evidence [0205] 1. 1,8 Cineole (compound “A”) [0206] 2. Naringin (compound “B”) [0207] 3. Betalain (compound “C”) Description of trial methodology and results [0208] 4. Trial I: Testing of life span extension in C. elegans worms [0209] 5. Trial II: In vitro testing of heat shock protein induction in CaCo2 cells [0210] 6. Trial III: In vitro testing of intestinal barrier function by transepithelial electrical resistance (TEER) in transwell CaCo2 cells [0211] 7. Trial IV: In vivo validation of beneficial effects of the naringin (Compound “A”) on growth performance in broilers under heat stress and evaluation of the optimum dose level [0212] 8. Trial V: In vivo validation of beneficial effects of combinations of naringin (Compound “B”) with different 1,8-cineole concentrations (Compound “A”) on growth performance, heat shock protein response and intestinal barrier integrity in broilers under heat stress [0213] 9. Trial VI: In vivo validation of beneficial effects of the combinations of naringin with 1,8-cineole (AB), or 1,8-cineole with betalain (AC).

DESCRIPTION

[0214] 1,8-CINEOL Analysis

[0215] GC MS

[0216] Gas chromatography analysis was carried out using a Focus GC coupled to a DSQII MS purchased from Thermo (Waltham, Mass., USA). The injection (injection volume 1 μl) was performed in the splitless mode at the injection temperature of 240° C. Separation was carried out on an Rxi-5 ms fused silica column (30 m×0.25 mm i.d.; 0.25 μm film thickness) from Restek (Bad Homburg, Germany) at constant flow. The oven program started at 50° C. and the temperature was increased in the first step to 190° C. at 5° C. min-1 and in the second step to 300° C. (held for 5 min) at 30° C. min-1. Helium (4.6) was used as carrier gas with a column flow rate of 1.0 ml min-1.

[0217] The mass spectrometer was operated in the selected ion monitoring (SIM) mode with the selected ions m/z 154, m/z 139, m/z 111, m/z 108. The ion source temperature was adjusted to 240° C. and transfer line temperature configured to 300° C.

[0218] Naringin Analysis

[0219] LC MS

[0220] The experiment was performed on an Agilent Series 1100 HPLC system. The separation column was a Poroshell 120 EC-C18 Eclipse (50×3 mm ID, 2.7 μm particle size) obtained from Agilent. The mobile phase consisted of a gradient of water and acetonitrile, both with 0.1% formic acid. Initial conditions were 15% acetonitrile for 5 minutes, then the gradient was run to 20% acetonitrile within 5 minutes. 100% acetonitrile within the next 5 minutes, and finally to 15% acetonitrile kept for another 5 min. The total chromatographic run time was 20 minutes. The flow rate was set to 0.8 mL min.sup.−1.

[0221] MS detection was carried out on an Agilent 6520 QTOF in the negative ion mode. The following ion source conditions were used: drying gas temperature 350° C., drying gas flow 10.5 L min-1, nebulizer pressure 45 psi, fragmentor voltage 125 V and capillary voltage 3750 V.

[0222] BETALAIN Analysis

[0223] LC MS (Pires Goncalves et al. 2012)

[0224] Reversed-phase chromatography was performed in a Waters (Milford, Mass.) 600 system equipped with a UV-Vis detector (dual-wavelength, Waters 2489) and a Jupiter-15 (300 Å, 15 μm, 250×21.2 mm, Phenomenex, Torrance, Calif.) C.sub.18 column. Gradients were formed between two helium-degassed solvents: solvent A: water with 1% v/v HOAc; linear gradient from 5% to 20% B: in 60 min at 25° C., flow rate: 10 mL/min.

[0225] A Bruker Daltonics Esquire 3000 Plus was used for the ESI-MS analyses. The vaporizer temperature was 325° C. and the voltage was maintained at 4.0 kV. The sheath gas was nitrogen, operated at a pressure of 26 psi (6.0 L/min). Compounds were ionized in the positive mode.

[0226] Description of Trial Methodologies

[0227] Trial I: Testing of Life Span Extension in C. elegans Worms

[0228] For this investigation a specific assay with C. elegans has been developed. Under regular temperature conditions C. elegans has a life span of about 14 days, including all larval stages. Worms reach their adult state after 3 to 4 days. Increasing temperature to 37° C. reduces the remaining life span of C. elegans drastically to 10 to 15 h. Consequently, in this assay the elongation of life span by control substances (Ascorbic acid and betaine) and numerous other phytogenic compounds (Mint oil, Eucalyptus oil), as well as some of their purified active constituents (Piperin, Capsaicin, Gingerol, Eugenol) has been screened. All tested substances have been used in concentrations (mg/L) which could be transferred later to dietary concentrations (mg/kg) of these substances in complete diets.

TABLE-US-00005 TABLE 5 Trial I: Concentration of compounds A, B and C and other active ingredients Equivalent concen- Concen- tration in feed tration (mg/kg) or water Ingredients (mg/L) (mg/L) Ascorbic acid 100 100 (water) Betaine 500 500 (feed) Citrus flavonoids 250 250 (feed) thereof Naringin (B) 50 50 (feed) Lamiaceae oil mixture 25 25 (feed) thereof 1,8-cineole (A) 7.5 7.5 (feed) Beetroot powder 250 250 (feed) thereof Betalain (C) 0.6875 0.6875 (feed) Mint oil 50 50 (feed) Eucalyptus oil 50 50 (feed) Eugenol 50 50 (feed) Pepper oleoresin 50 50 (feed) Capsicum oleoresin 50 50 (feed) Ginger oleoresin 50 50 (feed)

[0229] C. elegans Maintenance:

[0230] C. elegans wildtype strain N2, variation Bristol were obtained from the C. elegans Genetics Center, CGC (University of Minnesota, Minn., USA). Nematodes were maintained on nematode growth medium (NGM) agar plates seeded with E. coli OP50 at 20° C. according to standard protocols (Brenner S, 1974, The genetics of Caenorhabditis elegans. Methods such as freezing nematodes and obtaining synchronous populations using a bleaching method with hypochlorite treatment of egg-laying adults were also performed according to standard protocols (Stiernagle T, 2006; Maintenance of C. elegans. WormBook 1-11).

