Feed additive compositions and methods for making and using the same

Abstract

A feed additive mixture includes thymol, carvacrol, and at least two other active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes. A feed additive mixture includes thymol, and at least two other active compounds comprising eugenol and cinnamaldehyde and optionally one or more active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes. A feed additive mixture includes eugenol, cinnamaldehyde, and at least two other active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes. An animal feed includes a base feed and the feed additive mixture. The feed additive mixture has an anti-parasitic effect. A method for making the feed additive mixture, a method for making the animal feed, and a method for feeding an aquatic animal are also provided.

Description

PT-1970-WO-PCTFEED ADDITIVE COMPOSITIONS AND METHODS FOR MAKING AND USING THE SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 720.552, filed November 14, 2024, which is incorporated by reference herein in its entirety.FIELD

[0002] The present disclosure generally relates to animal feeds, in particular aquaculture feeds, and specifically fish feeds. In particular, the present disclosure relates to animal feeds, in particular aquaculture feeds, and specifically fish feeds containing feed additives that help deter parasites, and to compositions and methods for making such feeds.INTRODUCTION

[0003] Aquaculture of various fish species provides a food source for a growing world population. In 2021, it was estimated that about 175 million metric tons of fish were produced for global consumption. An increase in demand for fish by consumers has resulted in an increase in the number of aquaculture farms around the world. With an increase in aquaculture farms comes an increased need for fish feeds to nourish and maintain the volume of fish being produced and for sustainable technologies for safe and effective pest control in farmed fish populations.

[0004] There are multiple harmful pests that can target fish in aquaculture. The salmon louse, e.g., Lepeophtheirus salmonis or Caligus rogercresseyi, is an ectoparasite that has adapted evolutionarily to attach to salmon and remain unharmed by the fish’s immune system. Without wishing to be bound by any particular theory, it is believed that L. salmonis can modulate and evade the host’s immunity through the secretion of one or more molecules, including proteins, lipids, and the like, to induce a suppressive effect on the protective, beneficial immune responses of fish. Such downregulation of the protective host response can lead to opportunistic infection by other parasites, bacteria, viruses, protozoa, amoeba, and the like, which can, in turn, lead to an upregulation of inflammation. Such pests can modulate host immune responses to create an environment that favors the progression of infection or opens the fish to secondary co-infections with multiple opportunistic species. In some cases, the downregulation of the protective host response can itself lead to an upregulation of the non-protective or harmful immune responses. Furthermore, overstimulation of non-protective or harmful immune responses can have numerousPT-1970-WO-PCTadverse effects on fish at the physiological, histological, biochemical, and enzymatic levels. By way of example, the energetic cost of a non-protective immune or physiological response may interfere with the fish’s ability to clear toxic compounds, and deal with oxidative stress, heat stress, or other environmental assaults.[00051 Various copepod parasites, non-copepod parasites, viruses, and bacteria known to infect fish are thought to individually or collectively cause an upregulation of non-protective or harmful immune responses. By way of example, infection w ith one or more copepod species can suppress the immune system of fish, create open wounds, and provide an opportunity for co-infection with pests such as infectious salmon anemia virus or Piscirickettsia salmonis, the causative agent of salmon rickettsial syndrome. Further, an upregulation can lead to activation or overactivation of the host immune response in fish and can lead to undesirable inflammatory side effects that can cause tissue damage (i.e., immunopathology), decreased animal welfare, a loss in farm productivity, and even death in the fish.

[0006] There are various mechanisms available to manage parasitic diseases, including various synthetic medicinal agents, acid wash techniques, and vaccines. Such agents can be difficult to implement and be expensive in the long term. Additionally, resistance to various medicinal agents continues to be a problem in the field. Thus, a need exists to provide easy-to-use management methods by using simple and cost-effective methods and feed additive mixtures that can be administered to fish to manage co-infections brought on by multiple ty pes of copepod parasites, non-copepod parasites, and other pathogens.SUMMARY

[0007] The present disclosure provides animal feeds, in particular aquaculture feeds, and specifically fish feeds, and feed additives for use in such feeds.

[0008] A feed additive mixture includes, by weight of the feed additive mixture, 0.1 wt-% to 15 wt-% thymol, 0.1 wt-% to 45 wt-% carvacrol, and 5 wt-% to 80 wt-% of a total of at least two other active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

[0009] A feed additive mixture includes, by weight of the feed additive mixture, 0.1 wt-% to 5 wt-% thymol, and 2 wt-% to 95 wt-% of a total of at least two other active compounds including eugenol and cinnamaldehyde and optionally one or more active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.PT-1970-WO-PCT

[0010] A feed additive mixture includes, by weight of the feed additive mixture, 5 wt-% to 85 wt-% eugenol, 0.5 wt-% to 15 wt-% cinnamaldehyde, and 0.5 wt-% to 50 wt-% of a total of at least two other active compounds selected from thymol, monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

[0011] The feed additive mixture may include eugenol in an amount of 7 wt-% to 12.5 wt-%. The feed additive mixture may include cinnamaldehyde in an amount of 1 wt-% to 6 wt-%. The sum of the amount of the at least two other active compounds in the feed additive mixture may be 15 wt-% to 40 wt-%. The weight ratio of eugenol to cinnamaldehyde in the feed additive mixture may be from 3:1 to 6:1. The sum of the amounts of eugenol and cinnamaldehyde in the feed additive mixture may be 12 wt-% to 20 wt-%.

[0012] The at least two other active compounds may include one or more poly phenolic compounds. The polyphenolic compounds may include one or more flavonoids, tannins, or a combination thereof. The at least two other active compounds may include a bicyclic alcohol, an alkylbenzene, an allyl-substituted phenol, an aromatic aldehyde, or a combination of two or more thereof. The at least two other active compounds may include borneol, cymene, eugenol, cinnamaldehyde, or a combination of two or more thereof. The feed additive mixture may include eucalyptus essential oil, lemon essential oil, eugenol, and cinnamaldehyde. The feed additive mixture may include thyme oil, clove oil, and cinnamaldehyde. The feed additive mixture may include eugenol provided from clove oil and thymol provided from thyme oil.

[0013] The feed additive mixture may provide activation of animal immune response, reduction of animal oxidative stress, reduction of parasite host recognition, and regulation of iron metabolism, when administered to the animal.

[0014] The feed additive mixture may be formulated to target a parasite. The feed additive mixture may be formulated to target myxozoa, monogenean, sea lice, Benedenia seriolae, Neobenedenia species, Gyrodactylus, Amyloodinium, Neoparamoeba, Ichthyophthirius, or a combination of two or more thereof. The feed additive mixture may be formulated to target sea lice. The feed additive mixture may be formulated to target Caligus rogercresseyi.

[0015] An animal feed may include a base feed, and the feed additive mixture. The animal feed may include protein, starch, and fat. The animal feed may be a complete fish feed. The animal feed may include from 28 wt-% to 60 wt-% protein, 6 wt-% to 15 wt-% starch, and from 18 wt-% to 40 wt-% fat.

[0016] The animal feed may include one or more of pellets, extruded nuggets, steam pellets, flakes, tablets, granules, or powders. The animal feed may include at least two other activePT-1970-WO-PCTcompounds including a bicyclic alcohol, an alkylbenzene, an allyl -substituted phenol, an aromatic aldehyde, or a combination of two or more thereof. The animal feed may include borneol, cymene, eugenol, cinnamaldehyde, or a combination of two or more thereof. The animal feed may include thyme oil and clove oil.roo 171 The animal feed may include the feed additive mixture at 0.001 wt-% to 0.08 wt-% of the animal feed. The animal feed may include the feed additive mixture at 0.005 wt-% to 0.05 wt-% of the animal feed. The feed additive mixture in the animal feed may be configured for postextrusion oil-coating onto the animal feed.

[0018] The animal feed may be administered to an animal such that sea louse burden in the animal may be reduced relative to a control diet. The sea louse burden in the animal may be reduced by about 20 % four weeks post-infestation. The sea louse burden in the animal may be reduced by about 32 % four weeks post-infestation.

[0019] The animal feed may be administered to an animal such that the plasma total antioxidant capacity may increase relative to a control diet. The animal feed may be administered to an animal such that the indicators of iron metabolism such as bilirubin plasma levels and the hepatic and skin hepcidin antimicrobial peptide A (HampA) expression may be regulated relative to a control diet. The animal feed may be administered to an animal such that the skin tumor necrosis factor-a (TNF-a) expression may be regulated relative to a control diet.

[0020] A method for making the feed additive mixture for adding to a fish feed may include mixing thymol, carvacrol, and at least two other active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes. A method for making the feed additive mixture for adding to a fish feed may include mixing thymol, and at least two other active compounds including eugenol and cinnamaldehyde and optionally one or more active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes. A method for making the feed additive mixture for adding to a fish feed may include mixing eugenol, cinnamaldehyde. and at least two other active compounds selected from thymol, monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

[0021] A method for making an animal feed may include mixing the feed additive mixture with a base feed to form a feed composition.

[0022] A method of feeding an aquatic animal may include administering an amount of feed to the aquatic animal, where the feed includes a base feed and the feed additive mixture.PT-1970-WO-PCTBRIEF DESCRIPTION OF FIGURES

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

[0024] FIGS. 1A-1E and 2A-2D are graphical representations of experimental data from Example 1.

[0025] FIG 3. shows mean lice counts, by treatment, based on the inclusion rate of the feed additive mixture.

[0026] FIG 4. shows the efficacy %, by treatment, based on the inclusion rate of the feed additive mixture.

[0027] FIG 5. shows mean lice counts, by exposure, based on the inclusion rate of the feed additive mixture.

[0028] FIG 6. shows the efficacy %, by exposure, based on the inclusion rate of the feed additive mixture.

[0029] It should be understood that numerous other modifications and examples can be devised by those skilled in the art, which fall within the scope and spirit of the principles of this disclosure.DEFINITIONS

[0030] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0031] The term ‘‘about” is used here in conjunction with numeric values to include normal variations in measurements as expected by persons skilled in the art, and is understood to have the same meaning as “approximately” and to cover a typical margin of error, such as ±5 % of the stated value.

[0032] Terms such as “a,” “an,” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration.

[0033] The terms “a,” “an.” and “the” are used interchangeably with the term “at least one.” The phrases “at least one of’ and “comprises at least one of’ followed by a list refers to any one of the items in the list and any combination of two or more items in the list.

[0034] As used here, the term “or” is generally employed in its usual sense including “and / or” unless the content clearly dictates otherwise. The term “and / or” means one or all of the listed elements or a combination of any two or more of the listed elements.PT-1970-WO-PCT

[0035] The term “substantially” as used herein refers to a majority of, or mostly, as in at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.5 %, 99.9 %, 99.99 %, or at least about 99.999 % or more, or 100 %. The term “substantially free of’ as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that about 0 wt-% to about 5 wt-% of the composition is the material, or about 0 wt-% to about 1 wt-%, or about 5 wt-% or less, or less than, equal to, or more than about 4.5 wt-%, 4 wt-%, 3.5 wt-%, 3 wt-%, 2.5 wt-%, 2 wt-%, 1.5 wt-%, 1 wt-%, 0.9 wt-%, 0.8 wt-%, 0.7 wt-%, 0.6 wt-%, 0.5 wt-%, 0.4 wt-%, 0.3 wt-%, 0.2 wt-%, 0.1 wt-%. 0.01 wt-%. or about 0.001 wt-% or less, or about 0 wt-%.

[0036] Values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range were explicitly recited. For example, a range of “about 0.1 % to about 5 %” or “about 0.1 % to 5 %” should be interpreted to include not just about 0.1 % to about 5 %, but also the individual values (e.g., 1 %, 2 %, 3 %, and 4 %) and the sub-ranges (e.g., 0.1 % to 0.5 %, 1.1 % to 2.2 %, 3.3 % to 4.4 %) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z.” unless indicated otherwise.

[0037] Unless expressly stated, ppm (parts per million), percentage, and ratios are on a weight basis. Percentage on a weight basis is also referred to as wt-%.

[0038] As used here, “have,” “having,” “include,” “including,” “comprise,” “comprising,” or the like are used in their open-ended sense, and generally mean “including, but not limited to.” It will be understood that “consisting essentially of,” “consisting of,” and the like are subsumed in “comprising” and the like. As used herein, “consisting essentially of,” as it relates to a composition, product, method, or the like, means that the components of the composition, product, method, or the like are limited to the enumerated components and any other components that do not materially affect the basic and novel characterise c(s) of the composition, product, method, or the like.

[0039] The words “preferred” and “preferably” refer to embodiments that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferredPT-1970-WO-PCTembodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the disclosure, including the claims.DETAILED DESCRIPTION

[0040] The present disclosure generally relates to feed compositions, animal feeds, in particular aquaculture feeds, and specifically fish feeds. In particular, the present disclosure relates to animal feeds, in particular aquaculture feeds, and specifically fish feeds containing feed additives that help deter parasites, and to compositions and methods for making such feeds. The present disclosure further relates to feed additive mixtures, and specifically feed additive mixtures that have an anti-parasitic effect. The feed additive mixtures may be included in feed compositions or animal feeds. The terms feed composition and animal feed are used here interchangeably. The feed additive mixtures may have other beneficial effects, such as antioxidant, anti-inflammatory, and antimicrobial effects. The feed additive mixtures may be particularly useful for adding to fish feed but may also be useful for adding to the feed of other animals. While the beneficial effects are exemplified here in view of use of the feed additives in fish feed, similar anti-parasitic, antioxidant, anti-inflammatory, and / or antimicrobial effects may be achieved in other animals, as well.

[0041] Furthermore, the feed additive mixtures may promote mucin production in fish skin. Mucins are high-molecular weight glycoproteins that play a role in protecting and lubricating mucosal surfaces in fish. They contribute to the formation of the mucus layer which helps trap and remove debris, pathogens, and parasites from the skin surface of the fish. Additionally, the feed additive mixtures may promote immune responses such as the Thl / Th2 response. Thl and Th2 responses are two types of immune responses mediated by different subsets of T-helper cells (Th cells). Thl cells primarily produce cytokines such as interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). Thl response helps protect fish against intracellular pathogens such as viruses and certain bacteria. Th2 cells primarily produce cytokines such as interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin- 10 (IL- 10), and interleukin- 13 (IL- 13). Th2 response helps protect fish against extracellular pathogens such as helminths. The feed additive mixtures may aid in iron regulation in the fish. Iron is involved in several processes related to fish, for example, oxygen transport, enzyme function, immune response, pigmentation, and osmoregulation. Imbalances in iron regulation may lead to health issues such as metabolic disorders and impaired immune responses in fish.PT-1970-WO-PCT

[0042] According to embodiments, the present disclosure relates to feed additive mixtures that include thymol, carvacrol, and at least two other active compounds. According to embodiments, the present disclosure relates to feed additive mixtures that include thymol, and at least two other active compounds including eugenol and cinnamaldehyde and optionally one or more other active compounds. According to embodiments, the present disclosure relates to feed additive mixtures that include eugenol, cinnamaldehyde, and at least two other active compounds.

[0043] Thymol is a phenolic monoterpenoid found in the oil of thyme. Thymol can be extracted from various plants, including Thymus vulgaris (common thyme). Thymol is represented by the below formula:Thymol

[0044] Carvacrol is a phenolic monoterpenoid found in oil of thyme, as well as in the essential oil of Origanum vulgare (oregano). Carvacrol is represented by the below formula:Carvacrol

[0045] Citronellol is an acyclic monoterpenoid with two enantiomers that are found in different plants in nature. For example, (+)-citronellol ((3R)-3,7-dimethyloct-6-en-l-ol) is found in citronella oils (e.g., Cymbopogon nardus), while (-)-citronellol ((3S)-3,7-dimethyloct-6-en-l-ol) is found in the oils of rose and Pelargonium geraniums. The two enantiomers of citronellol are represented by the below formulas:(+)-citronellol (-)-citronellolPT-1970-WO-PCT

[0046] Cinnamaldehyde is an aromatic aldehyde found in various plants, including the bark of cinnamon trees. Cinnamaldehyde is represented by the below formula:Cinnamaldehyde

[0047] Eugenol is an allyl-substituted phenolic compound found in various plants, including in cloves. Eugenol is represented by the below formula:Eugenol

[0048] Flavonoids are a group of plant secondary metabolites with phenolic structures, found in the leaves, bark, roots, stems, or flowers of various fruits, vegetables, and grains. Flavonoids are represented by the below general structure:Flavonoid general structureEach of the benzene rings A and B are substituted with at least one hydroxy group and optionally alkoxy groups. The linker between the rings A and B may form a ring C, may include one or more double bonds, and may be substituted with various groups, in particular hydroxy and / or aldehyde groups.

[0049] Flavonoids are subdivided into different subgroups, including flavones, flavonols, flavanones, flavanols or catechins, anthocyanins, and chaicones.

[0050] Examples of flavones include apigenin, tangeretin, baicalein, and rhoifolin. Examples of flavonols include quercetin, myricetin, rutin, morin, and kaempherol. Examples of flavanones include hesperetin, naringin, naringenin, eriodictyol, and hesperidin. Examples of flavanols (catechins) include theaflavin. Examples of anthocyanins include cyanidin, malvidin, delphinidin, and peonidin. Examples of chaicones include phloretin, arbutin, phloridzin, and chalconaringenin. Isoflavonoids are a related group of compounds. Examples of isoflavonoids include genistin, genistein, daidzein, glycitein, and daidzin.PT-1970-WO-PCT

[0051] Fish feed used as fish diets in aquaculture applications may be provided in liquid or solid form, or as a combination of liquid and solid forms. Fish feeds in solid form can include pellets, extruded nuggets, steam pellets, flakes, tablets, powders, and the like. In various embodiments, the base feed can include a base feed pellet. In some embodiments, the base feed pellet can include a porous matrix distributed throughout. Fish feeds in liquid form can include aqueous solutions, oils, oil and water emulsions, slurries, suspensions, and the like. Most commonly, fish feed for use in aquaculture is provided in pelleted form. The fish feeds of the present disclosure are provided in solid form. The fish feed of the present disclosure may be referred to as pellets. However, it is understood that the fish feed may also be provided as flakes, tablets, granules, and the like.

[0052] The fish feed pellets may be produced using a base feed formulation using raw materials that may be chosen based on the application in which it is to be used and on the fish species. The base feed formulation may include solid raw materials or both solid and liquid raw materials.

[0053] The fish feed may include various ingredients that improve the stability of the feed and / or provide nutritional or health benefits for the fish. In some cases, antioxidants are used to help preserve the quality and nutritional properties of the feed.Feed additive mixture

[0054] The feed additive may be provided as a feed additive mixture. The feed additive mixture may include a mixture of desired active compounds for mixing into animal feed. The components of the feed additive mixture may be included in suitable concentrations so that a desired concentration in the animal feed is achieved upon mixing. According to embodiments, the feed additive mixture includes thymol and carvacrol as active compounds. The feed additive mixture may further include two or more active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes. According to embodiments, the feed additive mixture includes thymol and at least two other active compounds including eugenol and cinnamaldehyde. The feed additive mixture may further optionally include one or more other active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes. According to embodiments, the feed additive mixture includes eugenol and cinnamaldehyde as active compounds. The feed additive mixture may further include two or more active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.PT-1970-WO-PCT

[0055] The active compounds may provide beneficial properties to the feed additive mixture or the feed. The active compounds may be selected, at least in part, based on their capability to have an anti-parasitic effect. The active compounds may help alone or in combination of disrupting the parasite-host recognition, mitigation of oxidative stress, enhancement of immune response, regulation of iron homeostasis, or their antimicrobial properties.

[0056] Many of the active compounds of the feed additive may be obtained from essential oils. An essential oil or “EO” is a concentrated plant extract that is named after its source plant, and typically retains the natural flavor or scent of its source. Essential oils are often extracted using a solvent and include a wide variety of volatile chemical compounds. Reference to essential oils here includes both natural (plant extracted) essential oils, synthetic essential oils, and their mixtures. For example, “thyme essential oil” may include natural thyme oil (i.e., EO derived from plants in the Thymus genus) and / or synthetic thyme oil. Synthetic thyme oil can include synthetic thymol and optionally one or more other constituents of natural thyme oil. “Eucalyptus essential oil” may include natural eucalyptus oil (i.e., EO derived from plants in the Eucalyptus genus) and / or synthetic eucalyptus oil. “Lemon essential oil” may include natural lemon oil (i.e., EO derived from Citrus limon plants) and / or synthetic lemon oil. “Clove essential oil” may include natural clove oil (i.e., EO derived from Syzygium aromaticum) , synthetic clove oil, or mixtures thereof.Thymol

[0057] The feed additive mixture of the present disclosure includes thymol. Thymol is a natural monoterpene phenol compound found in several plants, including thyme (Thymus vulgaris , oregano (Origanum vulgare), and savory (Satureja sp.). Thymol may also be obtained synthetically by chemical synthesis. Thymol may exhibit a potent repellent effect against pests and parasites such as sea lice. Its antioxidant capability may help the affected animal to counteract with the oxidative stress caused by sea lice infection. Thymol may also prevent a secondary' bacterial infection as it has strong antimicrobial properties.

[0058] While thymol is primarily known for its antimicrobial and antioxidant properties, it is not commonly used in animal feeds as an additive due to several factors. The dosage of thymol or thyme essential oil supplementation in animal feeds is selected to ensure efficacy without adverse effects on animal health and performance. Thymol and essential oils containing thymol may be prone to oxidation and degradation, affecting their stability and bioavailability in animal feeds. The taste and odor of thymol may influence the palatability of animal feeds, potentially affectingPT-1970-WO-PCTfeed intake and acceptance by animal. The use of thymol-containing additives in commercial animal feeds may be subject to regulatory approval and safety assessments. The present disclosure, inter alia, describes the use of thymol and other natural compounds, such as essential oils containing thymol, as feed additives in aquaculture.

[0059] Thymol may be included in the feed additive mixture at any suitable concentration that will result in a desired concentration in the feed formulation, based on the inclusion rate of the feed additive mixture. For example, in some representative embodiments, the feed additive mixture includes thymol in a concentration of about 0.1 wt-% or greater, about 0.2 wt-% or greater, about 0.3 wt-% or greater, about 0.5 wt-% or greater, about 0.7 wt-% or greater, about 0.8 wt-% or greater, 1 wt-% or greater, 2 wt-% or greater, 3 wt-% or greater, 4 wt-% or greater, 5 wt-% or greater, 7.5 wt-% or greater, 10 wt-% or greater, or 12.5 wt-% or greater. The concentration of thymol in the feed additive mixture may be about 15 wt-% or lower, about 12.5 wt-% or lower, about 10 wt-% or lower, about 7.5 wt-% or lower, about 5 wt-% or lower, about 4 wt-% or lower, about 3 wt-% or lower, about 2 wt-% or lower, about 1 wt-% or lower, 0.8 wt-% or lower, about 0.7 wt-% or lower, about 0.5 wt-% or lower, about 0.3 wt-% or lower, or about 0.2 wt-% or lower. The concentration of thymol in the feed additive mixture may be from about 0.1 wt-% to about 15 wt-%, from about 1 wt-% to about 12.5 wt-%, or from about 5 wt-% to about 10 wt-%.

