Feed additive for improving production performance of bred animals

Abstract

The present invention is in the field of feed additives for animals (e.g., bred animals), in particular feed additives added to animal feed to achieve improved production performance (e.g., increased feed intake, increased average daily gain, increased feed efficiency (i.e., reduced feed conversion) and / or increased milk fat yield) in such animals.

Description

Technical Field

[0001] This invention relates to feed additives for animals (e.g., farmed animals), particularly feed additives added to animal feed to achieve improved production performance of such animals (e.g., increased feed intake, increased average daily weight gain, increased feed efficiency (i.e., reduced feed conversion ratio) and / or increased milk fat production). Background Technology

[0002] Antimicrobial agents have been used for decades as antibiotic growth promoters in livestock feed. However, resistance to these antimicrobial agents is now considered a serious threat to human health. Since January 2006, the European Union has completely banned the use of antimicrobial agents as growth promoters in livestock feed. Simultaneously, driven by population growth, the demand for animal-derived protein is increasing. Furthermore, food production needs to be more sustainable, requiring more output with less input. In short, animal production for food needs to become increasingly efficient.

[0003] These developments have sparked a surge in the development of feed additives that improve the production performance of farmed animals without inducing bacterial resistance. Plant-derived feed additives, also known as plant-based products, are substances of plant origin added to animal diets for the purpose of improving animal production performance. Essential oils, herbs, and spices are all used as sources of bioactive components such as phenols, polyphenols, flavonoids, terpenes, and others.

[0004] Glycyrrhiza glabra is a legume belonging to the Fabaceae family, native to Eurasia, Central and Southwest Asia, and the Mediterranean region. Specifically, the root of Glycyrrhiza glabra (with creeping stems and rhizomes) is called licorice. It has been used in various culinary applications for at least 4000 years. The root of Glycyrrhiza glabra is used as a medicinal plant with a variety of therapeutic effects (El-Saber Batiha et al. 2020. Biomolecules, 10): digestive system disorders, respiratory diseases, epilepsy, fever, decreased libido, paralysis, rheumatism, abnormal vaginal discharge, psoriasis, prostate cancer, malaria, hemorrhagic diseases, and jaundice. The main component in the root is glycyrrhizic acid (also known as glycyrrhizin, glycyrrhetinic acid glycoside, or 18-β-glycyrrhetinic acid). Glycyrrhizic acid accounts for approximately 10% of the dry weight of licorice root (Pastorino et al. 2018. PhytotherRes, 32: 2323-39).

[0005] In the latest literature review on licorice, the authors discussed the roots of the plant separately (Bisht et al. 2022. Phytomed Plus, 2:100206; Hejazi et al. 2021. Food Chem Toxicol, 150:112057). For example, in a review summarizing the anti-inflammatory effects of licorice (especially at the intestinal level), Leite et al. (2022. Int J Mol Sci, 23) focused on glycyrrhizin and glycyrrhetinic acid, both of which are found only in the roots of Glycyrrhiza glabra.

[0006] Licorice powder and licorice oil have previously been used in farmed animals. Alagawany et al. (2019. Animals (Basel), 9) summarized the benefits of licorice powder in improving the production performance of poultry, focusing on glycyrrhizic acid (a compound found only in the root). Therefore, in the art, licorice is equivalent to the root of the licorice plant.

[0007] Supra et al. (2018. Industrial Crops and Products, 112:217-24) compared two root samples and two leaf samples of *Glycyrrhiza glabra* using different chromatographic techniques and showed that glycyrrhizic acid was detected only in the root extract and not in the leaves. They also reported that the components of the roots and leaves were significantly different. Siracusa et al. (2011. Fitoterepia, 82:546-56) confirmed that the components of *Glycyrrhiza glabra* leaves were present in trace amounts or not at all in the roots of the same plant. The aerial parts of *Glycyrrhiza glabra* (such as the leaves) are considered agricultural chemical waste (Pastorino et al. 2018. Supra. Siracusa et al. 2011. Supra. Gowthaman et al. 2021. Letters in Animal Biology 01(2):14-20). They do not contain glycyrrhizic acid, which is considered the most important compound present in the roots. Zamiri et al. (Jan 2015. Trop Anim Health Prod.) used licorice leaves as forage as a source of so-called “condensed tannins” for lambs. Ashraf et al. (2017. Pak J Pharm Sci., 30: 567-72) injected licorice leaf extract into eggs to study its effects on Newcastle disease virus. The authors suggested that licorice leaf extract could be used to control Newcastle disease virus. Abarghuei and Salem (2021. Environ Sci Pollut Res Int.) aimed to evaluate the effects of licorice pulp and leaves on reducing rumen biogas production in sheep.

[0008] The object of this invention is to provide feed additives and / or animal feeds to improve the production performance of animals (preferably farmed animals, and more preferably farmed animals of many different species), for example, to increase feed intake, increase body weight gain, increase average daily weight gain, increase feed efficiency (i.e., reduce feed conversion ratio), increase specific growth rate, increase relative growth rate, increase milk fat production, and / or to prevent microbial (e.g., bacterial, viral, parasitic, fungal, and / or yeast) infections in these animals, reduce the severity of microbial infections, and / or treat microbial infections, and / or to prevent hepatotoxicity or liver damage due to mycotoxin exposure, reduce the severity of hepatotoxicity or liver damage due to mycotoxin exposure, and / or treat hepatotoxicity or liver damage due to mycotoxin exposure. Summary of the Invention

[0009] This disclosure provides animal feed comprising the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or extracts thereof.

[0010] In this embodiment, the above-ground portion of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) is dried and optionally pulverized.

[0011] In the embodiments, the feed includes the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or an equivalent amount of extract at an amount of about 1 mg / kg to about 10,000 mg / kg.

[0012] In this embodiment, the extract is selected from the group consisting of: aqueous extract, ethanol extract, methanol extract, isopropanol extract, ethyl acetate extract, propanol extract, hexane extract or supercritical CO2 extract, or any mixture thereof.

[0013] In a further aspect, this disclosure relates to the use of the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or extracts thereof as a feed additive.

[0014] In an embodiment, the feed additive is provided with written instructions to include the feed additive in animal feed at a rate of about 1 mg / kg to about 10,000 mg / kg of the above-ground part of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or an equivalent amount of extract.

[0015] In this implementation, feed additives are included in the premix.

[0016] In another aspect, this disclosure provides the use of the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or extracts thereof for increasing the production performance of animals, for example, for increasing feed intake, increasing average daily weight gain, increasing feed efficiency (i.e., reducing feed conversion ratio), increasing relative growth rate, increasing specific growth rate and / or increasing milk fat production.

