Supplements and compositions containing amino acids and IGF-1 and methods of use

Abstract

The present disclosure provides a supplement comprising the free amino acid L-glutamine or a salt thereof, L-leucine or a salt thereof, and L-arginine or a salt thereof, as well as active IGF-1. The L-glutamine or a salt thereof, L-leucine or a salt thereof, and L-arginine or a salt thereof may be present in a ratio of about 3:1:1. Compositions comprising the supplement, such as foods, are also provided. The present disclosure also includes, but is not limited to, methods of using these supplements, the methods including improving animal performance, improving animal processing coefficients, or enhancing animal intestinal health, wherein the method comprises administering to the animal a food having the supplement.

Description

Field of the Invention

[0001] This disclosure generally relates to supplements and compositions having IGF-1 and specific amino acids, and methods of using such supplements and compositions. Summary of the Invention

[0002] Nutrition in the early life of an animal is important for the development of the immune system and the skeletal muscle system. Typically, the feed provided ad libitum includes excessive components, which include grains, proteins, vitamins, minerals, etc. The inventors have found that adding a combination of active IGF-1 and three amino acids to food results in unexpected changes in certain useful properties. The combination of IGF-1 and three amino acids produces a nutritional synergism, wherein the combination of IGF-1 and three amino acids is nutritionally superior to any one of these components used alone at any test dose. In addition, the amount of these three amino acids is less than the amount typically used in animal food.

[0003] The terms used herein should be understood to have their ordinary meaning in the relevant art, unless otherwise specified. Several terms used herein and their meanings are set forth below.

[0004] The terms "weight percentage", "wt%", and "wt%" are used interchangeably herein and refer to the weight of a compound in a supplement or composition. For example, 1 gram of an amino acid in 100 grams of a composition is 1 wt% of the amino acid in the composition.

[0005] The term "and / or" means one or all of the recited elements or a combination of any two or more of the recited elements.

[0006] The words "preferred" and "preferably" refer to embodiments of this disclosure that may provide certain benefits in certain circumstances. However, in the same or other circumstances, other embodiments may also be preferred. Additionally, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of this disclosure.

[0007] When the terms "comprising" and its variants appear in the specification and claims, these terms do not have a limiting meaning.

[0008] It should be understood that when embodiments are described herein using language such as "include", "includes", or "including", other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided. The term "consisting of" means including and being limited to whatever is within the phrase "consisting of". That is, "consisting of" indicates that the listed elements are necessary or mandatory and that no other elements are present. The term "consisting essentially of" indicates including any elements listed after the phrase, and may include other elements in addition to the listed elements, provided that these elements do not interfere with or contribute to the activity or function specified for the listed elements in this disclosure.

[0009] Unless otherwise stated, "a / an", "the", and "at least one" are used interchangeably and mean one or more than one.

[0010] A condition that is "suitable" for an event to occur or "appropriate" is a condition that does not prevent such an event from occurring. Thus, these conditions allow, enhance, facilitate, and / or benefit the event.

[0011] As used herein, a "substantially free of" material means a supplement or composition having less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the material. In one embodiment, the presence of the material in the supplement or composition is undetectable.

[0012] As used herein, "providing" in the context of a supplement or composition means preparing the supplement or composition, purchasing the composition or supplement, or otherwise obtaining the supplement or composition.

[0013] Also herein, a numerical range recited by endpoints includes all the values included within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0014] Throughout the specification, references to "one embodiment", "an embodiment", "certain embodiments", or "some embodiments", etc., mean that a particular feature, configuration, supplement, composition, or property described in connection with that embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of such phrases throughout the specification are not necessarily all referring to the same embodiment of the present disclosure. Additionally, the particular features, configurations, supplements, compositions, or properties may be combined in any suitable manner in one or more embodiments.

[0015] For any method disclosed herein that includes discrete steps, those steps may be carried out in any feasible order. And, if appropriate, any combination of two or more steps may be carried out simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The illustrative embodiments of the present disclosure described in detail below may be best understood when read in conjunction with the following drawings.

[0017] Figure 1 Porcine satellite cells were shown treated with 1 of 8 treatments. The CON treatment consisted of low glucose Dulbecco's modified Eagle's medium supplemented with 2% (vol / vol) fetal bovine serum; IGF-1, CON medium plus 5 ng / mL active IGF-1; GLUT, CON medium plus 4 mM L-glutamine; LEU, CON medium plus 1 mM L-leucine; ARG, CON medium plus 1 mM L-arginine; LYS, CON medium plus 10 mM L-lysine; MET, CON medium plus 10 μM L-methionine. a、b、c、d Means with different superscripts were significantly different (P<0.05).

[0018] Figure 2 Porcine satellite cells were shown treated with 1 of 3 treatments. The CON treatment consisted of low glucose Dulbecco's modified Eagle's medium supplemented with 2% (vol / vol) fetal bovine serum; IGF-1, CON medium plus 5 ng / gml active IGF-1; COMBO, CON medium plus 5 ng / mL IGF-1, 4 mM L-glutamine, 1 mM L-leucine, and 1 mM L-arginine. a、b、c Means with different superscripts were highly significantly different (P<0.0001).

[0019] Figure 3Shows porcine satellite cells treated with 1 of 7 treatments. The CON treatment consisted of low-glucose Dulbecco's modified Eagle's medium supplemented with 2% (v / v) fetal bovine serum; IGF-1, CON medium plus 5 ng / mL active IGF-1; COMBO 0, CON medium plus 5 ng / mL active IGF-1, 0 mM L-glutamine, 1 mM L-leucine, and 1 mM L-arginine; COMBO 1, CON medium plus 5 ng / mL active IGF-1, 1 mM L-glutamine, 1 mM L-leucine, and 1 mM L-arginine; COMBO 2, CON medium plus 5 ng / mL active IGF-1, 2 mM L-glutamine, 1 mM L-leucine, and 1 mM L-arginine; COMBO 3, CON medium plus 5 ng / mL active IGF-1, 3 mM L-glutamine, 1 mM L-leucine, and 1 mM L-arginine; COMBO 4, CON medium plus 5 ng / mL active IGF-1, 4 mM L-glutamine, 1 mM L-leucine, and 1 mM L-arginine. a、b、c、d Means with different superscripts are significantly different (P < 0.05).

[0020] Figure 4 Shows porcine satellite cells treated with 1 of 5 treatments. The CON treatment consisted of low-glucose Dulbecco's modified Eagle's medium supplemented with 2% (v / v) fetal bovine serum; IGF-1, CON medium plus 5 ng / g active IGF-1; COMBO 0.5, CON medium plus 5 ng / mL IGF-1, 3 mM L-glutamine, 0.5 mM L-leucine, and 1 mM L-arginine; COMBO 1, CON medium plus 5 ng / mL IGF-1, 3 mM L-glutamine, 1 mM L-leucine, and 1 mM L-arginine; COMBO 2, CON medium plus 5 ng / mL IGF-1, 3 mM L-glutamine, 2 mM L-leucine, and 1 mM L-arginine. a、b、c、d、e Means with different superscripts are significantly different (P < 0.05).

[0021] Figure 5Shows porcine satellite cells treated with 1 of 9 treatments. The CON treatment consisted of low-glucose Dulbecco's modified Eagle's medium supplemented with 2% (vol / vol) fetal bovine serum; IGF-1, CON medium plus 5 ng / mL active IGF-1; COMBO 0, CON medium plus 5 ng / mL active IGF-1, 3 mM L-glutamine, 1 mM L-leucine, and 0 mM L-arginine; COMBO 0.1, CON medium plus 5 ng / mL active IGF-1, 3 mM L-glutamine, 1 mM L-leucine, and 0.1 mM L-arginine; COMBO 0.25, CON medium plus 5 ng / mL active IGF-1, 3 mM L-glutamine, 1 mM L-leucine, and 0.25 mM L-arginine; COMBO 0.5, CON medium plus 5 ng / mL active IGF-1, 3 mM L-glutamine, 1 mM L-leucine, and 0.5 mM L-arginine; COMBO 1, CON medium plus 5 ng / mL active IGF-1, 3 mM L-glutamine, 1 mM L-leucine, and 1 mM L-arginine; COMBO 2, CON medium plus 5 ng / mL active IGF-1, 3 mM L-glutamine, 1 mM L-leucine, and 2 mM L-arginine; COMBO 3, CON medium plus 5 ng / mL active IGF-1, 3 mM L-glutamine, 1 mM L-leucine, and 3 mM L-arginine. a、b、c、d、e、f Means with different superscripts are different P < 0.05.

[0022] Figure 6Shows porcine satellite cells treated with one of three treatments. The CON treatment consisted of low glucose Dulbecco's Modified Eagle's Medium supplemented with 2% (v / v) fetal bovine serum; IGF-1, CON medium plus 5 ng / mL active IGF-1; COMBO, CON medium plus 5 ng / mL active IGF-1, 3 mM L-glutamine, 1 mM L-leucine and 1 mM L-arginine; COMBO 0.5, CON medium plus 5 ng / mL active IGF-1, 3 mM L-glutamine, 1 mM L-leucine, 1 mM L-arginine and 0.5 mM L-methionine; COMBO 1, CON medium plus 5 ng / mL active IGF-1, 3 mM L-glutamine, 1 mM L-leucine, 1 mM L-arginine and 1 mM L-methionine; COMBO 2, CON medium plus 5 ng / mL active IGF-1, 3 mM L-glutamine, 1 mM L-leucine, 1 mM L-arginine and 2 mM L-methionine; COMBO 3, CON medium plus 5 ng / mL active IGF-1, 3 mM L-glutamine, 1 mM L-leucine, 1 mM L-arginine and 3 mM L-methionine. a、b、c、d、e Means with different superscripts were significantly different (P<0.05).

[0023] Figure 7 Shows porcine satellite cells treated with one of three treatments. The CON treatment consisted of low glucose Dulbecco's Modified Eagle's Medium supplemented with 2% (v / v) fetal bovine serum; IGF-1, CON medium plus 5 ng / mL active IGF-1; COMBO, CON medium plus 5 ng / mL active IGF-1, 3 mM L-glutamine, 1 mM L-leucine and 1 mM L-arginine; COMBO 0.5, CON medium plus 5 ng / mL active IGF-1, 3 mM L-glutamine, 1 mM L-leucine, 1 mM L-arginine and 0.5 mM L-lysine; COMBO 1, CON medium plus 5 ng / mL active IGF-1, 3 mM L-glutamine, 1 mM L-leucine, 1 mM L-arginine and 1 mM L-lysine; COMBO 2, CON medium plus 5 ng / mL active IGF-1, 3 mM L-glutamine, 1 mM L-leucine, 1 mM L-arginine and 2 mM L-lysine; COMBO 3, CON medium plus 5 ng / mL active IGF-1, 3 mM L-glutamine, 1 mM L-leucine, 1 mM L-arginine and 3 mM L-lysine. a、b、c、d Means with different superscripts were significantly different (P<0.05).

[0024] Figure 8 Porcine satellite cells were shown to be treated with one of three treatments (with or without 50 nM rapamycin). The CON treatment consisted of low glucose Dulbecco's modified Eagle's medium supplemented with 2% (v / v) fetal bovine serum; IGF-1, CON medium plus 5 ng / mL active IGF-1; COMBO, CON medium plus 5 ng / mL active IGF-1, 3 mM L-glutamine, 1 mM L-leucine, and 1 mM L-arginine. a、b、c Means with different superscripts were significantly different, P < 0.0001.

[0025] Figure 9 Avian satellite cells were shown to be treated with one of six treatments. The CON treatment consisted of low glucose Dulbecco's modified Eagle's medium supplemented with 2% (v / v) fetal bovine serum; IGF-1, CON medium plus 5 ng / g active IGF-1; COMBO, CON medium plus 5 ng / mL active IGF-1, 3 mM L-glutamine, 1 mM L-leucine, and 1 mM L-arginine; GLUT, CON medium plus 3 mM L-glutamine; ARG, CON medium plus 1 mM L-arginine; LEU, CON medium plus 1 mM L-leucine. a、b、c Means with different superscripts were significantly different, P < 0.05.

[0026] Figure 10 Body weight at day 42 was shown. The animals were fed a phase 1 diet from days 0 - 7, a phase 2 diet from days 8 - 21, and a phase 3 diet from days 22 - 42 of the trial. The control diet (CON) contained no additives above the basal diet; the positive control (BG) contained 2.5 kg / ton in phase 1 and 1.5 kg / ton in phase 2; one treatment (CT1) contained a supplement at 2.5 kg / ton in phase 1 and 1.5 kg / ton in phase 2; one treatment (CT2) contained a supplement at 350 g / ton in all three phases; and one treatment (CT3) contained a supplement at 175 g / ton in all three phases.