[0060] The amount of thymol in the feed additive mixture may be such that when the feed additive mixture is included in an animal feed, the amount of thymol in the animal feed may be about 0.01 ppm or greater, about 0.05 ppm or greater, about 0.1 ppm or greater, about 0.5 ppm or greater, about 1 ppm or greater, about 1.5 ppm or greater, about 2 ppm or greater, about 2.5 ppm or greater, about 3 ppm or greater, about 3.5 ppm or greater, about 4 ppm or greater, about 4.5 ppm or greater, about 5 ppm or greater, about 6 ppm or greater, about 7 ppm or greater, about 8 ppm or greater, about 9 ppm or greater, about 10 ppm or greater, about 11 ppm or greater, about 12 ppm or greater, about 13 ppm or greater, about 14 ppm or greater, about 15 ppm or greater, about 20 ppm or greater, about 25 ppm or greater, about 30 ppm or greater, about 35 ppm or greater, about 40 ppm or greater, about 45 ppm or greater, about 50 ppm or greater, about 60 ppm or greater, about 70 ppm or greater, about 80 ppm or greater, about 90 ppm or greater, about 100 ppm or greater, about 110 ppm or greater, or about 120 ppm or greater.

[0061] The amount of thymol in the animal feed may be about 125 ppm or lower, about 120 ppm or lower, about 110 ppm or lower, about 100 ppm or lower, about 90 ppm or lower, about 80 ppm or lower, about 70 ppm or lower, about 60 ppm or lower, about 50 ppm or lower, about 45 ppm or lower, about 40 ppm or low er, about 35 ppm or lower, about 30 ppm or lower, about 25PT-1970-WO-PCTppm or lower, about 20 ppm or lower, about 15 ppm or lower, about 14 ppm or lower, about 13 ppm or lower, about 12 ppm or lower, about 11 ppm or lower. about 10 ppm or lower, about 9 ppm or low er, about 8 ppm or low er, about 7 ppm or lower, about 6 ppm or lower, about 5 ppm or lower, about 4.5 ppm or lower, about 4 ppm or lower, about 3.5 ppm or lower, about 3 ppm or lower, about 2.5 ppm or lower, about 2 ppm or lower, about 1.5 ppm or lower, about 1 ppm or lower, about 0.5 ppm or lower, or about 0.05 ppm or lower.

[0062] The amount of thymol in the animal feed may be from about 0.01 ppm to about 125 ppm, from about 0.1 ppm to about 120 ppm, from about 0.5 ppm to about 100 ppm, from about 1 ppm to about 70 ppm, from about 2 ppm to about 50 ppm. from about 4 ppm to about 30 ppm, from about 4.5 ppm to about 25 ppm, from about 5 ppm to about 20 ppm, from about 6 ppm to about 15 ppm, or from about 7 ppm to about 14 ppm.

[0063] The active ingredients in the feed additive mixture may be provided in an inactive or significantly less active form. Once the feed is ingested, the compounds in an inactive or significantly less active form may be metabolized or transformed in vivo in the animal into an active compound. For example, an inactive or significantly less active form of a cyclic monoterpenoid phenol such as thymol, will typically be a compound wherein the alcohol function has been modified to, for example, an ether, ester or anhydride function. Inactive monoterpenoid esters can be prepared from the corresponding cyclic monoterpenoid phenol and an acid or acid derivative using standard methods in synthetic organic chemistry. Such methods include various active acid derivatives (for example, acid chlorides and acid anhydrides), various coupling agents (for example, carbodiimides and carbonyldiimidazole (CDI)) and simple acid catalyzed coupling. Some cyclic monoterpenoid phenol esters that may be used in compositions according to the present disclosure may include thymol formate, thymol acetate, thymol propionate, thymol butyrate, thymol valerate and other thymol esters with straight or branched, substituted or unsubstituted aliphatic acids (C3 to C20), thymol benzoate and other thymol esters of substituted aromatic acids.Carvacrol

[0064] The feed additive mixture of the present disclosure includes carvacrol. Carvacrol is classified as a phenolic monoterpene. Similar to thymol, carvacrol is a natural monoterpene phenol compound found in several plants, including thyme (Thymus vulgaris), oregano (Origanum vulgar e), and savory (Satureja sp.). Carvacrol may also be obtained synthetically by chemical synthesis. Carvacrol may exhibit repellent effects against sea lice. Additionally, carvacrol mayPT-1970-WO-PCTdemonstrate antioxidant properties and anti-inflammatory effects, contributing to its multiple biological benefits. Carvacrol suffers from similar issues as thymol for it to be considered as an additive in animal feed formulations. The present disclosure, inter alia, describes the use of carvacrol and other natural compounds, such as essential oils containing carvacrol, as feed additives in aquaculture.

[0065] Carvacrol may be included in the feed additive mixture at any suitable concentration that will result in a desired concentration in the feed formulation, based on the inclusion rate of the feed additive mixture. For example, in some representative embodiments, the feed additive mixture includes carvacrol in a concentration of about 0.1 wt-% or greater, about 0.2 wt-% or greater, about 0.3 wt-% or greater, about 0.5 wt-% or greater, about 0.8 wt-% or greater, about 1.0 wt-% or greater, about 1.2 wt-% or greater, about 1.5 wt-% or greater, about 1.7 wt-% or greater, about 2 wt-% or greater, about 3 wt-% or greater, about 4 wt-% or greater, about 5 wt-% or greater, about 10 wt-% or greater, about 20 wt-% or greater, about 30 wt-% or greater, or about 40 wt-% or greater. The concentration of carvacrol in the feed additive mixture may be about 45 wt-% or low er, about 40 wt-% or low er, about 30 wt-% or low er, about 20 wt-% or low er, about 10 wt-% or low er, about 5 wt-% or lower, about 4 wt-% or lower, about 3 wt-% or low er, about 2 wt-% or lower, about 1.7 wt-% or lower, about 1.5 wt-% or lower, about 1.2 wt-% or lower, about 1 wt-% or lower, about 0.8 wt-% or lower, about 0.5 wt-% or lower, or about 0.3 wt-% or lower. The concentration of carvacrol in the feed additive mixture may be from about 0.1 wt-% to about 45 wt-%, from about 1 wt-% to about 20 wt-%, or from about 5 wt-% to about 10 wt-%.

[0066] The amount of carvacrol in the feed additive mixture may be such that when the feed additive mixture is included in an animal feed, the amount of carvacrol in the animal feed may be about 0.01 ppm or greater, about 0.05 ppm or greater, about 0.1 ppm or greater, about 0.5 ppm or greater, about 1 ppm or greater, about 2 ppm or greater, about 3 ppm or greater, about 4 ppm or greater, about 5 ppm or greater, about 6 ppm or greater, about 7 ppm or greater, about 8 ppm or greater, about 9 ppm or greater, about 10 ppm or greater, about 15 ppm or greater, about 20 ppm or greater, about 30 ppm or greater, about 40 ppm or greater, about 50 ppm or greater, about 60 ppm or greater, about 70 ppm or greater, about 80 ppm or greater, about 90 ppm or greater, about 100 ppm or greater, about 150 ppm or greater, about 200 ppm or greater, or about 250 ppm or greater.

[0067] The amount of carvacrol in the animal feed may be about 300 ppm or lower, about 250 ppm or lower. about 200 ppm or lower, about 150 ppm or lower, about 100 ppm or lower, about 90 ppm or low er, about 80 ppm or lower, about 70 ppm or lower, about 60 ppm or lower, aboutPT-1970-WO-PCT50 ppm or lower, about 40 ppm or lower, about 30 ppm or lower, about 20 ppm or lower, about 15 ppm or lower, about 10 ppm or lower, about 9 ppm or lower, about 8 ppm or lower, about 7 ppm or lower, about 6 ppm or lower, about 5 ppm or lower, about 4 ppm or lower, about 3 ppm or lower, about 2 ppm or lower, about 1 ppm or lower, about 0.5 ppm or lower, or about 0.05 ppm or lower.

[0068] The amount of carvacrol in the animal feed may be from about 0.01 ppm to about 300 ppm, from about 0.5 ppm to about 250 ppm, from about 1 ppm to about 200 ppm, from about 2 ppm to about 150 ppm, from about 3 ppm to about 100 ppm, from about 4 ppm to about 90 ppm, from about 5 ppm to about 80 ppm, from about 6 ppm to about 70 ppm, from about 7 ppm to about 60 ppm, from about 8 ppm to about 50 ppm, from about 9 ppm to about 40 ppm, from about 10 ppm to about 30 ppm, or from about 15 ppm to about 20 ppm.

[0069] The carvacrol in the feed additive mixture may be provided in an inactive or significantly less active form. Some carvacrol esters that may be used in compositions according to the present disclosure may include carvacrol formate, carvacrol acetate, carvacrol propionate, carvacrol butyrate, carvacrol valerate and other carvacrol esters with straight or branched, substituted or unsubstituted aliphatic acids (C3 to C20), carvacrol benzoate and other carvacrol esters of substituted aromatic acids.Eugenol

[0070] The feed additive mixture of the present disclosure includes eugenol. Eugenol is a phenylpropanoid (allyl-substituted phenol). It occurs naturally in clove oil from Syzygium aromaticum and is also found in cinnamon Cinnamomum spp.) and bay Laurus nobilis). Eugenol may also be produced synthetically. Eugenol may exhibit repellent effects against sea lice and may contribute antioxidant and anti-inflammatory activity. The free phenolic hydroxyl of eugenol contributes to antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. As with other phenolics, eugenol presents formulation challenges for animal feeds, including volatility, oxidative degradation, strong odor and flavor that can affect palatability, and regulatory limits. The present disclosure, inter alia, describes the use of eugenol and eugenol -containing compositions as feed additives in aquaculture.

[0071] Eugenol may be included in the feed additive mixture at any suitable concentration that will result in a desired concentration in the feed formulation, based on the inclusion rate of the feed additive mixture. For example, in some representative embodiments, the feed additive mixture includes eugenol in a concentration of about 5 wt-% or greater, 6 wt-% or greater, 7 wt-%PT-1970-WO-PCTor greater, 8 wt-% or greater, 9 wt-% or greater, 10 wt-% or greater, 11 wt-% or greater, 12 wt-% or greater, 13 wt-% or greater, or 14 wt-% or greater.

[0072] The concentration of eugenol in the feed additive mixture may be 85 wt-% or lower, 75 wt-% or lower, 50 wt-% or lower, 40 wt-% or lower, 30 wt-% or lower, 20 wt-% or lower, 15 wt-% or lower, 14 wt-% or lower, 13 wt-% or lower, 12 wt-% or lower, 11 wt-% or lower, 10 wt-% or lower, 9 wt-% or lower, 8 wt-% or lower, 7 wt-% or lower, or 6 wt-% or lower.

[0073] The concentration of eugenol in the feed additive mixture may be from about 5 wt-% to about 85 wt-%, from about 20 wt-% to about 75 wt-%, from about 30 wt-% to about 60 wt-%, or from about 40 wt-% to about 50 wt-%.

[0074] The amount of eugenol in the feed additive mixture may be such that when the feed additive mixture is included in an animal feed, the amount of eugenol in the animal feed may be about 10 ppm or greater, about 15 ppm or greater, about 20 ppm or greater, about 25 ppm or greater, about 30 ppm or greater, about 35 ppm or greater, about 40 ppm or greater, about 45 ppm or greater, about 50 ppm or greater, about 55 ppm or greater, about 60 ppm or greater, about 65 ppm or greater, about 70 ppm or greater, about 75 ppm or greater, about 80 ppm or greater, about 85 ppm or greater, about 90 ppm or greater, or about 95 ppm or greater.

[0075] The amount of eugenol in the animal feed may be about 100 ppm or lower, about 95 ppm or lower, about 90 ppm or lower, about 85 ppm or lower, about 80 ppm or lower, about 75 ppm or lower, about 70 ppm or lower, about 65 ppm or lower, about 60 ppm or lower, about 55 ppm or lower, about 50 ppm or low er, about 45 ppm or lower, about 40 ppm or lower, about 35 ppm or lower, about 30 ppm or lower, about 25 ppm or lower, about 20 ppm or lower, or about 15 ppm or lower, about 10 ppm or lower.

[0076] The amount of eugenol in the animal feed may be from about 10 ppm to about 100 ppm, from about 15 ppm to about 95 ppm, from about 20 ppm to about 90 ppm, from about 25 ppm to about 85 ppm, from about 30 ppm to about 80 ppm, from about 35 ppm to about 75 ppm, from about 40 ppm to about 70 ppm, from about 45 ppm to about 65 ppm, or from about 50 ppm to about 60 ppm.Cinnamaldehyde

[0077] The feed additive mixture of the present disclosure includes cinnamaldehyde. Cinnamaldehyde is an aromatic aldehyde (phenylpropanoid) that occurs naturally in cinnamon bark oils from Cinnamomum spp. and can also be produced synthetically. Cinnamaldehyde may exhibit antiparasitic or repellent effects against ectoparasites such as sea lice and may contributePT-1970-WO-PCTantimicrobial, antioxidant, and anti-inflammatory activity. Cinnamaldehyde may display antibacterial and antibiofilm activity against Gram-positive and Gram-negative bacteria. As with other volatile phenylpropanoids, cinnamaldehyde presents formulation challenges in animal feeds, including reactivity of the aldehyde group (e.g., with amines), oxidative degradation, volatility, strong flavor / odor affecting palatability, and regulatory constraints on use levels. The present disclosure, inter alia, describes the use of cinnamaldehyde and cinnamaldehyde-containing compositions as feed additives in aquaculture.

[0078] Cinnamaldehyde may be included in the feed additive mixture at any suitable concentration that will result in a desired concentration in the feed formulation, based on the inclusion rate of the feed additive mixture. For example, in some representative embodiments, the feed additive mixture includes cinnamaldehyde in a concentration of about 0.5 wt-% or greater, 1 wt-% or greater, 1.5 wt-% or greater, 2 wt-% or greater, 2.5 wt-% or greater, 3 wt-% or greater, 3.5 wt-% or greater, 4 wt-% or greater, 4.5 wt-% or greater, 5 wt-% or greater, 5.5 wt-% or greater, 6 wt-% or greater, 6.5 wt-% or greater, 7 wt-% or greater, 7.5 wt-% or greater, 8 wt-% or greater, 8.5 wt-% or greater, 9 wt-% or greater, 9.5 wt-% or greater, 10 wt-% or greater, 10.5 wt-% or greater, 11 wt-% or greater, or 12.5 wt-% or greater.

[0079] The concentration of cinnamaldehyde in the feed additive mixture may be about 15 wt-% or lower. 12.5 wt-% or lower, 11 wt-% or lower, 10.5 wt-% or lower, 10 wt-% or lower, 9.5 wt-% or lower, 9 wt-% or lower, 8.5 wt-% or lower, 8 wt-% or lower, 7.5 wt-% or lower, 7 wt-% or lower, 6.5 wt-% or lower. 6 wt-% or lower, 5.5 wt-% or lower. 5 wt-% or lower, 4.5 wt-% or lower, 4 wt-% or lower, 3.5 wt-% or lower, 3 wt-% or lower, 2.5 wt-% or lower, 2 wt-% or lower, 1.5 wt-% or lower, or 1 wt-% or lower.

[0080] The concentration of cinnamaldehyde in the feed additive mixture may be from about 0.5 wt-% to about 15 wt-%, from about 0.5 wt-% to about 12.5 wt-%, from about 0.5 wt-% to about 10 wt-%, from about 1 wt-% to about 6 wt-%, from about 1 wt-% to about 11 wt-%, from about 1.5 wt-% to about 10.5 wt-%, from about 2 wt-% to about 10 wt-%, from about 2.5 wt-% to about 9.5 wt-%, from about 3 wt-% to about 9 wt-%. from about 3.5 wt-% to about 8.5 wt-%, from about 4 wt-% to about 8 wt-%, from about 4.5 wt-% to about 7.5 wt-%, from about 5 wt-% to about 7 wt-%, or from about 5.5 wt-% to about 6.5 wt-%, or from about 5.5 wt-% to about 6 wt-%.

[0081] The amount of cinnamaldehyde in the feed additive mixture may be such that when the feed additive mixture is included in an animal feed, the amount of cinnamaldehyde in the animal feed may be about 1 ppm or greater, about 1.5 ppm or greater, about 2 ppm or greater, about 2.5 ppm or greater, about 3 ppm or greater, about 3.5 ppm or greater, about 4 ppm or greater, aboutPT-1970-WO-PCT4.5 ppm or greater, about 5 ppm or greater, about 5.5 ppm or greater, about 6 ppm or greater, about 6.5 ppm or greater, about 7 ppm or greater, about 7.5 ppm or greater, about 8 ppm or greater, about 8.5 ppm or greater, about 9 ppm or greater, about 9.5 ppm or greater, about 10 ppm or greater, about 11 ppm or greater, about 12 ppm or greater, about 13 ppm or greater, about 14 ppm or greater, about 15 ppm or greater, about 20 ppm or greater, about 25 ppm or greater, about 30 ppm or greater, about 35 ppm or greater, about 40 ppm or greater, or about 45 ppm or greater.

[0082] The amount of cinnamaldehyde in the animal feed may be about 50 ppm or lower, about 45 ppm or lower, about 40 ppm or lower, about 35 ppm or lower, about 30 ppm or lower, about 25 ppm or lower, about 20 ppm or lower, about 15 ppm or lower, about 14 ppm or lower, about 13 ppm or lower, about 12 ppm or lower, about 11 ppm or lower, about 10 ppm or lower, about 9.5 ppm or lower, about 9 ppm or lower, about 8.5 ppm or lower, about 8 ppm or lower, about 7.5 ppm or lower, about 7 ppm or lower, about 6.5 ppm or lower, about 6 ppm or lower, about 5.5 ppm or lower, about 5 ppm or lower, about 4.5 ppm or lower, about 4 ppm or lower, about 3.5 ppm or lower, about 3 ppm or lower, about 2.5 ppm or lower, about 2 ppm or lower, or about 1.5 ppm or lower.

[0083] The amount of cinnamaldehyde in the animal feed may be from about 1 ppm to about 50 ppm, from about 1.5 ppm to about 45 ppm, from about 2 ppm to about 40 ppm, from about 2.5 ppm to about 35 ppm, from about 3 ppm to about 30 ppm, from about 3.5 ppm to about 25 ppm, from about 4 ppm to about 20 ppm, from about 4.5 ppm to about 15 ppm, from about 5 ppm to about 14 ppm, from about 5.5 ppm to about 13 ppm, from about 6 ppm to about 12 ppm, from about 6.5 ppm to about 11 ppm, from about 7 ppm to about 10 ppm, from about 7.5 ppm to about 9.5 ppm, or from about 8 ppm to about 9 ppm.Other Active Compounds

[0084] The feed additive mixture of the present disclosure also includes at least two other active compounds other than thymol and carvacrol. Such other active compounds may be selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

[0085] In an embodiment, a feed additive mixture of the present disclosure also includes at least two other active compounds other than thymol. Such other at least two other active compounds include eugenol and cinnamaldehyde. The feed additive mixture may optionally include one or more active compounds other than thymol, eugenol, and cinnamaldehyde. Such optional one or more other active compounds may be selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.PT-1970-WO-PCT

[0086] In an embodiment, a feed additive mixture of the present disclosure also includes at least two other active compounds other than eugenol and cinnamaldehyde. Such other active compounds may be selected from thymol, monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

[0087] Monoterpenes are a class of hydrocarbon compounds composed of two isoprene units, resulting in a total of ten carbon atoms and sixteen hydrogen atoms (CioHie). They are a subgroup of terpenes, which are naturally occurring organic compounds produced by plants, fungi, and some bacteria. Monoterpenes are characterized by their simple chemical structure and often possess strong aromatic properties. Monoterpenes are widely distributed in nature and are responsible for the characteristic scents and flavors of many plants. They are found in essential oils extracted from various plant parts, including leaves, flowers, fruits, and seeds. Some common examples of monoterpenes include limonene, a-pinene and [3-pinene, and terpinene (bicyclic monoterpene). In some embodiments, the feed additive mixture includes one or more of limonene, a-pinene and -pinene, or terpinene (bicyclic monoterpene). In some embodiments, the feed additive mixture is substantially free of monoterpenes.

[0088] While monoterpenes are hydrocarbon compounds composed solely of carbon and hydrogen atoms, monoterpenoids are derivatives of monoterpenes that have undergone chemical modifications (such as oxidation, or functional group additions). These modifications introduce additional oxygen-containing functional groups such as alcohols, ketones, or esters, into the molecule. Monoterpenoids are classified into different subgroups based on their chemical structures and functional groups. Some common types of monoterpenoids include menthol, citral, citronellol, linalool, thymol, carvacrol, and bicyclic monoterpenoids such as cineole and camphor. In some embodiments, the feed additive mixture includes one or more of menthol, citral, citronellol, linalool, thymol, carvacrol, or bicyclic monoterpenoids such as cineole and camphor. In some embodiments, the feed additive mixture is substantially free of monoterpenoids. In some embodiments, the feed additive mixture is substantially free of thymol. In some embodiments, the feed additive mixture is substantially free of carvacrol.

[0089] Polyphenolic compounds are natural compounds found abundantly in plants, fruits, vegetables, and other botanical sources. For example, plant extracts derived from sources such as grape seed, green tea, rosemary, and citrus fruits contain polyphenolic compounds with antioxidant properties. Certain herbs and spices used as feed ingredients in aquaculture may contain polyphenolic compounds. For example, oregano, thyme, cinnamon, and cloves are rich in polyphenols. Some microalgae, such as spirulina and chlorella, contain polyphenolic compoundsPT-1970-WO-PCTalong with other bioactive substances like carotenoids and phycocyanins. Polyphenolic compounds may include flavonoids and tannins. Tannins are polyphenolic compounds know n for their ability to bind and precipitate proteins. They are commonly found in various plant parts, including leaves, bark, fruits, and seeds. Tannins can be classified into two main types, hydrolyzable tannins and condensed tannins (also known as proanthocyanidins). Hydrolyzable tannins are esters of polyphenolic acids, such as gallic acid, while condensed tannins are polymers of flavan-3-ols (catechins and epicatechins). Tannins may contribute to the astringent taste of certain foods and beverages. In some embodiments, the feed additive mixture includes one or more flavonoids or tannins. In some embodiments, the feed additive mixture is substantially free of polyphenolic compounds.

[0090] Flavonoids are a diverse group of poly phenolic compounds known for their wide range of biological activities and health benefits. Flavonoids are synthesized by plants as secondary metabolites and play various roles in plant growth, development, defense against pathogens, and UV protection. Flavonoids consist of a 15-carbon skeleton with two aromatic rings. The linker between the rings A and B may form a ring C and may include one or more double bonds, and may be substituted with various groups, in particular hydroxy and / or aldehyde groups. Each of the benzene rings A and B may be substituted with at least one hydroxy group and optionally an alkoxy groups. The basic structure of flavonoids can undergo modifications such as hydroxylation, methylation, glycosylation, and prenylation, leading to a vast array of flavonoid compounds with diverse chemical structures and properties. Flavonoids are subdivided into different subgroups, including flavones, flavonols, flavanones, flavanols or catechins, anthocyanins, and chaicones. Some examples of flavonoids include apigenin and luteolin. In some embodiments, the feed additive mixture includes one or more of apigenin and luteolin. In some embodiments, the feed additive mixture is substantially free of flavonoids.