[0231] Treatment of Nematodes with Reference Substances (Ascorbic Acid, Betaine) and the Main Ingredients of the “New Feed Additive Supporting Animal Welfare Under Heat Stress Conditions”

[0232] Synchronous nematodes were raised in liquid culture using NGM liquid and packed E. coli HT115 according to Stiernagle. Carbenicillin was added to the NGM liquid in order to inactivate E. coli. A volume of 56 μl of NGM liquid was dispensed in each well of a 96-well microplate, to which 10 μl M9-buffer containing 10 synchronized L1 larvae were added. L1 larvae were maintained shaking at 20° C. and reached the adult stage within 3 days. All control substance (ascorbic acid, betaine) and test substances (Compounds “A” and “B”) were prepared as stock solutions in M9-buffer and sonicated for 5 min. Stock solutions (10-fold) had the following concentrations (Ascorbic acid 500/1000 mg/L, Betaine 5000 mg/L, Compound “A” 75 mg/L, Compound “B” 500 mg/L, Compound “C” 6.88 mg/L). 7 μl of each extract stock solution was added to the incubation medium to reach a final concentration of 100 mg/L (Ascorbic acid), 500 mg/L (Betaine), 7.5 mg/L (Compound “A”), 50 mg/L (Compound “B”) and 0,6875 mg/L (Compound “C”). Control nematodes were always treated with identical volumes of M9-buffer instead.

[0233] Determination of Survival Under Heat Stress

[0234] After incubation of young adult N2 nematodes for 48 h at 20° C. in the presence or absence of the control additives (ascorbic acid, betaine) and the New Feed Additive Supporting Animal Welfare Under Heat Stress Conditions components “A” and “B”

[0235] Survival was determined using a microplate thermotolerance assay as described (Gill M S, Olsen A, Sampayo J N, Lightgow G J, 2003; Free Radic Biol Med 35:558-565). In brief, nematodes were washed off the wells with M9-buffer/Tween®20 (1% v/v) into 15 ml tubes followed by additional three washing steps. In each well of a black 384-well low-volume microtitre plate 6.5 μl M9-buffer/Tween®20 (1% v/v) solution was added. Subsequently, one nematode was dispensed in 1 μl M9 buffer under a stereo-microscope (Breukhoven Microscope Systems) into each well and mixed with 7.5 μl SYTOX green to reach a final concentration of 1 μM. To prevent water evaporation the plates were sealed with Rotilab sealing film and covered with a lid. Heat shock (37° C.) was induced and fluorescence was measured with a Fluoroskan Ascent microtiter plate reader (Thermo Labsystems, Bonn, Germany) every 30 min. To detect SYTOX green fluorescence, excitation wavelength was set to 485 nm and emission was measured at 538 nm.

[0236] To determine the survival time for each nematode an individual fluorescence curve was generated. Time of death was defined as one hour after an increase in fluorescence over the baseline level was observed and was verified by touch provocation first. From the individual times of death Kaplan-Meier survival curves were drawn.

[0237] Results: Testing of Life Span Extension in C. elegans Worms (Trial I)

[0238] To investigate heat stress protective effects of both reference substances (Ascorbic acid, betaine) and of phytogenic compounds, including compound “A″, “B” and “C” of the “New Feed Additive Supporting Animal Welfare Under Heat Stress Conditions”, life span extension in C. elegans was tested under heat stress conditions (FIG. 1). Tested concentrations of the reference substances and of the phytogenic substances were chosen to reflect final dietary concentrations in animal diets (e.g. the reference substance betaine is used in animal diets in concentrations from 250 to 1000 mg/kg of diet; in the C. elegans model betaine was tested at a level of 500 mg/L=500 mg/kg=500 mg/kg). Both reference substances (ascorbic acid and betaine) increased life span for 0.5 to 1.5 hours. Compounds “A″ (FIG. 1D), “B” (FIG. 1C) and “C” (FIG. 1E) at the tested concentrations increased life span by 0,5 to 1,5 h.

[0239] Interestingly, comparative examples using pungent substances and cooling essential oils, frequently recommended against negative consequences of heat stress, and partially used in commonly available feed additive products, showed not benefits on life span extension (FIGS. 2 F, G, H, I, J, K).

[0240] Trial II: In Vitro Testing of Heat Shock Protein Induction in CaCo2 Cells

[0241] For this investigation a specific assay with CaCo2 monolayer cells has been developed. CaCo2 cells are regularly incubated at 37° C. Increasing the temperature to 41° C. causes a heat shock response in these cells (induction of heat shock proteins) to counteract cellular damage and to initiate repair mechanisms. Consequently, in this assay the potential of betaine (reference substance) and of compounds “A” and “B” has been studied in a dose-dependent manner to support and increase natural heat shock response.

[0242] Study with CaCo2 Cells

[0243] Materials

[0244] MEM with Earle's salts, fetal bovine serum (FBS), penicillin/streptomycin and trypsin-EDTA were purchased from Biochrom GmbH (Berlin, Germany). Entero-STIM Intestinal Epithelium Differentiation Medium and MITO+Serum Extender were obtained from Corning (Wiesbaden, Germany) and cell culture plates were purchased form Greiner Bio-One International GmbH (Kremsmūnster, Austria). RNeasy Mini Kit was obtained from Quiagen (Hilden, Germany), i Script cDNA Synthese Kit and iQ SYBR Green Supermixture from Bio-Rad (Munich, Germany) and oligo dT-primers from Eurofins Genomics (Ebersberg, Germany).