[0017] This disclosure further relates to compositions comprising the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or extracts thereof, or animal feeds as taught herein, for reducing mortality and / or for preventing, mitigating, and / or reducing microbial infections. In embodiments, the microbial infection is caused by Gram-negative bacteria, such as Gram-negative bacteria of the genus Vibrio, for example, Gram-negative bacteria of the species Vibrio parahaemolyticus.

[0018] In this embodiment, the extract is selected from the group consisting of: aqueous extract, ethanol extract, methanol extract, isopropanol extract, ethyl acetate extract, propanol extract, hexane extract or supercritical CO2 extract, or any mixture thereof.

[0019] In implementation, feed additives are intended to be included in the feed used for farmed animals or companion animals.

[0020] In this implementation, the farmed animals are selected from the group consisting of: poultry; pigs; ruminants, such as beef cattle and dairy cattle; fish, such as salmon, trout, sea bream, sea bass, tilapia and tuna; and crustaceans, such as shrimp.

[0021] In another embodiment, the companion animal is selected from the group consisting of: ornamental fish, cats, dogs, horses, rabbits, guinea pigs, and hamsters.

[0022] Conventional definition

[0023] Many terms are used in the following description and examples. The following definitions are provided to provide a clear and consistent understanding of the specification and claims (including the scope of such terms). Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All disclosures of publications, patent applications, patents, and other references cited herein are incorporated herein by reference in their entirety.

[0024] The term "licorice" ("licorice sugar") is the common name for Glycyrrhiza glabra. In the prior art, this term is used to refer to the root of the plant.

[0025] When referring to plants, the term "above-ground part" means anything that exists in the air or in the space above a solid surface. The above-ground part of a plant simply refers to the plant's structure above ground, including the stem, leaves, petioles, flowers, fruits, and seeds. The term may include only any above-ground part, for example, only leaves, or only stems, or the entire above-ground part (i.e., all plant parts above ground). In embodiments, when the term "above-ground part" is used herein, it may be replaced by the term "leaf."

[0026] When referring to plants, the term "leaf" refers to the typically flat, green growth that emerges from the stem of a vascular plant. As the primary site of photosynthesis, leaves produce nutrients for the plant, which in turn ultimately provide nourishment and sustain life for all terrestrial animals. Botanically speaking, leaves are an integral part of the stem system. They connect to the rest of the plant through a continuous vascular system, enabling the free exchange of nutrients, water, and the end products of photosynthesis (especially oxygen and carbohydrates) throughout the plant.

[0027] The term "farmed animal" refers to animals raised or bred for agricultural purposes, such as for consumption or to generate income through products like wool, meat, eggs, or dairy products. Farmed animals can be classified based on their digestive systems. This classification includes monogastric animals, ruminants, and pseudoruminants. Optionally or additionally, they can be classified based on their ecosystems. This classification includes aquaculture animals (when referring to animals raised in aquatic ecosystems, such as fish and shrimp) and livestock animals (when referring to animals raised on land, such as poultry, pigs, and bovines).

[0028] The term "companion animal" refers to a domesticated or tamed animal whose physiological, emotional, behavioral, and social needs can be met as a companion in the household or through a close, everyday relationship with a person. Species suitable as companion animals include dogs, cats, horses, rabbits, ferrets, birds, guinea pigs, and other small mammals, small reptiles, and ornamental fish.

[0029] As used herein, the term "pellets" or "feed pellets" refers to small particles or objects typically produced by pressing raw materials, such as feed ingredients (usually fermentable feed components like grains, cereals, legumes, and roughage). Feed pellets may also include other feed components, such as meat meal, fish meal, bone meal, by-products, oils, fats, fillers, or any mixture thereof, as well as minerals, vitamins, trace elements, and other substances. Depending on the nutritional needs, diet, digestive system (monogastronomical, ruminant, etc.) and living environment (e.g., aquatic, terrestrial, domesticated, etc.) of the animal to which the feed pellets are intended, animal feed pellets can vary in their composition and structural properties (e.g., hardness, density, durability, shape, size, etc.). Animal feed pellets can have any size, shape (e.g., round, rectangular, cylindrical, etc.), weight, and / or length. It should be understood that the weight of feed pellets will depend on the feed pellet components themselves (e.g., some components have a greater weight or density than others) and the shape, size, and length of the finished feed pellet. It is known that the size, shape, weight, and / or length of feed pellets will affect pellet durability. This is true for any method used to manufacture feed pellets, including those taught herein. It is also generally agreed in the fields of agriculture and animal nutrition that animals (e.g., young and adult livestock, such as beef cattle (e.g., beef calves), dairy cattle (e.g., dairy calves), and pigs) derive more benefits or achieve better growth (e.g., weight gain, increased height, enhanced growth curves) from pelleted feed compared to meal rations because pelleted feed is a more concentrated, palatable, and palatable form compared to meal rations. Pelleted feed has been shown to be easy to consume and minimize feed waste during consumption. It has also been shown that, given the opportunity to choose between the same feed in pellet or powder form, most animals will prefer the pelleted form. Animal feed pellets are typically produced on an industrial scale using processes such as pelleting. Processes used to produce animal feed pellets are well known to those skilled in the art.

[0030] As used herein, the term "premix" refers to a mixture of ingredients designed to be mixed with other ingredients (typically feed ingredients) before use. The ingredients in a premix typically consist of components added in small doses to animal feed (such as vitamins and minerals), and optionally antioxidants, pigments, and / or organic acids. For example, premixes for fish feed may include vitamins, minerals, antioxidants, and / or pigments, but typically do not include organic acids. In contrast, premixes for poultry or pig feed may include vitamins, minerals, and organic acids, as well as antioxidants, but do not include pigments.

[0031] As used herein, the term "feed conversion ratio" or "FCR" is a measure of the rate or speed at which a farmed animal converts animal feed into desired output (e.g., weight). The term "feed efficiency" as used herein is the reciprocal of FCR.

[0032] The terms “relative growth rate” or “RGR” used in this paper are expressed as a percentage and are calculated as follows: (final weight – initial weight) / initial weight.

[0033] The term “specific growth rate” or “SGR” is a coefficient that measures the percentage increase in a fish’s daily body weight: SGR = (Ln(Wt) - Ln(W0)) * 100 / t(d), where W0[g] = weight at the beginning of the cycle (in grams), Wt[g] = weight at the end of the cycle (in grams), t[d] = cycle, expressed in days; and Ln = natural logarithm.