[0091] Aromatic aldehydes are organic compounds characterized by a carbonyl group (C=O) bonded to an aromatic ring. Certain natural ingredients used in animal feed formulations may contain aromatic aldehydes as part of their chemical composition. These compounds may contribute to the overall flavor, aroma, and nutritional profile of the feed. Some examples of aromatic aldehydes include vanillin, benzaldehyde, cinnamaldehyde, and anisaldehyde. In some embodiments, the feed additive mixture includes one or more of vanillin, benzaldehyde, cinnamaldehyde. and anisaldehyde. In some embodiments, the feed additive mixture is substantially free of aromatic aldehydes.PT-1970-WO-PCT

[0092] In one or more embodiments, the active compounds other than thymol and carvacrol in the feed additive mixture of the present disclosure may include a bicyclic alcohol, an alkylbenzene, an allyl-substituted phenol, or a combination of two or more thereof. Some examples of these classes of compounds include, borneol (bicyclic alcohol), cymene (alkyl benzene), and eugenol (allyl-substituted phenol). Those skilled in the art would recognize that the specific examples of these broader classes of active compounds provided in the present disclosure may be presented as belonging to more than one class. For example, cymene (also know n as paracymene) may be classified as an alkyl benzene and as a monoterpene hydrocarbon.

[0093] In one or more embodiments, the two or more active compounds in the feed additive mixture include monoterpenoids, monoterpenes, polyphenolic compounds, aromatic aldehydes, bicyclic alcohols, alkyl benzenes, allyl-substituted phenols, or a combination of two or more thereof. Any of these active compounds may be included in the feed additive mixture at a suitable concentration that will result in a desired concentration in the feed formulation, based on the inclusion rate of the feed additive mixture. For example, in some representative embodiments, the two or more active compounds in the feed additive mixture include an active compound (such as a monoterpene, a monoterpenoid, a polyphenolic compound, an aromatic aldehyde, a bicyclic alcohol, an alkyl benzene, an allyl-substituted phenol) in a concentration of about 0.1 wt-% or greater, about 0.2 wt-% or greater, about 0.3 wt-% or greater, about 0.4 wt-% or greater, about 0.5 wt-% or greater, about 1 wt-% or greater, about 1.5 wt-% or greater, about 2 wt-% or greater, about 2.5 wt-% or greater, about 3 wt-% or greater, about 3.5 wt-% or greater, about 4 wt-% or greater, about 4.5 wt-% or greater, about 5 wt-% or greater, about 10 wt-% or greater, about 15 wt-% or greater, about 20 wt-% or greater, about 30 wt-% or greater, about 40 wt-% or greater, about 50 wt-% or greater, about 60 wt-% or greater, about 70 wt-% or greater, about 80 wt-% or greater, about 85 wt-% or greater, about 90 wt-% or greater, or about 95 wt-% or greater.

[0094] The concentration of the two or more active compounds (such as a monoterpene, a monoterpenoid, a polyphenolic compound, an aromatic aldehyde, a bicyclic alcohol, an alkyl benzene, an allyl-substituted phenol) in the feed additive mixture may be about 99 wt-% or lower, about 95 wt-% or low er, about 90 wt-% or lower, about 85 wt-% or lower, about 80 wt-% or lower, about 70 wt-% or low er, about 60 wt-% or low er, about 50 wt-% or low er, about 40 wt-% or lower, about 30 wt-% or low er, about 20 wt-% or lower, about 15 wt-% or low er, about 10 wt-% or lower, about 5 wt-% or lower, about 4.5 wt-% or lower, about 4 wt-% or lower, about 3.5 wt-% or lower, about 3 wt-% or lower, about 2.5 wt-% or lower, about 2 wt-% or lower, about 1.5 wt-% or lower,PT-1970-WO-PCTabout 1 wt-% or lower, about 0.5 wt-% or lower, about 0.4 wt-% or lower, about 0.3 wt-% or lower, or about 0.2 wt-% or lower.

[0095] The concentration of the two or more active compounds (such as a monoterpene, a monoterpenoid, a polyphenolic compound, an aromatic aldehyde, a bicyclic alcohol, an alkyl benzene, an allyl-substituted phenol) in the feed additive mixture may be from about 0.1 wt-% to about 99 wt-%, from about 1 wt-% to about 95 wt-%, from about 3 wt-% to about 90 wt-%, from about 4 wt-% to about 85 wt-%, from about 5 wt-% to about 80 wt-%, from about 6 wt-% to about 70 wt-%, from about 7 wt-% to about 60 wt-%, from about 8 wt-% to about 50 wt-%, from about 9 wt-% to about 40 wt-%, from about 10 wt-% to about 30 wt-%, or from about 15 wt-% to about 20 wt-%. In some embodiments, the concentration of an active compound in the feed additive mixture is from about 2 wt-% to about 95 wt-%, from about 5 wt-% to about 80 wt-%, from about 0.5 wt-% to about 50 wt-%, or from about 15 wt-% to about 40 wt-%.

[0096] The amount of an active compound (such as a monoterpene, a monoterpenoid, a polyphenolic compound, an aromatic aldehyde, a bicyclic alcohol, an alkyl benzene, an allylsubstituted phenol) in the feed additive mixture may be such that when the feed additive mixture is included in an animal feed, the amount of the active compound in the animal feed may be about 0.5 ppm or greater, about 1 ppm or greater, about 1.5 ppm or greater, about 2 ppm or greater, about 3 ppm or greater, about 4 ppm or greater, about 5 ppm or greater, about 10 ppm or greater, about 15 ppm or greater, about 20 ppm or greater, about 30 ppm or greater, about 40 ppm or greater, about 50 ppm or greater, about 100 ppm or greater, about 150 ppm or greater, about 200 ppm or greater, about 300 ppm or greater, about 400 ppm or greater, about 500 ppm or greater, about 600 ppm or greater, or about 700 ppm or greater.

[0097] The amount of an active compound (such as a monoterpene, a monoterpenoid, a polyphenolic compound, an aromatic aldehyde, a bicyclic alcohol, an alkyl benzene, an allylsubstituted phenol) in the animal feed may be about 800 ppm or lower, about 700 ppm or lower, about 600 ppm or lower, about 500 ppm or lower, about 400 ppm or lower, about 300 ppm or lower, about 200 ppm or lower, about 150 ppm or lower, about 100 ppm or lower, about 50 ppm or lower, about 40 ppm or lower, about 30 ppm or lower, about 20 ppm or lower, about 15 ppm or lower, about 10 ppm or lower, about 5 ppm or lower, about 4 ppm or lower, about 3 ppm or lower, about 2 ppm or lower, about 1.5 ppm or lower, or about 1 ppm or lower.

[0098] The amount of an active compound (such as a monoterpene, a monoterpenoid, a polyphenolic compound, an aromatic aldehyde, a bicyclic alcohol, an alkyl benzene, an allylsubstituted phenol) in the animal feed may be from about 10 ppm to about 100 ppm, from aboutPT-1970-WO-PCT15 ppm to about 95 ppm, from about 20 ppm to about 90 ppm, from about 25 ppm to about 85 ppm, from about 30 ppm to about 80 ppm, from about 35 ppm to about 75 ppm, from about 40 ppm to about 70 ppm, from about 45 ppm to about 65 ppm, or from about 50 ppm to about 60 ppm. In some embodiments, the amount of an active compound in the animal feed may be from about 0.5 ppm to about 400 ppm.

[0099] The active compounds (such as a monoterpene, a monoterpenoid, a polyphenolic compound, an aromatic aldehyde, a bicyclic alcohol, an alky l benzene, an allyl-substituted phenol) may be used in any combination, each active compound independently having an amount in the ranges discussed herein. The total amount of the at least two other active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes in the feed additive mixture may be about 0.5 to about 800 ppm, from about 1 ppm to about 700 ppm, from about 10 ppm to about 600 ppm, from about 20 ppm to about 500 ppm, from about 20 ppm to about 400 ppm, or from about 50 ppm to about 300 ppm.

[0100] In some embodiments, the other active compound is an aromatic aldehyde. In some embodiments, the other active compound is cinnamaldehyde.

[0101] In one or more embodiments, one or more components of the feed additive mixture of the present disclosure may be provided via the inclusion of an essential oil. Essential oil compositions may contain essential oils derived from plants (i.e.. "natural’' essential oils) and additionally or alternatively their synthetic analogues. In one or more embodiments, a combination of essential oils may be used. Some embodiments comprise a combination of natural and synthetic essential oils. In some embodiments, synthetic essential oils can be a “natures equivalent’’ synthetic blend, which generally mimics an essential oil assay of a natural essential oil. In one or more embodiments, an essential oil composition can include an essential oil fraction comprising two or more essential oils from one or more of the Lcimiciceae family, the Myrtaceae family, and Rutaceae family.

[0102] Lcimiciceae family, also known as the mint family or the deadnettle family, includes over 7.000 species of flowering plants, including herbs such as thyme, mint, basil, and oregano, as well as ornamental plants such as lavender and sage. In one or more embodiments, the Lamiaceae family essential oil in the feed additive mixture may include thyme essential oil (thyme EO). The main chemical components of thyme EO include thymol, carvacrol, borneol, cymene, linalool, apigenin, luteolin, and saponins and triterpenic acids. Saponins include a hydrophobic aglycone (sapogenin) attached to one or more hydrophilic sugar moieties (glycone) through glycosidic bonds. The aglycone portion of saponins can vary widely and may include steroid,PT-1970-WO-PCTtriterpene, or steroidal alkaloid structures. Triterpenic acids are a group of naturally occurring compounds derived from triterpenes, a subclass of terpenoids. They are formed when triterpenes undergo chemical modifications, such as oxidation or hydrolysis, resulting in the addition of functional groups such as carboxylic acids (-COOH). Some common triterpenic acids include oleanolic acid, ursolic acid, glycyrrhizic acid, and betulinic acid.

[0103] In addition to the beneficial properties of thymol, the other main components of thyme EO also display useful characteristics. For example, linalool may act as an antimicrobial and antiinflammatory agent, and cary ophyllene may provide immune system support by interacting with the CB2 receptors, furnishing potential pain and inflammation relief. Gamma-terpinene is a monoterpene. It is the precursor molecule for both thymol and carvacrol. It may display antiinflammatory' characteristics similar to carvacrol and thymol but through a different mechanism. The feed additive mixture may include linalool in an amount of about 0.1 ppm to about 20 ppm. The feed additive mixture may include caryophyllene in an amount of about 0.05 ppm to about 15 ppm. The feed additive mixture may include y-terpinene in an amount of about 0.1 ppm to about 25 ppm.

[0104] Myrtaceae family, also known as the myrtle family, includes over 5,800 species of flowering plants, including eucalyptus trees and shrubs, as well as other plants such as guava, clove, and allspice. In some embodiments, the feed additive mixture may include one or more essential oils of the Myrtaceae family, including eucalyptus essential oil (eucalyptus EO). The main components of eucalyptus EO include 1,8-cineole, cymene, pinene, and limonene. The feed additive mixture may include one or more of 1,8-cineole, cymene, pinene, and limonene, each independently in an amount of up to 25 ppm, or from 0.01 ppm to 25 ppm.

[0105] Rutaceae family, also known as the rue or citrus family, includes over 160 genera and around 2,000 species of flow ering plants, including citrus trees and shrubs, as well as other plants such as rue and bitter orange. In some embodiments, the feed additive mixture may include one or more essential oils of the Rutaceae family, including lemon essential oil (lemon EO). The main components of lemon EO include limonene and pinene. D-limonene is a monoterpene that may be applied as a fragrance ingredient in essential oils. It may display biological activity as an antioxidant, an anti -inflammation agent, and an immune modulator. Other constituents of lemon EO may include terpinene, citral, citronellal, myrcene, sabinene, and / or cymene. The feed additive mixture may include one or more of limonene, pinene, and terpinene, each independently in an amount of up to 25 ppm, or from 0.01 ppm to 25 ppm.PT-1970-WO-PCT

[0106] In one or more embodiments, the essential oil in the feed additive mixture may include thyme EO, clove EO, eucalyptus EO, lemon EO, or a combination of two or more thereof. Those skilled in the art would recognize that the main components of these essential oils are known but the exact composition of the essential oils would vary based on several factors such as the herb / tree / fruit variety, growing conditions, and extraction method. One or more of these essential oils may be included in the feed additive mixture at any suitable concentration that will result in a desired concentration in the feed formulation, based on the inclusion rate of the feed additive mixture. For example, in some representative embodiments, the feed additive mixture includes one or more essential oils, each independently at a concentration of about 1 wt-% or greater, about 2 wt-% or greater, about 3 wt-% or greater, about 4 wt-% or greater, about 5 wt-% or greater, about 10 wt-% or greater, about 15 wt-% or greater, about 20 wt-% or greater, about 25 wt-% or greater, about 30 wt-% or greater, about 35 wt-% or greater, about 40 wt-% or greater, or about 45 wt-% or greater.

[0107] In some representative embodiments, the feed additive mixture includes one or more essential oils, each independently at a concentration of about 50 wt-% or lower, about 45 wt-% or lower, about 40 wt-% or lower, about 35 wt-% or lower, about 30 wt-% or lower, about 25 wt-% or lower, about 20 wt-% or low- er, about 15 wt-% or lower, about 10 wt-% or lower, about 5 wt-% or lower, about 4 wt-% or lower, about 3 wt-% or lower, or about 2 wt-% or lower.

[0108] In some representative embodiments, the feed additive mixture includes one or more essential oils, each independently at a concentration from about 1 wt-% to about 50 wt-%, from about 2 wt-% to about 45 wt-%, from about 3 wt-% to about 40 wt-%, from about 4 wt-% to about 35 wt-%. from about 5 wt-% to about 30 wt-%, from about 10 wt-% to about 25 wt-%. or from about 15 wt-% to about 20 wt-%.

[0109] The amount of a one or more essential oils in the feed additive mixture may be such that w hen the feed additive mixture is included in an animal feed, the amount of the one or more essential oils in the animal feed may be about 1 ppm or greater, about 2 ppm or greater, about 5 ppm or greater, about 10 ppm or greater, about 15 ppm or greater, about 20 ppm or greater, about 25 ppm or greater, about 50 ppm or greater, about 75 ppm or greater, about 100 ppm or greater, about 125 ppm or greater, about 150 ppm or greater, about 175 ppm or greater, about 200 ppm or greater, about 225 ppm or greater, or about 250 ppm or greater. The amount of one or more essential oils in the animal feed may be about 500 ppm or lower, about 400 ppm or lower, about 300 ppm or lower, about 250 ppm or lower, about 200 ppm or lower, about 150 ppm or lower, about 125 ppm or lower, about 100 ppm or lower, about 75 ppm or lower, about 50 ppm or lower,PT-1970-WO-PCTabout 25 ppm or lower, or about 20 ppm or lower. The amount of one or more essential oils in the animal feed may be from about 1 ppm to about 500 ppm, from about 1 ppm to about 400 ppm, from about 1 ppm to about 300 ppm, from about 10 ppm to about 250 ppm, from about 10 ppm to about 200 ppm, or from about 10 ppm to about 100 ppm.

[0110] It is to be understood that the concentration of the essential oils may be independent from the concentration of any of its components included separately in the feed additive mixture. For example, the concentration of thyme EO or clove EO in the feed additive mixture may be independent from the concentration of thymol, carvacrol, eugenol, or cinnamaldehyde, alone or in a combination thereof, included separately in the feed additive mixture.

[0111] In some embodiments, the other active compound is thyme oil. The amount of thyme oil in the feed additive mixture may be such that when the feed additive mixture is included in an animal feed, the amount of thyme oil in the animal feed may be about 0.030 ppm or greater, about 0.050 ppm or greater, about 0.070 ppm or greater, about 0.090 ppm or greater, about 0.10 ppm or greater, about 0.13 ppm or greater, about 0.16 ppm or greater, about 0.19 ppm or greater, about 0.22 ppm or greater, about 0.25 ppm or greater, or about 0.28 ppm or greater. The amount of thyme oil in the animal feed may be about 0.30 ppm or lower, about 0.28 ppm or lower, about 0.25 ppm or lower, about 0.22 ppm or lower, about 0.19 ppm or lower, about 0.16 ppm or lower, about 0.13 ppm or lower, about 0.10 ppm or lower, about 0.090 ppm or lower, about 0.070 ppm or lower, or about 0.050 ppm or lower. The amount of thyme oil in the animal feed may be from about 0.030 ppm to about 0.30 ppm, from about 0.050 ppm to about 0.28 ppm, from about 0.070 ppm to about 0.25 ppm, from about 0.090 ppm to about 0.22 ppm, from about 0.10 ppm to about 0.19 ppm, from about 0.13 ppm to about 0.16 ppm, or from about 0.16 ppm to about 0.13 ppm.

[0112] In some embodiments, the other active compound is clove oil. The amount of clove oil in the feed additive mixture may be such that when the feed additive mixture is included in an animal feed, the amount of clove oil in the animal feed may be about 0.030 ppm or greater, about 0.050 ppm or greater, about 0.070 ppm or greater, about 0.090 ppm or greater, about 0.10 ppm or greater, about 0.13 ppm or greater, about 0.16 ppm or greater, about 0.19 ppm or greater, about 0.22 ppm or greater, about 0.25 ppm or greater, or about 0.28 ppm or greater. The amount of clove oil in the animal feed may be about 0.30 ppm or lower, about 0.28 ppm or lower, about 0.25 ppm or lower, about 0.22 ppm or lower, about 0.19 ppm or lower, about 0.16 ppm or lower, about 0.13 ppm or lower, about 0.10 ppm or lower, about 0.090 ppm or lower, about 0.070 ppm or lower, or about 0.050 ppm or lower. The amount of clove oil in the animal feed may be from about 0.030 ppm to about 0.30 ppm, from about 0.050 ppm to about 0.28 ppm, from about 0.070 ppm to aboutPT-1970-WO-PCT0.25 ppm, from about 0.090 ppm to about 0.22 ppm, from about 0.10 ppm to about 0.19 ppm, from about 0.13 ppm to about 0.16 ppm, or from about 0.16 ppm to about 0.13 ppm.

[0113] In some embodiments, the other active compound is lemon oil. The amount of lemon oil in the feed additive mixture may be such that when the feed additive mixture is mixed into an animal feed, the amount of lemon oil is 0.030 ppm or greater, 0.050 ppm or greater, 0.060 ppm or greater, 0.080 ppm or greater, 0.10 ppm or greater, 0.125 ppm or greater, or 0.15 ppm or greater. The amount of lemon oil in the animal feed may be 0.30 ppm or lower, 0.20 ppm or lower, 0.15 ppm or lower, 0.10 ppm or lower, or 0.080 ppm or lower.

[0114] In one or more embodiments, the two or more active compounds in the feed additive mixture may include citronellol. Citronellol is a terpene found in citronella plants and is commonly used as an insect repellent agent. Citronellol’s biological activities include inhibiting tick electrophysiological response and behavioral activity7by interfering the chemosensory system.

[0115] In one or more embodiments, the two or more active compounds in the feed additive mixture may include eugenol. In one or more embodiments, the two or more active compounds in the feed additive mixture may include cinnamaldehyde.

[0116] In one or more embodiments, the two or more active compounds in the feed additive mixture may include citronellol, cinnamaldehyde, eugenol, or a combination of two or more thereof. One or more of citronellol, cinnamaldehyde. and eugenol, may be included in the feed additive mixture at any suitable amount that will result in a desired amount in the feed formulation, based on the inclusion rate of the feed additive mixture. For example, in some representative embodiments, the feed additive mixture includes one or more of citronellol, cinnamaldehyde, eugenol in an amount of about 1 ppm or greater, about 2 ppm or greater, about 5 ppm or greater, about 10 ppm or greater, about 15 ppm or greater, about 20 ppm or greater, about 25 ppm or greater, about 50 ppm or greater, about 75 ppm or greater, about 100 ppm or greater, about 125 ppm or greater, about 150 ppm or greater, about 175 ppm or greater, about 200 ppm or greater, or about 250 ppm or greater. The amount of one or more of citronellol, cinnamaldehyde, eugenol in the feed additive mixture may be about 250 ppm or lower, about 200 ppm or lower, about 150 ppm or lower, about 125 ppm or lower, about 100 ppm or lower, about 75 ppm or lower, about 50 ppm or lower, about 25 ppm or lower, about 20 ppm or lower, about 15 ppm or lower, about 10 ppm or lower, about 5 ppm or lower, or about 2 ppm or lower. The amount of one or more of citronellol, cinnamaldehyde. eugenol in the feed additive mixture may7be from about 1 ppm to about 300 ppm, from about 10 ppm to about 250 ppm, from about 10 ppm to about 200 ppm, or from about 10 ppm to about 100 ppm.PT-1970-WO-PCT

[0117] In one or more embodiments, the two or more active compounds in the feed additive mixture may include cinnamaldehyde, eugenol, or a combination thereof. One or more of cinnamaldehyde and eugenol, may be included in the feed additive mixture at any suitable concentration that will result in a desired concentration in the feed formulation, based on the inclusion rate of the feed additive mixture. For example, in some representative embodiments, the feed additive mixture may include one or more of cinnamaldehyde and eugenol in a concentration of about 2 wt-% or greater, 5 wt-% or greater, about 10 wt-% or greater, about 15 wt-% or greater, about 20 wt-% or greater, about 25 wt-% or greater, about 30 wt-% or greater, about 35 wt-% or greater, about 40 wt-% or greater, about 45 wt-% or greater, about 50 wt-% or greater, about 55 wt-% or greater, about 60 wt-% or greater, about 65 wt-% or greater, about 70 wt-% or greater, about 75 wt-% or greater, about 80 wt-% or greater, about 85 wt-% or greater, or about 90 wt-% or greater.

[0118] In some representative embodiments, the feed additive mixture may include one or more of cinnamaldehyde and eugenol in a concentration of about 95 wt-% or lower, about 90 wt-% or lower, about 85 wt-% or lower, about 80 wt-% or lower, about 75 wt-% or lower, about 70 wt-% or lower, about 65 wt-% or lower, about 60 wt-% or low er, about 55 wt-% or lower, about 50 wt-% or low er, about 45 wt-% or lower, about 40 wt-% or lower, about 35 wt-% or lower, about 30 wt-% or lower, about 25 wt-% or lower, about 20 wt-% or lower, about 15 wt-% or lower, or about 10 wt-% or lower.

[0119] In some representative embodiments, the feed additive mixture may include one or more of cinnamaldehyde and eugenol in a concentration of from about 2 wt-% to about 95 wt-%, from about 10 wt-% to about 90 wt-%, from about 15 wt-% to about 85 wt-%, from about 20 wt-% to about 80 wt-%, from about 25 wt-% to about 75 wt-%, from about 30 wt-% to about 70 wt-%, from about 35 wt-% to about 65 wt-%, from about 40 wt-% to about 60 wt-%, or from about 45 wt-% to about 55 wt-%.