[0027] Figure 11 Feed conversion ratio at day 42 was shown. The animals were fed a phase 1 diet from days 0 - 7, a phase 2 diet from days 8 - 21, and a phase 3 diet from days 22 - 42 of the trial. The control diet (CON) contained no additives above the basal diet; the positive control (BG) contained It is 2.5 kg / ton in the first stage and 1.5 kg / ton in the second stage; one treatment (CT1) contains a supplement, which is 2.5 kg / ton in the first stage and 1.5 kg / ton in the second stage; one treatment (CT2) contains a supplement, which is 350 g / ton in all three stages; and one treatment (CT3) contains a supplement, which is 175 g / ton in all three stages.

[0028] Figure 12 The fecal diarrhea score is shown. The first-stage diet was fed from day 0 to day 7 of the trial, the second-stage diet was fed from day 8 to day 21, and the third-stage diet was fed from day 22 to day 42. The control diet (CON) contains no additives on top of the basal diet; the positive control (BG) contains It is 2.5 kg / ton in the first stage and 1.5 kg / ton in the second stage; one treatment (CT1) contains a supplement, which is 2.5 kg / ton in the first stage and 1.5 kg / ton in the second stage; one treatment (CT2) contains a supplement, which is 350 g / ton in all three stages; and one treatment (CT3) contains a supplement, which is 175 g / ton in all three stages.

[0029] Figure 13 The body weight at day 84 is shown. The starter diet was fed from day 0 to day 28 of the trial, the grower diet was fed from day 29 to day 56, and the finisher diet was fed from day 57 to day 84. The control diet (CON) contains no additives on top of the basal diet; one treatment (CT1) contains a supplement, which is 300 g / ton during the starter period and 50 g / ton in the second stage; one treatment (CT2) contains a supplement, which is 600 g / ton during the starter period and 100 g / ton in the second stage; one treatment (CT3) contains a supplement, which is 600 g / ton during the starter period and 50 g / ton in the second stage.

[0030] Figure 14 The feed conversion ratio at day 84 is shown. The starter diet was fed from day 0 to day 28 of the trial, the grower diet was fed from day 29 to day 56, and the finisher diet was fed from day 57 to day 84. The control diet (CON) contains no additives on top of the basal diet; one treatment (CT1) contains a supplement, which is 300 g / ton during the starter period and 50 g / ton in the second stage; one treatment (CT2) contains a supplement, which is 600 g / ton during the starter period and 100 g / ton in the second stage; one treatment (CT3) contains a supplement, which is 600 g / ton during the starter period and 50 g / ton in the second stage.

[0031] Figure 15Shows the lesion score and Escherichia coli prevalence at day 84. Starter diet was fed from day 0 - 28 of the trial, grower diet from day 29 - 56, and finisher diet from day 57 - 84. Control diet (CON) contained no additives on top of the basal diet; one treatment (CT1) contained a supplement at 300 g / ton during the starter period and 50 g / ton in the second phase; one treatment (CT2) contained a supplement at 600 g / ton during the starter period and 100 g / ton in the second phase; one treatment (CT3) contained a supplement at 600 g / ton during the starter period and 50 g / ton in the second phase.

[0032] Figure 16 Shows the Salmonella incidence and Clostridium perfringens prevalence. Starter diet was fed from day 0 - 28 of the trial, grower diet from day 29 - 56, and finisher diet from day 57 - 84. Control diet (CON) contained no additives on top of the basal diet; one treatment (CT1) contained a supplement at 300 g / ton during the starter period and 50 g / ton in the second phase; one treatment (CT2) contained a supplement at 600 g / ton during the starter period and 100 g / ton in the second phase; one treatment (CT3) contained a supplement at 600 g / ton during the starter period and 50 g / ton in the second phase.

[0033] Figure 17 Shows the ileal villus height at day 84. Starter diet was fed from day 0 - 28 of the trial, grower diet from day 29 - 56, and finisher diet from day 57 - 84. Control diet (CON) contained no additives on top of the basal diet; one treatment (CT1) contained a supplement at 300 g / ton during the starter period and 50 g / ton in the second phase; one treatment (CT2) contained a supplement at 600 g / ton during the starter period and 100 g / ton in the second phase; one treatment (CT3) contained a supplement at 600 g / ton during the starter period and 50 g / ton in the second phase.

[0034] Figure 18 Shows the feed conversion ratio at week 3. The control group (CON) had no additives on top of the complete basal diet, and the three treatments had supplements on top of the basal diet: 0.000050, 0.00015, and 0.00045 g / ton of feed (corresponding to CT 0.05, CT 0.15, and CT 0.45 respectively).

[0035] Figure 19Shows the hematocrit levels and serum protein levels at week 3. The control group (CON) had no additives on top of a complete basal diet, and the three treatments had supplements added on top of the basal diet: 0.000050, 0.00015, and 0.00045 g / ton of feed (corresponding to CT 0.05, CT 0.15, and CT 0.45 respectively).

[0036] Figure 20 Shows the lysozyme and cortisol concentrations at week 3. The control group (CON) had no additives on top of a complete basal diet, and the three treatments had supplements added on top of the basal diet: 0.000050, 0.00015, and 0.00045 g / ton of feed (corresponding to CT 0.05, CT 0.15, and CT 0.45 respectively).

[0037] Figure 21 Shows the sow body condition score at farrowing. The control diet (CON) had no additives on top of the basal diet; one diet (CT1) contained 1000 ppm or 1 - kg of supplement per ton of feed; one diet (CT2) contained 350 ppm or 0.35 - kg of supplement per ton of feed; and one diet (CT3) contained 100 ppm or 0.10 - kg of supplement per ton of feed. Sows had a 7 - 10 - day adaptation period before estrus, and day 0 of the trial was the estrus time.

[0038] Figure 22 Shows the number of days from weaning to estrus. The control diet (CON) had no additives on top of the basal diet; one diet (CT1) contained 1000 ppm or 1 - kg of supplement per ton of feed; one diet (CT2) contained 350 ppm or 0.35 - kg of supplement per ton of feed; and one diet (CT3) contained 100 ppm or 0.10 - kg of supplement per ton of feed. Sows had a 7 - 10 - day adaptation period before estrus, and day 0 of the trial was the estrus time.

[0039] Figure 23 Shows the average daily gain of piglets. The control diet (CON) had no additives on top of the basal diet; one diet (CT1) contained 1000 ppm or 1 - kg of supplement per ton of feed; one diet (CT2) contained 350 ppm or 0.35 - kg of supplement per ton of feed; and one diet (CT3) contained 100 ppm or 0.10 - kg of supplement per ton of feed. Sows had a 7 - 10 - day adaptation period before estrus, and day 0 of the trial was the estrus time. Piglets were fed a common diet to meet their nutritional requirements during the nursery and finishing periods.

[0040] Figure 24Shows the weight gain of piglets at day 126. The control diet (CON) contains no additives on top of the basal diet; one diet (CT1) contains 1000 ppm or 1 kg of supplement per metric ton of feed; one diet (CT2) contains 350 ppm or 0.35 kg of supplement per metric ton of feed; and one diet (CT3) contains 100 ppm or 0.10 kg of supplement per metric ton of feed. There is a 7 - 10 day adaptation period before the sow's estrus, and day 0 of the experiment is the estrus time. Piglets are fed a normal diet to meet their nutritional requirements during the nursery and fattening periods.

[0041] Figure 25 Shows the feed conversion rate of piglets at day 126. The control diet (CON) contains no additives on top of the basal diet; one diet (CT1) contains 1000 ppm or 1 kg of supplement per metric ton of feed; one diet (CT2) contains 350 ppm or 0.35 kg of supplement per metric ton of feed; and one diet (CT3) contains 100 ppm or 0.10 kg of supplement per metric ton of feed. There is a 7 - 10 day adaptation period before the sow's estrus, and day 0 of the experiment is the estrus time. Piglets are fed a normal diet to meet their nutritional requirements during the nursery and fattening periods. Detailed Description

[0042] The present disclosure provides a supplement that includes the amino acids L - glutamine (also referred to herein as Gln or Q), L - leucine (also referred to herein as Leu or L), and L - arginine (also referred to herein as Arg or R). These amino acids can be present in the supplement as free amino acids (e.g., not covalently linked to other amino acids to form a protein), as salts of the amino acids, or as a mixture of both the free form and the salt form of the amino acids. Thus, the glutamine in the supplement can be entirely in the free form, entirely in the salt form, or a mixture of both; the leucine in the supplement can be entirely in the free form, entirely in the salt form, or a mixture of both; and the arginine in the supplement can be entirely in the free form, entirely in the salt form, or a mixture of both. Unless the context indicates otherwise, amino acids referred to herein include free amino acids and salts of the amino acid. The amino acids glutamine, leucine, and arginine and their salt forms are readily available.

[0043] In one embodiment, these three amino acids are present in the supplement of the present disclosure in known ratios. In one embodiment, the ratios of these three amino acids are based on the molar ratios (mole ratios) of each amino acid.

[0044] The molar ratio of glutamine to leucine and arginine can vary between 2 and 4, including 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4. The molar ratio of leucine to glutamine and arginine can vary between 0.5 and 2, including 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2. The molar ratio of arginine to glutamine and leucine can vary between 0.5 and 3, including 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3. Non-limiting examples of the Q:L:R molar ratios that can be present in the supplements of the present disclosure include 2:1:1, 4:1:1, 3:0.5:1, 3:1.5:1, 3:1:0.5, and 3:1:1.5. In one embodiment, the ratio of Q:L:R is 3:1:1.

[0045] Typically, to determine the amount of each amino acid required to achieve the desired ratio, the number of moles of each amino acid can be converted to mass and the amounts of these amino acids can be combined. For example, when the ratio of Q:L:R is 3:1:1, 44 grams of L-glutamine, 13 grams of L-leucine, and 17 grams of L-arginine can be combined to produce the correct amino acid ratio.

[0046] The supplement also includes active insulin-like growth factor 1 (IGF-1, also referred to herein as IGF). One skilled in the art can readily determine whether a protein is IGF-1. For example, polyclonal and monoclonal antibodies that specifically bind to IGF-1 are commercially available and specifically react with IGF-1 from various species, including humans, horses, dogs, cows, swine, sheep, and fowl. These readily available antibodies lack cross-reactivity and / or interference from other closely related proteins and binding proteins. Single antibodies or groups of antibodies that recognize different regions of IGF (such as the N-terminus, C-terminus, or amino acids present between the two ends of the protein) can be used to determine whether a protein is an IGF-1 protein. Methods for determining whether an IGF-1 protein is active are known in the art and are conventional.

[0047] IGF is a protein that has a high degree of sequence similarity to insulin, but unlike insulin, IGF associates with different binding proteins present in serum and other biological fluids (Baxter, 2000, Am J Physiol Endocrinol Metab, 278:E967-E976; Hwa et al., 1999, Endocrine Reviews, 20(6):761-787). Most of the IGF present in products derived from animals (such as but not limited to blood and blood-derived products, milk and milk-derived products, and colostrum and colostrum-derived products) is bound to binding proteins. However, since these binding proteins inhibit the activity of IGF, most of the IGF present in animal-derived products is inactive due to its binding to binding proteins. For example, less than 1% of IGF-1 in plasma is not bound to binding proteins (Carel et al., Safety of Recombinant Human Growth Hormone, in: Current Indications for Growth Hormone Therapy, 2nd revised edition, volume editor: Hindmarsh, Karger Publishers, Switzerland, page 48).

[0048] If IGF is not bound to a binding protein, it is considered active, and if IGF is bound to a binding protein, it is considered inactive. Active IGF is commonly referred to in the art as free, unbound, bioactive, and / or active. Methods for measuring the concentration of active IGF are known to those skilled in the art and are routine. Assays (including solid-phase sandwich ELISA assays) are commercially available and allow the measurement of IGF that is not bound to binding proteins (e.g., R&D Systems, Minneapolis, Minnesota, catalog number DG100).

[0049] IGF that can be used in the supplements and methods of the present disclosure can be obtained from various sources. In one embodiment, the source is a natural source, such as a biological material from an animal. Examples of animals include but are not limited to vertebrates, which include but are not limited to bovine, porcine, avian, equine, and ovine animals. The amino acid sequences of IGF from different animals are highly conserved, and thus IGF from one type of animal typically has biological activity in other animals. For example, these examples show that porcine IGF functions in other animals, including chickens, turkeys, and fish, such as sea bass.

[0050] Examples of biomaterials that can be used as a source of IGF include, but are not limited to, blood and blood-derived products (e.g., whole blood, red blood cells, plasma and its derivatives); milk and milk products (e.g., liquid milk, milk powder, cheese, whey and whey products, curd, cheese, casein, lactose, milk fat and its derivatives); colostrum and colostrum-derived products (e.g., liquid colostrum, dried colostrum); eggs and egg-derived products (e.g., egg yolk, egg white, egg membranes), body fluids (e.g., saliva, semen), and tissues (e.g., mucosal tissue, intestinal tissue, embryonic tissue). Biomaterials that can be used to produce supplements with active IGF are readily available commercially.