[0034] As used herein, "secondary plant components" refers to specific compounds present in plants that do not function in plant growth and development but are essential for the plant's survival in its environment. These secondary plant components may be necessary for communication between plants and other organisms in symbiotic interactions (e.g., attracting beneficial organisms such as pollinators) or antagonistic interactions (e.g., defending against herbivores and pathogens). They may further help manage abiotic stresses (e.g., increased UV radiation). The broad functional scope of these specific metabolisms is not fully understood.

[0035] As used herein, the term “about” indicates a range of common tolerances in the art, such as within the standard deviation of the mean. The term “about” can be understood to encompass values ​​that deviate from the indicated value by up to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01%.

[0036] As used herein, the terms “comprising” or “to comprise” and their variations refer to situations where the terms are used in their non-restrictive sense to mean including the items immediately following the word, but not excluding items not specifically mentioned. This also encompasses the more restrictive verbs “consistently of” and “comprises of”.

[0037] The use of the indefinite articles "a" or "an" to refer to an element does not preclude the possibility of more than one element, unless the context clearly requires only one element. Therefore, the indefinite articles "a" or "an" generally mean "at least one". Attached Figure Description

[0038] Figure 1 Compare the chromatogram of the root sample of Glycyrrhiza glabra (clearly showing the glycyrrhizic acid peak) with the chromatogram of the leaf sample of Glycyrrhiza glabra (where the glycyrrhizic acid peak is absent).

[0039] Figure 2 The fecal oocyst excretion status of chicks is shown: not infected with E. maxima (NC) or infected with E. maxima (PC, MO, GG) (lacking further treatment (PC) or treated with monensin (MO), treated with 55 ppm licorice leaf (55 ppm GG) or treated with 160 ppm licorice leaf (160 ppm GG)). Detailed Implementation

[0040] This patent application has been divided into several parts. However, these parts should not be interpreted in isolation. Unless otherwise stated, each part should be interpreted in conjunction with the other parts. This means, for example, that all animal feed and / or feed additive compositions described in the "Animal Feed and / or Feed Additive Compositions" section are intended to be read in conjunction with the "Methods of Using Animal Feed and / or Feed Additives" section (i.e., all animal feeds set forth in the first part are suitable for the methods described in the latter part). Various optional and preferred features can be combined even when extracted from different parts of the specification. Similarly, all "Aspects" and "Descriptions" can be combined. Descriptions are not intended to be separated unless explicitly stated otherwise.

[0041] The inventors have unexpectedly discovered that including a relatively small amount of the aboveground parts (preferably leaves) of Glycyrrhiza glabra in broiler feed, shrimp feed, or salmon feed increases their production performance (e.g., in terms of weight gain or average daily weight gain and feed efficiency).

[0042] Animal feed and / or feed additive compositions

[0043] Therefore, this disclosure provides animal feed comprising the above-ground parts of Glycyrrhiza glabra or extracts thereof, preferably Glycyrrhiza glabra leaves or extracts thereof.

[0044] Animal feed can be ingested in any form suitable for the relevant animal. For example, poultry feed can be in the form of ground feed, powder, or pellets. Pig feed can be in the form of coarse powder or pellets. Feed for fish and crustaceans is typically in the form of extrusion (e.g., cook-extruded) or compressed pellets, but may also be in another form, such as flakes. In embodiments, animal feed can be in the form of ground feed, powder, coarse powder, flakes, or pellets, wherein the pellets may be extruded (e.g., cook-extruded) or compressed. In suitable embodiments, animal feed is in the form of pellets.

[0045] Animal feed typically includes protein, fat, carbohydrates, minerals, and vitamins. Animal feed may further include antioxidants, pigments, organic acids, enzymes, plant-derived compounds, and / or other feed additives. Those skilled in the art know how to formulate appropriate animal feed based on the target animal, its life stage, and / or its physiological condition.

[0046] The above-ground parts (preferably leaves) of Glycyrrhiza glabra can be derived from any species or subspecies of Glycyrrhiza glabra. Optionally, the above-ground parts or leaves may be pre-processed (e.g., fermented) before being used in animal feed or feed additives as taught herein.

[0047] The above-ground parts of licorice root (preferably licorice root leaves) can be used fresh or dried by any method known in the art. Those skilled in the art are familiar with suitable drying techniques. For example, drying can be performed using a drum dryer, belt dryer, dehydration drying, vacuum drying, microwave drying, infrared drying, or natural drying (sun drying). In a preferred embodiment, the above-ground parts of licorice root or licorice root leaves are dried.

[0048] Before or after drying (preferably after drying), the above-ground portion of licorice root (preferably licorice root leaves) can be pulverized to any suitable size. For example, fresh or dried leaves can be pulverized to less than 2 mm, preferably less than 1.5 mm, more preferably less than 1 mm, further preferably less than 0.8 mm, even more preferably less than 0.6 mm, most preferably less than 0.5 mm, less than 0.4 mm, or less than 0.3 mm, or even less than 0.2 mm or 0.150 mm in average particle size.

[0049] The above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) can be contained in amounts ranging from about 1 mg / kg to about 10,000 mg / kg of feed, preferably from about 5 mg / kg to about 8,000 mg / kg of feed, more preferably from about 10 mg / kg to about 6,000 mg / kg of feed, even more preferably from about 15 mg / kg to about 4,000 mg / kg of feed, even more preferably from about 20 mg / kg to about 3,000 mg / kg of feed, and even more preferably from about 25 mg / kg to about 2,000 mg / kg of feed. For example, the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) can be contained in amounts ranging from about 30 mg / kg to about 1,500 mg / kg of feed, about 40 mg / kg to about 1,200 mg / kg of feed, about 50 mg / kg to about 1,000 mg / kg of feed, or about 60 mg / kg to about 800 mg / kg of feed.

[0050] In implementation, the animal feed taught herein may be feed suitable for consumption by livestock animals (e.g., poultry, pigs, or ruminants such as dairy cows, beef cattle, sheep, and goats). Those skilled in the art know how to formulate feeds suitable for specific breeds of livestock and specific growth stages of livestock.

[0051] In embodiments, the animal feed taught herein may be aquaculture feed, i.e., feed suitable for consumption by fish or crustaceans. The feed may preferably be in pellet form, more preferably in the form of extruded (e.g., cook-extruded) pellets or compressed pellets. The feed may include protein, fat, carbohydrates, vitamins, minerals, and water, and may optionally further include pigments (e.g., astaxanthin) and / or antioxidants. The precise amounts of protein, fat, carbohydrates, vitamins, minerals, and water may depend on the species of fish or crustaceans fed the feed and the growth stage of the fish or crustaceans. Those skilled in the art know how to formulate feeds with appropriate compositions for fish or crustaceans.