[0245] Cell Culture and Differentiation of CaCo2 Cells

[0246] Human CaCo2 cells (DSMZ, Braunschweig, Germany) were maintained in MEM with Earle's salts supplemented with 10% FBS and 100 U/mL penicillin/100 μg/mL streptomycin and grown at 37° C. in a humidified atmosphere 95%) with 5% CO.sub.2. The cells were seeded in 12-well plates at 1.2×10.sup.6 cells per well to reach confluency the next day. The cells were further maintained in Entero-STIM Intestinal Epithelium Differentiation Medium supplemented with 100 U/mL penicillin/100 μg/mL streptomycin and 0.1% MITO+Serum Extender and the medium was changed daily. The experiment was carried out on day 5 when the cells were completely differentiated.

[0247] Induction of Heat Stress

[0248] To analyze the influence of betaine (reference substance) and compounds “A” “B” and “C” on the expression of the heat shock protein HSP70, the substances were added to the cells on day 4 and the cells were incubated with the extract overnight for 15 hours. The test substances were dissolved in complete differentiation medium and diluted to the following final concentrations: Betaine (250/500/1000/1500 mg/L); Compound “A”, (7,5, 15, 30 and 60 mg 1,8-cineole/L); Compound “B”, (6, 12, 18, 24, 30 and 60 mg Naringin/L); Compound “C”, (0,55, 1,10, 2,20 mg Betalain/L), respectively. To induce heat stress, the samples were incubated at 41° C. for 1 hour, while control samples were incubated at 37° C. for the same time.

[0249] Detection of HSP70 mRNA Expression by Real-Time PCR

[0250] The mRNA expression of HSP70 was measured quantitatively by real-time PCR (C1000 Thermal Cycler and CFX96 Real-Time System, Bio-Rad, Munich, Germany). Total RNA was isolated with RNeasy mini kit, followed by the transcription of 50 ng of total RNA into cDNA with the i Script cDNA Synthesis Kit (end volume: 20 μL) and the real-time PCR with the iQ SYBR Green Supermix according to manufacturer's instructions. Briefly, for real-time PCR 2 μL of cDNA was added to 18 μL master mixture (10 μL iQ SYBR Green supermix (2×), 2 μL primer [3 pmol/μL], 6 μL nuclease-free water). DNA denaturation and polymerase activation for 3 min at 95° C. was followed by 40 PCR cycles. One amplification cycle is divided into three parts: denaturation at 95° C. for 15 sec., annealing and extension at 60° C. for 60 sec. followed by a plate read after each cycle. Finally, a melt curve analysis was done by gradually increasing the temperature to 95° C., to exclude the formation of primer-dimers. The detected c.sub.T values were used for the calculation of the relative mRNA expression levels via the 2.sup.−ΔΔc.sub.T method (ZITAT LIVAK). Differences in cDNA amounts were normalized to the expression of β-actin mRNA. Statistical analysis was carried out in Graphpad Prism (version 6.02) using unpaired t test. The following primers were used for the amplification: β-actin forward: 5′-GCG GGA AAT CGT GCG TGA CAT T-3′ (SEQ ID NO:1); β-actin reverse: 5′-GAT GGA GTT GAA GGT AGT TTC GTG-3′(SEQ ID NO:2); HSP70 forward: 5′-CTA GCC TGA GGA GCT GCT GCG ACA G-3′ (SEQ ID NO:3); HSP70 reverse: 5′-GTT CCC TGC TCT CTG TCG GCT CGG CT-3′ (SEQ ID NO:4).

[0251] Results of In Vitro Testing of Heat Shock Protein Induction in CaCo2 Cells (Trial II)

[0252] To investigate the benefits of compounds “A”, “B” and “C” on the additional induction of heat shock response differentiated CaCo2 cells were exposed to increasing levels of compounds “A”, “B” and “C”, representing the above mentioned dose range applied in animal diets (FIGS. 3 B, C, D). As a reference substance, the frequently used anti heat stress feed additive betaine was tested accordingly (FIG. 3 A). Betaine showed neither a clear dose-response on additional heat shock protein 70 induction over the control. Whereas 100, 250 and 1000 mg/L betaine even decreased heat shock protein 70 induction compared to untreated control cells (−7 to −19%), 500 and 2000 mg/L resulted in a non-significant increase in heat shock protein 70 response (+10 to +12%) (FIG. 3 A). In contrast, all three compounds of the “New Feed Additive Supporting Animal Welfare Under Heat Stress Conditions” resulted in clear benefits over the control and indicated either a dose response or exceeding of optimum concentrations (FIGS. 3 B, C, D). For compound “B” already the lowest Naringin concentration tested, resulted in a clear but not significant increase of HSP 70 induction compared to untreated control cells (+16%). A significant increase in HSP 70 induction nearly at the same level could be observed for Naringin concentrations between 12 to 30 mg/L of compound “B″ (+24 to +27%). In contrast, 60 mg Naringin/L led to an adverse effect (−23%), indicating exceeding of the optimum dose range. Similarly already 7.5 mg 1,8 Cineole/L from compound “A″ resulted in a 15% increase in HSP 70 induction compared to control cells. 15 and 30 mg 1,8-Cineole/L from compound “A″ increased HSP 70 gene expression by 22 and 24%, respectively. The highest dose tested (45 mg 1,8-Cineole/L) increased HSP 70 expression even by 44%. However, due to price reasons of raw materials this concentration cannot be considered in a product formulation for a “New Feed Additive Supporting Animal Welfare Under Heat Stress Conditions”. The highest concentration tested was in the range, reported from Turk et al. (2016). To test if there are additional effects of the single compounds, as an example HSP 70 induction by compound“A” and “C” alone, as well as of the combination of both substances is given in FIG. 3 E. At the tested levels of substance “A” and “C” alone with 20.8% and 21,1% a nearly significant or significant increase of HSP 70 expression could be achieved, which was at a comparable level, as reported in FIGS. 3 C and D. The combination of both substances nearly doubled HSP 70 induction to 40,2% and clearly showed the improved effect of both substances (FIG. 3 E).