[0120] The amount of one or more of cinnamaldehyde and eugenol in the feed additive mixture may be such that when the feed additive mixture is included in an animal feed, the amount of the active compound in the animal feed may be about 0.5 ppm or greater, about 1 ppm or greater, about 1.5 ppm or greater, about 2 ppm or greater, about 3 ppm or greater, about 4 ppm or greater, about 5 ppm or greater, about 10 ppm or greater, about 15 ppm or greater, about 20 ppm or greater, about 30 ppm or greater, about 40 ppm or greater, about 50 ppm or greater, about 100 ppm or greater, about 150 ppm or greater, about 200 ppm or greater, about 300 ppm or greater, about 400 ppm or greater, about 500 ppm or greater, about 600 ppm or greater, or about 700 ppm or greater.PT-1970-WO-PCT

[0121] The amount of one or more of cinnamaldehyde and eugenol in the animal feed may be about 800 ppm or lower, about 700 ppm or lower, about 600 ppm or lower, about 500 ppm or lower, about 400 ppm or lower, about 300 ppm or lower, about 200 ppm or lower, about 150 ppm or lower, about 100 ppm or lower, about 50 ppm or lower, about 40 ppm or lower, about 30 ppm or lower, about 20 ppm or lower, about 15 ppm or lower, about 10 ppm or lower, about 5 ppm or lower, about 4 ppm or lower, about 3 ppm or lower, about 2 ppm or lower, about 1.5 ppm or lower, or about 1 ppm or lower.

[0122] The amount of one or more of cinnamaldehyde and eugenol in the animal feed may be from about 10 ppm to about 100 ppm, from about 15 ppm to about 95 ppm, from about 20 ppm to about 90 ppm, from about 25 ppm to about 85 ppm, from about 30 ppm to about 80 ppm, from about 35 ppm to about 75 ppm, from about 40 ppm to about 70 ppm, from about 45 ppm to about 65 ppm, or from about 50 ppm to about 60 ppm. In some embodiments, the amount of an active compound in the animal feed may be from about 0.5 ppm to about 400 ppm.

[0123] In some embodiments, the other active compound is cinnamaldehyde. The amount of cinnamaldehyde in the feed additive mixture may be such that when the feed additive mixture is mixed into an animal feed, the amount of cinnamaldehyde is 1 ppm or greater, 1.5 ppm or greater, 2 ppm or greater, 3 ppm or greater, 4 ppm or greater, 6 ppm or greater, 8 ppm or greater 10 ppm or greater, 15 ppm or greater 20 ppm or greater, 30 ppm or greater, or 40 ppm or greater. The amount of cinnamaldehyde in the animal feed may be 50 ppm or lower, 40 ppm or lower, 30 ppm or lower, 20 ppm or lower, 15 ppm or lower, 10 ppm or lower, 8 ppm or lower, 6 ppm or lower, 4 ppm or lower, 3 ppm or lower, 2 ppm or lower, or 1.5 ppm or low er.

[0124] In some embodiments, the other active compound is citronellol. The amount of citronellol in the feed additive mixture may be such that when the feed additive mixture is mixed into an animal feed, the amount of citronellol is 0.1 ppm or greater, 0.5 ppm or greater, 1 ppm or greater, 1.5 ppm or greater, 2 ppm or greater, 3 ppm or greater, or 4 ppm or greater. The amount of citronellol in the animal feed may be 20 ppm or lower, 15 ppm or lower, 10 ppm or lower, 8 ppm or lower, 6 ppm or lower, or 5 ppm or lower.

[0125] In one or more embodiments, the two or more active compounds in the feed additive mixture may include citrus flavonoids. Citrus flavonoids (for example, naringin), may have multiple biological functions. They may display strong anti-inflammatory and antioxidant activities and modulate Thl / Th2 cytokine and inflammatory mediators. Citrus flavonoids may be included in the feed additive mixture at any suitable concentration that will result in a desired concentration in the feed formulation, based on the inclusion rate of the feed additive mixture. ForPT-1970-WO-PCTexample, in some representative embodiments, the feed additive mixture includes citrus flavonoids in a concentration of about 1 ppm or greater, about 2 ppm or greater, about 5 ppm or greater, about 10 ppm or greater, about 15 ppm or greater, about 20 ppm or greater, about 25 ppm or greater, about 50 ppm or greater, about 75 ppm or greater, about 100 ppm or greater, about 125 ppm or greater, about 150 ppm or greater, about 175 ppm or greater, about 200 ppm or greater, about 225 ppm or greater, or about 250 ppm or greater. The concentration of citrus flavonoids in the feed additive mixture may be about 500 ppm or lower, about 400 ppm or lower, about 300 ppm or lower, about 250 ppm or lower, about 200 ppm or lower, about 150 ppm or lower, about 125 ppm or lower, about 100 ppm or lower, about 75 ppm or lower, about 50 ppm or lower, about 25 ppm or lower, or about 20 ppm or lower. The concentration of citrus flavonoids in the feed additive mixture may be from about 1 ppm to about 500 ppm, from about 1 ppm to about 400 ppm, from about 1 ppm to about 300 ppm, from about 10 ppm to about 250 ppm, from about 10 ppm to about 200 ppm, or from about 10 ppm to about 100 ppm.

[0126] When the feed additive mixture is added to (e.g., mixed into) a feed formulation, the active compounds may be distributed throughout the feed. The feed may be provided as pellets. The active compounds may be included in and distributed throughout the pellets.

[0127] The feed additive mixture may include a carrier. Any suitable carriers may be used. The earner may include flour, meal, or starch prepared from a grain or a legume. Examples of suitable carriers include wheat, barley, soybean meal, dehulled fava bean, pea starch, dehulled pea, and the like, and combinations of two or more thereof.Animal Feeds

[0128] The feed additive mixture of the present disclosure may be included in an animal feed. The animal feed may be an aquaculture feed, such as a fish feed, to be used as fish diets in aquaculture applications. According to an embodiment, the feed additive mixture is capable of decreasing the degradation of astaxanthin during manufacturing and / or storage. The animal feeds disclosed herein may include a base feed. The feed additive mixture may be mixed with the base feed. The feed additive mixture may be mixed with the base feed prior to manufacturing of feed pellets from the base feed.

[0129] The animal feed (e.g., fish feed) may include a feed additive mixture of desired active compounds for mixing into the feed. The components of the animal feed may be included in suitable amounts so that a desired amount in the animal feed is achieved upon mixing. According to embodiments, the animal feed (made up of a base feed and the feed additive composition asPT-1970-WO-PCTdiscussed here) includes thymol and carvacrol as active compounds. The animal feed may further include one or more active compounds, such as one or more compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes. The active compounds may provide beneficial properties to the animal feed. The active compounds may be selected, at least in part, based on their capability to have an anti-parasitic effect. The active compounds may be selected to provide one or more of disrupting the parasite-host recognition, mitigation of oxidative stress, enhancement of immune response, regulation of iron homeostasis, or their antimicrobial properties.

[0130] The animal feeds of the present disclosure include thymol and carvacrol. Thymol and carvacrol may be included in the animal feed at any suitable amount that will result in a desired amount in the feed formulation, based on the inclusion rate of the animal feed. For example, in some representative embodiments, the animal feed includes thymol in an amount of about 0.01 ppm or greater, about 0.05 ppm or greater, about 0.1 ppm or greater, about 0.5 ppm or greater, about 1 ppm or greater, about 5 ppm or greater, about 10 ppm or greater, about 13 ppm or greater, about 20 ppm or greater, about 30 ppm or greater, about 50 ppm or greater, about 75 ppm or greater, about 100 ppm or greater, about 110 ppm or greater, or about 120 ppm or greater, by weight of the animal feed.

[0131] The amount of thymol in the animal feed may be about 125 ppm or lower, about 120 ppm or lower, about 110 ppm or lower, about 100 ppm or lower, about 80 ppm or lower, about 60 ppm or lower, about 52 ppm or lower, about 50 ppm or lower, about 40 ppm or lower, about 30 ppm or lower, about 20 ppm or lower, about 13 ppm or lower, about 10 ppm or lower, about 5 ppm or lower, about 1 ppm or lower, about 0.5 ppm or lower, or about 0.05 ppm or lower, by weight of the animal feed.

[0132] The amount of thymol in the animal feed may be from about 0.01 ppm to about 125 ppm, from about 0.1 ppm to about 120 ppm, from about 1 ppm to about 100 ppm, from about 10 ppm to about 75 ppm, from about 12 ppm to about 55 ppm, or from about 13 ppm to about 52 ppm. by weight of the animal feed.

[0133] Furthermore, for example, in some representative embodiments, the animal feed includes carvacrol in an amount of about 0.01 ppm or greater, about 0.05 ppm or greater, about 0.1 ppm or greater, 1 ppm or greater, about 2 ppm or greater, about 5 ppm or greater, about 10 ppm or greater, about 20 ppm or greater, about 50 ppm or greater, about 75 ppm or greater, about 100 ppm or greater, about 125 ppm or greater, about 150 ppm or greater, about 175 ppm or greater,PT-1970-WO-PCTabout 200 ppm or greater, about 225 ppm or greater, about 250 ppm or greater, or about 275 ppm or greater, by weight of the animal feed.

[0134] The amount of carvacrol in the animal feed may be about 300 ppm or lower, about 275 ppm or lower, about 250 ppm or lower, about 225 ppm or lower, about 200 ppm or lower, about 175 ppm or lower, about 150 ppm or lower, about 125 ppm or lower, about 100 ppm or lower, about 75 ppm or lower, about 50 ppm or lower, about 20 ppm or lower, about 10 ppm or lower, about 5 ppm or lower, about 2 ppm or lower, 0.5 ppm or lower, or about 0.05 ppm or lower, by weight of the animal feed.

[0135] The amount of carvacrol in the animal feed may be from about 0.01 ppm to about 300 ppm, from about 1 ppm to about 250 ppm, from about 10 ppm to about 200 ppm, from about 15 ppm to about 150 ppm, from about 20 ppm to about 100 ppm, by weight of the animal feed.

[0136] In some embodiments, the animal feed of the present disclosure also includes at least two active compounds other than thymol and carvacrol. In one or more embodiments, the two or more active compounds in the animal feed include monoterpenoids, monoterpenes, polyphenolic compounds, aromatic aldehydes, bicyclic alcohols, alkyl benzenes, allyl-substituted phenols, or a combination of two or more thereof. Any of these active compounds may be included in the animal feed at any suitable amount that will result in a desired amount in the feed formulation, based on the inclusion rate of the animal feed. The active compounds (such as a monoterpene, a monoterpenoid, a polyphenolic compound, an aromatic aldehyde, a bicyclic alcohol, an alkyl benzene, an allyl-substituted phenol) may be used in any combination, each active compound independently having an amount in the ranges discussed herein.

[0137] The amount of an active compound (such as a monoterpene, a monoterpenoid, a polyphenolic compound, an aromatic aldehyde, a bicyclic alcohol, an alkyl benzene, an allyl-substituted phenol) in the animal feed may be about 0.5 ppm or greater, about 1 ppm or greater, about 2 ppm or greater, about 5 ppm or greater, about 10 ppm or greater, about 15 ppm or greater, about 20 ppm or greater, about 25 ppm or greater, about 50 ppm or greater, about 75 ppm or greater, about 100 ppm or greater, about 125 ppm or greater, about 150 ppm or greater, about 175 ppm or greater, about 200 ppm or greater, about 225 ppm or greater, about 250 ppm or greater, about 300 ppm or greater, about 400 ppm or greater, about 500 ppm or greater, about 600 ppm or greater, or about 700 ppm or greater, by weight of the animal feed.

[0138] The amount of an active compound (such as a monoterpene, a monoterpenoid, a polyphenolic compound, an aromatic aldehyde, a bicyclic alcohol, an alkyl benzene, an allyl-substituted phenol) in the animal feed may be about 800 ppm or lower, about 700 ppm or lower,PT-1970-WO-PCTabout 600 ppm or lower, about 500 ppm or lower, about 400 ppm or lower, about 300 ppm or lower, about 250 ppm or lower, about 200 ppm or lower, about 150 ppm or lower, about 125 ppm or lower, about 100 ppm or lower, about 75 ppm or lower, about 50 ppm or lower, about 25 ppm or lower, about 20 ppm or lower, about 15 ppm or lower, about 10 ppm or lower, about 5 ppm or lower, about 2 ppm or lower, or about 1 ppm or lower, by weight of the animal feed.

[0139] The amount of an active compound (such as a monoterpene, a monoterpenoid, a polyphenolic compound, an aromatic aldehyde, a bicyclic alcohol, an alkyl benzene, an allylsubstituted phenol) in the animal feed may be from about 0.5 ppm to about 800 ppm, from about 1 ppm to about 700 ppm, from about 5 ppm to about 600 ppm. from about 10 ppm to about 500 ppm, from about 20 ppm to about 400 ppm, or from about 30 ppm to about 300 ppm, by weight of the animal feed. In some embodiments, the amount of an active compound (such as a monoterpene, a monoterpenoid, a polyphenolic compound, an aromatic aldehyde, a bicyclic alcohol, an alkyl benzene, an allyl-substituted phenol) in the animal feed may be from about 0.5 ppm to about 400 ppm, by weight of the animal feed.

[0140] The total amount of the at least two other active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes in the animal feed may be from about 0.5 ppm to about 800 ppm, from about 0.5 ppm to about 400 ppm, from about 1 ppm to about 700 ppm, from about 10 ppm to about 600 ppm, from about 20 ppm to about 500 ppm, from about 20 ppm to about 400 ppm, or from about 50 ppm to about 300 ppm, by weight of the animal feed.

[0141] The animal feed of the present disclosure includes thymol. Thymol may be included in the animal feed at any suitable amount that will result in a desired amount in the feed formulation, based on the inclusion rate of the animal feed. For example, in some representative embodiments, the animal feed includes thymol in an amount of about 0.01 ppm or greater, about 0.05 ppm or greater, about 0.1 ppm or greater, about 0.5 ppm or greater, about 1 ppm or greater, about 5 ppm or greater, about 10 ppm or greater, about 13 ppm or greater, about 20 ppm or greater, about 30 ppm or greater, about 50 ppm or greater, about 75 ppm or greater, or about 100 ppm or greater, by weight of the animal feed.

[0142] The amount of thymol in the animal feed may be about 125 ppm or lower, about 120 ppm or lower, about 110 ppm or lower, about 100 ppm or lower, about 80 ppm or lower, about 60 ppm or lower, about 52 ppm or lower, about 50 ppm or lower, about 40 ppm or lower, about 30 ppm or lower, about 20 ppm or lower, about 13 ppm or lower, about 10 ppm or lower, about 5PT-1970-WO-PCTppm or lower, about 1 ppm or lower, about 0.5 ppm or lower, or about 0.05 ppm or lower, by weight of the animal feed.

[0143] The amount of thymol in the animal feed may be from about 0.01 ppm to about 125 ppm, from about 0.1 ppm to about 120 ppm, from about 1 ppm to about 100 ppm, from about 10 ppm to about 75 ppm. from about 12 ppm to about 55 ppm. or from about 13 ppm to about 52 ppm, by weight of the animal feed.

[0144] In some embodiments, the animal feed of the present disclosure also includes at least two active compounds other than thymol. In one or more embodiments, the two or more active compounds in the animal feed include eugenol and cinnamaldehyde. In some embodiments, the animal feed further optionally includes one or more other active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, aromatic aldehydes, bicyclic alcohols, alkyl benzenes, allyl-substituted phenols, or a combination of two or more thereof. Any of these active compounds may be included in the animal feed at any suitable amount that will result in a desired amount in the feed formulation, based on the inclusion rate of the animal feed. The active compounds (such as eugenol, cinnamaldehyde, a monoterpene, a monoterpenoid, a polyphenolic compound, an aromatic aldehyde, a bicyclic alcohol, an alkyl benzene, an allyl-substituted phenol) may be used in any combination, each active compound independently having an amount in the ranges discussed herein.

[0145] The amount of an active compound (such as eugenol, cinnamaldehyde, a monoterpene, a monoterpenoid, a polyphenolic compound, an aromatic aldehyde, a bicyclic alcohol, an alkyl benzene, an allyl-substituted phenol) in the animal feed may be about 0.5 ppm or greater, about 1 ppm or greater, about 2 ppm or greater, about 5 ppm or greater, about 10 ppm or greater, about 15 ppm or greater, about 20 ppm or greater, about 25 ppm or greater, about 50 ppm or greater, about 75 ppm or greater, about 100 ppm or greater, about 125 ppm or greater, about 150 ppm or greater, about 175 ppm or greater, about 200 ppm or greater, about 225 ppm or greater, about 250 ppm or greater, about 300 ppm or greater, about 400 ppm or greater, about 500 ppm or greater, about 600 ppm or greater, or about 700 ppm or greater, by weight of the animal feed.

[0146] The amount of an active compound (such as eugenol, cinnamaldehyde, a monoterpene, a monoterpenoid, a polyphenolic compound, an aromatic aldehyde, a bicyclic alcohol, an alkyl benzene, an allyl-substituted phenol) in the animal feed may be about 800 ppm or lower, about 700 ppm or lower, about 600 ppm or lower, about 500 ppm or lower, about 400 ppm or lower, about 300 ppm or lower, about 250 ppm or lower, about 200 ppm or lower, about 150 ppm or lower, about 125 ppm or lower, about 100 ppm or lower, about 75 ppm or lower, about 50 ppm orPT-1970-WO-PCTlower, about 25 ppm or lower, about 20 ppm or lower, about 15 ppm or lower, about 10 ppm or lower, about 5 ppm or lower, about 2 ppm or lower, or about 1 ppm or lower, by weight of the animal feed.

[0147] The amount of an active compound (such as eugenol, cinnamaldehyde. a monoterpene, a monoterpenoid, a polyphenolic compound, an aromatic aldehyde, a bicyclic alcohol, an alkyl benzene, an allyl -substituted phenol) in the animal feed may be from about 0.5 ppm to about 800 ppm, from about 1 ppm to about 700 ppm, from about 5 ppm to about 600 ppm, from about 10 ppm to about 500 ppm, from about 20 ppm to about 400 ppm, or from about 30 ppm to about 300 ppm, by weight of the animal feed. In some embodiments, the amount of an active compound (such as eugenol, cinnamaldehyde, a monoterpene, a monoterpenoid, a polyphenolic compound, an aromatic aldehyde, a bicyclic alcohol, an alkyl benzene, an allyl-substituted phenol) in the animal feed may be from about 0.5 ppm to about 400 ppm, by weight of the animal feed.

[0148] The total amount of the at least two other active compounds selected from eugenol, cinnamaldehyde, monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes in the feed additive mixture may be from about 0.5 ppm to about 800 ppm, from about 0.5 ppm to about 400 ppm, from about 1 ppm to about 700 ppm, from about 10 ppm to about 600 ppm, from about 20 ppm to about 500 ppm, from about 20 ppm to about 400 ppm, or from about 50 ppm to about 300 ppm, by weight of the animal feed.

[0149] The animal feed of the present disclosure includes eugenol and cinnamaldehyde. Eugenol and cinnamaldehyde may be included in the animal feed at any suitable amount that will result in a desired amount in the feed formulation, based on the inclusion rate of the animal feed. For example, in some representative embodiments, the animal feed includes eugenol in an amount of about 10 ppm or greater, about 15 ppm or greater, about 20 ppm or greater, about 25 ppm or greater, about 30 ppm or greater, about 35 ppm or greater, about 40 ppm or greater, about 45 ppm or greater, about 50 ppm or greater, about 55 ppm or greater, about 60 ppm or greater, about 65 ppm or greater, about 70 ppm or greater, about 75 ppm or greater, about 80 ppm or greater, about 85 ppm or greater, about 90 ppm or greater, or about 95 ppm or greater, by weight of the animal feed.

[0150] The amount of eugenol in the animal feed may be about 100 ppm or lower, about 95 ppm or lower, about 90 ppm or lower, about 85 ppm or lower, about 80 ppm or lower, about 75 ppm or lower, about 70 ppm or lower, about 65 ppm or lower, about 60 ppm or lower, about 55 ppm or lower, about 50 ppm or lower, about 45 ppm or lower, about 40 ppm or lower, about 35PT-1970-WO-PCTppm or lower, about 30 ppm or lower, about 25 ppm or lower, about 20 ppm or lower, or about 15 ppm or lower, about 10 ppm or lower, by weight of the animal feed.

[0151] The amount of eugenol in the animal feed may be from about 10 ppm to about 100 ppm, from about 15 ppm to about 95 ppm, from about 20 ppm to about 90 ppm, from about 25 ppm to about 85 ppm, from about 30 ppm to about 80 ppm, from about 35 ppm to about 75 ppm, from about 40 ppm to about 70 ppm, from about 45 ppm to about 65 ppm, or from about 50 ppm to about 60 ppm, by weight of the animal feed.

[0152] Furthermore, for example, in some representative embodiments, the animal feed includes cinnamaldehyde in an amount of about 1 ppm or greater, about 1.5 ppm or greater, about 2 ppm or greater, about 2.5 ppm or greater, about 3 ppm or greater, about 3.5 ppm or greater, about 4 ppm or greater, about 4.5 ppm or greater, about 5 ppm or greater, about 5.5 ppm or greater, about 6 ppm or greater, about 6.5 ppm or greater, about 7 ppm or greater, about 7.5 ppm or greater, about 8 ppm or greater, about 8.5 ppm or greater, about 9 ppm or greater, about 9.5 ppm or greater, about 10 ppm or greater, about 11 ppm or greater, about 12 ppm or greater, about 13 ppm or greater, about 14 ppm or greater, about 15 ppm or greater, about 20 ppm or greater, about 25 ppm or greater, about 30 ppm or greater, about 35 ppm or greater, about 40 ppm or greater, or about 45 ppm or greater, by weight of the animal feed.

[0153] The amount of cinnamaldehyde in the animal feed may be about 50 ppm or lower, about 45 ppm or lower, about 40 ppm or lower, about 35 ppm or lower, about 30 ppm or lower, about 25 ppm or lower, about 20 ppm or lower, about 15 ppm or lower, about 14 ppm or lower, about 13 ppm or lower, about 12 ppm or lower, about 11 ppm or lower, about 10 ppm or lower, about 9.5 ppm or lower, about 9 ppm or lower, about 8.5 ppm or lower, about 8 ppm or lower, about 7.5 ppm or lower, about 7 ppm or lower, about 6.5 ppm or lower, about 6 ppm or lower, about 5.5 ppm or lower, about 5 ppm or lower, about 4.5 ppm or lower, about 4 ppm or lower, about 3.5 ppm or lower, about 3 ppm or lower, about 2.5 ppm or lower, about 2 ppm or low er, or about 1.5 ppm or low er, by w eight of the animal feed.