[0052] When producing feed to meet the nutritional requirements of marine fish (such as cod, pollock, sea bass, and sea bream), the feed contains at least 10% by weight, at least 12.5% ​​by weight, or at least 15% by weight of fat. Optionally, when intended for feeding salmon, the feed may contain at least 18% by weight, at least 20% by weight, at least 23% by weight, at least 25% by weight, at least 26% by weight, or at least 28% by weight of fat. Preferably, the fat used in feeds suitable for fish or crustaceans may include omega-3 fatty acids (such as DHA and / or EPA).

[0053] In implementation, the animal feed taught herein can be feed suitable for companion animals (e.g., dogs, cats, horses, etc.). Those skilled in the art know how to formulate feed for companion animals according to their type and stage of growth.

[0054] As a substitute for the above-ground parts or leaves, or additionally, an equal amount of extracts of the above-ground parts or leaves of Glycyrrhiza glabra can be used.

[0055] Plant extraction is the process of extracting specific components present in plants (so-called secondary phytochemicals, including but not limited to alkaloids, terpenes, saponins, phenolic compounds, flavonoids, and / or tannins). Plant extraction is a solid / liquid separation operation: a solid object (the aboveground portion of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves), optionally dried and / or pulverized) is contacted with a fluid (solvent). The target plant component is dissolved and contained in the solvent. The resulting solution is the desired extract. The solvent may eventually be removed, although not necessarily. The solvent can be any solvent suitable for preparing plant extracts for animal feed. Suitable solvents include, but are not limited to, polar solvents (e.g., water, alcohols (such as ethanol, methanol, and isopropanol)), moderately polar solvents (e.g., acetone, dichloromethane), and nonpolar solvents (e.g., ethyl acetate, hexane, ether, chloroform). Those skilled in the art can select a single solvent or two or more solvents according to this disclosure to prepare the extract. Generally, extraction processes include maceration, digestion, decoction, infusion, percolation, Soxhlet extraction, supercritical extraction, ultrasound-assisted and microwave-assisted extraction, supercritical fluid extraction (e.g., using supercritical carbon dioxide), accelerated solvent extraction, and solid-phase extraction. Fractionation and purification of phytochemicals can be achieved through the application of various techniques (e.g., but not limited to, liquid-liquid partitioning, cross-flow filtration, and chromatography (e.g., paper chromatography, thin-layer chromatography, gas chromatography, high-performance liquid chromatography, and centrifugal partition chromatography)). Finally, the obtained compounds are characterized using various identification techniques (e.g., mass spectrometry, infrared spectroscopy, ultraviolet spectroscopy, and nuclear magnetic resonance spectroscopy, or any combination thereof). Those skilled in the art can select appropriate extraction methods.

[0056] The amount of extract of the aboveground parts or leaves of *Glycyrrhiza glabra* added to the animal feeds and / or feed additives taught herein can be determined by making the content of one or more secondary plant components in the extract equal to the indicated amount of one or more secondary plant components in the aboveground parts or leaves of *Glycyrrhiza glabra*. An amount of extract providing the content of one or more secondary plant components equal to the indicated amount in the aboveground parts or leaves of *Glycyrrhiza glabra* can thus be added to the animal feed. For example, secondary plant components present in *Glycyrrhiza glabra* leaves include, but are not limited to, phenolic compounds, such as flavanones, isopentenyl dihydrostilbene, isopentenyl isoflavones, isopentenyl flavanones, dihydrochalcones, chalcones, flavonoid-C-glycosides, flavonol-O-glycosides, hydroxycinnamic acid, and coumarins, which exist in their glycosidic forms or as aglycones.

[0057] In this embodiment, the extract is selected from the group consisting of: aqueous extract, ethanol extract, methanol extract, isopropanol extract, ethyl acetate extract, propanol extract, hexane extract, or supercritical CO2 extract, or any mixture thereof. Optionally, as stated above, a mixture of solvents may be used for extraction.

[0058] This disclosure further provides a feed additive comprising the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or extracts thereof as taught herein. Additionally, this disclosure provides the use of the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or extracts thereof as a feed additive.

[0059] The feed additive may further include vitamins, minerals, organic acids, antioxidants, and / or pigments. The feed additive may be provided in the form of a premix. The feed additive is preferably intended to be included in the feed for farmed animals or companion animals.

[0060] The feed additive may be provided with written instructions to use the feed additive at a concentration of about 1 mg / kg to about 10,000 mg / kg of the feed, preferably about 5 mg / kg to about 8,000 mg / kg of the feed, more preferably about 10 mg / kg to about 6,000 mg / kg of the feed, even more preferably about 15 mg / kg to about 4,000 mg / kg of the feed, even more preferably about 20 mg / kg to about 3,000 mg / kg of the feed, and even more preferably about 25 mg / kg of the feed. The amount of *Glycyrrhiza glabra* top part (preferably *Glycyrrhiza glabra* leaves) in the animal feed is included in the animal feed, such as about 30 mg / kg to about 1500 mg / kg, about 40 mg / kg to about 1200 mg / kg, about 50 mg / kg to about 1000 mg / kg, or about 60 mg / kg to about 800 mg / kg. Alternatively, if an extract of *Glycyrrhiza glabra* top part or *Glycyrrhiza glabra* leaves is used, the same amount of extract as taught above is used.

[0061] Methods of using animal feed and / or feed additives

[0062] In one aspect, this disclosure provides methods for feeding animals (e.g., raising animals such as chickens, pigs, cows, fish or shrimp, etc.) or companion animals such as dogs or cats) with animal feed as taught herein.

[0063] In a further aspect, this disclosure relates to the use of the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or extracts thereof, or animal feed or feed additives as taught herein, for increasing the production performance of animals.

[0064] In another aspect, this disclosure relates to the use of the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or extracts thereof, or animal feed or feed additives as taught herein, for increasing feed intake, increasing body weight gain, increasing average daily weight gain, increasing feed efficiency (i.e., reducing feed conversion ratio), increasing relative growth rate, increasing specific growth rate, and / or increasing milk fat production.

[0065] In embodiments, the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or extracts thereof, or feed additives or animal feeds as taught herein, may be used as antibacterial agents, antiviral agents, antifungal agents, or antiparasitic agents.

[0066] In another embodiment, the above-ground portion of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or its extracts or feed additives as taught herein can be used as preservatives or as cell-protecting agents.

[0067] Furthermore, this disclosure provides the use of compositions comprising the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or extracts thereof, as taught herein, or animal feeds or feed additives as taught herein, in reducing mortality rates (e.g., mortality rates of diseased animals, such as, mortality rates of diseased shrimp, such as, mortality rates of shrimp suffering from (e.g., acute hepatopancreatic necrosis disease (AHPND) caused by bacteria of the genus Vibrio (e.g., Gram-negative bacteria of the species Vibrio parahaemolyticus)).