[0051] In one embodiment, the IGF that can be used in the methods described herein is produced using recombinant techniques or chemically or enzymatically synthesized. Recombinant production can be accomplished in substantially any expression system, including but not limited to prokaryotic systems, such as bacteria (e.g., Escherichia coli) and eukaryotic (e.g., yeast) systems. Polynucleotide sequences encoding active IGF are readily available, and methods for producing recombinant IGF are known and conventional. Typically, IGF produced using recombinant techniques or chemically or enzymatically synthesized is active because it has not been exposed to binding proteins.

[0052] In one embodiment, the amount of the amino acid glutamine in the supplement can be at least 0.5 grams of glutamine per kilogram (g / kg) of supplement, at least 5 g / kg, at least 10 g / kg, at least 20 g / kg, at least 30 g / kg, at least 40 g / kg, at least 50 g / kg, or at least 60 g / kg of supplement. In one embodiment, the amount of the amino acid glutamine in the supplement can be no greater than 500 g / kg of supplement, no greater than 400 g / kg, no greater than 300 g / kg, no greater than 200 g / kg, no greater than 100 g / kg, no greater than 80 g / kg, no greater than 60 g / kg, no greater than 50 g / kg, no greater than 40 g / kg, or no greater than 30 g / kg of supplement. For example, the amount of glutamine in the supplements of the present disclosure can be in the range from 0.5 g / kg to 500 g / kg, 10 g / kg to 100 g / kg, 20 g / kg to 80 g / kg, or 30 g / kg to 60 g / kg.

[0053] In one embodiment, the amount of the amino acid leucine in the supplement can be at least 0.1 g leucine / kg supplement, at least 1 g / kg, at least 5 g / kg, at least 10 g / kg, at least 15 g / kg, at least 20 g / kg, or at least 25 g / kg supplement. In one embodiment, the amount of the amino acid leucine in the supplement can be no greater than 100 g / kg supplement, no greater than 75 g / kg, no greater than 50 g / kg, no greater than 40 g / kg, no greater than 30 g / kg, no greater than 25 g / kg, no greater than 20 g / kg, no greater than 15 g / kg, or no greater than 10 g / kg supplement. For example, the amount of leucine in the supplement of the present disclosure can be in the range from 0.1 g / kg to 100 g / kg, 1 g / kg to 75 g / kg, or 5 g / kg to 50 g / kg.

[0054] In one embodiment, the amount of the amino acid arginine in the supplement can be at least 0.1 g arginine / kg supplement, at least 1 g / kg, at least 10 g / kg, at least 20 g / kg, at least 30 g / kg, at least 40 g / kg, or at least 50 g / kg supplement. In one embodiment, the amount of the amino acid arginine in the supplement can be no greater than 200 g / kg supplement, no greater than 100 g / kg, no greater than 75 g / kg, no greater than 50 g / kg, no greater than 40 g / kg, no greater than 30 g / kg, no greater than 20 g / kg, no greater than 10 g / kg, or no greater than 1 g / kg supplement. For example, the amount of arginine in the supplement of the present disclosure can be in the range from 0.1 g / kg to 200 g / kg or 1 g / kg to 100 g / kg.

[0055] In one embodiment, the amount of active IGF-1 in the supplement can be at least 100 nanograms per kilogram (ng / kg) of the supplement, at least 200 μg / kg, at least 300 μg / kg, at least 400 μg / kg, at least 500 μg / kg, at least 600 μg / kg, or at least 700 μg / kg of the supplement. In one embodiment, the amount of active IGF-1 in the supplement can be no greater than 2000 micrograms per kilogram (μg / kg) of the supplement, no greater than 1750 μg / kg, no greater than 1500 μg / kg, no greater than 1250 μg / kg, no greater than 1000 μg / kg, no greater than 800 μg / kg, no greater than 700 μg / kg, no greater than 600 μg / kg, no greater than 500 μg / kg, no greater than 400 μg / kg, no greater than 300 μg / kg, or no greater than 200 μg / kg of the supplement. In one embodiment, the amount of IGF-1 in the supplement of the present disclosure can be in the range from 100 μg / kg to 2000 μg / kg, 200 μg / kg to 1750 μg / kg, 300 μg / kg to 1500 μg / kg, or 400 μg / kg to 1250 μg / kg.

[0056] The supplement of the present disclosure can also include other components, including but not limited to cereals, proteins, vitamins, minerals, preservatives, antibiotics, pigments, stabilizers (including heat stabilizers), and other additives. In one embodiment, the supplement includes yeast. In one embodiment, the component includes an extract of cells expressing IGF. For example, if recombinant IGF is expressed in yeast, the yeast containing IGF can be lysed and the resulting extract can be used. In one embodiment, the component can be inactive.

[0057] In one embodiment, the supplement includes 30 g / kg to 60 g / kg (e.g., 44 g / kg) of glutamine, 5 g / kg to 50 g / kg (e.g., 13 g / kg) of leucine, 1 g / kg to 100 g / kg (e.g., 17 g / kg) of arginine, 300 μg / kg to 1500 μg / kg (e.g., 450 μg / kg) of active IGF, and yeast (e.g., 93 wt%).

[0058] In one embodiment, the supplement includes active IGF produced using a recombinant system, such as a prokaryotic expression system (e.g., Escherichia coli) or a eukaryotic system (e.g., yeast). The IGF can be from any source, such as horse, dog, bovine, porcine, sheep, or avian. In one embodiment, the recombinant IGF is porcine IGF.

[0059] In one embodiment, the supplement of the present disclosure is used as a supplement for adding to the food or water of an animal. Thus, the supplement of the present disclosure can take any form that can be used for adding to food or water. For example, the supplement can be in the form of a liquid, an emulsion, a powder (e.g., spray-dried powder), a cake, a meal, a pill, a crumb, a granule, etc.

[0060] The present disclosure also provides a composition comprising the supplement. In one embodiment, the composition is a food. As used herein, "food" is a compound or mixture of compounds that can be consumed by an animal and provides nutrition for the animal. In one embodiment, the food is feed for use by an animal, e.g., for feeding domesticated animals such as companion animals (including but not limited to canines and felines) and livestock, which include but are not limited to cattle, swine, poultry, horses, and ovine animals. In another embodiment, the animal is an aquatic animal used in aquaculture, such as but not limited to fish (including sea bass, shrimp, or eels). The food can be solid, semi-solid, or liquid. A variety of foods available for administration to animals are obtainable. In one embodiment, the food is a food designed for feeding animals for meat production. In one embodiment, the food is a food designed for feeding animals for egg production. In one embodiment, the supplement is added to the water source of the animal.

[0061] The amount of the supplement of the present disclosure added to a composition (such as food) is sufficient for use in the methods described herein.

[0062] In one embodiment, the amount of the supplement added to food (such as animal feed) or water is at least 0.0000005 grams per ton (g / ton) of food, at least 0.000005 g / ton, at least 0.00005 g / ton, at least 0.0005 g / ton, or at least 0.005 g / ton. In one embodiment, the amount of the supplement added to food is no greater than 0.05 g / ton of supplement, no greater than 0.005 g / ton, no greater than 0.0005 g / ton, no greater than 0.00005 g / ton, or no greater than 0.000005 g / ton. In one embodiment, the amount of the supplement of the present disclosure in the food can be in the range from 0.000005 g / ton to 0.005 g / ton, 0.00005 g / ton to 0.005 g / ton, or 0.00005 g / ton to 0.0005 g / ton.

[0063] In one embodiment, the amount of the supplement added to the food is at least 5 grams per ton (g / ton) of food, at least 25 g / ton, at least 50 g / ton, at least 100 g / ton, at least 250 g / ton, at least 500 g / ton, or at least 750 g / ton. In one embodiment, the amount of the supplement added to the food is not greater than 2750 g / ton of the supplement, not greater than 2600 g / ton, not greater than 2400 g / ton, not greater than 2250 g / ton, not greater than 2000 g / ton, not greater than 1750 g / ton, not greater than 1500 g / ton, not greater than 1250 g / ton, not greater than 1000 g / ton, not greater than 750 g / ton, not greater than 500 g / ton, not greater than 250 g / ton, not greater than 100 g / ton, or not greater than 50 g / ton. In one embodiment, the amount of the supplement disclosed herein in the food can be in the range from 5 g / ton to 2750 g / ton or from 50 g / ton to 2600 g / ton.

[0064] In one embodiment, the amount of the amino acid glutamine in the food can be at least 0.0000002 wt% of the food, at least 0.000002 wt% of the food, at least 0.00002 wt% of the food, or at least 0.0002 wt% of the food. In one embodiment, the amount of the amino acid glutamine in the food can be not greater than 0.1 wt% of the supplement, not greater than 0.01 wt% of the food, or not greater than 0.001 wt% of the food. In one embodiment, the amount of glutamine in the food disclosed herein can be in the range from 0.0000002 wt% to 0.1 wt%, from 0.000002 wt% to 0.1 wt%, from 0.00002 wt% to 0.1 wt%, from 0.0002 wt% to 0.1 wt%, from 0.002 wt% to 0.1 wt%, or from 0.02 wt% to 0.1 wt%.

[0065] In one embodiment, the amount of the amino acid leucine in the food can be at least 0.00000006 wt% of the food, at least 0.0000006 wt% of the food, at least 0.000006 wt% of the food, at least 0.00006 wt% of the food, or at least 0.0006 wt% of the food. In one embodiment, the amount of the amino acid leucine in the food can be not greater than 0.03 wt% of the supplement, not greater than 0.003 wt% of the food, or not greater than 0.0003 wt% of the food. In one embodiment, the amount of leucine in the food disclosed herein can be in the range from 0.00000006 wt% to 0.03 wt%, from 0.0000006 wt% to 0.03 wt%, from 0.000006 wt% to 0.03 wt%, from 0.00006 wt% to 0.03 wt%, from 0.0006 wt% to 0.03 wt%, or from 0.006 wt% to 0.03 wt%.

[0066] In one embodiment, the amount of the amino acid arginine in the food can be at least 0.00000006 wt%, at least 0.0000006 wt%, at least 0.000006 wt%, at least 0.00006 wt% or at least 0.0006 wt% of the food. In one embodiment, the amount of the amino acid arginine in the food can be no greater than 0.06 wt% of the supplement, no greater than 0.006 wt% of the food, no greater than 0.0006 wt% of the food. In one embodiment, the amount of arginine in the food disclosed herein can be in the range from 0.00000006 wt% to 0.06 wt%, from 0.0000006 wt% to 0.06 wt%, from 0.000006 wt% to 0.06 wt%, from 0.00006 wt% to 0.06 wt%, from 0.0006 wt% to 0.06 wt% or from 0.006 wt% to 0.06 wt%.

[0067] In one embodiment, the total weight percentage (wt%) of L-glutamine or its salt, L-leucine or its salt, and L-arginine or its salt added through the supplement in the composition is less than the total wt% of any other free natural amino acid or its salt in the composition. In one embodiment, the total weight percentage (wt%) of L-glutamine or its salt, L-leucine or its salt, and L-arginine or its salt added through the supplement in the composition is less than the total wt% of the combination of other free natural amino acids or their salts in the composition.

[0068] The present disclosure also provides a method of using the supplement described herein. In one embodiment, the method includes manufacturing the food described herein. The method includes combining the supplement with the food in an amount as described herein.

[0069] In one embodiment, the method includes administering the food to an animal. Administering includes making the food available to the animal. Administration can be started as soon as possible after birth. For example, the food can be provided immediately after the birth of a non-mammal (such as poultry). In the case of mammals, the food can be provided when the animal is weaned. The food can be administered to the animal throughout its life cycle or at different stages (such as before weaning, after weaning, before adulthood, and / or adulthood). In some embodiments, the food can be administered as one or more diets. The diets of livestock can be divided into diets provided to the animal at different times during its life, and are commonly referred to in the art as pre-starter diets, starter diets, grower diets, and finisher diets. In one embodiment, the food can be available ad libitum.