[0154] The amount of cinnamaldehyde in the animal feed may be from about 1 ppm to about 50 ppm, from about 1.5 ppm to about 45 ppm, from about 2 ppm to about 40 ppm, from about 2.5 ppm to about 35 ppm, from about 3 ppm to about 30 ppm, from about 3.5 ppm to about 25 ppm, from about 4 ppm to about 20 ppm, from about 4.5 ppm to about 15 ppm, from about 5 ppm to about 14 ppm, from about 5.5 ppm to about 13 ppm. from about 6 ppm to about 12 ppm, from about 6.5 ppm to about 11 ppm, from about 7 ppm to about 10 ppm, from about 7.5 ppm to about 9.5 ppm, or from about 8 ppm to about 9 ppm, by w eight of the animal feed.PT-1970-WO-PCT

[0155] In some embodiments, the animal feed of the present disclosure also includes at least two active compounds other than eugenol and cinnamaldehyde. In one or more embodiments, the two or more active compounds in the animal feed include thymol, monoterpenoids, monoterpenes, polyphenolic compounds, aromatic aldehydes, bicyclic alcohols, alkyl benzenes, allyl-substituted phenols, or a combination of two or more thereof. Any of these active compounds may be included in the animal feed at any suitable amount that will result in a desired amount in the feed formulation, based on the inclusion rate of the animal feed. The active compounds (such as thymol, a monoterpene, a monoterpenoid, a polyphenolic compound, an aromatic aldehyde, a bicyclic alcohol, an alkyl benzene, an allyl-substituted phenol) may be used in any combination, each active compound independently having an amount in the ranges discussed herein. The amount of an active compound (such as thymol, a monoterpene, a monoterpenoid, a polyphenolic compound, an aromatic aldehyde, a bicyclic alcohol, an alkyl benzene, an allyl-substituted phenol) in the animal feed may be about 0.5 ppm or greater, about 1 ppm or greater, about 2 ppm or greater, about 5 ppm or greater, about 10 ppm or greater, about 15 ppm or greater, about 20 ppm or greater, about 25 ppm or greater, about 50 ppm or greater, about 75 ppm or greater, about 100 ppm or greater, about 125 ppm or greater, about 150 ppm or greater, about 175 ppm or greater, about 200 ppm or greater, about 225 ppm or greater, about 250 ppm or greater, or about 300 ppm or greater, by weight of the animal feed.

[0156] The amount of an active compound (such as thymol, a monoterpene, a monoterpenoid, a polyphenolic compound, an aromatic aldehyde, a bicyclic alcohol, an alkyl benzene, an allyl-substituted phenol) in the animal feed may be about 400 ppm or lower, about 300 ppm or lower, about 250 ppm or lower, about 200 ppm or lower, about 150 ppm or lower, about 125 ppm or lower, about 100 ppm or lower, about 75 ppm or lower, about 50 ppm or lower, about 25 ppm or lower, about 20 ppm or lower, about 15 ppm or lower, about 10 ppm or lower, about 5 ppm or lower, about 2 ppm or lower, or about 1 ppm or lower, by weight of the animal feed.

[0157] The amount of an active compound (such as thymol, a monoterpene, a monoterpenoid, a polyphenolic compound, an aromatic aldehyde, a bicyclic alcohol, an alkyl benzene, an allyl-substituted phenol) in the animal feed may be from about 0.5 ppm to about 400 ppm, from about 1 ppm to about 300 ppm, from about 5 ppm to about 250 ppm, from about 10 ppm to about 200 ppm, from about 20 ppm to about 100 ppm, or from about 30 ppm to about 50 ppm, by weight of the animal feed.

[0158] The total amount of the at least two other active compounds selected from thymol, monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes in the feedPT-1970-WO-PCTadditive mixture may be about 0.5 to about 400 ppm, from about 1 ppm to about 300 ppm, from about 10 ppm to about 200 ppm, or from about 20 ppm to about 100 ppm, by weight of the animal feed.

[0159] The animal feeds of the present disclosure include the feed additive mixture. The feed additive mixture may be included in the animal feed at any suitable inclusion rate (suitable amount) that will result in a desired amount in the animal feed. For example, in some representative embodiments, the animal feed includes feed additive mixture in an amount of about 1 ppm or greater, about 1.5 ppm or greater, about 2 ppm or greater, about 5 ppm or greater, about 7.5 ppm or greater, about 8 ppm or greater, about 10 ppm or greater, about 12 ppm or greater, about 16 ppm or greater, about 20 ppm or greater, about 30 ppm or greater, about 40 ppm or greater, about 50 ppm or greater, about 60 ppm or greater, about 64 ppm or greater, about 70 ppm or greater, about 80 ppm or greater, about 90 ppm or greater, about 100 ppm or greater, about 110 ppm or greater, about 120 ppm or greater, about 130 ppm or greater, about 140 ppm or greater, about 150 ppm or greater, about 160 ppm or greater, about 170 ppm or greater, about 180 ppm or greater, about 190 ppm or greater, about 200 ppm or greater, about 225 ppm or greater, about 250 ppm or greater, about 275 ppm or greater, about 300 ppm or greater, about 350 ppm or greater, about 400 ppm or greater, about 450 ppm or greater, about 500 ppm or greater, about 550 ppm or greater, about 600 ppm or greater, about 650 ppm or greater, about 700 ppm or greater, or about 750 ppm or greater, by weight of the animal feed.

[0160] In some representative embodiments, the animal feed includes feed additive mixture in an amount of about 800 ppm or lower, about 750 ppm or lower, about 700 ppm or lower, about 650 ppm or lower, about 600 ppm or lower, about 550 ppm or lower, about 500 ppm or lower, about 450 ppm or lower, about 400 ppm or lower, about 350 ppm or lower, about 300 ppm or lower, about 275 ppm or lower, about 250 ppm or lower, about 225 ppm or lower, about 200 ppm or lower, about 190 ppm or lower, about 180 ppm or lower, about 170 ppm or lower, about 160 ppm or lower, about 150 ppm or lower, about 140 ppm or lower, about 130 ppm or lower, about 120 ppm or lower, about 110 ppm or lower, about 100 ppm or lower, about 90 ppm or lower, about 80 ppm or lower, about 70 ppm or lower, about 64 ppm or lower, about 60 ppm or lower, about 50 ppm or lower, about 40 ppm or lower, about 30 ppm or lower, about 20 ppm or lower, about 16 ppm or lower, about 12 ppm or lower, about 10 ppm or lower, about 8 ppm or lower, about 7.5 ppm or lower, about 5 ppm or lower, about 2 ppm or lower, or about 1.5 ppm or lower, by weight of the animal feed.PT-1970-WO-PCT

[0161] In some representative embodiments, the animal feed includes feed additive mixture in an amount from about 1 ppm to about 800 ppm, from about 1.5 ppm to about 750 ppm, from about 2 ppm to about 700 ppm, from about 5 ppm to about 650 ppm, from about 7.5 ppm to about 600 ppm, from about 10 ppm to about 550 ppm, from about 12 ppm to about 500 ppm, from about 16 ppm to about 450 ppm, from about 20 ppm to about 400 ppm. from about 40 ppm to about 350 ppm, from about 64 ppm to about 300 ppm, from about 150 ppm to about 200 ppm from about 160 ppm to about 275 ppm, from about 170 ppm to about 250 ppm, from about 180 ppm to about 225 ppm, or from about 190 ppm to about 200 ppm, by weight of the animal feed. In some representative embodiments, the animal feed includes feed additive mixture in an amount from about 50 ppm to about 800 ppm, or from about 150 ppm to 200 ppm, by weight of the animal feed.

[0162] It will be appreciated that the animal feeds disclosed herein can include suitable types of animal feed specific for a given animal. It will also be appreciated that the animal feeds disclosed herein can include suitable types of fish feed specific for a given fish species. The fish feeds may be used as a component of a diet fed to any species belonging to the families Cyprinidae, Cichlidae, Pangasiidae, Sciaenidae, Serranidae, Carangidae, Sparidae, Lateolabracidae, Moronidae, Mugilidae, Cypriniformes, Latidae, Eleotridae, Tilapiini and Salmonidae. In various aspects, this disclosure provides a fish feed or fish feed diet for species within the family Salmonidae. The fish feeds provided herein may be used to feed wild fish or farmed fish. In various aspects, both wild fish and farmed fish may be fed simultaneously. Further, the fish feed may be used to feed freshwater fish or salt water (e.g., marine) fish, or both. In some other embodiments, the animal feed may be formulated for other farmed animals, such as various farmed land animals. In some embodiments, the animal feed is formulated for shrimp.

[0163] The animal feed of the present disclosure may include a number of different ingredients or raw materials that can sustain life, growth, and reproduction of the animal. The animal feeds disclosed herein may be fish feeds and may include a number of different ingredients or raw materials that can sustain life, growth, and reproduction of the fish. The animal feeds can include any substrate that is edible to the animal. For example, an edible substrate can provide a source of nutrition to the animal or may be an inert substrate with no nutritive value to the animal. In various aspects, the animal feeds disclosed herein can include feeds that are either nutritional animal feeds or non-nutritional feeds. Nutritional animal feeds can include a nutritional food stuff formulated for fish as part of its diet as the main source of nutrition, growth, and reproduction. Suitable nutritional animal feeds can include one or more of proteinaceous material as a source of proteins, peptides, and amino acids; carbohydrates; and fats, as described below. Non-nutritional animalPT-1970-WO-PCTfeeds can include any substrate that is edible to the animal but does not provide nutrition to sustain life, grow th, or reproduction. In various aspects, the nutritional or non-nutritional fish feeds herein can include one or more compounds designed to alter the quality, quantity, or appearance of a fish and fish tissue. For example, a nutritional or non-nutritional fish feed can include a carotenoid compound to improve the appearance (e.g.. color) of the muscle tissue. By way of example, the carotenoid compound can include compounds such as astaxanthin.

[0164] The animal feed may be a complete fish feed. A complete fish feed may be a feed for fish that is compounded to be fed as the sole ration and that can maintain life, promote growth, and sustain reproduction without any additional substances being consumed except water. Complete fish feeds can include compounded mixtures containing various energy sources such as carbohydrates, proteins, and fats. In various aspects, the fish feeds herein can include at least a protein and a starch. Additional ingredients may be included, such as vitamins and minerals as necessary to support the life, growth, and reproduction of fish. A complete fish feed can include ingredients such as, but not limited to, fish meal, poultry meal, plant meal, vegetable meal, com meal, com gluten meal, soy meal, soy protein concentrate, single cell protein, insect meal, algae meal, algae oil, krill meal, krill oil, meat meal, blood meal, feather meal, starches, tapioca starch, wheat, wheat gluten, guar meal, guar protein concentrate, peas, pea protein concentrate, pea starch, beans, fava beans, sunflower meal, vegetable oil, canola oil, poultry oil, rapeseed oil. fish oil, soy¬ oil, linseed oil, camelina oil, lecithin, macro-minerals, minerals, vitamins, amino acids, pigments, and any combinations thereof. It will be appreciated that the fish feeds herein can include fish meal that can include plant-derived or animal-derived matter. Any animal-derived matter present in the fish meal may be derived from the same species of fish or a different species of fish (e.g., heterologous species or non-fish species).

[0015] The animal feed of the present disclosure may- include appropriate amount of protein for the target animal. In some representative embodiments, the total protein content in the animal feed may be about 10 wt-% or greater, about 20 wt-% or greater, about 25 wt-% or greater, about 30 wt-% or greater, about 35 wt-% or greater, about 40 wt-% or greater, about 45 wt-% or greater, about 50 wt-% or greater, about 55 wt-% or greater, about 60 wt-% or greater, or about 65 wt-% or greater, by weight of the animal feed. In some representative embodiments, the total protein content in the animal feed may be about 70 wt-% or lower, about 65 wt-% or lower, about 60 wt-% or lower, about 55 wt-% or lower, about 50 wt-% or lower, about 45 wt-% or lower, about 40 wt-% or lower, about 35 wt-% or lower, about 30 wt-% or lower, or about 25 wt-% or lower, by weight of the animal feed. In some representative embodiments, the total protein content in thePT-1970-WO-PCTanimal feed may be from about 10 wt-% to about 70 wt-%, from about 20 wt-% to about 60 wt-%, from about 28 wt-% to about 60 wt-%, from about 30 wt-% to about 50 wt-%, or from about 35 wt-% to about 45 wt-%, by weight of the animal feed. The total protein in the animal feed may be variable depending on the formulation, species, age, and intended use of the feed. It will be appreciated that the various protein requirements of the species of animal receiving the animal feed may be adjusted to meet the protein requirements of that species. Typically, the desired concentration of protein in a fish feed is about 40 wt-%, by weight of the animal feed.

[0166] The protein in the animal feeds disclosed herein may be from any suitable source including, but not limited to, one or more of fish meal, land-animal protein (e.g., poultry meal), plant-based protein (e.g., vegetable meal), or any combinations thereof.

[0017] In some representative embodiments, the amount of fish meal in the animal feed may be about 0 wt-% or greater, about 5 wt-% or greater, about 10 wt-% or greater, about 20 wt-% or greater, about 25 wt-% or greater, about 30 wt-% or greater, about 40 wt-% or greater, or about 50 wt-% or greater, by weight of the animal feed. In some representative embodiments, the amount of fish meal in the animal feed may be about 80 wt-% or lower, about 70 wt-% or lower, about 60 wt-% or lower, about 50 wt-% or lower, or about 40 wt-% or lower, by w eight of the animal feed. In some representative embodiments, the amount of fish meal in the animal feed may be from about 0 wt-% to about 80 wt-%. from about 10 wt-% to about 70 wt-%. from about 20 wt-% to about 60 wt-%, or from about 25 wt-% to about 50 wt-%, by weight of the animal feed.

[0018] In some representative embodiments, the land-animal protein in the animal feed may be about 0 wt-% or greater, about 5 wt-% or greater, about 10 wt-% or greater, about 20 wt-% or greater, about 25 wt-% or greater, about 30 wt-% or greater, about 40 wt-% or greater, or about 50 wt-% or greater, by w eight of the animal feed. In some representative embodiments, the landanimal protein in the animal feed may be about 80 wt-% or low er, about 70 wt-% or lower, about 60 wt-% or low er, about 50 wt-% or low er, or about 40 wt-% or low er, by w eight of the animal feed. In some representative embodiments, the land-animal protein in the animal feed may be from about 0 wt-% to about 80 wt-%. from about 10 wt-% to about 70 wt-%. from about 20 wt-% to about 60 wt-%, or from about 25 wt-% to about 50 wt-%, by weight of the animal feed.

[0019] In some representative embodiments, the plant-based protein in the animal feed may be about 0 wt-% or greater, about 5 wt-% or greater, about 10 wt-% or greater, about 20 wt-% or greater, about 25 wt-% or greater, about 30 wt-% or greater, about 40 wt-% or greater, or about 50 wt-% or greater, by w eight of the animal feed. In some representative embodiments, the plantbased protein in the animal feed may be about 80 wt-% or lower, about 70 wt-% or lower, aboutPT-1970-WO-PCT60 wt-% or lower, about 50 wt-% or lower, or about 40 wt-% or lower, by weight of the animal feed. In some representative embodiments, the plant-based protein in the animal feed may be from about 0 wt-% to about 80 wt-%, from about 10 wt-% to about 70 wt-%, from about 20 wt-% to about 60 wt-%, or from about 25 wt-% to about 50 wt-%, by weight of the animal feed.[01701 The animal feed of the present disclosure may include appropriate amount of total fat (e.g., oil, fat, and / or lipids). Tn some representative embodiments, the total fat content in the animal feed may be about 5 wt-% or greater, about 10 wt-% or greater, about 20 wt-% or greater, or about 25 wt-% or greater, by w eight of the animal feed. In some representative embodiments, the total fat content in the animal feed may be about 50 wt-% or lower, about 40 wt-% or lower, about 30 wt-% or lower, about 20 wt-% or lower, or about 10 wt-% or lower, by weight of the animal feed. In some representative embodiments, the total fat content in the animal feed may be from about 5 wt-% to about 50 wt-%, from about 10 wt-% to about 40 wt-%, or from about 18 wt-% to about 40 wt-%, by weight of the animal feed. The total fat in the animal feed may be variable depending on the formulation, target fish species, and intended use of the animal feed. It will be appreciated that the various fat requirements of the target species of animal and may be adjusted to meet the fat requirements of that species. Suitable fats for use herein may include, but are not to be limited to, those provided by canola oil, poultry oil, rapeseed oil, fish oil, soy oil, linseed oil, camelina oil. palm oil, lecithin, or any combinations or fractions thereof.

[0171] EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) are omega-3 fatty acids that are components of the total fatty acid content in certain foods or supplements. The term “EPA / DHA of total fatty' acids'’ refers to the percentage or amount of EPA and DHA present relative to the total amount of fatty acids in the product. The animal feed of the present disclosure may include appropriate amount of EPA / DHA of total fatty acids. In some representative embodiments, the EPA / DHA of total fatty acids in the animal feed may be about 2 wt-% or greater, about 3 wt-% or greater, about 4 wt-% or greater, about 5 wt-% or greater, about 6 wt-% or greater, or about 10 wt-% or greater, by weight of the animal feed. In some representative embodiments, the EPA / DHA of total fatty acids in the animal feed may be about 25 wt-% or lower, about 20 wt-% or lower, about 17.5 wt-% or lower, about 15 wt-% or lower, about 12.5 wt-% or lower, or about 10 wt-% or lower, by w eight of the animal feed. In some representative embodiments, the EPA / DHA of total fatty acids in the animal feed may be from about 2 wt-% to about 25 wt-%, from about 4 wt-% to about 15 wt-%, or from about 5 wt-% to about 10 wt-%, by weight of the animal feed. Typically, the desired EPA / DHA of total fatty acids in the animal feed is about 7 wt-%, by w eight of the animal feed.PT-1970-WO-PCT

[0172] The animal feed of the present disclosure may include appropriate amount of starch. In some representative embodiments, the starch content in the animal feed may be about 2 wt-% or greater, about 3 wt-% or greater, about 4 wt-% or greater, about 5 wt-% or greater, about 6 wt-% or greater, or about 10 wt-% or greater, by weight of the animal feed. In some representative embodiments, the starch content in the animal feed may be about 25 wt-% or lower, about 20 wt-% or lower, about 17.5 wt-% or lower, about 15 wt-% or lower, about 12.5 wt-% or lower, or about 10 wt-% or lower, by weight of the animal feed. In some representative embodiments, the starch content in the animal feed may be from about 2 wt-% to about 25 wt-%, from about 4 wt-% to about 15 wt-%, or from about 6 wt-% to about 15 wt-%, by weight of the animal feed. Typically, the desired starch content in the animal feed is about 8 wt-%, by weight of the animal feed.

[0173] The animal feed of the present disclosure may include appropriate amount of dry matter. In some representative embodiments, the dry7matter content of the animal feed may be about 80 wt-% or greater, about 85 wt-% or greater, about 90 wt-% or greater, or about 95 wt-% or greater, by weight of the animal feed. In some representative embodiments, the dry matter content of the animal feed may be about 99 wt-% or lower, about 98 wt-% or lower, or about 95 wt-% or lower, by w eight of the animal feed. In some representative embodiments, the dry matter content of the animal feed may be from about 80 wt-% to about 99 wt-%, from about 85 wt-% to about 99 wt-%, or from about 90 wt-% to about 98 wt-%, by weight of the animal feed. Typically, the dry matter content in the animal feed is about 95 wt-%, by weight of the animal feed.

[0174] The moisture content of the animal feeds disclosed herein may vary depending on the contents and preparation method of the feed. The animal feed of the present disclosure may include appropriate amount of moisture content. In some representative embodiments, the moisture content in the animal feed may be about 2 wt-% or greater, about 3 wt-% or greater, about 4 wt-% or greater, about 5 wt-% or greater, about 10 wt-% or greater, or about 15 wt-% or greater, by weight of the animal feed. In some representative embodiments, the moisture content in the animal feed may be about 20 wt-% or lower, about 15 wt-% or lower, about 10 wt-% or lower, or about 8 wt-% or lower, by weight of the animal feed. In some representative embodiments, the moisture content in the animal feed may be from about 2 wt-% to about 20 wt-%, from about 4 wt-% to about 15 wt-%, or from about 5 wt-% to about 10 wt-%, by w eight of the animal feed.PT-1970-WO-PCTAdditional Components

[0175] The animal feed may also include additional components. Additional components may include components that provide nutritional benefits, health benefits, or aid in processing, handling, or storage. Such additional components may include, for example, vitamins, minerals, fatty acids, amino acids, thickeners, fillers, pigments, and any combinations thereof.Target Animals, Fish

[0176] In one or more embodiments, the animal feed of the present disclosure is a complete fish feed. Animal feeds provided herein may be fed as a fish feed diet or used to feed any fish that is susceptible to infection, infestation, or co-infection by one or more pests. For example, the fish feed may be used in aquaculture as a component of a diet fed to any farmed fish including, for example, commercially relevant fish species. For example, the fish feeds provided herein may form part of diet fed to any of freshwater fish, brackish fish, or saltwater fish. The fish feeds may be used as a component of a diet fed to any species belonging to the families Cyprinidae, Cichlidae, Pangasiidae, Sciaenidae, Serranidae, Carangidae, Sparidae, Lateolabracidae, Moronidae, Mugilidae, Cypriniformes, Latidae, Eleotridae, Tilapiini and Salmonidae. As such, the fish feeds herein may be used to feed species belonging to any of the genera within these families and in particular, those species that are farmed for human or animal consumption. For example, and without limitation, the fish feeds described herein may be used to feed species belonging to the genera Salmo and / or Oncorhynchus . In particular, the fish feeds herein may be used to control pests in populations of wild or farmed salmon or trout species, including, for example, any of Atlantic salmon (Salmo salary Pacific salmon, Char, or Rainbow trout. Moreover, the fish feed may be used for other fish species within the aquaculture industry such as sea bass, bream, grouper, pompano, and tuna, as well as in the pet and decorative fish industries, for example for pest control in koi (Cyprinus rubrofuscus) and goldfish (Carassius auratus).Target Copepod Pests

[0177] The feed additive mixture of the present disclosure may be formulated to target specific pests affecting the target animals. In some embodiments, the feed additive mixture is formulated to target copepod pests. As used herein, the term “copepod” refers to a group of crustaceans found in fresh water and in seawater, and which have one or more parasitic phases of their life cycle. Unless otherwise noted, the term copepod may refer to any of the various species of sea lice asPT-1970-WO-PCTdescribed herein. Copepod pests of the target animals (e.g., fish) may include ectoparasites including species belonging to the phylum Arthropoda. Susceptible arthropods include various copepods that include many species of sea lice that inhabit fish hosts. By way of example, in the case of fish belonging to the family Salmonidae (e.g., Salmo and / or Oncorhynchus spp.), the feed additive mixture may help control sea lice infections, sea lice infestations, copepod infections, copepod infestations, or any combinations thereof. The feed additive mixture may help control ectoparasitic crustaceans, including branchiurans such as Argulus spp. (fish lice; order Arguloida, subclass Branchiura) and copepods such as Caligus spp. (sealice; subclass Copepodaln particular, the feed additive mixture may be effective at controlling copepod infections and infestations of one or more types of farmed fish.