[0068] This disclosure further provides the use of compositions comprising the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or extracts thereof, as taught herein, or animal feeds or feed additives as taught herein, in preventing microbial infections and / or diseases caused by such microbial infections, alleviating the severity of microbial infections and / or diseases caused by such microbial infections, and / or reducing microbial infections and / or diseases caused by such microbial infections.

[0069] This disclosure also provides methods for preventing, mitigating, and / or reducing microbial infections in animals, the methods comprising administering to the animal a composition comprising the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or extracts thereof, or an animal feed or feed additive as taught herein. Microbial infections can be caused by any microorganism, such as bacteria, viruses, parasites, yeasts, or fungi.

[0070] Microbial infections can be caused by, for example, Gram-positive or Gram-negative bacteria.

[0071] Non-limiting examples of bacteria that can cause microbial infections include *Escherichia*, *Salmonella*, *Vibrio*, *Flavobacterium*, *Campylobacter*, *Clostridium*, *Streptococcus*, *Piscirickettsia* (such as *Piscirickettsia salmonis*, which causes salmon rickettsial syndrome (SRS)), and others such as *Escherichia coli*, *Salmonella typhimurium*, *Salmonella dublin*, *Salmonella enterica*, *Salmonella enteritidis*, *Clostridium perfringens*, and *Pasteurella*. Listeria monocytogenes, Mycoplasma bovis, Haemophilus somnus, Campylobacter jejunum, Campylobacter hepaticus, and Campylobacter bilis are among the bacteria found in this strain.

[0072] In this embodiment, the microbial infection is caused by Gram-negative bacteria (e.g., Gram-negative bacteria of the genus Vibrio).

[0073] In aquaculture, several species of Vibrio spp. are currently considered pathogenic or opportunistic pathogens in farmed fish, shellfish, and shrimp. These include Vibrio anguillarum, Vibrio ordalii, Vibrio vulnificus, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio salmonicida, Vibrio harveyi, Vibrio tubiashii, and Vibrio cholerae. Vibriosis caused by these species is a common and highly destructive bacterial disease in fish farming and aquaculture.

[0074] In this embodiment, the microbial infection is caused by *Vibrio anguillarum*, *Vibrio ordalii*, *Vibrio vulnificus*, *Vibrio alginolyticus*, *Vibrio parahaemolyticus*, *Aliivibrio salmonicida*, *Vibrio harveyi*, *Vibrio tubiashii*, and / or *Vibrio cholerae*. In this case, the infected animal may be a fish or a crustacean (e.g., shrimp). In this embodiment, the microbial infection is caused by *Vibrio parahaemolyticus*. The infected animal may be a crustacean (e.g., shrimp). The disease caused by *Vibrio parahaemolyticus* infection may be vibriosis.

[0075] Other Gram-negative bacteria that can cause microbial infections include members of the family Flavobacteriaceae (e.g., the genus Flavobacterium, such as those selected from the group consisting of: *Flavobacterium columnnaris*, *Flavobacter johnsoniae*, *Tenacibaculum maritimum*, *Flavobacter maritimus*, *Flavobacterium psychrophilum*, *Flavobacterium branchiophilum*, *Tenacibaculum ovolyticum*, and *Chryseobacterium scopthalmum*). Diseases caused by infections with these bacteria can be called flavibacterial infections.

[0076] In another embodiment, the microbial infection is caused by Gram-positive bacteria, such as Gram-positive Streptococcus spp., for example, Streptococcus agalactiae.

[0077] Members of the genus *Streptococcus* cause mild to severe bacterial diseases in animals. These organisms typically colonize one or more species as symbiotics and can cause opportunistic infections in those hosts. *Streptococcus agalactiae* is a common cause of subclinical mastitis in bovines. In some regions, it also appears to be common in clinical or subclinical mastitis in sheep and goats. *Streptococcus canis* is a major opportunistic pathogen affecting dogs and cats. It can cause a variety of diseases, including skin and soft tissue infections, arthritis, reproductive system diseases, mastitis, pneumonia, sepsis, and streptococcal toxic shock-like syndrome, as well as cervical lymphadenitis in kittens aged 3–6 months and otitis externa in dogs. In cats, this organism sometimes causes neonatal sepsis. *Streptococcus dysgalactiae* subsp. *Dysgalactiae* is commonly associated with clinical or subclinical mastitis in cattle, but the bacterium has also been detected in other diseases in this species, including severe cellulitis and toxic shock-like syndrome. Some reports of *Streptococcus dysgalactiae* subsp. *Dysgalactiae* in other hosts describe mastitis in sheep and goats, suppurative polyarthritis in lambs, septicemia in fish and dogs, and septicemia and encephalitis in bats. In this embodiment, the Streptococcus species are selected from the group consisting of: *Streptococcus agalactiae*, *Streptococcus canis*, *Streptococcus dysgalactiae subsp. Dysgalactiae*, *Streptococcus equisubsp. Zooepidemicus*, *Streptococcus halichoeri*, *Streptococcus iniae*, and *Streptococcus suis*. Diseases caused by infection with these bacteria can include skin and soft tissue infections, arthritis, reproductive system diseases, mastitis, pneumonia and sepsis, and streptococcal toxic shock-like syndrome.

[0078] Non-limiting examples of viruses that can cause microbial infections, depending on the type of farmed or companion animal, include: White Spot Syndrome Virus (WSSV) causing white spot syndrome (also known as white spot disease); Infectious Hypodermic and Hematopoietic Necrosis Virus (IHHNV) causing infectious hypodermic and hematopoietic necrosis; Tora Syndrome Virus (TSV) causing Tora syndrome; Infectious Myonecrosis Virus (Myo / IMNV) causing infectious myonecrosis; Fish Myocarditis Virus (PMCV) causing fish myocarditis; and other viruses that cause viral infections. Viral hemorrhagic septicemia virus; infectious hematopoietic necrosis virus (IHHV) causing infectious hematopoietic necrosis disease; salmon infectious anemia virus (ISAV) causing infectious anemia in salmon; fish ororeovirus (PRV) causing heart and skeletal muscle inflammation (HSMI), jaundice syndrome, proliferative melanosis syndrome, and erythrocyte inclusion body syndrome in fish; tilapia lake virus; latent lethal nodavirus; shrimp hemocyte iridovirus; abalone herpesvirus; and porcine reproductive and respiratory syndrome virus (PRRSV). Porcine epidemic diarrhea virus (PRRSV), porcine epidemic diarrhea virus (PEDV), African swine fever virus (ASFV), classical swine fever virus (CSFV), Nipah virus, swine vesicular disease virus (VVV), porcine transmissible gastroenteritis virus (TGEV), avian influenza virus (IHV), infectious bursal disease virus (IBDV), Marek's disease virus (MDV), avian metapneumovirus, infectious bronchitis virus (IBBV), and other infectious diseases. Infectious laryngotracheitis virus causing laryngotracheitis, duck hepatitis virus causing duck hepatitis, pseudorabies virus causing pseudorabies, bluetongue virus causing bluetongue, foot-and-mouth disease virus causing foot-and-mouth disease (serotypes A, O, C, SAT1, SAT2, SAT3, Asia1), Japanese encephalitis virus causing Japanese encephalitis, rabies virus causing rabies, Rift Valley fever virus causing Rift Valley fever, rinderpest virus causing rinderpest, vesicular stomatitis virus causing vesicular stomatitis, West Nile fever virus causing West Nile fever, and others.