[0070] In one embodiment, when the animal is a porcine (such as a piglet, sow, gilt, and / or boar), the supplement in the composition is present in an amount ranging from 10 grams of supplement per ton of feed to 2.75 kg / ton, or from 100 g / ton of feed to 2.5 kg / ton. In one embodiment, when the animal is a turkey, the supplement in the composition is present in an amount ranging from 50 grams of supplement per ton of feed to 1200 g / ton, or from 100 g / ton to 600 g / ton. In one embodiment, when the animal is a laying hen, the supplement in the composition is present in an amount ranging from 30 grams of supplement per ton of feed to 90 g / ton, or from 50 g / ton to 70 g / ton. In one embodiment, when the animal is a broiler, the supplement in the composition is present in an amount ranging from 25 grams of supplement per ton of feed to 600 g / ton, or from 50 g / ton to 300 g / ton. In one embodiment, when the animal is a fish (such as Asian sea bass), the supplement in the composition is present in an amount ranging from 0.000050 grams of supplement per ton of feed to 0.0005 g / ton, or from 0.000050 g / ton to 0.00045 g / ton.

[0071] In one embodiment, the method is for improving animal performance, improving animal processing factor, increasing animal gut health factors, or a combination thereof. In one embodiment, the animal is livestock housed in a high-stress environment. As used herein, "high-stress environment" refers to an environment that is not optimal for animal growth. Examples of high-stress environmental conditions include, but are not limited to, undesirable environmental temperatures (too high or too low), and the presence of pathogens in the environment. As used herein, "low-stress environment" refers to an environment that is optimal for animal growth. Optimal environmental conditions for animal growth are known to those of ordinary skill in the art.

[0072] Examples of performance include, but are not limited to, increased body weight, improved feed conversion rate, reduced mortality, and improved flock uniformity (such as as measured by, for example, weight variation). Typically, to determine whether there is a change in body weight, feed conversion rate, mortality, flock uniformity, immune response, gut health, and / or the ability to respond to systemic stress, a group of animals receiving the supplement is compared to a group of animals not receiving the supplement. In one embodiment, the method of the present disclosure results in an increase in body weight of an animal compared to an animal in a similar environment but not fed a food containing the supplement. In one embodiment, the method of the present disclosure results in an improvement in feed conversion rate of an animal compared to an animal in a similar environment but not fed a food containing the supplement. In one embodiment, the method of the present disclosure results in a reduction in mortality of an animal compared to an animal in a similar environment but not fed a food containing the supplement. In one embodiment, the method of the present disclosure results in an improvement in flock uniformity of an animal compared to an animal in a similar environment but not fed a food containing the supplement.

[0073] In one embodiment, compared to animals in a similar environment but not fed a food containing a supplement, there are statistically significant changes in the increase in animal body weight, the improvement in feed conversion rate, the improvement in herd uniformity, the reduction in mortality, the immune response, intestinal health, and / or the ability to respond to systemic stress. In one embodiment, compared to animals in a similar environment but not fed a food containing a supplement, the increase in body weight, the improvement in feed conversion rate, the improvement in herd uniformity, the reduction in mortality, the immune response, intestinal health, and / or the ability to respond to systemic stress have a change of at least 0.1%, at least 0.25%, at least 0.5%, at least 1%, at least 3%, at least 5%, at least 7%, at least 9%, at least 11%, or at least 13%.

[0074] Examples of processing coefficients include, but are not limited to, the percentage of chilled carcass yield and total breast meat yield relative to live weight. Typically, the change in chilled carcass yield and / or the increase in total breast meat yield are determined by comparing a group of animals receiving the supplement to a group of animals not receiving the supplement. In one embodiment, the methods of the present disclosure result in an increase in chilled carcass yield in animals compared to animals in a similar environment but not fed a food containing a supplement. In one embodiment, the methods of the present disclosure result in an increase in the percentage of total breast meat yield relative to live weight in animals compared to animals in a similar environment but not fed a food containing a supplement.

[0075] In one embodiment, compared to animals in a similar environment but not fed a food containing a supplement, there are statistically significant changes in the increase in chilled carcass yield and / or the increase in the percentage of total breast meat yield relative to live weight. In one embodiment, compared to animals in a similar environment but not fed a food containing a supplement, the increase in chilled carcass yield and / or the increase in the percentage of total breast meat yield relative to live weight in animals have a change of at least 0.1%, at least 0.25%, at least 0.5%, at least 1%, at least 3%, at least 5%, at least 7%, at least 9%, at least 11%, or at least 13%.

[0076] Examples of gut health factors include, but are not limited to, villus development, gut lesions, gut bacteria, and diarrhea. Typically, changes in villus development, gut lesions, gut bacteria, and / or diarrhea are determined by comparing a population of animals receiving a supplement to a population of animals not receiving the supplement. In one embodiment, the methods of the present disclosure result in an increase in villus cell height and / or an increase in crypt depth in an animal compared to an animal in a similar environment that is not fed a food containing the supplement. In one embodiment, the methods of the present disclosure result in a decrease in gut lesions in an animal compared to an animal in a similar environment that is not fed a food containing the supplement. In one embodiment, the methods of the present disclosure result in a decrease in gut bacteria (e.g., Escherichia coli, Salmonella, and / or Clostridium) in an animal compared to an animal in a similar environment that is not fed a food containing the supplement. In one embodiment, the methods of the present disclosure result in a decrease in diarrhea in an animal compared to an animal in a similar environment that is not fed a food containing the supplement.

[0077] In one embodiment, the change in villus development, the decrease in gut lesions, and / or the decrease in gut bacteria are statistically significant changes compared to an animal in a similar environment that is not fed a food containing the supplement. In one embodiment, the change in villus development, the decrease in gut lesions, and / or the decrease in gut bacteria are changes of at least 0.1%, at least 0.25%, at least 0.5%, at least 1%, at least 3%, at least 5%, at least 7%, at least 9%, at least 11%, or at least 13% compared to an animal in a similar environment that is not fed a food containing the supplement.

[0078] In one embodiment, a food containing the supplement is provided to an animal (such as a sow) after weaning of the offspring until estrus. The food can shorten the interval between weaning and estrus. The shortening of the interval between weaning and estrus can be a change of at least 0.1%, at least 0.25%, at least 0.5%, at least 1%, at least 3%, at least 5%, at least 7%, at least 9%, at least 11%, or at least 13% compared to an animal in a similar environment that is not fed a food containing the supplement, or can be at least 0.5, at least 1, at least 1.5, or at least 2 days compared to an animal in a similar environment that is not fed a food containing the supplement.

[0079] In one embodiment, a food containing a supplement is provided to an animal (such as a piglet). Adding the supplement to the diet can result in an increase in average daily gain, an increase in body weight at day 126, a reduction in fecal diarrhea, and / or an improvement in feed conversion rate. Compared to animals in a similar environment but not fed the food containing the supplement, the increase in average daily gain, the increase in body weight (such as body weight at day 126 after weaning), the reduction in fecal diarrhea, and / or the improvement in feed conversion rate can be a change of at least 0.1%, at least 0.25%, at least 0.5%, at least 1%, at least 3%, at least 5%, at least 7%, at least 9%, at least 11%, or at least 13%.

[0080] In one embodiment, a food containing a supplement is provided to an animal (such as an egg-laying avian, such as a laying hen). Adding the supplement to the diet can result in an increase in body weight, an improvement in health as measured by a reduction in intestinal lesions and / or an increase in the percentage of fat pads in body weight, an increase in egg production, an increase in the total number of eggs produced, and / or an improvement in feed conversion rate. Surprisingly, the increase in egg production and the increase in the total number of eggs produced do not result in a decrease in egg weight and eggshell weight. Compared to animals in a similar environment but not fed the food containing the supplement, the increase in body weight, the improvement in health as measured by a reduction in intestinal lesions and / or an increase in the percentage of fat pads in body weight, the increase in egg production, the increase in the total number of eggs produced, and / or the improvement in feed conversion rate can be a change of at least 0.1%, at least 0.25%, at least 0.5%, at least 1%, at least 3%, at least 5%, at least 7%, at least 9%, at least 11%, or at least 13%.

[0081] In one embodiment, a food containing a supplement is provided to an animal (such as a fish, such as Asian sea bass). Adding the supplement to the diet can result in an increase in immune response (as indicated by a higher hematocrit level), a higher serum protein concentration, an improvement in intestinal health (as indicated by a higher lysozyme concentration), and / or a higher responsiveness to systemic stress (as indicated by an increased serum cortisol concentration). Compared to animals in a similar environment but not fed the food containing the supplement, the increase in immune response, the higher serum protein concentration, the improvement in intestinal health, and / or the higher responsiveness to systemic stress can be a change of at least 0.1%, at least 0.25%, at least 0.5%, at least 1%, at least 3%, at least 5%, at least 7%, at least 9%, at least 11%, or at least 13%.

[0082] Exemplary embodiments

[0083] Example 1. A supplement comprising free amino acid L-glutamine or a salt thereof, L-leucine or a salt thereof, and L-arginine or a salt thereof and active IGF-1, wherein the L-glutamine or a salt thereof, L-leucine or a salt thereof, and L-arginine or a salt thereof are present in a ratio of 2-4:1:1, 3:0.5-2:1, or 3:1:0.5-3.

[0084] Example 2. The supplement as described in Example 1, wherein at least one of the L-glutamine, L-leucine, and L-arginine is in the form of a salt.

[0085] Example 3. The supplement as described in any one of Examples 1-2, wherein the amount of L-glutamine or its salt is at least 0.5 grams to no more than 500 grams of amino acid per kilogram of supplement.

[0086] Example 4. The supplement as described in any one of Examples 1-3, wherein the amount of L-leucine or its salt is at least 0.1 grams to no more than 100 grams of amino acid per kilogram of supplement.

[0087] Example 5. The supplement as described in any one of Examples 1-4, wherein the amount of L-arginine or its salt is at least 0.1 grams to no more than 200 grams of amino acid per kilogram of supplement.

[0088] Example 6. The supplement as described in any one of Examples 1-5, wherein the amount of active IGF-1 is at least 100 micrograms to no more than 2000 micrograms of active IGF-1 per kilogram of supplement.

[0089] Example 7. The supplement as described in any one of Examples 1-6, wherein the amount of L-glutamine or its salt is at least 0.5 grams to no more than 500 grams of amino acid per kilogram of supplement, the amount of L-leucine or its salt is at least 0.1 grams to no more than 100 grams of amino acid per kilogram of supplement, the amount of L-arginine or its salt is at least 0.1 grams to no more than 200 grams of amino acid per kilogram of supplement, and the amount of active IGF-1 is at least 100 grams to no more than 2000 micrograms of IGF-1 per kilogram of supplement.

[0090] Example 8. The supplement as described in any one of Examples 1-7, wherein the active IGF-1 is recombinant IGF-1.

[0091] Example 9. The supplement as described in any one of Examples 1-8, which further comprises food.

[0092] Example 10. A food product, which comprises the supplement as described in any one of Examples 1-9, and the supplement is present in the food product in an amount of at least 0.000005 g / ton and no more than 0.005 g / ton.

[0093] Example 11. A food product, which comprises the supplement as described in any one of Examples 1-9, and the supplement is present in the food product in an amount of at least 50 grams per ton (g / ton) of the food product and no more than 2600 g / ton.

[0094] Example 12. The food as described in Example 10 or 11, wherein the total weight percentage (wt%) of the L-glutamine or its salt, L-leucine or its salt, and L-arginine or its salt is less than the total wt% of the other amino acids in the supplement.

[0095] Example 13. The food as described in any one of Examples 10 - 12, wherein one, two, or all three of the L-glutamine, L-leucine, and L-arginine are in the form of a salt.

[0096] Example 14. The food as described in any one of Examples 10 - 13, wherein the amount of L-glutamine or its salt is at least 0.0000002 wt%.

[0097] Example 15. The food as described in any one of Examples 10 - 14, wherein the amount of L-leucine or its salt is at least 0.00000006 wt%.

[0098] Example 16. The food as described in any one of Examples 10 - 15, wherein the amount of L-arginine or its salt is at least 0.00000006 wt%.

[0099] Example 17. A method comprising administering to an animal an effective amount of (i) a food comprising a supplement as described in any one of Examples 1 - 9, or (ii) a food as described in any one of Examples 10 - 16.

[0100] Example 18. A method for improving animal performance, the method comprising administering to the animal (i) a food comprising a supplement as described in any one of Examples 1 - 9 or (ii) a food as described in any one of Examples 10 - 16, wherein the improved performance comprises an increase in body weight, an improvement in feed conversion rate, a decrease in mortality, an improvement in herd uniformity, or a combination thereof.

[0101] Example 19. A method for improving animal processing coefficient, the method comprising administering to the animal (i) a food comprising a supplement as described in any one of Examples 1 - 9 or (ii) a food as described in any one of Examples 10 - 16, wherein the improved processing coefficient is an increase in chilled carcass yield, an increase in total breast meat yield, or a combination thereof.

[0102] Example 20. A method for improving animal intestinal health, the method comprising administering to the animal (i) a food comprising a supplement as described in any one of Examples 1 - 9 or (ii) a food as described in any one of Examples 10 - 16, wherein the improved intestinal health is a reduction in intestinal bacteria, a reduction in intestinal lesions, a reduction in fecal diarrhea, or a combination thereof.