[0178] A species of copepod that is an ectoparasite of Atlantic salmon belongs to the Lepeophtheirus genus and is known as the salmon louse, Lepeophtheirus salmonis. As used herein, it will be appreciated that the term ”sea louse” refers to the singular form and the term “sea lice” refers to the plural form. Each term may be used interchangeably unless otherwise noted. Lepeophtheirus salmonis are a species of copepod ectoparasites that primarily live on salmon, including Atlantic and Pacific salmon, and sometimes on sea trout. Other types of copepod ectoparasites infecting fish belonging to the family Salmonidae include Caligus clemensi, Caligus elongatus, and Caligus rogercresseyi. Sea lice, including those from the genera Lepeophtheirus and Caligus, are ectoparasites that feed off the blood, mucus, muscle, and skin of various salmon species.

[0179] On average, L. salmonis may live for approximately up to 215 days for a full life cycle. It will be appreciated that each stage of the L. salmonis life cycle may be dependent on temperature, salinity- of the water, water currents, pollution levels, and various additional environmental factors. Thus, the complete life cycle of L. salmonis may be from 32 days up to 215 days depending on the fluctuations in such external factors.

[0180] In various aspects, the fish feed including the feed additive mixture is capable of any of reducing, preventing, or controlling Lepeophtheirus or Caligus infections or infestations. A Lepeophtheirus infection or infestation may be caused or contributed by the salmon louse, Lepeophtheirus salmonis. A Caligus infection or infestation may be caused or contributed to by the sea lice, Caligus clemensi, Caligus elongatus, and Caligus rogercresseyi. In various aspects, the fish feed may be for species belonging to the family Salmonidae. For example, the fish feeds herein may be formulated for Salmo and / or Oncorhynchus spp. However, it will be appreciated that the fish feeds herein may be given to any fish that is susceptible to a Lepeophtheirus or CaligusPT-1970-WO-PCTinfection or infestation. It will be appreciated that an infection or infestation with Lepeophtheirus or Caligus species may leave fish vulnerable to secondary infection with one or more additional pests, including parasites, viruses, bacteria, and the like, leading to a co-infection in the fish.Target Non-Copepod Parasites

[0181] In some embodiments, the feed additive mixture is formulated to target non-copepod pests. Pests of the target fish herein may include non-copepod parasites that may cause a number of diseases in fish. The non-copepod parasites that are suitable for managing with the feed additive mixtures described herein may include one or more of Amyloodinium ocellatum, Car dicola forsteri, Cryptocaryon irritans, Diplectanum spp., Gyrodactylus salaris, Henneguya salminicola, Ichthyophthirius multifiliis, Kudoa thyrsites, Myxobolus cerebralis, Neoparamoeba perurans, Saprolegnia spp. including Saprolegnia parasitica, Sparicotyle chrysophrii, Trichodina spp., or Zeylanicobdella arugamensis. These organisms may all cause one or more clinical manifestations of disease that may decrease the wellbeing of the fish, affect the edible muscle quality of the fish, may lower farm productivity by decreasing the fish available for market, increase mortality in the fish populations, and may lead to significant economic losses for farmers.

[0182] The dinoflagellate ectoparasite Amyloodinium ocellatum is known to cause amyloodiniosis in farmed and aquarium fish alike. Amyloodiniosis is characterized primarily as an infestation of the gills of fish, but may also infest the skin, fins, and eyes. Clinical symptoms of amyloodiniosis may include anorexia, decreased activity', labored breathing, and poor overall wellbeing in the fish.

[0183] The parasitic ciliate Ichthyophthirius multifiliis may cause the disease in fish often called ‘lch.”f. multifiliis shows low host specificity and infests the interior of the organism on the epithelial layers of the gills, skin, and fins. I. multifliis may cause significant damage to the skin, gills, and fins of its host, and thus the fish experience distress and lead to anorexia, respiratory distress, discoloration and color spotting, inactivity, scratching against objects, and upside-down swimming. If left uncontrolled, I. multifiliis may lead to 100 % mortality rates.

[0184] The ciliate Cryptocaryon irritans is known to cause marine white spot disease in wild and farm-raised fish. C. irritans organisms feed on the skin, eyes, and gills of fish and may deteriorate the function of these tissues. The marine white spot disease caused by C. irritans may lead to whiteish nodules on the fish and to an overproduction of mucus, discoloration of the skin, anorexia, hyperactivity, poor overall wellbeing, and respiratory distress in the fish.PT-1970-WO-PCT

[0185] The organisms of the genus Diplectanum are parasites that infect the gill of fish species. Diplectanum suitable as targets for the feed additive mixtures herein may include but are not to be limited to, Diplectanum aculeatum, Diplectanum aequans, Diplectanum banyulense, Diplectanum belengeri, Diplectanum bocqueti, Diplectanum chabaudi, Diplectanum copiosum, Diplectanum dollfusi, Diplectanum femineum. or Diplectanum flagritubus . The Diplectanum spp. may infest fish by feeding on the gills and causing diplectanosis in the fish. Fish with diplectanosis may present with damaged gill tissue, respiratory distress, poor overall wellbeing, and secondary infection due to viruses and bacteria.

[0186] The salmon fluke, Gyrodactylus salaris, is a parasite species that lives on the surface of the body of many freshwater fish. G. salaris feeds on the body of many freshwater fish and may cause skin wounds and open the fish to co-infection with one or more additional pests.

[0187] Cardicola forsteri is a blood fluke that may infect bluefin tunas. C. forsteri may cause severe bronchitis, or swelling of the gills, and myocarditis in fish and may lead to high mortality rates in fish populations.

[0188] Kudoa thyr sites is a myxozoan parasite that infects the muscles of a broad range of marine fishes and has a global distribution. K. thyrsites may cause nodules or cysts within the muscle tissue of the fish. The infection may lead to an exaggerated immune response that may lead to tissue degeneration whereby the tissue becomes soft due to enzymatic digestion of the muscle fibers. Fish infected with K. thyrsites may become damaged to the point that they are no longer of sufficient quality for marketing to consumers, thus causing significant losses to farmers.

[0189] The myxosporean Henneguya salminicola (also known as Henneguya zschokkei) may infect or infest fish and cause a disease called milky flesh or tapioca disease. Milky’ flesh or tapioca disease as caused by H. salminicola, may be characterized in that the muscle of the fish is intermittently spotted with small, white cysts filled with the spores of the organism. The milky flesh disease may disrupt farm productivity, because while generally the disorder is harmless, it causes a loss in economic profit due to unsightly flesh caused by the disorder.

[0190] The myxosporeanA7yxo6o / M5 cerebralis causes whirling disease in various fish species. Whirling disease may be characterized as a chronic disease that infects salmon, and particularly juvenile salmon, and causes severe deformities in the cartilage, bone, and muscle of the fish. When the parasite infects the cartilage of the fish its presence may create pressure against the spinal cord and cause a characteristic swimming pattern in the form of a whirl. The disease may be particularly uncomfortable for the fish and may lead to severe economic loss for farmers.PT-1970-WO-PCT

[0191] The protozoan Neoparamoeba perurans (synonym. Paramoeba perurans) causes amoebic gill disease in various fish species. The protozoan attaches to the gills of fish and causes tissue damage through increased mucus production and multifocal patches of swollen tissue. Fish experience respiratory distress due to gill damage. The disease causes extreme distress in fish and may lead to significant economic losses for fish farms.

[0192] The organism Sparicotyle chrysophrii is a parasite that infects the gills of various fish species. Once the organisms attach to the gills it causes tissue damage that may lead to respiratory distress, anemia, and death. The disease causes extreme distress in fish and may lead to significant economic losses for fish farms.

[0193] The single-celled flagellates of the genus Trichodina may infect or infest various fish, including those common to ornamental fishponds such as koi and goldfish. Trichodina attaches to the skin and gills of fish and causes damage to these tissues as well as respiratory7distress, anorexia, and loss of scales.

[0194] The fungus of the genus Saprolegnia spp. , including Saprolegnia parasitica, may infect or infest various fish ty pes and may cause saprolegniasis. Saprolegniasis may be characterized in fish by the presence of greyish-white patches having filamentous mycelium on the body or fins of fish. The disorder may cause tissue damage and in extreme cases, death.

[0195] Zeylanicobdella arugamensis is a species of marine leeches that may parasitize a number of fish species reared in aquaculture. Z. arugamensis attaches to the skin, gills, fins, and mouth surfaces of various fish species and causes wounds in areas of attachment. Wounds caused by attachment and feeding of Z. arugamensis may predispose the fish to secondary infection with other pests.

[0196] Monogeneans are a group of parasitic flatworms belonging to the class Monogenea. They are primarily^ ectoparasites that typically7attach to the external surfaces of fish, amphibians, and aquatic reptiles. Monogeneans are characterized by their simple, flattened bodies and a posterior attachment organ known as the haptor, which bears hooks, clamps, or both for attaching to the host.

[0197] Benedenia seriolae is a species of monogenean parasite belonging to the family Capsalidae. It is a parasitic flatworm that primarily infects marine fish, particularly species of the family Sparidae (seabreams) and Seriolidae (amber] acks and yellowtails). Benedenia seriolae is known to cause a condition called “Benedenia disease” or “fluke disease” in affected fish. Infestations by B. seriolae may lead to significant damage to the gills, skin, and fins of the host fish, resulting in respiratory7distress, tissue damage, and secondary infections.PT-1970-WO-PCT

[0198] Neobenedenia is a genus of monogenean parasites within the family Capsalidae. These parasites primarily infect marine fish, causing skin and gill infections. Neobenedenia species are similar to Benedenia species, and they are often collectively referred to as “skin flukes” or “gill flukes” due to their parasitic lifestyle and effects on fish health.

[0199] Gyrodactylus is a genus of monogenean parasites belonging to the family Gyrodactylidae. These parasites are known for infecting a wide range of freshwater and marine fish species worldwide. Gyrodactylus infections may cause significant damage to the skin, fins, and gills of the host fish. Infestations may lead to irritation, inflammation, tissue damage, and respiratory distress, particularly in heavily infected individuals or species that are sensitive to the parasite. Infestations may be introduced through infected fish, contaminated water, or equipment, and they may also spread through the movement of infected fish between different locations.

[0200] In one or more embodiments, the feed additive mixture of the present disclosure is formulated to target myxozoa, monogenean, sea lice, Benedenia seriolae, Neobenedenia species, Gyrodactylus, Amyloodinium, Neoparamoeba, Ichthyophthirius, or a combination of two or more thereof.Methods of Making

[0201] The present disclosure provides methods for making an animal feed that includes a feed additive mixture. In some embodiments, the present disclosure provides methods for making feed additive mixtures for adding to animal feed. The present disclosure provides methods for making an animal feed and a feed additive mixture for adding to the animal feed, which include thymol, carvacrol, and at least two other active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes. The present disclosure provides methods for making animal feed, and feed additive mixtures for adding to the animal feed, with formulations that may have an anti-parasitic effect. The components of the feed additive mixture may be included in any suitable concentration so that a desired final concentration in the animal feed is achieved upon mixing.

[0202] The method may include manufacturing a feed additive mixture. The method may further optionally include manufacturing an animal feed that includes the feed additive mixture. Manufacturing may include various processes, including mixing, grinding, extrusion, drying, or a combination of two or more thereof. At least some of the processing steps may include application of heat and / or steam to the feed additive composition. In some cases, the feed additive compositionPT-1970-WO-PCTthat includes the feed additive may be extruded into pellets during manufacturing. The feed additive composition may further be processed into a feed.

[0203] The feed additive mixture may be made by mixing the components of the feed additive mixture. Some of the components of the feed additive mixture may be pre-mixed and ground prior to mixing with other components. The feed additive mixture may optionally be granulated, pelleted, and / or dried. In some embodiments, the feed additive mixture includes one or more of thymol, carvacrol, and at least two other active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes, mixed and ground prior to addition of the remaining ingredients.

[0204] The method for making an animal feed may include the preparation of raw materials and further processing to make the animal feed. The method includes mixing of the various components to prepare a feed additive composition. The method may further include processing the feed additive composition into the feed using any commonly used processing steps. The feed additive composition may include the feed additive mixture and a base feed. The method includes mixing the feed additive mixture and the base feed. The method may involve mixing the components to improve nutrient distribution and consistency in the production process.

[0205] The preparation of the raw materials may include various mixing and grinding steps. For example, certain raw materials may be mixed together and ground to a desired particle size. In some embodiments, raw materials may be ground to a desired particle size separately, prior to being mixed together. Any suitable means to grind the material may be used, for example, a hammer mill.

[0206] A base feed may be formed from various raw materials described elsewhere herein. The base feed may include macro-ingredients and numerous micro-ingredients. The base feed ingredients may be loaded into a mixer, where they undergo pre-grind mixing. Pre-grind mixing may be done to improve homogeneity of the base feed. After pre-grind mixing, the base feed may be subjected to grinding to refine the particle size. Mixing and grinding may be used to improve the feed’s texture and digestibility and its suitability’ for consumption by the animal. In some embodiments, additional micro-ingredients may be added to the base feed before or after the pregrind mixing, and before or after grinding. In some embodiments, the additional micro-ingredients may be separately ground prior to their addition to the base feed. In some embodiments, the additional micro-ingredients may not be ground prior to the addition to the base feed.

[0207] The feed additive mixture may be added to the base feed. The feed additive mixture may be added to the base feed after the pre-grind mixing and grinding. In some embodiments, allPT-1970-WO-PCTmacro-ingredients, along with micro-ingredients, and feed additive mixture, including astaxanthin, may be loaded into a mixer, where they undergo pre-grind mixing. The mixture may then be ground to obtain the desirable particle size. The mixture may again be mixed after being ground.

[0208] The prepared feed additive mixture may be mixed into the base feed for preparing the feed additive composition. The feed additive composition may also include additional ingredients in addition to the base feed and the feed additive mixture. The base feed or any additional ingredients may undergo further grinding to facilitate their incorporation into the feed. In some embodiments, the base feed or any additional ingredients do not undergo additional grinding. After the feed additive mixture is added to the base feed, the mixture may undergo mixing to distribute the feed additive mixture throughout the base feed. After mixing of the based feed and feed additive mixture, the mixture may be preconditioned to adjust its moisture content. The moisture content of the mixture may be brought to about 10 wt-% or greater, about 15 wt-% or greater, about 18 wt-% or greater, or about 20 wt-% or greater. The moisture content of the mixture may be brought to about 50 wt-% or lower, about 40 wt-% or lower, about 30 wt-% or lower, or about 25 wt-% or lower. The moisture content of the mixture may be brought to from about 18 wt-% to about 40 wt-%.

[0209] The mixture may be processed through extrusion or pressing to form pellets or other desired shapes, such as nuggets, steam pellets, or tablets. The moisture addition may occur during the extrusion process. The extruded feed may be cooled to ambient temperature. Extrusion may be followed by dry ing to remove excess moisture. The extruded feed may be dried to a desired final moisture content to improve the stability and shelf-life of the final product. One or more additives may be included in or added to the pellets. To further enhance the quality of the feed, the feed pellets may be coated. The pellets may be coated by one or more coating agents or oils. By way of example, the pellet may be sealed by the application of a coating agent, such as a layer or coating of oil, such as fish oil. In various aspects, one or more further layers or coatings of animal feed may be applied to the (optionally sealed) coating or layer of animal feed. In this way, any given animal feed flake or pellet may include multiple layers of animal feed and / or sealing substance layers. In some embodiments, a thin layer of coating may be applied to improve appearance and nutritional content. Examples of suitable coating methods include vacuum coating, spray coating, dip coating, and the like. Alternatively, the feed mixture may be provided as a powder or as flakes. Finally, the feed may be packed into appropriate packaging materials to help preserve their freshness and quality until use.PT-1970-WO-PCTMethods of Using

[0210] The present disclosure provides methods of using the animal feed that includes the feed additive mixture. The present disclosure provides methods of feeding the animal feed to an animal. The present disclosure provides methods of feeding the animal feed to fish. The animal feed includes a feed additive mixture that includes thymol, carvacrol, and at least two other active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes. The animal feed is formulated to have an anti-parasitic effect. The animal feed and the feed additive mixtures may have other beneficial effects, such as antioxidant, antiinflammatory, and antimicrobial effects. The animal feed and the feed additive mixture may disrupt parasite-host recognition, mitigate oxidative stress, enhance immune response, regulate iron homeostasis in the animal. The animal feed may be a fish feed.

[0211] The method of using the animal feed includes administering the feed to the animal. The method of using animal feed may include administering the feed to fish. In some embodiments, the present disclosure provides methods for feeding an aquatic animal. The method of feeding an aquatic animal may include administering a specific amount of feed. Further, the method may include administering a specific amount of the active compounds to the animal. The animal feed may be fed at a rate that achieves the desired antiparasitic effect. For example, the animal feed may be administered to the animal through the animal feed at a rate that results in the administration of the active compounds at a rate of about 1 mg to 120 mg per kg body weight / day (mg / kg / day), about 1 to about 90 mg / kg / day, about 1 to about 80 mg / kg / day, about 1 to about 70 mg / kg / day. about 1 to about 60 mg / kg / day, about 5 to about 50 mg / kg / day, about 10 to about 40 mg / kg / day, about 15 to about 35 mg / kg / day, about 20 to about 30 mg / kg / day, about 0.01 to about 10 mg / kg / day, or about 0.01 to about 5.0 mg / kg / day.EXEMPLARY EMBODIMENTS

[0212] The following is a list of exemplary embodiments, the numbering of which is not to be construed as designating levels of importance:

[0213] Embodiment 1 is a feed additive mixture comprising, by weight of the feed additive mixture: 0.1 wt-% to 15 wt-%, 1 wt-% to 12.5 wt-%, or 5 wt-% to 10 wt-% thymol; 0.1 wt-% to 45 wt-%. 1 wt-% to 20 wt-%, or 5 wt-% to 10 wt-% carvacrol; and 0.1 wt-% to 99 wt-%, 1 wt-% to 95 wt-%, 5 wt-% to 80 wt-%, 10 wt-% to 30 wt-%, or 15 wt-% to 20 wt-% of a total of at leastPT-1970-WO-PCTtwo other active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

[0214] Embodiment 2 is a feed additive mixture comprising, by weight of the feed additive mixture: 0.1 wt-% to 15 wt-%, 1 wt-% to 12.5 wt-%, or 5 wt-% to 10 wt-% thymol; and 0.1 wt-% to 99 wt-%, 1 wt-% to 95 wt-%, 2 wt-% to 95 wt-%, 5 wt-% to 80 wt-%. 10 wt-% to 30 wt-%. or 15 wt-% to 20 wt-% of a total of at least two other active compounds comprising eugenol and cinnamaldehyde and optionally one or more active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

[0215] Embodiment 3 is a feed additive mixture comprising, by weight of the feed additive mixture: 5 wt-% to 85 wt-%, 20 wt-% to 75 wt-%, 30 wt-% to 60 wt-%, or 40 wt-% to 50 wt-% eugenol; 0.5 wt-% to 15 wt-%, 0.5 wt-% to 12.5 wt-%, 0.5 wt-% to 10 wt-%, 1 wt-% to 11 wt-%, 1.5 wt-% to 10.5 wt-%, 2 wt-% to 10 wt-%, 2.5 wt-% to 9.5 wt-%, 3 wt-% to 9 wt-%, 3.5 wt-% to 8.5 wt-%, 4 wt-% to 8 wt-%, 4.5 wt-% to 7.5 wt-%. 5 wt-% to 7 wt-%. or 5.5 wt-% to 6.5 wt-%, or 5.5 wt-% to 6 wt-% cinnamaldehyde; and 0.1 wt-% to 99 wt-%. 0.5 wt-% to 50 wt-%, 1 wt-% to 95 wt-%, 5 wt-% to 80 wt-%, 10 wt-% to 30 wt-%, or 15 wt-% to 20 wt-% of a total of at least two other active compounds selected from thymol, monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

[0216] Embodiment 4 is the feed additive mixture of any one of embodiments 2 or 3, wherein eugenol is present in an amount of 7 wt-% to 12.5 wt-%.

[0217] Embodiment 5 is the feed additive mixture of any one of embodiments 2 or 3, w herein cinnamaldehyde is present in an amount of 1 wt-% to 6 wt-%.

[0218] Embodiment 6 is the feed additive mixture of any one of the preceding embodiments, w herein the sum of the amount of the at least two other active compounds is 15 wt-% to 40 wt-%.

[0219] Embodiment 7 is the feed additive mixture of any one of embodiments 2 or 3, w h erein the weight ratio of eugenol to cinnamaldehyde is from 3:1 to 6:1.

[0220] Embodiment 8 is the feed additive mixture of Embodiment 7, wherein the weight ratio of eugenol to cinnamaldehyde is from 3.5:1 to 5: 1.

[0221] Embodiment 9 is the feed additive mixture of any one of the preceding embodiments, wherein the feed additive mixture comprises thyme oil, clove oil, and cinnamaldehyde.

[0222] Embodiment 10 is the feed additive mixture of Embodiment 9, wherein eugenol is provided from clove oil and thymol is provided from thyme oil.

[0223] Embodiment 11 is the feed additive mixture of any one of the preceding embodiments, wherein the at least tw o other active compounds comprise one or more monoterpenoids.PT-1970-WO-PCT

[0224] Embodiment 12 is the feed additive mixture of Embodiment 11, wherein the monoterpenoids comprise citronellol, linalool, cineole, or a combination of two or more thereof.

[0225] Embodiment 13 is the feed additive mixture of any one of the preceding embodiments, wherein the at least two other active compounds comprise one or more monoterpenes.

[0226] Embodiment 14 is the feed additive mixture of Embodiment 13. wherein the monoterpenes comprise pinene, limonene, or a combination thereof.

[0227] Embodiment 15 is the feed additive mixture of any one of the preceding embodiments, wherein the at least two other active compounds comprise one or more polyphenolic compounds.

[0228] Embodiment 16 is the feed additive mixture of Embodiment 15. wherein the polyphenolic compounds comprise one or more flavonoids, tannins, or a combination thereof.

[0229] Embodiment 17 is the feed additive mixture of any one of the preceding embodiments, wherein the at least two other active compounds comprise a bicyclic alcohol, an alkylbenzene, an allyl-substituted phenol, an aromatic aldehyde, or a combination of two or more thereof.

[0230] Embodiment 18 is the feed additive mixture of Embodiment 17, wherein the at least two other active compounds comprise borneol, cymene, eugenol, cinnamaldehyde, or a combination of two or more thereof.

[0231] Embodiment 19 is the feed additive mixture of any one of the preceding embodiments, wherein the at least two other active compounds comprise citronellol and one or more flavonoids.

[0232] Embodiment 20 is the feed additive mixture of any one of the preceding embodiments, wherein the feed additive mixture provides activation of animal immune response, reduction of animal oxidative stress, reduction of parasite host recognition, and regulation of iron metabolism, when administered to the animal.

[0233] Embodiment 21 is the feed additive mixture of any one of the preceding embodiments, wherein the feed additive mixture provides reduction of available iron to parasite in or on an animal when administered to the animal.

[0234] Embodiment 22 is the feed additive mixture of any one of the preceding embodiments, wherein the feed additive mixture has an antimicrobial effect.

[0235] Embodiment 23 is the feed additive mixture of any one of the preceding embodiments, wherein the feed additive mixture is formulated to target a parasite.