[0079] In this implementation, the microbial infection is caused by parasites, such as internal or external parasites. The parasites may be members of the phylum Apicomplexa (including the genera Cryptosporidium spp., Plasmodium spp., Eimeria spp., Neospora, Babesia, and Theileria).

[0080] Non-limiting examples of parasites taught in this article include parasites of the genus *Eimeria* that cause coccidiosis in various animal species, parasites of the genus *Cryptosporidium* that cause cryptosporidiosis, and *Enterocytozoon* parasites (*Enterocytozoon hepatopenaei*) that can cause early death syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) in shrimp.

[0081] Parasites of the genus *Eimeria* include, but are not limited to, *Eimeria tenella*, *Eimeria acervulina*, *Eimeria praecox*, *Eimeria mitis*, *Eimeria necatrix*, and *Eimeria maxima*, which cause coccidiosis in birds; *Eimeria zuernii*, *Eimeria alabamensis*, and *Eimeria bovis*, which cause coccidiosis in ruminants (such as cattle); and any other *Eimeria* species that cause coccidiosis in various animal species.

[0082] Parasites of the genus Cryptosporidium include, but are not limited to, all of the following that cause cryptosporidiosis: Cryptosporidium parvum, Cryptosporidium meleagridis, Cryptosporidium felis, Cryptosporidium canis, and Cryptosporidium hominis.

[0083] This disclosure further provides a method for improving the production performance of animals (e.g., farmed animals) (e.g., increasing feed intake, increasing average daily weight gain, increasing feed efficiency (i.e., reducing feed conversion ratio) and / or increasing milk yield), the method comprising the step of administering the animal the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or an extract thereof or an animal feed or feed additive as taught herein.

[0084] Furthermore, this disclosure provides a method for reducing mortality in animals, the method comprising the step of administering to the animals a composition comprising the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or an extract thereof, or an animal feed or feed additive as taught herein.

[0085] Furthermore, the compositions taught herein have been found to provide protection for cells, particularly hepatocytes, against fungal toxins such as aflatoxin B1. Absorbed fungal toxins are known to be detoxified in the liver, as with all toxins. Even short-term exposure to uninhibited fungal toxins is sufficient to cause significant liver damage and loss of liver function. Therefore, the compositions taught herein can also be used as preservatives or as cell-protective agents. Animal feeds or compositions taught herein can be used to prevent hepatotoxicity or liver injury due to fungal toxin exposure, reduce the severity of hepatotoxicity or liver injury due to fungal toxin exposure, and / or treat hepatotoxicity or liver injury due to fungal toxin exposure.

[0086] Animal feeds and / or feed additives used in the above methods may be as described above.

[0087] Extraction can be performed as described above.

[0088] Animals can be selected from domesticated animals and companion animals. Domesticated animals can be selected from the following groups: poultry; pigs; ruminants, such as beef cattle and dairy cattle; fish, such as salmon, trout, sea bream, sea bass, tilapia, and tuna; and crustaceans, such as shrimp. Companion animals can be selected from the following groups: ornamental fish, cats, dogs, horses, rabbits, guinea pigs, and hamsters.

[0089] Animals may be animals affected by a microbial infection (also known as "infected animals") or animals at risk of being infected by a microbial infection (also known as "animals at risk of infection"). Infected animals or animals at risk of infection may be aquatic species (e.g., fish or shrimp), or livestock species (e.g., chickens, pigs, or cows), or any other animal species mentioned herein.

[0090] In practice, the performance benefits obtained in animals fed with the feed additives or animal feeds taught herein are relative to the results obtained from animals (of the same species) fed with the same animal feeds without such feed additives.

[0091] The invention is further illustrated by the following embodiments, but is not intended to be limiting. Based on the above discussion and these embodiments, those skilled in the art can determine the essential features of the invention and, without departing from its teachings and scope, can make various changes and modifications to adapt it to various uses and conditions. Therefore, various modifications to the invention, other than those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0092] Appropriate implementation methods:

[0093] 1. An animal feed comprising the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or an extract thereof, wherein the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or an equal amount of the extract are included in an amount of about 1 mg / kg to about 10 g per kg of feed.

[0094] 2. The animal feed according to embodiment 1, wherein the aboveground portion of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) is dried and optionally pulverized.

[0095] 3. An animal feed according to any one of embodiments 1 or 2, wherein the extract is selected from the group consisting of: aqueous extract, ethanol extract, methanol extract, isopropanol extract, ethyl acetate extract, propanol extract, hexane extract or supercritical CO2 extract, or any mixture thereof.

[0096] 4. Use of the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or its extracts as a feed additive, wherein the feed additive is provided with written instructions to include the feed additive in animal feed at an amount of about 1 mg / kg to about 10,000 mg / kg of the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or an equivalent amount of the extract.

[0097] 5. Use of the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or its extracts as antibacterial agents, antiviral agents, antifungal agents, antiyeast agents, antiparasitic agents or feed preservatives.

[0098] 6. As per embodiment 4 or 5, the feed additive, antibacterial agent, antiviral agent, antifungal agent, antiyeast agent, antiparasitic agent, or feed preservative is included in the premix.

[0099] 7. A method of feeding an animal, the method comprising the step of administering an animal feed according to any one of embodiments 1 to 3 to the animal.

[0100] 8. Use of the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or extracts thereof, or animal feed according to any one of embodiments 1 to 3, for increasing the production performance of animals.

[0101] 9. Use of the above-ground parts of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or extracts thereof, or animal feed according to any one of embodiments 1 to 3, for increasing feed intake, increasing average daily weight gain, increasing feed efficiency (i.e., reducing feed conversion ratio), increasing relative growth rate, increasing specific growth rate, and / or increasing milk fat production.