[0253] Trial III: In Vitro Testing of Intestinal Barrier Function by Transepithelial Electrical Resistance (TEER) in Transwell CaCo2 Cells

[0254] In order to test, if the compounds “A”, “B”, and “C” help to support intestinal barrier integrity under heat stress conditions experiments with transwell CaCo2 cell layers have been performed. In a sterile, biosafety level 2 cell culture hood 0.8 mm pore PET transwell inserts were placed into a 24-well plate (4 wells per condition) All materials entering were sterilized with ethanol before.

[0255] Cell culture medium containing DMEM medium supplemented with 4.5 g/L glucose, 15% fetal bovine serum, and 1% Pen Strep, was prepared and heated to 37° C. in a water bath.

[0256] 200-400 μl of the cell solution of human epithelial colorectal adenocarcinoma (Caco-2) cells in cell medium were placed the upper (apical) chamber of the transwell insert, at a density of 1.5×105 cells/cm2. The lower (basolateral) chamber was filled with 700-900 μl of cell medium.

[0257] Cells were cultured at 37° C., 5% CO.sub.2, and 95% humidity for 16 days, and medium in the upper and lower chamber was replaced every 4 days. Following this incubation cells were incubated for additional 24h with tissue culture medium, containing compounds “A”, “B” or “C” were added at levels comparable to Trial II. After this incubation time, basal TEER was measured according to Ghaffarian and Muro 2013. Subsequently, temperature was increased to heat stress conditions (42° C.) and TEER measurement was continued for up to 6 h. From the comparison of TEER values of untreated control cells, carried along in every experiment were compared with those of cells treated with compounds “A”, “B” or “C” conclusions on protective effects of the compounds on intestinal barrier function could be drawn.

[0258] Results of In Vitro Testing of Intestinal Barrier Function by Transepithelial Electrical Resistance (TEER) in Transwell CaCo2 Cells (Trial III)

[0259] Since betalain could been shown for the first time to improve HSP 70 induction in CaCo2 cell monolayers (FIG. 3 D), its protective effects on intestinal barrier integrity have been examined. It could be shown that betalain pretreatment of CaCo2 cells grown in transwell culture plates led to a significantly lower decrease of TEER under heat stress conditions compared to untreated control cells (FIG. 4). From this fact, it could be concluded for the first time, that betalain unrolls protective effects on intestinal barrier integrity.

[0260] TRIAL IV: In Vivo Validation of Beneficial Effects of the Naringin (Compound “B”) on Growth Performance in Broilers Under Heat Stress and Evaluation of the Optimum Dose Level

[0261] Beneficial effects of compound “B″ have not yet been reported to improve animal welfare (improved performance, heat shock response and intestinal barrier integrity) in vivo. Since compound “B” showed a beneficial response in all in vitro tests, the first in vivo trial consequently aimed to investigate beneficial effects of this compound on performance and heat shock response in broilers under heat stress. Betaine, frequently used as an additive, improving performance of animals under heat stress was carried along in this trial as a positive control group.

[0262] Trial Design

[0263] 1152 one-day old Ross 308 male broilers from a total of 1400 were used in the experiment. Any day-old broilers, showing signs of health problems, injury, or of being too small or in poor condition were excluded in the selection process. Broilers were sexed at the hatchery. All day-old chicks were individually weighted and grouped according to weight. The birds were then assigned to the 36 pens (6 repetitions of 6 treatments), and each pen initially consisted of 34 broilers. On day 4 of the experiment in each pen, the weakest 2 broilers were removed and all pens were equated to 32 birds per pen by negative selection. Birds of the negative control group were fed the basal diet without any further phytogenic additive. The reference group was fed a diet providing 500 mg/kg feed betaine from a natural betaine source (Actibeet®, Agrana). To the diets of groups 3, 4, 5 and 6 the phytogenic premixes with compound “B” were added to achieve final dietary Naringin concentrations of 12/18/24/30 mg/kg of feed.

TABLE-US-00006 TABLE 5 Trial IV: Experimental treatment groups Betaine (positive Naringin Treat- control), (compound B), ment mg/kg feed mg/kg feed T1 — — T2 500 — T3 — 12 T4 — 18 T5 — 24 T6 — 30

[0264] Animal Housing and Management

[0265] The trial was carried out at the Delacon Research Facility (Stošíkovice na Louce, Czech Republic). Birds were kept in floor pens, each having an area of 2.1 m.sup.2 (1,65×1,275 m; 2.03 m.sup.2 of usable area=not counting the space occupied with the feeder), with fresh wood shavings as bedding material. Stocking density at the end of the trial was 14,78 chicks/m.sup.2 (38.7 kg/m.sup.2). The building was supplied with artificial, programmable lights, automated central heating and forced ventilation. Temperature was set according to the breeders' recommendations from day 1 to day 21 of age. The onset of heat stress was on day 22 of age. Thereby a cyclic heat stress regime was applied. The heat stress period was from 09:00 in the morning until 17:00 in the afternoon. The average house temperature in this period was 34° C. The “cooling night period” was from 19:00 in the evening until 07:00 in the morning. The average room temperature in this period was 26° C. The time intervals from 07:00 to 09:00 in the morning and from 17:00 to 19:00 in the evening was used to gradually increase or decrease the temperature to 34° C. and 26° C., respectively. In order to increase the humidity of the chicken house under the heat stress regime, towels on the heat panels were moistened 5 times per hour during the heat stress period. Relative humidity was maintained at about 70-80%.

[0266] Experimental Diets

[0267] Birds were fed a basal diet, based on wheat, corn, and soybean meal. For each feeding period (starter/grower, grower/finisher) feed was calculated to be iso-nutritive (Table 6). Dietary nutrient concentrations were calculated to fulfill the current recommendations of the broilers according to the breeders' recommendations. According to the applied 2-phase-feeding, a starter/grower diet was offered from days 1 to 21 and a grower/finisher diet from days 22 to 42. The composition of the diets, used in trials IV, V, VI, for the single phases and the calculated nutrient contents are given in Table. All diets were offered to the birds as pelleted feed.