[0236] Embodiment 24 is the feed additive mixture of any one of the preceding embodiments, wherein the feed additive mixture is formulated to target myxozoa, monogenean, sea lice, Benedenia seriolae, Neobenedenia species. Gyrodactylus, Amyloodinium, Neoparamoeba, Ichthyophthirius, or a combination of two or more thereof.PT-1970-WO-PCT

[0237] Embodiment 25 is the feed additive mixture of any one of the preceding embodiments, wherein the feed additive mixture is formulated to target sea lice.

[0238] Embodiment 26 is the feed additive mixture of any one of the preceding embodiments, wherein the feed additive mixture is formulated to target Caligus rogercresseyi.

[0239] Embodiment 27 is an animal feed comprising: a base feed; and a feed additive mixture comprising, by weight of the animal feed: 0.01 ppm to 125 ppm, 0.1 ppm to 120 ppm, 1 ppm to 100 ppm, 10 ppm to 75 ppm, 12 ppm to 55 ppm, or 13 ppm to 52 ppm thymol; 0.01 ppm to 300 ppm, 1 ppm to 250 ppm, 10 ppm to 200 ppm, 15 ppm to 150 ppm, 20 ppm to 100 ppm carvacrol; and a total amount of 0.5 ppm to 800 ppm, 0.5 ppm to 400 ppm, 1 ppm to 700 ppm, 10 ppm to 600 ppm, 20 ppm to 500 ppm, 20 ppm to 400 ppm, or 50 ppm to 300 ppm of at least two other active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

[0240] Embodiment 28 is an animal feed comprising: a base feed; and a feed additive mixture comprising, by weight of the animal feed: 0.01 ppm to 125 ppm, 0.1 ppm to 120 ppm, 1 ppm to 100 ppm, 10 ppm to 75 ppm, 12 ppm to 55 ppm, or 13 ppm to 52 ppm thymol; and a total amount of 0.5 ppm to 800 ppm, 0.5 ppm to 400 ppm, 1 ppm to 700 ppm, 10 ppm to 600 ppm, 20 ppm to 500 ppm, 20 ppm to 400 ppm, or 50 ppm to 300 ppm of at least two other active compounds comprising eugenol and cinnamaldehyde and optionally one or more active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

[0241] Embodiment 29 is an animal feed comprising: a base feed; and a feed additive mixture comprising, by weight of the animal feed: 10 ppm to 100 ppm, 15 ppm to 95 ppm, 20 ppm to 90 ppm, 25 ppm to 85 ppm, 30 ppm to 80 ppm. 35 ppm to 75 ppm. 40 ppm to 70 ppm, 45 ppm to 65 ppm, or 50 ppm to 60 ppm eugenol; 1 ppm to 50 ppm, 1.5 ppm to 45 ppm, 2 ppm to 40 ppm, 2.5 ppm to 35 ppm, 3 ppm to 30 ppm, 3.5 ppm to 25 ppm, 4 ppm to 20 ppm, 4.5 ppm to 15 ppm, 5 ppm to 14 ppm, 5.5 ppm to 13 ppm, 6 ppm to 12 ppm, 6.5 ppm to 11 ppm, 7 ppm to 10 ppm, 7.5 ppm to 9.5 ppm, or 8 ppm to 9 ppm cinnamaldehyde; and a total amount of 0.5 to 400 ppm, 1 ppm to 300 ppm, 10 ppm to 200 ppm, or 20 ppm to 100 ppm of at least two other active compounds selected from thymol, monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

[0242] Embodiment 30 is the animal feed of any one of embodiments 27 to 29, comprising a carrier wherein the carrier further comprises a flour, meal, or starch prepared from a grain or a legume.PT-1970-WO-PCT

[0243] Embodiment 31 is the animal feed of any one of embodiments 27 to 30, wherein the animal feed is a complete fish feed.

[0244] Embodiment 32 is the animal feed of any one of embodiments 27 to 31, wherein the base feed comprises protein, starch, and fat.

[0245] Embodiment 33 is the animal feed of Embodiment 32, wherein the animal feed comprises from 28 wt-% to 60 wt-% protein, 6 wt-% to 15 wt-% starch, and from 18 wt-% to 40 wt-% fat.

[0246] Embodiment 34 is the animal feed of embodiments 32 or 33, wherein the animal feed comprises land-animal protein, fish meal, plant-based protein, or a combination of two or more thereof.

[0247] Embodiment 35 is the animal feed of any one of embodiments 27 to 34, wherein the animal feed comprises one or more of pellets, extruded nuggets, steam pellets, flakes, tablets, granules, or powders.

[0248] Embodiment 36 is the animal feed of any one of embodiments 27 to 35, wherein the at least two other active compounds comprise one or more monoterpenoids.

[0249] Embodiment 37 is the animal feed of Embodiment 36, wherein the monoterpenoids comprise citronellol, linalool, cineole, or a combination of two or more thereof.

[0250] Embodiment 38 is the animal feed of any one of embodiments 27 to 37, wherein the at least two other active compounds comprise one or more monoterpenes.

[0251] Embodiment 39 is the animal feed of Embodiment 38, wherein the monoterpenes comprise pinene, limonene, or a combination thereof.

[0252] Embodiment 40 is the animal feed of any one of embodiments 27 to 39, wherein the at least two other active compounds comprise one or more polyphenolic compounds.

[0253] Embodiment 41 is the animal feed of Embodiment 40, wherein the polyphenolic compounds comprise one or more flavonoids, tannins, or a combination thereof.

[0254] Embodiment 42 is the animal feed of any one of embodiments 27 to 41, wherein the at least two other active compounds comprise a bicyclic alcohol, an alkylbenzene, an allylsubstituted phenol, an aromatic aldehyde, or a combination of two or more thereof.

[0255] Embodiment 43 is the animal feed of Embodiment 42, wherein the at least two other active compounds comprise borneol, cymene, eugenol, cinnamaldehyde, or a combination of two or more thereof.

[0256] Embodiment 44 is the animal feed of any one of embodiments 27 to 43, wherein the at least two other active compounds comprise citronellol and one or more flavonoids.PT-1970-WO-PCT

[0257] Embodiment 45 is the animal feed of any one of embodiments 27 to 44, wherein the animal feed comprises thyme oil and clove oil.

[0258] Embodiment 46 is the animal feed of any one of embodiments 27 to 45, wherein the feed additive mixture provides activation of animal immune response, reduction of animal oxidative stress, reduction of parasite host recognition, and regulation of iron metabolism, when administered to the animal.

[0259] Embodiment 47 is the animal feed of any one of embodiments 27 to 46, wherein the feed additive mixture provides reduction of available iron to parasite in or on an animal when administered to the animal.

[0260] Embodiment 48 is the animal feed of any one of embodiments 27 to 47, wherein the feed additive mixture has an antimicrobial effect.

[0261] Embodiment 49 is the animal feed of any one of embodiments 27 to 48, wherein the feed additive mixture is formulated to target a parasite.

[0262] Embodiment 50 is the animal feed of any one of embodiments 27 to 49, wherein the feed additive mixture is formulated to target myxozoa, monogenean, sea lice, Benedenia seriolae, Neobenedenia species, Gyrodactylus, Amyloodinium, Neoparamoeba, Ichthyophthirius, or a combination of two or more thereof.

[0263] Embodiment 51 is the animal feed of any one of embodiments 27 to 50, wherein the feed additive mixture is formulated to target sea lice.

[0264] Embodiment 52 is the animal feed of any one of embodiments 27 to 51 , wherein the feed additive mixture is formulated to target Caligus rogercresseyi.

[0265] Embodiment 53 is the animal feed of any one of embodiments 27 to 52, wherein the feed additive mixture is included in the animal feed from 1 ppm to 800 ppm, 1.5 ppm to 750 ppm, 2 ppm to 700 ppm, 5 ppm to 650 ppm, 7.5 ppm to 600 ppm, 10 ppm to 550 ppm, 12 ppm to 500 ppm, 16 ppm to 450 ppm, 20 ppm to 400 ppm, 40 ppm to 350 ppm, 64 ppm to 300 ppm, 160 ppm to 275 ppm, 170 ppm to 250 ppm, 180 ppm to 225 ppm, or 190 ppm to 200 ppm, by weight of the animal feed.

[0266] Embodiment 54 is the animal feed of Embodiment 53, wherein the feed additive mixture is included in the animal feed from 150 ppm to 200 ppm, by weight of the animal feed.

[0267] Embodiment 55 is the animal feed of any one of embodiments 27 to 54, wherein the feed additive mixture is configured for post-extrusion oil-coating onto the animal feed.PT-1970-WO-PCT

[0268] Embodiment 56 is the animal feed of any one of embodiments 27 to 55, wherein when the animal feed is administered to an animal, sea louse burden in the animal is reduced relative to a control diet.

[0269] Embodiment 57 is the animal feed of Embodiment 56, wherein sea louse burden in the animal is reduced by at least about 20 % four weeks post-infestation.

[0270] Embodiment 58 is the animal feed of Embodiment 56, wherein sea louse burden in the animal is reduced by at least about 32 % four weeks post-infestation.

[0271] Embodiment 59 is the animal feed of any one of embodiments 27 to 58, wherein when the animal feed is administered to an animal, plasma total antioxidant capacity increases relative to a control diet.

[0272] Embodiment 60 is the animal feed of any one of embodiments 27 to 59, wherein when the animal feed is administered to an animal, plasma total bilirubin decreases relative to a control diet.

[0273] Embodiment 61 is the animal feed of any one of embodiments 27 to 60, wherein when the animal feed is administered to an animal, hepatic and skin hepcidin antimicrobial peptide A (HampA) expression is regulated relative to a control diet.

[0274] Embodiment 62 is the animal feed of any one of embodiments 27 to 61, wherein when the animal feed is administered to an animal, skin tumor necrosis factor-a (TNF-a) expression decreases relative to a control diet.

[0275] Embodiment 63 is a method for making a feed additive mixture for adding to a fish feed, the method comprising: mixing thymol, carvacrol, and at least two other active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

[0276] Embodiment 64 is a method for making a feed additive mixture for adding to a fish feed, the method comprising: mixing thymol and at least two other active compounds comprising eugenol and cinnamaldehyde and optionally one or more compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

[0277] Embodiment 65 is a method for making a feed additive mixture for adding to a fish feed, the method comprising: mixing eugenol and cinnamaldehyde and at least two other active compounds selected from thymol, monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

[0278] Embodiment 66 is the method of any one of embodiments 63 to 65, further comprising dissolving or dispersing the feed additive mixture in a coating oil.PT-1970-WO-PCT

[0279] Embodiment 67 is a method for making an animal feed, the method comprising: mixing a feed additive mixture with a base feed to form a feed composition, the feed additive mixture comprising, by weight of the animal feed: 0.01 ppm to 125 ppm thymol; 0.01 ppm to 300 ppm carvacrol; and a total amount of 0.5 ppm to 800 ppm of at least two other active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

[0280] Embodiment 68 is a method for making an animal feed, the method comprising: mixing a feed additive mixture with a base feed to form a feed composition, the feed additive mixture comprising, by weight of the animal feed: 0.01 ppm to 125 ppm thymol: and a total amount of 0.5 ppm to 800 ppm of at least two other active compounds comprising eugenol and cinnamaldehyde and optionally one or more active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

[0281] Embodiment 69 is a method for making an animal feed, the method comprising: mixing a feed additive mixture with a base feed to form a feed composition, the feed additive mixture comprising, by weight of the animal feed: 10 ppm to 100 ppm eugenol; 1 ppm to 50 ppm cinnamaldehyde; and a total amount of 0.5 ppm to 400 ppm of at least two other active compounds selected from thymol, monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

[0282] Embodiment 70 is the method of any one of embodiments 67 to 69, wherein the animal feed is a complete fish feed.

[0283] Embodiment 71 is the method of any one of embodiments 67 to 70, wherein the base feed comprises protein, starch, and fat.

[0284] Embodiment 72 is the method of embodiment 71, wherein the animal feed comprises from 28 wt-% to 60 wt-% protein, 6 wt-% to 15 wt-% starch, and from 18 wt-% to 40 wt-% fat.

[0285] Embodiment 73 is the method of any one of embodiments 67 to 72, wherein the animal feed comprises land-animal protein, fish meal, plant-based protein, or a combination of two or more thereof.

[0286] Embodiment 74 is the method of any one of embodiments 67 to 73. wherein the method further comprises extruding or pressing the feed composition to form pellets, extruded nuggets, steam pellets, flakes, tablets, or granules.

[0287] Embodiment 75 is a method of feeding an aquatic animal, the method comprising administering an amount of animal feed to the aquatic animal, the feed comprising: a base feed; and a feed additive mixture comprising active compounds comprising, by weight of the animal feed: 0.01 ppm to 125 ppm, 0.1 ppm to 120 ppm, 1 ppm to 100 ppm, 10 ppm to 75 ppm, 12 ppmPT-1970-WO-PCTto 55 ppm, or 13 ppm to 52 ppm thymol; 0.01 ppm to 300 ppm, 1 ppm to 250 ppm, 10 ppm to 200 ppm, 15 ppm to 150 ppm, 20 ppm to 100 ppm carvacrol; and a total amount of 0.5 ppm to 800 ppm, 0.5 ppm to 400 ppm, 1 ppm to 700 ppm, 10 ppm to 600 ppm, 20 ppm to 500 ppm, 20 ppm to 400 ppm, or 50 ppm to 300 ppm of at least two other active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

[0288] Embodiment 76 is a method of feeding an aquatic animal, the method comprising administering an amount of animal feed to the aquatic animal, the animal feed comprising: a base feed; and a feed additive mixture comprising, by weight of the animal feed: 0.01 ppm to 125 ppm, 0.1 ppm to 120 ppm. 1 ppm to 100 ppm, 10 ppm to 75 ppm, 12 ppm to 55 ppm, or 13 ppm to 52 ppm thymol; and a total amount of 0.5 ppm to 800 ppm, 0.5 ppm to 400 ppm, 1 ppm to 700 ppm, 10 ppm to 600 ppm, 20 ppm to 500 ppm, 20 ppm to 400 ppm, or 50 ppm to 300 ppm of at least two other active compounds comprising eugenol and cinnamaldehyde and optionally one or more active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

[0289] Embodiment 77 is a method of feeding an aquatic animal, the method comprising administering an amount of animal feed to the aquatic animal, the animal feed comprising: a base feed; and a feed additive mixture comprising, by weight of the animal feed: 10 ppm to 100 ppm, 15 ppm to 95 ppm. 20 ppm to 90 ppm. 25 ppm to 85 ppm, 30 ppm to 80 ppm, 35 ppm to 75 ppm, 40 ppm to 70 ppm, 45 ppm to 65 ppm, or 50 ppm to 60 ppm eugenol; 1 ppm to 50 ppm, 1.5 ppm to 45 ppm, 2 ppm to 40 ppm, 2.5 ppm to 35 ppm, 3 ppm to 30 ppm, 3.5 ppm to 25 ppm, 4 ppm to 20 ppm, 4.5 ppm to 15 ppm, 5 ppm to 14 ppm, 5.5 ppm to 13 ppm, 6 ppm to 12 ppm, 6.5 ppm to 11 ppm, 7 ppm to 10 ppm, 7.5 ppm to 9.5 ppm, or 8 ppm to 9 ppm cinnamaldehyde; and a total amount of 0.5 to 400 ppm, 1 ppm to 300 ppm, 10 ppm to 200 ppm, or 20 ppm to 100 ppm of at least two other active compounds selected from thymol, monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

[0290] Embodiment 78 is the method of any one of embodiments 75 to 77, wherein the administering provides activation of animal immune response, reduction of animal oxidative stress, reduction of parasite host recognition and regulation of iron metabolism.

[0291] Embodiment 79 is the method of any one of embodiments 75 to 78, wherein the administering provides reduction of available iron to parasite in or on an animal.

[0292] Embodiment 80 is the method of any one of embodiments 75 to 79. wherein the aquatic animal is European sea bass, sea bream, amberjack, barramundi, shrimp, tilapia, carp, trout, or golden pompano.PT-1970-WO-PCT

[0293] Embodiment 81 is the method of any one of embodiments 75 to 80, wherein the administering reduces a parasite load.

[0294] Embodiment 82 is the method of Embodiment 81, wherein the parasite comprises myxozoa, monogenean, sea lice, Benedenia seriolae, Neobenedenia species, Gyrodactylus, Amyloodinium, Neoparamoeba. Ichthyophthirius. or a combination of two or more thereof.

[0295] Embodiment 83 is the method of any one of embodiments 75 to 82, wherein the amount of feed administered provides about 1 mg to 120 mg of the active compounds per kg body weight of the aquatic animal per day.

[0296] The present invention can be better understood by reference to the following examples which are offered by way of illustration. The present invention is not limited to the examples given herein.EXAMPLESExample 1

[0297] The efficacy of various phytogenic compounds was compared to synthetic antiparasitics, calculating LD50 values for comparison. The evaluated phytogenic compounds included Comparative Sample 1, Comparative Sample 2, Comparative Sample 3, citronellol, thyme oil, carvacrol, citrus flavonoids, chamomile, eucalyptus oil, eugenol, lemon oil, and cinnamaldehyde. The components of Comparative Samples 1-3 are shown in TABLE 1.Copepods and exposure assays

[0298] Ovigerous C. rogercresseyi females kept under laboratory conditions produced the 3500 copepods used in the exposure assays. The copepods were grouped (n = 30) and exposed to one of the following phytogenies: Comparative Sample 1, Comparative Sample 2, Comparative Sample 3, citronellol, thyme oil, carvacrol, citrus flavonoids, chamomile, eucalyptus oil, eugenol, lemon oil, and cinnamaldehyde (TABLE 1). For each compound, a stock solution including 0.5 g of the compound in 5 mL DMSO was prepared to ensure homogeneous solubilization. From this stock solution, 7 dilutions were prepared. For the first dose (Dose 1), 0.2 mL of stock solution was diluted in 98 mL of filtered seawater, and subsequent dilutions were prepared until the seven dilutions were completed (see TABLE 2). Each of these dilutions was then further diluted in 1 L of seawater for the exposure assays, resulting in final test concentrations ranging from 0.2 ppm to 0.02 ppm, as shown in TABLE 2. Two controls were included in the dilution set, one with DMSO and the other with seawater.PT-1970-WO-PCTTABLE 1. Samples.TABLE 2. Phytogenic doses.*ppm = mg solute per liter of seawater after the final dilution step as described hereinPT-1970-WO-PCT

[0299] Four replicates of each group were used. Each group was incubated in a petri dish with 100 mL doses of the solutions. All copepods were incubated with constant aeration at 14 °C for 48 h. Then, the water was filtered and live, dead, and lethargic copepods were counted.

[0300] If a very high mortality rate was observed with the first stock solution, a second stock solution was prepared by diluting 1 mL of the first stock solution in 1 mL of DMSO. The same calculation was then performed for the 7 doses using the second stock solution. This process was repeated if the second stock solution also showed a high mortality rate.Statistical analysis.

[0301] Lice survival as a function of the dose with 95 % confidence interval and an estimate of the LD50 w as modelled with a binomial GAM (generalized additive model) with a “replicate” random effect. The confidence interval is constructed by including all doses for which the 50 % survival rate is included in the 95 % confidence interval for the mean response. All graphs and analyses were performed using R statistical software packages (available from The R Project for Statistical Computing, The R Foundation).Results and Discussion

[0302] The results of the tests are shown in FIGURES 1A-2D and TABLE 3 below. In the figures, the relative percentages of alive, lethargic, and dead lice are shown as a function of the dose with a LOESS smoother to show the general pattern.

[0303] It was observed that at the tested doses, chamomile, Comparative Sample 3, and Comparative Sample 1 had a limited or no significant effect on the lice. The number of live lice remained at nearly 100 %, while the number of dead lice was close to 0 %, exhibiting only a slight increase at the highest dose. The number of lethargic lice w as nearly 0 % at all tested doses.

[0304] On the other hand, cinnamaldehyde, carvacrol, thyme oil, eugenol, and Comparative Sample 2 were observed to have a strong effect on the lice, each resulting in a sharp decrease in live lice at approximately between 0.02 and 0.08 ppm, with no live copepods observed beyond a dose of 0.08 ppm. The number of lethargic lice reached between 12.5 % and 40 % at doses below 0.05 ppm for carvacrol, thyme oil, eugenol, and Comparative Sample 2. At doses above 0.05 ppm, the lethargic copepods disappear and transition to the dead group. The results for cinnamaldehyde, carvacrol, thyme oil, eugenol, and Comparative Sample 2 are shown in FIGURES 1A-1E, respectively.PT-1970-WO-PCT

[0305] Citrus flavonoids, citronellol, lemon oil, and eucalyptus oil were observed to have a less pronounced effect compared to cinnamaldehyde, carvacrol, thyme oil, eugenol, and Comparative Sample 2. Live individuals were observed even at high doses. However, a decreasing trend was observed at the higher doses. The number of dead individuals was observed to increase at higher doses but does not reach 100 % within the evaluated dose range in most cases. Lemon oil reached 100 % kill rate at a dose of 0.15 ppm. Very few lethargic individuals were observed in lemon oil and eucalyptus oil. A significant number of lethargic individuals were observed in citrus flavonoids and citronellol, reaching a relative percentage above 50 %, peaking at intermediate doses before decreasing again. This suggests that the percentage of lethargic individuals is higher at intermediate doses and decreases at higher doses. The results for citrus flavonoids, citronellol, lemon oil, and eucalyptus oil are shown in FIGURES 2A-2D, respectively.

[0306] The LD50 doses and associated confidence intervals for the tested compounds were calculated using dose-response curve modeling. For this modeling, lethargic and dead individuals were grouped into a new “dead’7category if lethargic individuals tended to decrease as the dose of the phytogenic compound increases. For the phytogenic compounds cinnamaldehyde and carvacrol, the confidence interval was wide, covering the entire range of doses evaluated, suggesting high uncertainty in this estimate, with an approximate LD50 value of 0.002 ppm and 0.018 ppm, respectively. This uncertainty could be associated with the strong effect these two phytogenic compounds had on mortality. For the other three phytogenic compounds, the confidence interval was narrower, as the observed mortality curve was not as steep.

[0307] The LD50 values are shown in TABLE 3 below.TABLE 3. LD50 valuesPT-1970-WO-PCT

[0308] It was concluded that phytogenic compounds can be effective compared to synthetic antiparasitics. The phytogenic compounds cinnamaldehyde, carvacrol, thyme oil, eugenol, and Comparative Sample 2 showed a significant decrease in the survival of C. rogercresseyi copepods at low doses (0.02-0.05 ppm). At doses above 0.05 ppm, no live copepods were observed, and the lethargic transition to dead. Citrus flavonoids, citronellol, lemon oil. and eucalyptus oil. decreased the survival of live individuals with higher doses. Lemon oil reached 100 % mortality at a dose of 0.15 ppm.

[0309] Without wishing to be bound by theory, it is hypothesized that at least some of the variability in the effects of phytogenic compounds can be attributed to differences in the absorption, distribution, metabolism, and / or excretion of the active compounds by the parasites. Some may be more easily absorbed and utilized by the organism than others. Different phytogenies may act through various biological mechanisms. Some can directly interfere with vital processes of the target organism, while others may have more indirect or less potent effects. Compounds within a phytogenic can interact synergistically (enhancing the effect) or antagonistically (reducing the effect). These interactions can influence the overall efficacy of the phytogenic. The susceptibility of the target organism (in this case, lice) can vary. Some organisms may be more resistant to certain compounds due to genetic or adaptive factors.