[0102] 10. A composition for use in reducing mortality, comprising a composition of the above-ground portion of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or an extract thereof, or an animal feed according to any one of embodiments 1 to 3.

[0103] 11. A composition for use in preventing, alleviating, and / or reducing microbial infection, comprising a composition of the above-ground portion of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or an extract thereof, or an animal feed according to any one of embodiments 1 to 3.

[0104] 12. The composition used according to embodiment 11, wherein the microbial infection is caused by a microorganism selected from the group consisting of bacteria, viruses, fungi, yeasts or parasites.

[0105] 13. A composition for use according to any one of embodiments 11 or 12, wherein the microbial infection is caused by Gram-negative bacteria, such as Gram-negative bacteria of the genus Vibrio, such as Gram-negative bacteria of the species Vibrio parahaemolyticus.

[0106] 14. A composition for use according to any one of embodiments 11 or 12, wherein the microbial infection is caused by a virus, for example, vitiligo syndrome virus.

[0107] 15. A composition for use according to any one of embodiments 11 or 12, wherein the microbial infection is caused by parasites, such as parasites of the genus Eimeria, parasites of the genus Cryptosporidium, or parasites of the genus Enterocytozoa.

[0108] 16. A composition for preventing hepatotoxicity or liver injury caused by exposure to mycotoxins (e.g., exposure to aflatoxin B1), reducing the severity of hepatotoxicity or liver injury caused by mycotoxins exposure, and / or treating hepatotoxicity or liver injury caused by mycotoxins exposure, comprising the above-ground portion of Glycyrrhiza glabra (preferably Glycyrrhiza glabra leaves) or an extract thereof, or animal feed according to any one of embodiments 1 to 3.

[0109] 17. The use according to any one of embodiments 4 to 6, or the composition for use according to any one of embodiments 10 to 16, wherein the extract is selected from the group consisting of: aqueous extract, ethanol extract, methanol extract, isopropanol extract, ethyl acetate extract, propanol extract, hexane extract or mixtures of these solvents or supercritical CO2 extract, or any mixture thereof.

[0110] 18. A composition for use according to any one of embodiments 4 to 6, or for use according to any one of embodiments 10 to 17, wherein the feed additive is intended to be included in the feed for farmed animals or companion animals.

[0111] 19. According to the use of embodiment 18, the farmed animals are selected from the group consisting of: poultry; pigs; ruminants, such as beef cattle and dairy cattle; fish, such as salmon, trout, sea bream, sea bass, tilapia and tuna; and crustaceans, such as shrimp.

[0112] 20. According to the use of embodiment 18, the companion animal is selected from the group consisting of: ornamental fish, cats, dogs, horses, rabbits, guinea pigs and hamsters.

[0113] Example

[0114] Example 1

[0115] In this embodiment, the presence of 18-β-glycyrrhizic acid was analyzed in samples of the roots and leaves of Glycyrrhiza glabra, which is a typical component of the roots of Glycyrrhiza glabra and a major reason for the use of licorice in food and feed applications.

[0116] The material used for analysis was an Atlantis T3 column (3.5 μm, 4.6 × 150 mm (number 1.7)), and the solvents were acetonitrile and 3% aqueous acetic acid.

[0117] The reference solution was prepared as follows: 10.5 mg of glycyrrhizic acid monoammonium salt was dissolved in 0.8% ammonium hydroxide aqueous solution and then diluted to 50.0 ml. 25 ml of this stock solution was diluted to 50 ml with 0.8% ammonium hydroxide solution. The stock solution was prepared as follows: 0.4 g of powdered roots and leaves (using a laboratory grinder and passing through a 2.0 mm sieve) was weighed and extracted with 50.0 ml of 0.8% ammonium hydroxide aqueous solution, then sonicated for 30 minutes and centrifuged at 4400 rpm for 5 minutes. The supernatant was used as the stock solution. The diluted sample solution was prepared as follows: 2 ml of this stock solution was diluted to 10 ml with 0.8% ammonium hydroxide aqueous solution to prepare the diluted sample solution.

[0118] The results of the analysis are summarized in Table 1 below.

[0119] Table 1. 18-β-glycyrrhizic acid content in roots and leaves.

[0120]

[0121] Figure 1 The chromatograms of the root sample of *Glycyrrhiza glabra* (top, clearly showing the glycyrrhizic acid peak) were compared with those of the leaf sample of *Glycyrrhiza glabra* (bottom) (where the glycyrrhizic acid peak was absent). Table 1 summarizes the findings. Figure 1The results showed that glycyrrhizic acid was not detected or was detected at trace levels in the leaves of *Glycyrrhiza glabra*. Conversely, 4.13% glycyrrhizic acid was detected in the root samples of the same plant.

[0122] This experiment was repeated three times using leaves from different Glycyrrhiza glabra plants, and the same results were obtained. The typical peak of glycyrrhizic acid was not observed.

[0123] Example 2

[0124] The effects of dietary supplementation of Glycyrrhiza glabra leaves on the production performance of shrimp were studied.

[0125] Licorice root leaves are cultivated. Cultivation and drying follow Good Agricultural and Collecting Practices. Licorice root leaves are ground into particles <0.3 mm in size. Basic dietary components are stated in Table 2.

[0126] Table 2. Basic dietary components of extruded shrimp feed.

[0127] raw material Addition level (in kg / 100kg) Soy products / by-products 35-40 Wheat products / by-products 35-40 Fish meal / byproducts 8-15 Major minerals 4-6 Soybean phospholipids / oil 1.5-2.5 Fish oil NA 1.5-2 Crustacean attractants 1.5-3 Hydrolysis products 1.5-3 amino acid mixture 0.5-1.2 Vitamin-mineral premix 0.3-0.5

[0128] The diet was made into extruded granules of 1.4 ± 0.1 mm.

[0129] The feed production process follows standard guidelines for shrimp feed extrusion. First, all dried raw materials (except trace components) are passed through a 1mm (50Hz) pulverizer sieve. Next, the components are weighed to 0.01g accuracy on an electronic scale according to the formulation specifications. After extrusion, the pellets are dried until the moisture content is below 10%. The final feed is then packaged and stored at 4°C.

[0130] The experimental treatments were conducted as follows. The "control group" shrimp received a basal shrimp diet. The "treatment group" shrimp received a basal diet supplemented with 520 g of dried and ground licorice leaves per metric ton of feed (520 ppm).

[0131] The control and treatment groups were each placed in a separate plastic box. Each 290L box was equipped with an individual filtration system and filled with artificial seawater with a salinity of 20g / L. 75 shrimp were introduced into each box. The shrimp were Penaeus vannamei larvae.