[0103] Example 21. The method according to any one of Examples 17 to 20, wherein the food comprises a supplement in an amount of at least 50 g / ton.

[0104] Example 22. The method according to any one of Examples 17 to 21, wherein the animal is a porcine animal or an avian animal.

[0105] Example 23. The method according to any one of Examples 17 to 22, wherein the animal is housed in a high-stress environment.

[0106] Example 24. A method of shortening the time from weaning of offspring to re-estrus, the method comprising administering to the animal (i) a food comprising a supplement as described in any one of Examples 1-9 or (ii) a food as described in any one of Examples 10-16, wherein the time from weaning of offspring to re-estrus is shortened compared to an animal in a similar environment but not fed the composition.

[0107] Example 25. The method according to Example 24, wherein the animal is a sow.

[0108] Example 26. The method according to Example 24 or 25, wherein the supplement is present at 100 grams of supplement / ton of food to 2.5 kg / ton.

[0109] Example 27. A method of increasing average daily gain, increasing body weight, reducing fecal diarrhea, and / or improving feed conversion rate in an animal, the method comprising administering to the animal (i) a food comprising a supplement as described in any one of Examples 1-9 or (ii) a food as described in any one of Examples 10-16, wherein the increase in average daily gain, increase in body weight, reduction in fecal diarrhea, and / or improvement in feed conversion rate is a change compared to an animal in a similar environment but not fed a food containing the supplement.

[0110] Example 28. The method according to Example 27, wherein the animal is a piglet.

[0111] Example 29. The method according to Example 27 or 28, wherein the supplement is present at 100 grams of supplement / ton of food to 2.5 kg / ton.

[0112] Example 30. A method for increasing body weight, improving health as measured by reduction of intestinal lesions and / or increase in the percentage of body weight accounted for by fat pads, increasing egg production, increasing total egg production, and / or improving feed conversion rate in laying poultry, the method comprising administering to the animal (i) a foodstuff comprising a supplement as described in any one of Examples 1-9 or (ii) a foodstuff as described in any one of Examples 10-16, wherein the increase in body weight, improvement in health as measured by reduction of intestinal lesions and / or increase in the percentage of body weight accounted for by fat pads, increase in egg production, increase in total egg production, and / or improvement in feed conversion rate is a change compared to an animal in a similar environment but not fed a foodstuff containing the supplement.

[0113] Example 31. The method according to Example 30, wherein the laying poultry is a chicken.

[0114] Example 32. The method according to Example 30 or 31, wherein the supplement is present at 30 grams of supplement / ton of food to 90 g / ton.

[0115] Example 33. A method for increasing hematocrit level, increasing serum protein concentration, increasing lysozyme concentration, and / or increasing serum cortisol concentration in fish, the method comprising administering to the animal (i) a foodstuff comprising a supplement as described in any one of Examples 1-9 or (ii) a foodstuff as described in any one of Examples 10-16, wherein the increase in hematocrit level, increase in serum protein concentration, increase in lysozyme concentration, and / or increase in serum cortisol concentration is a change compared to an animal in a similar environment but not fed a foodstuff containing the supplement.

[0116] Example 34. The method according to Example 33, wherein the fish is Asian sea bass.

[0117] Example 35. The method according to Example 33 or 34, wherein the supplement is present at 0.000050 grams of supplement / ton of food to 0.0005 g / ton.

[0118] Examples

[0119] The present disclosure is illustrated by the following examples. It should be understood that the specific examples, materials, amounts, and procedures should be broadly interpreted in accordance with the scope and spirit of the present disclosure as set forth herein.

[0120] Example 1

[0121] Combined effects of IGF-1, L-glutamine, L-leucine, and L-arginine on skeletal muscle-driven growth in avian and porcine species

[0122] The use of bioactive amino acids (AAs) and insulin-like growth factor-1 (IGF-1) as individual AAs or in specific combinations has been studied in depth. Amino acid supplementation typically includes essential amino acids, with lysine and methionine most commonly used as supplements in commercial livestock feeds. This report presents a surprising finding that a combination of two non-essential amino acids, L-glutamine and L-arginine, along with L-leucine and active IGF-1 can be used in commercial production of livestock species.

[0123] Materials and Methods

[0124] Porcine and avian satellite cells were isolated as previously described (Vaughn et al., 2017). Porcine satellite cells (SCs) were plated at a density of 5000 cells / cm 2 and cultured in growth medium (GM) for 3 days to allow the SCs to reach 70% confluence. The growth medium consisted of high-glucose Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% (v / v) fetal bovine serum (GE Healthcare, Pittsburgh, PA), 100 U penicillin / mL, 100 μg streptomycin / mL, and 20 μg gentamicin / mL. At this point, the medium was replaced with differentiation medium (DM) for 4 days to induce myotube development. The differentiation medium consisted of low-glucose Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 2% (v / v) fetal bovine serum (GE Healthcare, Pittsburgh, PA), 100 U penicillin / mL, 100 μg streptomycin / mL, and 20 μg gentamicin / mL. At this point, differentiated SC treatments were applied in DM for a treatment period of 72 hours as described below.

[0125] The first series of experiments was conducted on porcine satellite cells, with the negative control consisting of DM. The supplementation of yeast broth (5 ng / mL, active IGF-1 produced by yeast), L-leucine (1 mM), L-arginine (1 mM), L-glutamine (4 mM), L-lysine (10 mM), and L-methionine (10 μM) was evaluated by adding them to DM, with the doses based on those reported in the literature. The data are as Figure 1 shown. Based on these results, subsequent experiments were conducted by combining glutamine, arginine, and leucine at the same doses used in the first experiment. The data are as Figure 2 shown. The next series of experiments was conducted by titrating each individual amino acid into the amino acid combination to determine the useful concentration of each amino acid, as Figures 3 - 7 outlined.

[0126] In previous experiments, the concentration and inclusion of amino acids were determined to be 1 mM L-leucine, 1 mM arginine, 3 mM glutamine, added to DM containing 5 ng / mL of yeast-produced active IGF-1. The key cell signaling pathway as a possible mode of action here is mammalian target of rapamycin (mTOR) pathway signaling. To test whether this is the mode of action of this amino acid combination, rapamycin was added to the culture medium, and the data are as Figure 8 outlined.

[0127] To evaluate cross-species efficacy, experiments were conducted on avian satellite cells. For avian satellite cells, the control consisted of DM, and the treatment contained 5 ng / mL of yeast-produced IGF-1; or a combination of 5 ng / mL of yeast-produced IGF-1, L-leucine (1 mM), L-arginine (1 mM), L-glutamine (3 mM), and each amino acid was evaluated individually by adding the amino acid to DM, and the data are as Figure 9 shown.

[0128] Results

[0129] As Figure 1 outlined, compared with the control treatment, active IGF-1 and individual amino acids were able to increase the size of myotubes, and L-glutamine, L-leucine, and L-arginine had a stronger growth response of myotubes compared with L-methionine or L-lysine. This finding led to the next experiment, in which a combination of yeast-produced active IGF-1, L-glutamine, L-leucine, and L-arginine was applied to satellite cells. This specific combination of active IGF-1 and amino acids led to an additive response in myotube growth.

[0130] The next series of experiments aimed to determine the useful inclusion amount of each individual amino acid. The useful inclusion amount of each amino acid is the lowest amount of each amino acid required to produce the maximum growth response. The data collected are as Figures 3 - 5 outlined, and the mixture of amino acids is 3 mM L-glutamine, 1 mM L-leucine, and 1 mM L-arginine. To confirm that these amino acids are a useful amino acid combination, experiments were conducted by titrating L-methionine and L-lysine. As Figure 6 and Figure 7 shown, the addition of L-methionine and L-lysine did not increase the growth of myotubes. Finally, we hypothesized that the mode of action of this unique amino acid combination is by triggering mTOR pathway signaling, and this was confirmed because after adding rapamycin, the treated SC had a similar myotube size to the control culture ([[]] Figure 8 ).

[0131] Experiments conducted using avian satellite cells confirmed the evaluation of cross-species efficacy.Figure 9 The data outlined in Figure 9 show that each component that effectively improves the growth of porcine satellite cells also effectively improves the growth of avian satellite cells. In addition, a specific combination of 5 ng / mL of yeast-produced active IGF-1, 3 mM L-glutamine, 1 mM L-leucine, and 1 mM L-arginine provides an effective cellular response. Previous mechanistic studies using satellite cells as an animal growth model have shown that a combination containing active IGF-1, L-glutamine, L-leucine, and L-arginine will effectively promote growth when used as a feed supplement for pigs and chickens (Shappell et al., 2000; Vaughn et al., 2017).

[0132] References cited in Example 1

[0133] Shappell, N.W., V.J. Feil, D.J. Smith, G.L. Larsen, and D.C. McFarland. 2000. Response of C2C12 mouse and turkey skeletal muscle cells to the beta-adrenergic agonist ractopamine. Journal of animal science 78(3):699 - 708.

[0134] Vaughn, M., K. Phelps, and J. Gonzalez. 2017. In vitro supplementation with the porcine plasma product, stimulates activity of porcine fetal myoblasts and neonatal satellite cells in a divergent manner. Animal: an international journal of animal bioscience: 1 - 9.

[0135] Example 2

[0136] Evaluation of the effect of supplements on broiler performance

[0137] A 42-day feeding trial was conducted at a commercial research facility to determine the efficacy of a supplement (CT). Supplement CT contained 450 ng / g of yeast-derived active IGF-1, 93 wt% yeast, 4.4 wt% L-glutamine, 1.7 wt% L-arginine, and 1.3 wt% L-leucine. Chickens were grown in two different environments. One environment was a "low-stress" environment using clean wood shavings bedding, and the other was a "high-stress" environment where used wood shavings were obtained from a commercial farm that had experienced high mortality; the debris was also inoculated with coccidia oocysts and Clostridium to simulate a commercial environment challenge. The supplement was fed at the following rates in the diets: 300 g CT / ton of starter chick feed (days 0-14), 50 g CT / ton of grower chick feed (days 14-28), and 50 g CT / ton of finisher chick feed (days 28-42). Another group of chickens in the low-stress environment was fed the supplement only in the starter chick feed: 300 g of supplement / ton of starter chick feed.

[0138] Growing chickens in a high-stress environment had a negative impact on the overall performance of the chickens; however, the addition of CT successfully mitigated the hazards of the high-stress environment, and in many cases, the performance was equal to or better than that of chickens on the negative control diet (complete diet without supplement) in the low-stress environment. Due to the addition of CT, significant improvements in body weight and feed conversion rate were observed in chicks grown in both environments on day 42. Chickens fed CT grew more uniformly than chickens raised on their respective negative control diets, which is an important factor in the processing. The addition of CT significantly reduced the mortality rate of chickens in the severely stressed environment. The addition of CT significantly increased the total carcass yield and breast meat yield (both expressed as a percentage of carcass weight and total production). This was observed under both environmental conditions.

[0139] Problem description

[0140] The post-hatch period of broiler chickens is crucial for the development of the small intestine and adaptation to nutritional changes (Whitten et al., 2012). Additionally, during the post-hatch period, the immune system of broiler chickens is still immature, and the metabolic demands of skeletal muscle are greatest. Post-hatch nutrition may affect lifelong immunity and muscle metabolism. Restriction of energy and amino acid density during the first 14 days impairs performance and exacerbates muscle myopathy (Meloche et al., 2018), thus suggesting the importance of the first 14 days of life for skeletal muscle development. Furthermore, post-hatch feed restriction inhibits the myogenic signaling regulatory pathway (Velleman et al., 2010; Velleman et al., 2014). For many years, spray-dried plasma (SDP) protein has been widely used in the diet of nursery pigs. SDP contains functional proteins, including immunoglobulins and bioactive peptides, which may play a key role in cell signaling. Recently, studies have evaluated the use of SDP in poultry diets and have obtained positive results (Campbell et al., 2003; Bregendahl et al., 2005; Jamroz et al., 2012; Henn et al., 2013). These positive results include greater weight gain and significant benefits during exposure to pathogenic bacteria (King et al., 2005; Beski et al., 2015; Beski et al., 2016).

[0141] Materials and Methods

[0142] A study was conducted to evaluate the performance of broiler chickens fed a diet with a supplement (CT). The supplement CT was prepared containing 450 ng / g of yeast-derived IGF-1, 93% yeast, 4.4% L-glutamine, 1.7% L-arginine, and 1.3% L-leucine. The trial was conducted at a well-known commercial testing laboratory in the United States to simulate a commercial environment.