[0310] All references and publications cited herein are expressly incorporated herein by¬ reference in their entirety into this disclosure, except to the extent they may directly contradict this disclosure. Although specific embodiments have been illustrated and described herein, it will be appreciated by those of ordinary skill in the art that a variety of alternate and / or equivalent implementations maybe substituted for the specific embodiments shown and described without departing from the scope of the present disclosure. It should be understood that this disclosure is not intended to be unduly limited by the illustrative embodiments and examples set forth herein and that such examples and embodiments are presented by way of example only with the scope of the disclosure intended to be limited only by the claims set forth here.Example 2

[0311] The in vivo efficacy of D-Bug PT2, a phytogenic feed additive mixture, was evaluated against sealice (Caligus rogercresseyi) infestation in Atlantic salmon (Salmo salar). Three dietary¬ inclusion levels were tested to assess dose-response relationships and identify the optimal effective range.PT-1970-WO-PCTFish Trial

[0312] Approximately 500 juvenile post-smolt Atlantic salmon (about 150 gm) were obtained from a pathogen-free certified fish farm and acclimated for three weeks at the Cargill Innovation Center in Colaco, Chile. Fish were maintained in 200 L seawater tanks (32 practical salinity units or psu, 12 ± 1 °C, 12: 12 h lightdark photoperiod) with continuous flow-through system. Fish were fed experimental diets for four weeks prior to lice challenge, followed by a three-day fasting period. Each dietary group was tested in triplicate tanks with 40 fish per tank.Feed

[0313] The base diet was EWOS CALIBER 100 (MicroG 100) (available from Cargill Aqua Nutrition - EWOS Chile Alimentos Ltd., Coronel, Chile). The PT2 feed additive was dispersed in the coating oil and applied post-extrusion at 0.2 kg / ton, 0.4 kg / ton, or 0.8 kg / ton feed (0.02 %, 0.04 %, or 0.08 % inclusion corresponding to Test Diet 1, Test Diet 2, and Test Diet 3, respectively), with a control diet receiving coating oil only and no PT2, as shown in Table 4. Comparative Diet 1 without the additive was included as a control diet. Diet compositions were matched for proximate nutrients. Fish were fed test diets for 4 weeks pre-challenge and 4 weeks post-challenge. Copepodids were introduced at 35 per fish, and lice were counted 4 weeks postinfestation.TABLE 4. Inclusion levels of PT2 in experimental dietsSea Lice and Sampling

[0314] Copepodids (3-5 days old, about 90 % free-living population) were introduced at 35 copepodids per fish. Water flow was halted and tank levels reduced to two-thirds during a 4-hour dark incubation period. Fish continued receiving experimental diets for four weeks postinfestation.PT-1970-WO-PCT

[0315] Sampling was conducted on day 28 prior to infestation (Week 2 or W2) and day 56 at trial conclusion (Week-End or WEND). Ten fish per tank were euthanized, measured, and examined for parasites. Lice were counted and classified by sex four weeks post-infestation when all reached adult stage. Plasma was collected for biochemical markers, including total antioxidant capacity and total bilirubin. Head kidney, liver, and skin were sampled for exploratory gene expression. Skin and liver histology were scored. Performance endpoints included weight gain, specific grow th rate (SGR), and feed conversion ratio (FCR). Blood, skin / muscle tissue, and liver samples were collected and stored at -80 °C. Remaining fish were examined for attached and detached lice.Statistical analysis

[0316] Two complementary statistical approaches were employed. First, treatments were analyzed as discrete groups (“by treatment”) using marginal means estimation. Second, a generalized additive model (GAM) treated inclusion levels as a continuous exposure gradient (“by exposure”) to detect non-linear dose-response patterns. Growth parameters (weight gain, specific growth rate or SGR, feed conversion ratio or FCR), lice counts, plasma biomarkers, and histological scores were analyzed. Efficacy was calculated as percentage reduction in lice burden relative to control with 95 % confidence intervals.Results and discussion

[0317] No significant differences (P>0.05) in weight gain, specific growth rate (SGR), or feed conversion ratio (FCR) were observed among treatment groups. Small numerical differences between the highest dose and other groups did not reach statistical significance.

[0318] Test Diet 1 with an inclusion level of 200 ppm demonstrated significant efficacy when analyzed by treatment (FIGURE 3). Adult male, adult female, and total lice counts were reduced compared to control. The estimated reduction in total parasite load was approximately 32 % with wide confidence intervals (3 % to 53 % efficacy. FIGURE 4). Higher inclusion levels (0.04 % and 0.08 %) showed no significant efficacy.

[0319] When analyzed using a GAM with continuous dose gradient (FIGURE 5), a nonmonotonic relationship emerged. The 0.02 % group showed approximately 20 % efficacy but w as not statistically different from other treatments (FIGURE 6). This non-linear pattern suggests the effective dose range lies near 0.02 %, with diminished efficacy at higher concentrations. Without wishing to be bound by theory, the lack of increased protection at elevated doses challengesPT-1970-WO-PCTassumptions about direct antiparasitic mechanisms and instead suggests host-mediated protective responses.

[0320] Fish fed 0.02 % D-Bug PT2 showed significantly (P < 0.05) lower hepcidin antimicrobial peptide A (HampA) gene expression in liver and skin, indicating reduced parasite burden. Skin tumor necrosis factor alpha (TNF-a) levels decreased (P < 0.05) at both 0.02 % and 0.08 % inclusion rates, demonstrating anti-inflammatory effects. Genes for immune response and mucin production showed more pronounced changes at higher doses (0.04 % and 0.08 %).

[0321] Total bilirubin was significantly lower (P < 0.05) in the 0.02 % group at WEND compared to control. Without wishing to be bound by theory, this selective effect at the effective dose suggests involvement of iron and heme metabolism in protection. Plasma creatine tended lower (P < 0.1) with 0.02 % treatment.

[0322] All D-Bug PT2-supplemented diets significantly increased (P < 0.05) total antioxidant capacity at WEND regardless of inclusion level. Before infestation (W2), no differences existed between groups. Without wishing to be bound by theory, this enhanced antioxidant status suggests improved capacity to manage oxidative stress during parasitic challenges, potentially maintaining cellular integrity and supporting immune function.

[0323] Histological evaluation revealed no significant differences in skin or liver scores between treated and control groups at either time point. When analyzed as continuous exposure, increasing D-Bug PT2 levels associated with linearly decreased skin scores at both W2 and WEND, suggesting improved skin condition independent of fice burden. Visual scoring showed fish receiving 0.02 % had numerically higher percentage of normal skin (10 %) versus control and higher doses (4-6 %) at WEND. No adverse histological changes were observed in liver across all doses, confirming safety.

[0324] No significant changes were observed in plasma markers of tissue damage (aspartate aminotransferase or AST, alanine aminotransferase or ALT, lactate dehydrogenase or LDH, creatine kinase or CK, lipase, urea, uric acid) across treatment groups, further supporting the safety profile of D-Bug PT2.Conclusions

[0325] D-Bug PT2 demonstrated protective efficacy against Caligus rogercresseyi at 0.02 % inclusion (0.2 kg / ton feed) with approximately 32 % lice burden reduction (95 % CI: 3-53 %). All inclusion levels were safe, with no adverse effects on growth performance or tissue health markers. No adverse effects on grow th parameters, tissue integrity, or metabolic markers werePT-1970-WO-PCTobserved at any inclusion level. Importantly, all three D-Bug PT2-supplemented diets significantly enhanced antioxidant capacity, indicating that even at doses that did not reduce parasite burden, the additive provided beneficial physiological effects that may support overall fish health during parasitic challenges.

[0326] It was noted that when analyzed using an alternative statistical model, the reduction in lice counts was estimated to be approximately 20 % to 22 % and not statistically significant. Thus, depending on the statistical model applied, the observed reduction ranged from 20 % to 32 %.

[0327] Enhanced total antioxidant capacity across all doses was observed. Without wishing to be bound by theory, it is hypothesized that reduced bilirubin and HampA expression specifically at 0.02 %, implicates the involvement of iron homeostasis. Without wishing to be bound by theory, it is hypothesized that decreased TNF-a expression indicates anti-inflammatory effects of the formulation in test diets as compared to the control diet (Comparative Diet 1). Without wishing to be bound by theory, it is hypothesized that D-Bug PT2 protection involves coordinated host mechanisms including modulation of iron and heme metabolism, enhancement of antioxidant defenses, regulation of inflammatory responses, and strengthening of mucosal immunity.

Claims

1. PT-1970-WO-PCT2.CLAIMS1. A feed additive mixture comprising, by weight of the feed additive mixture:0.1 w t-% to 15 wt-% thymol;0.1 wt-% to 45 wt-% carvacrol; and6.5 wt-% to 80 wt-% of a total of at least two other active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

2. A feed additive mixture comprising, by weight of the feed additive mixture:0.1 wt-% to 5 wt-% thymol; and9.2 wt-% to 95 wt-% of a total of at least two other active compounds comprising eugenol and cinnamaldehyde and optionally one or more active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

3. A feed additive mixture comprising, by weight of the feed additive mixture:11.5 wt-% to 85 wt-% eugenol;0.5 wt-% to 15 wt-% cinnamaldehyde; and0.5 wt-% to 50 wt-% of a total of at least two other active compounds selected from thymol, monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

4. The feed additive mixture of any one of claims 2 or 3, wherein eugenol is present in an amount of 7 wt-% to 12.5 wt-%.

5. The feed additive mixture of any one of claims 2 or 3, wherein cinnamaldehyde is present in an amount of 1 wt-% to 6 wt-%.

6. The feed additive mixture of any one of the preceding claims, wherein the sum of the amount of the at least two other active compounds is 15 wt-% to 40 wt-%.

7. The feed additive mixture of any one of claims 2 or 3, wherein the weight ratio of eugenol to cinnamaldehyde is from 3:1 to 6:1.PT-1970-WO-PCT8. The feed additive mixture of claim 7, wherein the weight ratio of eugenol to cinnamaldehyde is from 3.5:1 to 5:1.

9. The feed additive mixture of any one of the preceding claims, wherein the feed additive mixture comprises thyme oil, clove oil, and cinnamaldehyde.

10. The feed additive mixture of claim 9, wherein eugenol is provided from clove oil and thymol is provided from thyme oil.

11. The feed additive mixture of any one of the preceding claims, wherein the at least two other active compounds comprise one or more monoterpenoids.

12. The feed additive mixture of claim 11, wherein the monoterpenoids comprise citronellol, linalool, cineole, or a combination of two or more thereof.

13. The feed additive mixture of any one of the preceding claims, wherein the at least two other active compounds comprise one or more monoterpenes.

14. The feed additive mixture of claim 13, wherein the monoterpenes comprise pinene, limonene, or a combination thereof.

15. The feed additive mixture of any one of the preceding claims, wherein the at least two other active compounds comprise one or more poly phenolic compounds.

16. The feed additive mixture of claim 15, wherein the polyphenolic compounds comprise one or more flavonoids, tannins, or a combination thereof.

17. The feed additive mixture of any one of the preceding claims, wherein the at least two other active compounds comprise a bicyclic alcohol, an alkylbenzene, an allyl-substituted phenol, an aromatic aldehyde, or a combination of two or more thereof.

18. The feed additive mixture of claim 17, wherein the at least two other active compounds comprise borneol, cymene, eugenol, cinnamaldehyde, or a combination of two or more thereof.PT-1970-WO-PCT19. The feed additive mixture of any one of the preceding claims, wherein the at least two other active compounds comprise citronellol and one or more flavonoids.

20. The feed additive mixture of any one of the preceding claims, wherein the feed additive mixture provides activation of animal immune response, reduction of animal oxidative stress, reduction of parasite host recognition, and regulation of iron metabolism, when administered to the animal.

21. The feed additive mixture of any one of the preceding claims, wherein the feed additive mixture provides reduction of available iron to parasite in or on an animal when administered to the animal.

22. The feed additive mixture of any one of the preceding claims, wherein the feed additive mixture has an antimicrobial effect.

23. The feed additive mixture of any one of the preceding claims, wherein the feed additive mixture is formulated to target a parasite.

24. The feed additive mixture of any one of the preceding claims, wherein the feed additive mixture is formulated to target myxozoa, monogenean, sea lice, Benedenia seriolae, Neobenedenia species. Gyrodactylus, Amyloodinium. Neoparamoeba, Ichthyophthirius, or a combination of two or more thereof.

25. The feed additive mixture of any one of the preceding claims, wherein the feed additive mixture is formulated to target sea lice.

26. The feed additive mixture of any one of the preceding claims, wherein the feed additive mixture is formulated to target Caligus rogercresseyi.

27. An animal feed comprising:38.a base feed; and39.a feed additive mixture comprising, by weight of the animal feed: PT-1970-WO-PCT0.01 ppm to 125 ppm thymol;0.01 ppm to 300 ppm carvacrol; and42.a total amount of 0.5 ppm to 800 ppm of at least two other active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

28. An animal feed comprising:44.a base feed; and45.a feed additive mixture comprising, by weight of the animal feed:0.01 ppm to 125 ppm thymol; and47.a total amount of 0.5 ppm to 800 ppm of at least two other active compounds comprising eugenol and cinnamaldehyde and optionally one or more active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

29. An animal feed comprising:49.a base feed; and50.a feed additive mixture comprising, by weight of the animal feed:0.01 ppm to 100 ppm eugenol;52.1 ppm to 50 ppm cinnamaldehyde; and53.a total amount of 0.5 ppm to 400 ppm of at least two other active compounds selected from thymol, monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

30. The animal feed of any one of claims 27 to 29, comprising a carrier wherein the carrier further comprises a flour, meal, or starch prepared from a grain or a legume.

31. The animal feed of any one of claims 27 to 30, wherein the animal feed is a complete fish feed.

32. The animal feed of any one of claims 27 to 31, wherein the base feed comprises protein, starch, and fat.PT-1970-WO-PCT33. The animal feed of claim 32, wherein the animal feed comprises from 28 wt-% to 60 wt-% protein, 6 wt-% to 15 wt-% starch, and from 18 wt-% to 40 wt-% fat.

34. The animal feed of claims 32 or 33, wherein the animal feed comprises land-animal protein, fish meal, plant-based protein, or a combination of two or more thereof.

35. The animal feed of any one of claims 27 to 34, wherein the animal feed comprises one or more of pellets, extruded nuggets, steam pellets, flakes, tablets, granules, or powders.

36. The animal feed of any one of claims 27 to 35, wherein the at least two other active compounds comprise one or more monoterpenoids.

37. The animal feed of claim 36. wherein the monoterpenoids comprise citronellol, linalool, cineole, or a combination of two or more thereof.

38. The animal feed of any one of claims 27 to 37, wherein the at least two other active compounds comprise one or more monoterpenes.

39. The animal feed of claim 38, wherein the monoterpenes comprise pinene, limonene, or a combination thereof.

40. The animal feed of any one of claims 27 to 39, wherein the at least two other active compounds comprise one or more polyphenolic compounds.

41. The animal feed of claim 40, wherein the poly phenolic compounds comprise one or more flavonoids, tannins, or a combination thereof.

42. The animal feed of any one of claims 27 to 41 , wherein the at least two other active compounds comprise a bicyclic alcohol, an alkylbenzene, an allyl-substituted phenol, an aromatic aldehyde, or a combination of two or more thereof.

43. The animal feed of claim 42, wherein the at least two other active compounds comprise borneol, cymene, eugenol, cinnamaldehyde, or a combination of two or more thereof.PT-1970-WO-PCT44. The animal feed of any one of claims 27 to 43, wherein the at least two other active compounds comprise citronellol and one or more flavonoids.

45. The animal feed of any one of claims 27 to 44, wherein the animal feed comprises thyme oil and clove oil.

46. The animal feed of any one of claims 27 to 45, wherein the feed additive mixture provides activation of animal immune response, reduction of animal oxidative stress, reduction of parasite host recognition, and regulation of iron metabolism, when administered to the animal.

47. The animal feed of any one of claims 27 to 46, wherein the feed additive mixture provides reduction of available iron to parasite in or on an animal when administered to the animal.

48. The animal feed of any one of claims 27 to 47, wherein the feed additive mixture has an antimicrobial effect.

49. The animal feed of any one of claims 27 to 48, wherein the feed additive mixture is formulated to target a parasite.

50. The animal feed of any one of claims 27 to 49, wherein the feed additive mixture is formulated to target myxozoa, monogenean, sea lice, Benedenia seriolae, Neobenedenia species, Gyrodactylus. Amyloodinium, Neoparamoeba, Ichthyophthirius, or a combination of two or more thereof.

51. The animal feed of any one of claims 27 to 50, wherein the feed additive mixture is formulated to target sea lice.

52. The animal feed of any one of claims 27 to 51, wherein the feed additive mixture is formulated to target Caligus rogercresseyi.PT-1970-WO-PCT53. The animal feed of any one of claims 27 to 52, wherein the feed additive mixture is included in the animal feed at 1 ppm to 800 ppm, by weight of the animal feed.

54. The animal feed of claim 53, wherein the feed additive mixture is included in the animal feed at 150 ppm to 200 ppm. by weight of the animal feed.

55. The animal feed of any one of claims 27 to 54, wherein the feed additive mixture is configured for post-extrusion oil-coating onto the animal feed.

56. The animal feed of any one of claims 27 to 55, wherein when the animal feed is administered to an animal, sea louse burden in the animal is reduced relative to a control diet.

57. The animal feed of claim 56. wherein sea louse burden in the animal is reduced by at least about 20 % four weeks post-infestation.

58. The animal feed of claim 56, wherein sea louse burden in the animal is reduced by at least about 32 % four weeks post-infestation.

59. The animal feed of any one of claims 27 to 58, wherein when the animal feed is administered to an animal, plasma total antioxidant capacity increases relative to a control diet.

60. The animal feed of any one of claims 27 to 59, wherein when the animal feed is administered to an animal, plasma total bilirubin decreases relative to a control diet.

61. The animal feed of any one of claims 27 to 60, wherein when the animal feed is administered to an animal, hepatic and skin hepcidin antimicrobial peptide A (HampA) expression is regulated relative to a control diet.

62. The animal feed of any one of claims 27 to 61, wherein when the animal feed is administered to an animal, skin tumor necrosis factor-a (TNF-a) expression decreases relative to a control diet.PT-1970-WO-PCT63. A method for making a feed additive mixture for adding to a fish feed, the method comprising:88.mixing thymol, carvacrol, and at least two other active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

64. A method for making a feed additive mixture for adding to a fish feed, the method comprising:90.mixing thymol and at least two other active compounds comprising eugenol and cinnamaldehyde and optionally one or more compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

65. A method for making a feed additive mixture for adding to a fish feed, the method comprising:92.mixing eugenol and cinnamaldehyde and at least two other active compounds selected from thymol, monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

66. The method of any one of claims 63 to 65. further comprising dissolving or dispersing the feed additive mixture in a coating oil.

67. A method for making an animal feed, the method comprising:95.mixing a feed additive mixture with a base feed to form a feed composition, the feed additive mixture comprising, by weight of the animal feed:0.01 ppm to 125 ppm thymol;0.01 ppm to 300 ppm carvacrol; and98.a total amount of 0.5 ppm to 800 ppm of at least two other active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

68. A method for making an animal feed, the method comprising:100.mixing a feed additive mixture with a base feed to form a feed composition, the feed additive mixture comprising, by weight of the animal feed:0.01 ppm to 125 ppm thymol; andPT-1970-WO-PCT102.a total amount of 0.5 ppm to 800 ppm of at least two other active compounds comprising eugenol and cinnamaldehyde and optionally one or more active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

69. A method for making an animal feed, the method comprising:104.mixing a feed additive mixture with a base feed to form a feed composition, the feed additive mixture comprising, by weight of the animal feed:0.01 ppm to 100 ppm eugenol;106.1 ppm to 50 ppm cinnamaldehyde; and107.a total amount of 0.5 ppm to 400 ppm of at least two other active compounds selected from thymol, monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

70. The method of any one of claims 67 to 69, wherein the animal feed is a complete fish feed.

71. The method of any one of claims 67 to 70. wherein the base feed comprises protein, starch, and fat.

72. The method of claim 71, wherein the animal feed comprises from 28 wt-% to 60 wt-% protein, 6 wt-% to 15 wt-% starch, and from 18 wt-% to 40 wt-% fat.

73. The method of any one of claims 67 to 72, wherein the animal feed comprises landanimal protein, fish meal, plant-based protein, or a combination of two or more thereof.

74. The method of any one of claims 67 to 73. wherein the method further comprises extruding or pressing the feed composition to form pellets, extruded nuggets, steam pellets, flakes, tablets, or granules.

75. A method of feeding an aquatic animal, the method comprising administering an amount of animal feed to the aquatic animal, the feed comprising:114.a base feed; and PT-1970-WO-PCT115.a feed additive mixture comprising active compounds comprising, by weight of the animal feed:0.01 ppm to 125 ppm thymol;0.01 ppm to 300 ppm carvacrol; and118.a total amount of 0.5 ppm to 800 ppm of at least two other active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

76. A method of feeding an aquatic animal, the method comprising administering an amount of animal feed to the aquatic animal, the animal feed comprising:120.a base feed; and121.a feed additive mixture comprising, by w eight of the animal feed:0.01 ppm to 125 ppm thymol; and123.a total amount of 0.5 ppm to 800 ppm of at least two other active compounds comprising eugenol and cinnamaldehyde and optionally one or more active compounds selected from monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

77. A method of feeding an aquatic animal, the method comprising administering an amount of animal feed to the aquatic animal, the animal feed comprising:125.a base feed; and126.a feed additive mixture comprising, by weight of the animal feed:0.01 ppm to 100 ppm eugenol;128.1 ppm to 50 ppm cinnamaldehyde; and129.a total amount of 0.5 ppm to 400 ppm of at least tw o other active compounds selected from thymol, monoterpenoids, monoterpenes, polyphenolic compounds, and aromatic aldehydes.

78. The method of any one of claims 75 to 77, w herein the administering provides activation of animal immune response, reduction of animal oxidative stress, reduction of parasite host recognition and regulation of iron metabolism.PT-1970-WO-PCT79. The method of any one of claims 75 to 78, wherein the administering provides reduction of available iron to parasite in or on an animal.

80. The method of any one of claims 75 to 79, wherein the aquatic animal is European sea bass, sea bream, amberjack, barramundi, shrimp, tilapia, carp, trout, or golden pompano.

81. The method of any one of claims 75 to 80, wherein the administering reduces a parasite load.

82. The method of claim 81, wherein the parasite comprises myxozoa, monogenean, sea lice, Benedenia seriolae, Neobenedenia species, Gyrodactylus, Amyloodinium, Neoparamoeba, Ichthyophthirius, or a combination of two or more thereof.

83. The method of any one of claims 75 to 82, wherein the amount of feed administered provides about 1 mg to 120 mg of the active compounds per kg body weight of the aquatic animal per day.

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