TABLE-US-00007 TABLE 6: Trials IV, V, VI: Composition and calculated analyses of the experimental diets Feed formulas A) Starter/ B) Grower/ Raw material Unit Grower Finisher Wheat % 31,750 34,190 Corn % 23,500 21,500 Rape meal % 4,000 4,500 Wheat flour % 2,500 4,500 Maizegerm % 1,500 Soya meal % 29,000 25,000 Animal fat % 4,000 Hydrolysed protein % 3,000 Soya oil % 1,000 L-Lysine HCl 98 % 0,370 0,300 L-Threonine 98 % 0,070 0,100 DL-Methionine liquid % 0,460 0,380 Limestone % 1,500 1,300 Salt % 0,200 0,230 Monocalciumphosphate % 1,000 0,800 Sodium carbonate % 0,150 0,200 Vitamin and mineral % 0,500 0,500 premixture trial premixture with active % 1.000 1.000 compounds A,B,C Total % 100.00 100.00 Calculated nutrients Dry matter g/kg 889,614 892,194 MEp MJ 12,145 13,023 Crude protein g/kg 226,606 200,375 Fibre g/kg 27,203 27,776 Fat g/kg 44,392 74,913 Ash g/kg 60,557 53,039 Lysine g/kg 14,433 12,077 Methionine g/kg 6,967 5,915 Met + Cys g/kg 10,475 9,508 Threonine g/kg 8,929 8,127 Arginine g/kg 14,405 12,462 Ca g/kg 9,243 8,039 P non phytate g/kg 3,341 2,907 Na g/kg 1,920 1,578 Cl g/kg 1,800 1,960

[0268] The premixes were prepared at the Delacon Biotechnik GmbH facility in Steyregg (Austria). They were prepared to provide the intended final dietary concentrations at an inclusion level of 0.1% (1.0 mg/kg). The concentration of active substances in the premixes was done immediately after preparation of the premixes.

[0269] The production of the complete pelleted diets with the addition of the premixes to the diets was carried by the feedmill Biosta s.r.o. Blučina (Czech Republic).

[0270] Samples were taken directly after manufacturing. 1 kg feed of each treatment and period will be stored at the trial facility at cool and dry conditions till the approval of the final report.

[0271] Diets were analyzed for nutrient content at Zemědělská oblastní laboratoř, Chotýšany. Diets were analyzed for dry matter, crude protein, crude fat, ash, sugar, starch, Ca, and P. Moreover, the active ingredients were analysed in the diets.

[0272] Tissue Sampling

[0273] On day 21 and 42 of the experiment 2 chickens of each repetition was killed for collecting liver and jejunum to measure gene expression.

[0274] Statistical Analysis of the Data

[0275] The statistical analyses were performed with the software package SAS. After checking the homogeneity of the data, means were compared by the usual test procedures (Tukey test). Statistical significance was declared at P 0.05, with 0.05<P 0.10 considered as a near-significant trend.

[0276] Results of the In Vivo Validation of Beneficial Effects of the Naringin (Compound “B”) on Growth Performance in Broilers Under Heat Stress and Evaluation of the Optimum Dose Level (Trial IV)

[0277] Table 7 shows the analysed concentrations of naringin in the diets of Trial IV. In the diets recovery of the active substance Naringin was nearly 100%, thus the specified contents appear also in the final diets. A similarly high recovery rate could be also analysed for the reference substance betaine.

TABLE-US-00008 TABLE 7 Trial IV - Betaine and Naringin content of the trial diets Betaine (positive control) Naringin (compound “B”) Treat- mg/kg feed mg/kg feed ment analysed expected analysed expected T1 — — — — T2 470 500 — — T3 — — 12.3 12 T4 — — 17.4 18 T5 — — 24.2 24 T6 — — 29.4 30

[0278] Since positive effects of compound “B” (Naringin) on animal welfare aspects under heat stress conditions (e.g. protection of intestinal barrier function, linked to an increase in performance) have not been reported yet, the aim of the trial was to evaluate dose-dependent effects of compound “B” versus a standard dose (500 mg/kg) of the reference substance betaine. Already in the pre-heat stress period (d 0 to 21), both the reference substance (+4.9%) and all doses (12, 18, 24 and 30 mg/kg diet) of compound “B” (+2.79 to +13%) resulted in beneficial effects on body weight and daily body weight gain. Compared to the negative control this effect was even significant (+13%) for the lowest compound “B” dose tested (12 mg/kg) (FIGS. 5 and 6). The positive effect of the reference substance and of compound “B” on body weight development and daily gain continued also under heat stress conditions (d22 to 42) (FIGS. 5 and 6). However, under heat stress the effects of the reference substance betaine on body weight development were 2 to 5% higher compared to those of compound “B” (FIGS. 5 and 6). In contrast to the pre heat stress period a clear dose-response of compound “B” could be observed under heat stress (FIGS. 5 and 6). Due to the fact that additives against negative effects of heat stress are fed through the entire mast period, the strong preconditioning effect of already the lowest dose of compound “B” was maintained until the end of the trial. At day 42 birds of the groups treated with the reference substance (+170 g) and with compound “B” (+80 to +140 g) had distinctly higher body weights compared with birds of the untreated control (FIGS. 5 and 6). The highest final body weight compared (+140 g) to the negative control could be obtained with the lowest compound “B” dose. Analogous results as found for body weight development could also be observed for feed conversion ratio (FIG. 7).

[0279] Trial V: In Vivo Validation of Beneficial Effects of Combinations of Naringin (Compound “B”) with Different 1,8-Cineole Concentrations (Compound “A”) on Growth Performance, Heat Shock Protein Response and Intestinal Barrier Integrity in Broilers Under Heat Stress

[0280] In the dose response trial for compound “B” (Trial IV) beneficial effects on the performance of the broilers under heat stress could be observed. It turned out that the lowest dose of 12 mg/kg Naringin complete diet resulted in performance values (body weight/weight gain/FCR) which were slightly better than in the betaine reference group. Higher concentrations of compound “B” did not result in further benefits for growth performance. In the dose response trial for compound “B” beneficial effects on the performance of the broilers under heat stress could be observed. Consequently in this trial the additional value of adding 1,8-Cineole (compound “A”) in a dose-dependent manner (6/12/24 mg/kg feed) to compound “B” at constantly 12 mg Naringin/kg diet was evaluated. The addition of 500 mg/kg betaine alone or 500 mg betaine+12 mg 1,8-Cineole/kg served as reference groups.