[0143] These products were evaluated in two environmental scenarios: a low-stress environment using clean wood shavings bedding and a severe stress environment where the bedding was obtained from a commercial farm that had previously experienced high mortality. Additionally, Clostridium and coccidia oocysts were dispersed in the bedding of these pens. CT was fed at the following rates: 300 g / ton in the starter (days 0 - 14) diet, 50 g / ton in the grower (days 14 - 28) diet, and 50 g / ton in the finisher (days 28 - 42) diet. A common basal diet was used as the carrier for the test article (Table 1). The diets were fortified with a nutritionally complete vitamin and trace mineral mixture. A common basal diet was prepared and aliquots were used when preparing the test diets.

[0144] Table 1. Composition (%) and nutritional analysis of the diets

[0145]

[0146]

[0147] The test period started on Day 0 (hatch day of chicks) of the trial and ended on Day 42 of the trial. Each pen contained 52 mixed-sex Cobb broilers (ratio 50:50), and 12 replicates were randomly assigned to each group. On Day 0, 66 chicks were placed in each pen, and the number was reduced to 52 after crop fill measurement of each chick and verification of sex by feather inspection on Day 1. No chickens were replaced during the remaining study. Chicks were observed daily for signs of abnormal growth patterns or health problems. All chickens were vaccinated against coccidiosis at the hatchery. No antibiotics were used throughout the trial.

[0148] Chickens were weighed at 1, 7, 14, 28, and 42 days of age, and feed consumption was determined. Salmonella incidence was tested (two males and two females per pen at 14 days, and five males and five females per pen at 42 days) to simulate the counts required by USDA / FSIS at processing. In addition, intestinal samples were collected from two males and two females per pen at 14 and 42 days. Samples were taken from two intestinal regions of each chicken, one region at the distal end of the duodenal loop and the second region approximately two inches in front of the Meckel's diverticulum. Lesion scores were determined by the method of Johnson and Reid (1970), and the numbers of various organisms were determined. Ileal villus height and crypt depth were measured. At 42 days of age, 10 chickens per pen were processed, and part yields were determined.

[0149] Data were analyzed using SAS 9.4. Grubb's test was used to observe and remove potential "outliers", which were not found in these studies. Treatments were listed as categorical fixed effects. Supplemental groups were compared to the untreated control group used as a reference. All data sets for each time period were analyzed using an ordinal regression model using the SAS statistical software package. Residual plots were examined to evaluate model fit. Statistical significance was determined as P ≤ 0.05.

[0150] Results and Discussion

[0151] The performance of broiler chickens is generally significantly affected by the growth environment (Table 2). Almost every production measurement is negatively affected by a severely stressful environment. However, adding CT at 300 g / ton in the starter diet and 50 g / ton in the grower and finisher diets (300 - 50 - 50) helps overcome the performance decline. By adding CT at 7, 14, 28, and 42 days of age, body weight was significantly improved. This improvement was particularly evident in chickens in the group subjected to severe environmental stress. In chickens grown in a low-stress environment, at 42 days of age, chickens fed CT were 175 g heavier than chickens fed the negative control. In chickens grown in a severely stressful environment, chickens fed CT were 262 g heavier than the corresponding negative control chickens. Chickens in the poor environment group fed CT were significantly heavier than chickens fed the negative control in the low-stress environment.

[0152] Table 2. Live performance of chickens fed betaGRO and supplements under different environmental conditions

[0153]

[0154]

[0155] 1 Twelve replicate pens per treatment, with 52 mixed-sex chickens per pen.

[0156] 2 Clean wood shavings bedding.

[0157] 3 Wood shavings from a chicken house with high mortality, inoculated with Clostridium spores and coccidial oocysts. 4 Supplement.

[0158] 5 EPEF = (grams of average weight gain per day x % survival rate) / (feed conversion rate x 10)

[0159] abc Means with a common superscript within a row are not significantly different (P < 0.05).

[0160] A similar response in feed conversion ratio was observed. The feed conversion ratio of chickens grown in a severe stress environment was significantly lower than that of the same group of chickens grown in a low stress environment. However, adding CT to the diet at 300 g / ton in the starter diet and 50 g / ton in the grower and finisher diets significantly improved the feed conversion ratio in both environmental scenarios. The feed conversion ratio of chickens fed CT in the low stress environment was 0.07 points lower than that of chickens fed the negative control from day 0 to 42. The feed conversion ratio of chickens fed CT in the severe stress environment was 0.14 and 0.13 points lower than that of chickens fed the negative control at day 42. There was no significant difference between chickens fed a diet with CT in a harsh environment and chickens fed the negative control in a low stress environment.

[0161] Mortality was greatly affected by the growth environment of the chickens. The mortality of chickens grown in a low stress environment was the lowest, and there was no significant difference between chickens with different diet treatments. However, as expected, the mortality of chickens grown in a severe stress environment was high. Adding CT significantly reduced the mortality of chickens grown in this environment by nearly half compared to chickens fed the negative control diet.

[0162] Flock uniformity is an important coefficient in the processing process and was significantly improved in both environments. The flock uniformity of chickens fed CT was significantly better than that of chickens fed the corresponding negative control. Severe environmental stress conditions reduced the flock uniformity of chickens fed the negative control diet, but had no significant effect on chickens fed a diet with CT.

[0163] The European Production Efficiency Factor (EPEF) is a formula that takes into account weight gain, feed conversion ratio, and mortality to arrive at a single value that can reflect the overall performance. Due to better weight gain, improved feed conversion ratio, and reduced mortality, the EPEF value of chickens fed a diet with CT was significantly higher than that of chickens fed the negative control diet under both environmental conditions.

[0164] The two environmental conditions of chicken growth and the inclusion or exclusion of CT significantly affected economically important processing coefficients (Table 3). For chickens grown under low stress conditions, the cooled carcass yield of chickens fed CT was 0.95% higher than that of chickens fed the control diet. The cooled carcass yield of chickens fed the negative control diet in a severe stress environment was 2.93% lower than that of chickens fed the negative control diet in a low stress environment. However, the cooled carcass yield of chickens fed diet CT in high stress conditions was similar to that of chickens fed CT in low stress conditions and was 4.45% higher than that of chickens fed the negative control diet in harsh environmental conditions.

[0165] Table 3. 42-day processing yields of chickens fed betaGRO and supplements under different environmental conditions.

[0166]

[0167]

[0168] 1 Clean wood shavings bedding.

[0169] 2 Wood shavings from a chicken house with high mortality, inoculated with Clostridium spores and coccidian oocysts.

[0170] 3 There were 12 replicates per treatment and 10 chickens per replicate.

[0171] 4 Supplement.

[0172] abc Means with common superscripts within a row were not significantly different (P < 0.05).

[0173] The rearing environmental conditions of chickens also significantly affected the total breast meat yield. When grown under low-stress conditions, the percentage of intact breast meat yield of chickens fed CT was 1.39% higher. When grown under high-stress conditions, chickens fed the negative control diet had 1.84% less intact breast meat than those fed the same diet under low-stress conditions. The intact breast meat yield of chickens fed a diet with CT under severe stress conditions was 2.47% higher than that of chickens fed the negative control diet, and the yield was significantly higher than that of chickens fed the negative control diet under low-stress conditions.

[0174] If studying important breast meat marketing factors, adding CT at 300 g / ton in the starter diet and 50 g / ton in the grower and finisher diets resulted in significantly higher pectoralis major, pectoralis minor, and total breast meat yields than those of chickens fed the negative control in any environmental condition. In fact, the breast meat amount of broilers fed CT under severe stress environments was equal to or greater than that of chickens fed the negative control in low-stress environments.

[0175] For chickens grown under low-stress environments, there were no significant differences in villus development and intestinal lesion scores between chickens fed the negative control diet or the diet with CT (Table 4). However, under severe stress environments, the villus cell height and crypt depth of chickens fed CT were significantly greater than those of chickens fed the negative control. As expected, there were significant differences in lesion scores between chickens fed under low-stress and severe stress environments. Among chickens fed under the severe stress program, chickens supplemented with CT had significantly lower lesion scores than those fed the negative control diet. These improvements should contribute to better digestion of dietary nutrients.

[0176] Table 4. Intestinal measurements of chickens fed betaGRO and supplements under different environmental conditions

[0177]

[0178] abcd There was no significant difference in the mean values with a common superscript within the row (P < 0.05).

[0179] In addition, chickens fed in a severe stress environment had a higher number of Escherichia coli at 14 days and 42 days than those fed in a low stress environment, and the number of Escherichia coli in chickens fed CT was significantly reduced. The incidence of Salmonella was higher in chickens fed in a severe stress environment, and the number of Salmonella cases at 14 and 42 days of age was significantly reduced when fed a diet with CT. The incidence of Clostridium organisms closely related to necrotic enteritis was significantly higher in chickens fed in a severe stress environment, but this incidence was significantly reduced in chickens fed CT. Neither diet nor environment had a significant effect on antigen-presenting cells (APC) or oocyst counts.

[0180] The improvements in body weight and feed conversion ratio visible from the addition of CT were consistent with the results of Campbell et al. (2003), Bregendahl et al. (2005), Jamroz et al. (2012), and Henn et al. (2013) (fed diets containing porcine or bovine plasma proteins). The greater response to CT under challenge conditions was consistent with the findings of Henn et al., (2013). The improvement in intestinal morphology was consistent with the results of King et al. (2005) and Beski et al. (2015). The improvement in the performance of chickens fed CT during exposure to highly pathogenic bacteria was consistent with the work of Beski et al. (2016). Overall, these comparisons pointed to the unique and unexpected properties of CT, whose biological effects as a non-animal-derived product were previously associated with feeding animal-derived protein mixtures.

[0181] Many of the responses to supplemental CT observed in the current study are consistent with data previously reported for broilers supplemented with spray-dried plasma (SDP) (Campbell et al., 2003; Bregendahl et al., 2005; King et al., 2005; Jamroz et al., 2012; Henn et al., 2013; Beski et al., 2015; Beski et al., 2016); however, the inclusion rate of CT in the diet was significantly lower; only 1.5%-6% of the SDP inclusion rate. Therefore, the response to CT supplementation may not be nutritional but due to improved metabolism. Recent studies have shown that betaGRO (BG), an animal protein-derived product fed at a similar rate to CT, can promote immune cell function and skeletal muscle growth in vitro (Vaughn et al., 2017; Vaughn et al., 2018). Inclusion of 10 mg / mL of BG in the culture medium resulted in a significant increase in myotube size, mediated by positive changes in the mechanistic target of rapamycin (mTOR) signaling protein (Vaughn et al., 2017). An immunological study conducted by Vaughn et al. (2018) observed more than a two-fold increase in the respiratory metabolism of actively growing β lymphocytes, mediated by mTOR pathway signaling, as addition of rapamycin abolished all positive treatment effects. This, combined with the current data, suggests that supplementation with CT in a severely stressed environment can enhance the immune system of broilers to mitigate bacterial interference with the small intestine (which causes performance disruption), while promoting excellent lean tissue growth.

[0182] Conclusions and Applications

[0183] Adding the yeast-based product CT to the diet fed at a rate of 300 g / ton from day 0 - 14 and then 50 g / ton until day 42 significantly improved the growth rate, feed conversion ratio, survival rate, dressing percentage, and breast meat yield of broilers.

[0184] Chickens grown in a severely stressed environment responded to added CT to a greater extent than chickens fed in a lower stress environment.

[0185] Improvements in villus height and crypt depth in chickens fed CT may be related to improvements in performance.

[0186] Compared to chickens fed a negative control diet, chickens fed CT had a lower incidence of Salmonella in the carcass, fewer intestinal bacteria, and a lower lesion score.

[0187] The low inclusion rate of CT indicates that performance improvements are driven by improved mechanistic metabolism, making broilers healthier and growing faster.

[0188] References Cited in Example 2

[0189] Beski, S. S. M., R. A. Swick, and P. A. Iji. 2015. Subsequent growth performance and digestive physiology of broilers fed on starter diets containing spray-dried porcine plasma as a substitute for meat meal. Br. Poult. Sci. 56:559 - 568.

[0190] Beski, S. S. M., R. A. Swick, and P. A. Iji. 2016. Effect of dietary inclusion of spray-dried porcine plasma on performance, some physiological and immunological response of broiler chickens challenged with salmonella. J. Anim. Physiol. Anim. Nutr. 100:957 - 966.

[0191] Bregendahl, K D., U. Ahn, D. W. Trampel, and J. M. Campbell. 2005. Effects of dietary spray-dried bovine plasma protein on broiler growth performance and breast-meat yield. J. Appl. Poult. Res. 14:560 - 568.

[0192] Campbell, J. M., J. Quigley, L. Russel, and M. Kidd. 2003. Effect of spray-dried bovine serum on intake, health, and growth of broilers housed in different environments. J. Anim. Sci. 81:2776 - 2782.