[0281] 1224 one-day old Ross 308 male broilers from a total of 1400 were used in the experiment. Any day-old broilers, showing signs of health problems, injury, or of being too small or in poor condition were excluded in the selection process. Broilers were sexed at the hatchery. All day-old chicks were individually weighted and grouped according to weight. The birds were then assigned to the 36 pens (6 repetitions of 6 treatments), and each pen initially consisted of 34 broilers. On day 4 of the experiment in each pen, the weakest 2 broilers were removed and all pens were equated to 32 birds per pen by negative selection.

[0282] Birds of the negative control group were fed the basal diet without any further phytogenic additive. The reference groups 2 and 6 were fed a diet providing 500 mg/kg betaine from a natural betaine source (Actibeet®, Agrana) or 500 mg/kg betaine from a natural betaine source (Actibeet®, Agrana)+12 mg/kg 1,8 Cineole (compound “A”). To the diets of groups 3, 4 and 5 the phytogenic premixes were added providing 12 mg/kg of Naringin plus 6, 12 or 24 mg 1,8-Cineole/kg (Table 8).

TABLE-US-00009 TABLE 8 Trial V: Experimental treatment groups of the validation trial Betaine Naringin (positive (compound 1,8-cineole Treat control), B), (compound A) ment mg/kg feed mg/kg feed mg/kg feed T1 — — — T2 500 — — T3 — 12  6 T4 — 12 12 T5 — 12 24 T6 500 — 12

[0283] Trial V methodology, including animal housing and management, preparation and composition of experimental diets, as well as the organ sampling and statistical procedures were analogous to trial IV.

[0284] Results of the In Vivo Validation of Beneficial Effects of Combinations of Naringin (Compound “B”) with Different 1,8-Cineole Concentrations (Compound “A”) on Growth Performance, Heat Shock Protein Response and Intestinal Barrier Integrity in Broilers Under Heat Stress (Trial V)

[0285] Table 9 shows the analysed concentrations of Betaine, Naringin and 1,8 cineole in the diets of Trial V. The analysed values show that the minimum specifications were reached or exceeded for all experimental groups.

TABLE-US-00010 TABLE 9 Trial V: calculated Betaine, Naringin and 1,8-cineole content of the trial diets Betaine Naringin (positive 1,8-cineole (com- Treat- control) (compound A) pound B) Ratio ment mg/kg feed mg/kg feed mg/kg feed (A:B) T1 — — — — T2 500 — — — T3 —  6 12 0.5:1   T4 — 12 12 1:1 T5 — 18 12 1.5:1   T6 — 24 12 2:1 T7 500 12 — 1:0

[0286] Since compound “B” in the first in vivo trial showed beneficial effects on performance parameters of broilers under heat stress, which were slightly better compared to the reference substance betaine, the aim of this trial was to test a synergistic/beneficial effects of combinations of a fixed dose of compound “B” (12 mg/kg) plus increasing doses of compound “A” (6 to 24 mg/kg). The intention of this study consists in exploring combinations of compound “A” and “B” for the New Feed Additive Supporting Animal Welfare Under Heat Stress Conditions which exceed the effects of the reference substance betaine. In addition, a combination of betaine with the intermediate dose of compound “A” (12 mg/kg) was chosen as a second reference in order to test, if there exist synergistic effects of betaine and compound “A”. Similarly, as in trial 1, all additives (betaine and combinations of compounds “A” and “B”) unrolled already growth promoting effects in the pre-heat stress period (FIGS. 8 and 9). Whereas betaine alone increased daily weight gain by 2.5% compared to the negative control, the addition of 12 mg/kg compound “A” reduced this effect to nearly 1%. In contrast all combinations of compound “B” plus compound “A” exceeded the growth promoting effect of betaine alone and a fortiori that of the combination of betaine plus substance A (FIGS. 8 and 9). Moreover, a clear dose-response effect could be observed for the combinations “AB” (+2,1%; +2.5; +4.7%) (FIGS. 8 and 9). As observed in the first trial, the reference substance betaine developed a better growth promoting effect under heat stress conditions (+2.0%) compared to the negative control. Under heat stress conditions all combinations “AB” exceeded the growth performing effects of betaine alone distinctly. The dose response effect, found in the pre heat stress period, could not be observed under heat stress for the tested combinations “AB”. All tested combinations “AB” increased growth performance by 5.2 to 5.6% compared to the negative control and by 3.0% to 3.5% compared to the reference substance betaine (FIGS. 8 and 9), respectively. Most interestingly, the combination of the reference substance betaine with the medium dose of compound “A”, which failed in the pre heat stress period, unrolled in the heat stress period even slightly better growth promoting effects compared to the combinations “AB”. However, since feed additives counteracting heat stress are used during the entire mast period, the combinations “AB” overall resulted in the highest improvement of final body weight compared to the negative control (+4.8%, +5.2%, +4.9%), to the reference substance betaine (+1.6%, +2.0%, +1.8%) and also compared to the combination of betaine plus the medium concentration of compound “A” (+0.2%, +0.7%, +0.4%). Very similar effects, as observed for growth performance could also be found for FCR (food conversion rate) (FIG. 10).

[0287] Trial VI: In Vivo Validation of Beneficial Effects of the Final Prototype Combinations of 1,8-Cineole with Naringin (AB), or 1,8-Cineole with Betalain (AC).