[0193] Henn, J.D., J. Bockor, M.S. Viera, A.M.L. Riberio, A.M. Kessler, L. Albino, H. Rostagno, J.D. Crenshaw, J.M. Campbell, and L.F.S. Rangel. 2013. Inclusion of porcine spray - dried plasma in broiler diets. J. Appl. Poult. Res. 22:229 - 237.

[0194] Jamroz, D., A. Wiliczkiewicz, J. Orda, J. Kuryszko, and T. Stefaniak. 2012. Use of spray - dried porcine blood by - products in diets for young chickens. J. Anim. Physiol. Anim. Nutr. 96:319 - 333.

[0195] Johnson, J., and M. Reid. 1970. Anticoccidal drugs: lesion scoring techniques in battery and floor pen experiments with chickens. Exp. Parasitol. 28:30 - 36.

[0196] King, M.R., V. Ravindran, P.C.H. Morel, D.V. Thomas, M.J. Birtles, and J.R. Pluske, and L. Bocktor. 2005. Effects of spray - dried colostrum and plasma on the performance and gut morphology of broiler chicks. Aust. J. Agric. Res. 56:811 - 817.

[0197] Meloche, K., B. Fancher, D. Emmerson, S. Bilgili, and W. Dozier III. 2018. Effects of reduced dietary energy and amino acid density on Pectoralis major myopathies in broiler chickens at 36 and 49 days of age1. Poult. Sci. 97(5):1794 - 1807.

[0198] Vaughn, M., K. Phelps, and J. Gonzalez. 2017. In vitro supplementation with the porcine plasma product, stimulates activity of porcine fetal myoblasts and neonatal satellite cells in a divergent manner. Animal.: an international journal of animal bioscience: 1 - 9.

[0199] Vaughn, M., M. Rahe, J. Loughmiller, M. Murtaugh. 2018. Targeting immune cell energetics to produce healthy pigs. National hog farmer, Industry voice. https: / / www.nationalhogfarmer.com / animal - health / targeting - immune - cell - energetics - produce - healthy - pigs.

[0200] Velleman, S., C. Coy, and D. Emmerson. 2014. Effect of the timing of posthatch feed restrictions on broiler breast muscle development and muscle transcriptional regulatory factor gene expression. Poult. Sci. 93(6):1484 - 1494.

[0201] Velleman, S.G., K. Nestor, C. Coy, I. Harford, and N. Anthony. 2010. Effect of posthatch feed restriction on broiler breast muscle development and muscle transcriptional regulatory factor gene and heparan sulfate proteoglycan expression. Int. J. Poult. Sci 9:417 - 425.

[0202] Whitten, P.J.A., D.J. Langhout, and M.W.A. Verstegen. 2012. Small intestine development in chicks after hatch and in pigs around the time of weaning in relation to nutrition: a review. Acta. Agric. Scand. Section A, Vol. 62, pp 1 - 12.

[0203] Example 3

[0204] Evaluation of the Effect of Supplements on Piglet Performance

[0205] Materials and Methods

[0206] The ability of the feed supplement described in Example 2 to affect the growth performance, feed efficiency, and intestinal health of nursery pigs was evaluated. A 42-day trial was conducted at a commercial research facility. The diets were formulated in three phases to best meet the nutritional requirements of the piglets. The piglets were weaned at 19 days of age (considered day 0 of the trial) and fed a phase 1 diet from day 0 to 7 of the trial, a phase 2 diet from day 8 to 21, and a phase 3 diet from day 22 to 42. The control diet (CON) contained no additives above the basal diet; the positive control (BG) contained 2.5 kg / ton in phase 1 and 1.5 kg / ton in phase 2; one treatment (CT1) contained the supplement at 2.5 kg / ton in phase 1 and 1.5 kg / ton in phase 2; one treatment (CT2) contained the supplement at 350 g / ton in all three phases; and one treatment (CT3) contained the supplement at 175 g / ton in all three phases. The response criteria included weight gain, feed conversion ratio, and fecal score.

[0207] Results ( Figures 10 - 12 )

[0208] Adding betaGRO and the supplement resulted in higher body weights on day 42 of the trial compared to the control (P < 0.05). Among all treatments, pigs in the CT2 treatment group had the greatest body weight on day 42 (P < 0.05). Pigs in the BG and CT1 treatment groups had similar body weights on day 42, which were greater than those in the CT3 and CON groups (P < 0.05). The body weight on day 42 of the CT3 group was greater compared to the CON treatment.

[0209] Adding betaGRO and the supplement to the diet improved the feed conversion ratio (P < 0.05). Pigs fed BG, CT1, and CT2 had better feed conversion ratios compared to CT3 (P < 0.05), and CT3 had a better feed conversion ratio compared to CON. Fecal diarrhea scores were collected as an indicator of intestinal health.

[0210] On days 21 and 42 of the trial, the fecal diarrhea scores of piglets fed betaGRO or the supplement were lower compared to CON (P < 0.05). On day 21 of the trial, the fecal scores of BG and CT2 were lower compared to CT3 (P < 0.05), while the fecal score of CT1 was moderate (P > 0.05). On day 42 of the trial, the fecal scores of CT1 and CT2 piglets were lower compared to all other treatments (P < 0.05), and the fecal scores of BG and CT3 were lower than those of CON (P < 0.05).

[0211] Example 4

[0212] Evaluation of the effect of the supplement on skeletal muscle-driven growth in avian species

[0213] Materials and Methods

[0214] The ability of the feed supplement described in Example 2 to affect the growth performance, feed efficiency, and intestinal health of turkey poults was evaluated. An 84-day trial was conducted at a commercial research facility. The diet was formulated in three phases to best meet the nutritional requirements of the turkeys.

[0215] The starter diet was fed from day 0 - 28 of the trial, the grower diet from day 29 - 56, and the finisher diet from day 57 - 84.

[0216] The control diet (CON) contained no additives above the basal diet; one treatment (CT1) contained the supplement at 300 g / ton during the starter period and 50 g / ton during the second phase; one treatment (CT2) contained the supplement at 600 g / ton during the starter period and 100 g / ton during the second phase; one treatment (CT3) contained the supplement at 600 g / ton during the starter period and 50 g / ton during the second phase. The response criteria included weight gain, feed conversion ratio, intestinal health, and intestinal bacterial prevalence.

[0217] Results( Figures 13 - 17 )

[0218] When the supplement was administered to turkey poults, weight gain and feed conversion ratio were increased at all supplement doses compared to the CON group (P < 0.05). Among all treatments, turkeys in the CT2 treatment had the greatest weight (P < 0.05), while turkeys fed CT1 and CT3 had similar weights (P > 0.05), which were greater than the weights of CON turkeys (P < 0.05). Additionally, CT2 had a better feed conversion ratio compared to CT1 and CT3 (P < 0.05), and CT3 had a better feed conversion ratio than CT1 (P < 0.05).

[0219] On the objective scale of intestinal lesions, turkeys fed all doses of the supplement had lower lesion scores (P<0.05) compared to turkeys in the CON treatment group, and all supplement treatments had similar lesion scores (P>0.05). When measuring the presence of Escherichia coli in the small intestine, the prevalence of E. coli in turkeys in all supplement treatment groups was reduced (P<0.05) compared to CON-fed turkeys. The percentage of turkeys with Salmonella in the small intestine was lower in all supplement treatment groups compared to the CON treatment (P<0.05). The percentage of turkeys infected with Salmonella was reduced (P<0.05) in turkeys fed the CT2 dose compared to CT3 turkeys, where the percentage of CT1 turkeys infected with Salmonella was similar to CT2 and CT3 (P>0.05). The log formation of Clostridium perfringens was lower (P<0.05) in turkeys fed all doses of the supplement compared to turkeys in the CON treatment group, and was similar at all supplement doses (P>0.05). Ileal villus height was measured, and all doses of the supplement resulted in greater ileal villus height (P<0.05) compared to the CON treatment.

[0220] Example 6

[0221] Evaluation of the effect of supplements on the performance of Asian sea bass

[0222] Materials and methods

[0223] Ability to evaluate the effects of the supplements described in Example 2 on the growth performance, immunity, and health of Asian sea bass (Lates calcalifer). A 12-week study was conducted at a university research facility. There was a control group that received no additives on a complete basal diet, and three treatments that received supplements in addition to the basal diet: 0.000050, 0.00015, and 0.00045 g / ton of feed. Response criteria included growth rate, feed consumption, feed efficiency, immunity, and intestinal health.

[0224] Results( Figures 18 - 20 )

[0225] When sea bass were fed the supplement, the feed conversion rate increased significantly (P<0.05) during the first 3 weeks of the trial, thus improving the growth efficiency. The supplement also enhanced the immune response, as indicated by higher hematocrit levels at 0.00045 g / ton (P<0.05); serum protein concentration was higher (P<0.05) when the supplement was added to the basal diet at 0.00015 and 0.00045 g / ton. All levels of supplementation resulted in higher lysozyme concentration (P<0.05), indicating improved intestinal health of sea bass through supplementation. Serum cortisol concentration indicates the ability to respond to systemic stress; cortisol levels increased (P<0.05) when supplemented at 0.00045 g / ton compared to the control. Supplementation with 0.00005 and 0.00015 had similar cortisol concentrations (P>0.05) compared to the other treatments.

[0226] Example 6

[0227] Evaluation of the effect of supplement on sow pregnancy and live performance of newborn piglets

[0228] Today, many test products are almost routinely fed in modern sow and piglet production to assist in the sow's parturition period and improve piglet quality (especially through weaning). Since live performance and intestinal health directly affect piglet weight uniformity, we determined whether the body weight of piglets during the nursery period was affected when the supplement was administered.

[0229] Materials and methods

[0230] The trial was conducted at a commercial research facility, and the performance of 20 sows per treatment and 160 piglets per treatment was tracked throughout the nursery period. The supplement described in Example 2 was used.

[0231] The control diet (CON) contained no additives above the basal diet; one diet (CT1) contained 1000 ppm or 1 kg of supplement per metric ton of feed; one diet (CT2) contained 350 ppm or 0.35 kg of supplement per metric ton of feed; and one diet (CT3) contained 100 ppm or 0.10 kg of supplement per metric ton of feed. Sows had a 7 - 10 day adaptation period before estrus, and day 0 of the trial was the time of estrus. Piglets were fed a normal diet to meet their nutritional requirements during the nursery and finishing periods.

[0232] The standard test parameters for sows were body condition score and the interval from weaning to estrus. The response criteria for piglets included average daily gain, body weight at day 126, and feed conversion rate.

[0233] Results( Figures 21 - 25 )

[0234] Sows responded to supplementation, with CT1 and CT2 having higher body condition scores at the end of farrowing compared to CT3 and CON (P<0.05). Additionally, the interval from weaning to estrus was shortened in sows treated with all supplements compared to CON sows (P<0.05). The interval from weaning to estrus was shorter in sows in the CT1 treatment group compared to sows in the CT3 treatment group (P<0.05), and the interval from weaning to estrus in sows in CT2 was similar to that in CT1 and CT3 treatments (P>0.05).

[0235] During the 126-day feeding period, piglets fed by sows supplemented with supplements had greater average daily weight gain compared to piglets fed by CON sows (P<0.05). Piglets from CT1 sows had greater average daily weight gain compared to piglets from CT3 sows (P<0.05), although piglets from CT2 sows had similar average daily weight gain to piglets from CT1 and CT3 sows (P>0.05). Additionally, piglets fed by sows supplemented with supplements had greater body weight on day 126 compared to piglets fed by CON sows (P<0.05). The body weight of piglets from CT1 sows on day 126 was higher than that of piglets from CT3 sows (P<0.05), although the body weight of piglets from CT2 sows on day 126 was similar to that of piglets from CT1 and CT3 sows (P>0.05). During the 126-day feeding period, piglets from CT1 sows had the best feed conversion rate compared to all other treatments (P>0.05). The feed conversion rate of piglets from CT2 sows was improved compared to piglets from CT3 and CON sows (P>0.05), and piglets from CT3 sows had a better feed conversion rate compared to piglets from CON sows (P>0.05).

[0236] Example 7

[0237] Evaluation of the effects of supplements on egg production, growth performance and feed efficiency in laying hens

[0238] Materials and methods

[0239] The ability of the supplement described in Example 2 to affect egg production, growth performance, and feed efficiency in laying hens was evaluated. A 140-day trial was conducted at a commercial research facility. Each replicate in each treatment (or experimental) group contained three commercial laying hens, and 20 replicates were randomly assigned to each group, containing 60 commercial laying hens, for a total of 120 animals in the study. The trial period started on Day 0, and laying hens at 18 weeks of age were randomly assigned. The trial was completed on Day 140, and a consistent industry-standard diet was fed throughout the trial period to best meet the nutritional requirements of the laying hens. The control diet (CON) contained no additives above the basal diet, and the treatment diet (CT) contained 60 g / t of the supplement. The response criteria included hen body weight, hen fat pad (% of body weight), hen intestinal lesion score, egg production (%), egg weight, eggshell weight, eggshell thickness, total number of eggs produced, and feed conversion ratio of egg production.

[0240] Results

[0241] Body weight (Table 5). When the supplement was provided to commercial laying hens, body weight was improved at Day 28 (p < 0.05), Day 56 (p < 0.05), Day 84 (p < 0.05), Day 112, and Day 140 (p < 0.05).

[0242] Table 5.

[0243]

[0244]

[0245] Note: Rows without a common superscript are significantly different (P < 0.05) as determined by the least significant difference.

[0246] (P < 0.05).

[0247] Health of the chickens (Table 6). At the end of the study, a gross necropsy was performed on all remaining hens to determine the intestinal lesion score and measure the percentage of fat pad to body weight. The fat pad was improved by the supplement (p < 0.05), and the intestinal lesion score was reduced and improved.

[0248] Table 6.

[0249]

[0250] Note: Rows without a common superscript are significantly

[0251] different (P < 0.05).

[0252] Egg production (Table 7). Egg production (%) was measured as the percentage of eggs laid per hen per treatment group per day. Egg production (%) increased during the periods of 0 - 28 days (p < 0.05), 29 - 56 days (p < 0.05), 57 - 84 days (p < 0.05), 85 - 112 days (p < 0.05), 113 - 140 days (p < 0.05) when hens were fed supplementary feed and during the experimental period of 0 - 140 days (p < 0.05).

[0253] Table 7.

[0254]

[0255]

[0256] Note: Rows without a common superscript are significantly different (P < 0.05) as determined by the least significant difference.

[0257] (P < 0.05).

[0258] Egg weight (Table 8). Egg weight was measured as the average weight of eggs in grams per treatment group. The average egg weight improved during the measurement periods of 0 - 28 days (p < 0.05), 29 - 56 days, 57 - 84 days, 85 - 112 days, and 113 - 140 days after the hens' diet was supplemented.

[0259] Table 8.

[0260]

[0261] Note: Rows without a common superscript are significantly different (P < 0.05) as determined by the least significant difference.

[0262] (P < 0.05).

[0263] Eggshell weight (Table 9). Eggshell weight was measured as the average weight of eggs in grams per treatment group. The average eggshell weight improved during the measurement periods of 29 - 56 days, 57 - 84 days, 85 - 112 days, and 113 - 140 days.

[0264] Table 9.

[0265]

[0266]

[0267] Note: Rows without a common superscript are significantly different (P < 0.05) as determined by the least significant difference.

[0268] (P < 0.05).

[0269] Total number of eggs laid, total kilograms of eggs laid (Table 10). The total number of eggs laid and the total weight (kilograms) of eggs laid by hens fed the supplement improved.

[0270] Table 10. Days 0 - 140 of the trial

[0271]

[0272] Note: Rows without a common superscript are significantly different (P < 0.05) as determined by the least significant difference.

[0273] (P < 0.05).

[0274] Feed conversion ratio (kilograms of feed / dozen eggs) (Table 11). The feed conversion ratio per dozen eggs was calculated by dividing the total amount of feed consumed by each treatment group by the number of dozens of eggs produced by each treatment group. A lower number means that more eggs are produced assuming the same amount of feed consumed (or, in other words, hens that consume less feed can produce the same number of eggs). During the trial: days 0 - 28 (p < 0.05), days 29 - 56 (p < 0.05), days 57 - 84 (p < 0.05), days 85 - 112 (p < 0.05), days 113 - 140 (p < 0.05), and overall days 0 - 140 (p < 0.05), the feed conversion ratio (kg feed / dozen eggs) of hens fed the supplement improved.

[0275] Table 11. Feed conversion ratio (kg feed / dozen eggs)

[0276]

[0277]

[0278] Note: Rows without a common superscript are significantly different

[0279] (P < 0.05).

[0280] Feed conversion ratio (kg feed / kg eggs) (Table 12). The kg feed conversion ratio per kg of eggs produced was calculated by dividing the total amount of feed consumed (in kg) by each treatment group by the total kg of eggs produced by each treatment group. A lower number means that assuming the same amount of feed consumed, the total weight of eggs produced is greater (or in other words, hens that consume less feed can produce the same weight of eggs). During the trial: days 0 - 28 (p < 0.05), days 29 - 56 (p < 0.05), days 57 - 84 (p < 0.05), days 85 - 112 (p < 0.05), days 113 - 140 (p < 0.05), and overall days 0 - 140 (p < 0.05), the feed conversion ratio (kg feed / kg eggs) of laying hens fed the supplement increased.

[0281] Table 12. Feed conversion ratio (kg feed / kg eggs)

[0282]

[0283] Note: As determined by the least significant difference, rows without a common superscript are significantly

[0284] different (P < 0.05).

[0285] The complete disclosures of all patents, patent applications, and publications cited herein, as well as materials that are electronically available, are hereby incorporated by reference in their entirety. Supplementary materials cited in publications (such as supplementary tables, supplementary figures, supplementary materials and methods, and / or supplementary experimental data) are likewise incorporated by reference in their entirety. In the event of any inconsistencies between the disclosure of this application and the disclosure of any document incorporated by reference herein, the disclosure of this application shall govern. The above detailed description and examples are given for purposes of clear understanding only. They should not be construed as unnecessary limitations. This disclosure is not limited to the exact details shown and described, and variations that are obvious to those skilled in the art will be included within the disclosure defined by the claims.

[0286] Unless otherwise indicated, all numerical values representing amounts of components, molecular weights, etc. used in this specification and the claims should be understood to be modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending on the desired properties sought to be obtained by this disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0287] While the numerical ranges and parameters setting forth the broad scope of this disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains a range necessarily resulting from the standard deviation found in its respective testing measurements.

[0288] All headings are for the convenience of the reader and shall not be used to limit the meaning of the text following the heading unless so specified.

Claims

1. A supplement for feeding animals, which comprises free amino acid L-glutamine or its salt, L-leucine or its salt, and L-arginine or its salt, and active insulin-like growth factor 1 (IGF-1), wherein the L-glutamine or its salt, L-leucine or its salt, and L-arginine or its salt are present in a molar ratio of 2-4:1:1, 3:0.5-2:1, or 3:1:0.5-3.

2. The supplement according to claim 1, wherein at least one of the L-glutamine, the L-leucine, and the L-arginine is in the form of a salt.

3. The supplement according to claim 1, wherein the amount of L-glutamine or its salt is 0.5 g to 500 g of amino acid / kg of supplement.

4. The supplement according to claim 1, wherein the amount of L-leucine or its salt is 0.1 g to 100 g of amino acid / kg of supplement.

5. The supplement according to claim 1, wherein the amount of L-arginine or its salt is 0.1 g to 200 g of amino acid / kg of supplement.

6. The supplement according to claim 1, wherein the amount of active IGF-1 is 100 μg to 2000 μg of active IGF-1 / kg of supplement.

7. The supplement according to claim 1, wherein the amount of L-glutamine or its salt is 0.5 g to 500 g of amino acid / kg of supplement, the amount of L-leucine or its salt is 0.1 g to 100 g of amino acid / kg of supplement, the amount of L-arginine or its salt is 0.1 g to 200 g of amino acid / kg of supplement, and the amount of active IGF-1 is 100 μg to 2000 μg of IGF-1 / kg of supplement.

8. The supplement according to claim 1, wherein the active IGF-1 is recombinant IGF-1.

9. The supplement according to claim 1, which further comprises animal food.

10. The supplement according to claim 1, wherein the L-glutamine or its salt, L-leucine or its salt, and L-arginine or its salt are present in a molar ratio of 3:1:

1.

11. The supplement according to claim 1, wherein the supplement comprises 30 g / kg to 60 g / kg of L-glutamine, 5 g / kg to 50 g / kg of L-leucine, 1 g / kg to 100 g / kg of L-arginine, 300 μg / kg to 1500 μg / kg of active IGF, and yeast.

12. An animal food, which comprises the supplement according to any one of claims 1-11, and the supplement is present in the animal food in an amount of 0.000005 g / ton of the animal food to 0.005 g / ton of the animal food.

13. An animal food, which comprises the supplement according to any one of claims 1-11, and the supplement is present in the animal food in an amount of 50 g / ton of the animal food to 2600 g / ton of the animal food.

14. The animal food according to claim 12 or 13, wherein the total weight percentage (wt%) of the L-glutamine or its salt, the L-leucine or its salt, and the L-arginine or its salt is less than the total wt% of the other amino acids in the supplement.

15. The animal food according to claim 13, wherein one, two, or all three of the L-glutamine, the L-leucine, and the L-arginine are in the form of a salt.

16. The animal food according to claim 13, wherein the amount of L-glutamine or its salt is at least 0.0000002 wt%.

17. The animal food according to claim 13, wherein the amount of L-leucine or its salt is at least 0.00000006 wt%.

18. The animal food according to claim 13, wherein the amount of L-arginine or its salt is at least 0.00000006 wt%.

19. A method for non-therapeutic purposes, which comprises administering to an animal an effective amount of (i) an animal food comprising the supplement according to any one of claims 1-11, or (ii) an animal food according to any one of claims 12-18.

20. The method according to claim 19, wherein the animal food comprises the supplement in an amount of at least 50 grams per ton of the animal food.

21. The method according to claim 19, wherein the animal is a porcine animal or a poultry animal.

22. The method according to claim 19, wherein the animal is raised in a high-stress environment.

23. A method for improving animal performance, the method comprising administering to the animal (i) an animal food supplemented with the supplement according to any one of claims 1-11 or (ii) an animal food according to any one of claims 12-18, wherein the improved performance is an increase in body weight, an improvement in feed conversion rate, an improvement in herd uniformity, or a combination thereof.

24. Use of (i) an animal food supplemented with the supplement according to any one of claims 1-11 or (ii) an animal food according to any one of claims 12-18 in the preparation of an animal product for reducing animal mortality.

25. A method for improving animal processing coefficient, the method comprising administering to the animal (i) a composition supplemented with the supplement according to any one of claims 1-11 or (ii) an animal food according to any one of claims 12-18, wherein the improved processing coefficient is an increase in chilled carcass yield, an increase in total breast meat yield, or a combination thereof.

26. Use of (i) a composition supplemented with the supplement according to any one of claims 1-11 or (ii) an animal food according to any one of claims 12-18 in the preparation of a product for improving animal intestinal health, wherein the improved intestinal health is a reduction in intestinal bacteria, a reduction in intestinal lesions, a reduction in fecal diarrhea, or a combination thereof.

27. The use according to claim 26, wherein the animal is a laying poultry.

28. A method for shortening the time from weaning of offspring to re - estrus, the method comprising administering to an animal (i) a composition supplemented with a supplement as described in any one of claims 1 - 11 or (ii) an animal food as described in any one of claims 13 - 18, wherein the time from weaning of offspring to re - estrus is shortened compared to an animal in a similar environment but not fed the composition or animal food.

29. The method according to claim 28, wherein the animal is a sow.

30. The method according to claim 29, wherein the supplement is present in an amount of 100 grams per ton to 2.5 kilograms per ton of the composition or animal food.

31. A method for increasing average daily gain, increasing body weight, and / or improving feed conversion rate in an animal, the method comprising administering to the animal (i) a composition supplemented with a supplement as described in any one of claims 1 - 11 or (ii) an animal food as described in any one of claims 13 - 18, wherein the increase in average daily gain, increase in body weight, and / or improvement in feed conversion rate is a change compared to an animal in a similar environment but not fed a composition or animal food containing the supplement.

32. The method according to claim 31, wherein the animal is a piglet.

33. The method according to claim 31, wherein the supplement is present in an amount of 100 grams per ton to 2.5 kilograms per ton of the composition or animal food.

34. A method for increasing body weight, improving health as measured by an increase in the percentage of fat pads in body weight, increasing egg production, increasing the total number of eggs produced, and / or improving feed conversion rate in egg - laying poultry, the method comprising administering to the animal (i) a composition supplemented with a supplement as described in any one of claims 1 - 11 or (ii) an animal food as described in any one of claims 13 - 18, wherein the increase in body weight, improvement in health as measured by an increase in the percentage of fat pads in body weight, increase in egg production, increase in the total number of eggs produced, and / or improvement in feed conversion rate is a change compared to an animal in a similar environment but not fed a composition or animal food containing the supplement.

35. The method according to claim 34, wherein the egg - laying poultry is a chicken.

36. The method according to claim 35, wherein the supplement is present in an amount of 30 grams per ton to 90 grams per ton of the composition or animal food.

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