[0288] In the in vivo validation trial of beneficial effects of combinations of Naringin (compound “B”) with different 1,8-cineole concentrations (compound “A”) it turned out, that all tested combinations of “AB” were much more effective than the positive control betaine alone, and even as the combination of betaine+compound “A”. Thus, consequently the aim of the last in vivo trial of the series was to test, if in addition to the combination “AB”, as well as the combination “AC” unrolls stronger protection and growth performance under heat stress conditions than 500 mg/kg betaine as the positive control.

[0289] 816 one-day old Ross 308 male broilers from a total of 960 were used in the experiment. Any day-old broilers, showing signs of health problems, injury, or of being too small or in poor condition were excluded in the selection process. Broilers were sexed at the hatchery. All day-old chicks were individually weighted and grouped according to weight. The birds were then assigned to the 36 pens (6 repetitions of 6 treatments), and each pen initially consisted of 34 broilers. On day 4 of the experiment in each pen, the weakest 2 broilers were removed and all pens were equated to 32 birds per pen by negative selection.

[0290] Birds of the negative control group were fed the basal diet without any further phytogenic additive. The reference group 2 was fed a diet providing 500 mg/kg betaine from a natural betaine source (Actibeet®, Agrana). To the diets of groups 3 and 4 the phytogenic premixes were added providing either 12 mg Naringin/kg from citrus flavonoids, or 0.55 mg Betalain/kg from beetroot and barbary, each in combination with 12 mg 1,8-Cineole/kg from the laminaceae oil mixture (Table 10).

TABLE-US-00011 TABLE 10 Trial VI: Experimental treatment groups Betaine 1,8-cineole Naringin Betalaine Treat- (+ control) (A) (B) (C) Ratio ment mg/kg feed mg/kg feed mg/kg feed mg/kg feed A:B/C T1 — — — — — T2 500 — — — — T3 — 12 12 — 1:1 T4 — 12 — 55 0.2:1  

[0291] Trial VI methodology, including animal housing and management, preparation and composition of experimental diets, as well as the organ sampling and statistical procedures were analogous to trial IV.

[0292] Results of the In Vivo Validation of Beneficial Effects of the Combination of the 1,8-Cineole with Naringin (AB), or 1,8-Cineole with Betalain (AC) on Growth Performance, Heat Shock Protein Response and Intestinal Barrier Integrity in Broilers Under Heat Stress Compared to Betaine as the Positive Control (Trial VI)

[0293] In Table 11 the analysed values of the concentrations of Betaine, 1,8-Cineole, Naringin and Betalain in Trial VI are displayed.

TABLE-US-00012 TABLE 11 Trial VI—Betaine, 1,8-cineole, Naringin and betalain content of the trial diets Betaine (+control) 1,8-cineole Naringin (B) Betalain (C) mg/kg (A) mg/kg feed mg/kg feed feed mg/kg feed ratio ratio Treatment calculated calculated calculated A:B calculated A:C T1 — — — — — — T2 50 — — — — — T3 — 30 30 1:1 — — T4 — 1.37 — — 1.37 1:1

[0294] Due to the positive findings on additional effects of substances “B” and “C” on heat shock response in CaCo2 cells (FIG. 3E) in the last trial of this series it has been evaluated, if under heat stress conditions not only combinations of substances “AB”, but also combinations of substances “AC” show beneficial effects on growth performance of heat stressed broilers. In the pre-heat stress phase the positive control (betaine) and both combinations “AB” and “AC” already improved body weight by 3,60 to 4,65% compared to the untreated negative control (FIG. 11). “AC” turned out as the most effective combination, whereas in this trial “AB” was slightly less effective than betaine (FIG. 11). However, during the heat stress period both combinations “AB” and “AC” unrolled their high potential to improve the performance of broilers. Both additives improved final body weight by about 1%, compared to the positive control, corresponding to a 50 g higher final body weight. Daily weight gain during the heat stress phase could be even improved by about 1,5% by both combinations of “AB” and “AC” compared to the positive control betaine (FIG. 12). Improvement in final body weight and daily gain was also reflected by FCR (FIG. 13), which was improved to a higher extent by both combinations of “AB” and “AC” compared to the positive control (betaine).

REFERENCES

[0295] Brand W, van der Wel P A, Rein M J, Barron D, Williamson G, van Bladeren P J, Rietjens I M. Metabolism and transport of the citrus flavonoid hesperetin in Caco-2 cell monolayers. Drug Metab Dispos. 2008 September;36(9):1794-802. doi: 10.1124/dmd.107.019943.

[0296] Chen R C, Sun G B, Wang J, Zhang H J, Sun X B. Naringin protects against anoxia/reoxygenation-induced apoptosis in H9c2 cells via the Nrf2 signaling pathway. Food Funct. 2015; 6: 1331-1344.

[0297] Esatbeyoglu T, Wagner A E, Schini-Kerth V B, Rimbach G. Betanin—a food colorant with biological activity. Mol Nutr Food Res. 2015 January;59(1):36-47.

[0298] Ghaffarian R, Muro S. Models and methods to evaluate transport of drug delivery systems across cellular barriers. J Vis Exp. 2013 Oct. 17;(80):e 50638.

[0299] Hosseini S M, Farhangfar H, Nourmohammadi R. Effects of a blend of essential oils and overcrowding stress on the growth performance, meat quality and heat shock protein gene expression of broilers. Br Poult Sci. 2018 February;59(1):92-99. doi: 10.1080/00071668.2017.1390209.

[0300] FangFang, Li H, Qin T, Li M, Ma S. Thymol improves high-fat diet-induced cognitive deficits in mice via ameliorating brain insulin resistance and upregulating NRF2/HO-1 pathway. Metab Brain Dis. 2017; 32: 385-393.

[0301] Kamboh A A, Hang S Q, Bakhetgul M, Zhu W Y. Effects of genistein and hesperidin on biomarkers of heat stress in broilers under persistent summer stress. Poult Sci. 2013; 92: 2411-2418.

[0302] Kluth D, Banning A, Paur I, Blomhoff R, Brigelius-Flohé R. Modulation of pregnane X receptor- and electrophile responsive element-mediated gene expression by dietary polyphenolic compounds. Free Radic Biol Med. 2007 Feb. 1;42(3):315-25.

[0303] König J, Wells J, Cani P D, García-Ródenas C L, MacDonald T, Mercenier A, Whyte J, Troost F, Brummer R J. Human Intestinal Barrier Function in Health and Disease. Clin Transl Gastroenterol. 2016 Oct. 20;7(10):e196. doi: 10.1038/ctg.2016.54

[0304] Lee C H, Wettasinghe M, Bolling B W, Ji L L, Parkin K L. Betalains, phase II enzyme-inducing components from red beetroot (Beta vulgaris L.) extracts. Nutr Cancer. 2005; 53(1):91-103.

[0305] Maria Miguel, Antioxidants 2018; 7(4):53.

[0306] Noda S, Tanabe S, Suzuki T. Differential effects of flavonoids on barrier integrity in human intestinal Caco-2 cells. J Agric Food Chem. 2012 May 9;60(18):4628-33. doi: 10.1021/jf300382h.

[0307] Pearce S C, Mani V, Boddicker R L, Johnson J S, Weber T E, Ross J W, Rhoads R P, Baumgard L H, Gabler N K. Heat stress reduces intestinal barrier integrity and favors intestinal glucose transport in growing pigs. PLoS One. 2013 Aug. 1;8(8):e70215. doi: 10.1371/journal.pone.0070215 (A).

[0308] Pearce S C, Mani V, Weber T E, Rhoads R P, Patience J F, Baumgard L H, Gabler N K. Heat stress and reduced plane of nutrition decreases intestinal integrity and function in pigs. J Anim Sci. 2013 November; 91(11):5183-93. doi: 10.2527/jas.2013-6759 (B).

[0309] Pires Goncalves J C, de Souza Trassi M A, Lopes N B, Dōrr F A, dos Santos M T, Baader W J, Olivera Jr. V X, Bastos E L. A comparative study of the purification of betanin. Food Chemistry. 2012; 131:231-238

[0310] Placha I, Takacova J, Ryzner M, Cobanova K, Laukova A, Strompfova V, Venglovska K, Faix S. Effect of thyme essential oil and selenium on intestine integrity and antioxidant status of broilers. Br Poult Sci. 2014 February;55(1):105-14. doi: 10.1080/00071668.20130.

[0311] Rani N, Bharti S, Manchanda M, Nag T C, Ray R, Chauhan S S, Kumari S, Arya D S. Regulation of heat shock proteins 27 and 70, p-Akt/p-eNOS and MAPKs by Naringin Dampens myocardial injury and dysfunction in vivo after ischemia/reperfusion. PLoS One. 2013 Dec. 6;8(12):e82577. doi: 10.1371/journal.pone.0082577.

[0312] Saeed M, Babazadeh D, Naveed M3, Arain M A, Hassan F U, Chao S. Reconsidering betaine as a natural anti-heat stress agent in poultry industry: a review. Trop Anim Health Prod. 2017 October;49(7):1329-1338. doi: 10.1007/s11250-017-1355-z. Epub 2017 Jul. 21.

[0313] Sharma K, Mahato N, Cho M H, Lee Y R. Converting citrus wastes into value-added products: Economic and environmently friendly approaches. Nutrition. 2017 February;34:29-46. doi: 10.1016/j.nut.2016.09.006.

[0314] Suntar I, Khan H, Patel S, Celano R, Rastrelli L. An Overview on Citrus aurantium L.: Its Functions as Food Ingredient and Therapeutic Agent. Oxid Med Cell Longev. 2018 May 2; 2018:7864269. doi: 10.1155/2018/7864269.

[0315] Türk G, Çeribaşi AO, Şimşek ÜG, Çeribaşi S, Güvenç M, Özer Kaya Ş, Çiftçi M, Sōnmez M, Yüce A, Bayrakdar A, Yaman M, Tonbak F. Dietary rosemary oil alleviates heat stress-induced structural and functional damage through lipid peroxidation in the testes of growing Japanese quail. Anim Reprod Sci. 2016 January;164:133-43. doi: 10.1016/j.anireprosci.2015.11.021.

[0316] Uritu C M, Mihai C T, Stanciu G D, Dodi G, Alexa-Stratulat T, Luca A, Leon-Constantin M M, Stefanescu R, Bild V, Melnic S, Tamba B I. Medicinal Plants of the Family Lamiaceae in Pain Therapy: A Review. Pain Res Manag. 2018 May 8; 2018:7801543. doi: 10.1155/2018/7801543.

[0317] Wieten L, van der Zee R, Spiering R, Wagenaar-Hilbers J, van Kooten P, Broere F, van Eden W. A novel heat-shock protein coinducer boosts stress protein Hsp70 to activate T cell regulation of inflammation in autoimmune arthritis. Arthritis Rheum. 2010 April; 62(4):1026-35. doi: 10.1002/art.27344.

[0318] Zhu C, Dong Y, Liu H, Ren H, Cui Z. Hesperetin protects against H2O2-triggered oxidative damage via upregulation of the Keap1-Nrf2/HO-1 signal pathway in ARPE-19 cells. Biomed Pharmacother. 2017; 88:124-133.

[0319] Zou Y, Wang J, Peng J, Wei H. Oregano Essential Oil Induces SOD1 and GSH Expression through Nrf2 Activation and Alleviates Hydrogen Peroxide-Induced Oxidative Damage in IPEC-J2 Cells. Oxid Med Cell Longev. 2016; 2016:5987183. doi: 10.1155/2016/5987183 (A).

[0320] Zou Y, Xiang Q, Wang J, Peng J, Wei H. Oregano Essential Oil Improves Intestinal Morphology and Expression of Tight Junction Proteins Associated with Modulation of Selected Intestinal Bacteria and Immune Status in a Pig Model. Biomed Res Int. 2016; 2016:5436738. doi: 10.1155/2016/5436738 (B).