[0132] Total weight of each group was obtained at the start of the experiment (day 0) and the end of the experiment (day 18). The average weight of the shrimp in each group was calculated at the start and end. The daily feed intake rate for each group was calculated based on the average weight of the shrimp and adjusted daily according to desired shrimp growth and mortality. Feed was automatically dispensed 6 times daily over the 18-day period. Water quality was maintained through a filtration system and regular water changes. The water temperature was maintained at 24°C. The room where the tanks were placed received 12 hours of light daily.

[0133] Growth performance was assessed using the following parameters: weight gain (WG) and feed conversion ratio (FCR).

[0134] Table 3. Shrimp growth performance

[0135] Production response (units) control group Processing group Treatment effectiveness (%) Weight gain (g) 2.12 2.30 +8.3％ FCR 0.95 0.88 -7.4％

[0136] The results shown in Table 3 demonstrate that adding Glycyrrhiza glabra leaves to the shrimp diet improved production performance. The addition of Glycyrrhiza glabra leaves increased weight gain by 8.3%. The addition of Glycyrrhiza glabra leaves reduced feed conversion ratio (meaning improved feed efficiency) by 7.4%.

[0137] Example 3

[0138] The effects of two dietary supplement doses of dried Glycyrrhiza glabra leaves on the production performance of broiler chickens were investigated.

[0139] Licorice glabra leaves are cultivated. Cultivation and drying follow Good Agricultural and Collecting Practices. Licorice glabra leaves are ground into pellets <0.3 mm in size. A feed in powder form is provided according to the chickens' age: starter feed from day 0 to day 14, growing feed from day 15 to day 28, and finisher feed from day 29 to day 42. The basic diet composition is described in Table 4.

[0140] Table 4. Composition of basic starter feed, growing feed and finishing feed.

[0141] Raw materials, in kg / 100kg Open-end material Growth feed Fattening feed cereals 58.44 60.36 64.56 soybean meal 30.469 23.82 19.87 Dried lees and solubles 4.00 8.00 8.00 meat meal and bone meal 4.00 4.00 3.821 soybean oil 0.972 2.024 2.256 limestone 0.309 0.287 0.221 dicalcium phosphate 0.609 0.281 0.110 Salt 0.391 0.375 0.379 choline 0.064 0.069 0.069 Lysine 0.218 0.273 0.259 Methionine 0.287 0.266 0.236 threonine 0.107 0.106 0.085 A mixture that supplies other minerals and vitamins 0.143 0.143 0.143

[0142] The experimental treatments were conducted as follows. Control group chickens received basal starter feed, basal growing feed, and basal finishing feed. Treatment group 1 chickens received a basal diet supplemented with 39g of dried, ground licorice root leaves per metric ton of feed. Treatment group 2 chickens received a basal diet supplemented with 78g of dried, ground licorice root leaves per metric ton of feed.

[0143] Each experimental treatment was assigned to 12 enclosures per treatment. Each enclosure (4'×5') contained one drinker and one feeder. Chickens were raised on fresh wood chips. Thirty-four day 0 (1-day-old) Ross 308 chicks were assigned to each enclosure. Chicks were evenly distributed into 108 study enclosures based on weight. Continuous lighting was provided from day 0 to day 42.

[0144] When the chickens were 0, 14, 28, and 42 days old, all broilers were weighed by pen. Feed was added as needed by weighing, and any remaining feed was weighed when the chickens were weighed. The following data were collected during the study: broiler body weight on day 0 and day 42 (to calculate weight gain), feed intake on day 42, feed conversion ratio on day 42, and mortality. Based on these results, feed conversion ratio and adjusted feed conversion ratio were calculated.

[0145] Calculate the feed conversion ratio for a single pen by dividing (the sum of the total weight of all chickens in the pen + the sum of the total weight of all dead broilers in the pen) by the total feed consumption.

[0146] The feed conversion ratio after final BW adjustment on day 42 is calculated as follows: Feed conversion ratio + (2.22kg - actual body weight) / 7.

[0147] Table 5. Production performance of broiler chickens

[0148]

[0149] 1 FCR = Feed Conversion Rate

[0150] The results shown in Table 5 indicate that the addition of licorice root powder to the diet of broiler chickens improved production performance. For both levels of licorice root powder addition, the addition resulted in a 3.2% increase in final body weight (P = 0.053). The addition of licorice root powder reduced feed conversion ratio. For example, with 39 ppm of licorice root powder, the adjusted feed conversion ratio decreased by 4.1% (P = 0.005), and with 78 ppm of licorice root powder, the adjusted feed conversion ratio decreased by 5% (P = 0.005). Mortality rates were the same in all treatment groups.

[0151] Example 4

[0152] In this experiment, the effects of a diet including dried licorice leaves on shrimp's response to disease challenge were investigated. The disease challenge was acute hepatopancreatic necrosis disease (AHPND) induced by Vibrio parahaemolyticus.

[0153] Licorice root leaves are cultivated. Cultivation and drying follow Good Agricultural and Collecting Practices. Licorice root leaves are ground into particles <0.3 mm in size. The components of the basic diet are shown in Table 6 below.

[0154] Table 6. Basic dietary components of extruded shrimp feed.

[0155] raw material Addition level (in kg / 100kg) Soy products / by-products 35-40 Wheat products / by-products 35-40 Fish meal / byproducts 8-15 Major minerals 4-6 Soybean phospholipids / oil 1.5-2.5 Fish oil NA 1.5-2 Crustacean attractants 1.5-3 Hydrolysis products 1.5-3 amino acid mixture 0.5-1.2 Vitamin-mineral premix 0.3-0.5

[0156] The diet was made into 1.4±0.1mm extruded pellets.

[0157] The feed production process follows standard guidelines for shrimp feed extrusion. First, all dried raw materials (except trace components) are passed through a 1mm (50Hz) pulverizer sieve. Next, the components are weighed to 0.01g accuracy on an electronic scale according to the formulation specifications. After extrusion, the pellets are dried until the moisture content is below 10%. The final feed is then packaged and stored at 4°C.

[0158] During the first part of this experiment, shrimp were housed in groups and fed either a "control group" diet or a "treatment group" diet. These two diets were obtained as follows: the control group received a basal diet; the treatment group received a control diet supplemented with 1040 g of dried licorice leaves per metric ton of feed (1040 ppm).

[0159] The control and treatment groups were each placed in a separate plastic tank. Each 290L tank was equipped with an individual filtration system and filled with artificial seawater with a salinity of 20g / L. 75 shrimp were introduced into each tank. The shrimp were Penaeus vannamei larvae.

[0160] After 18 days of growth, shrimp from both groups were transferred to a disease challenge facility and placed in infection units (one shrimp per tank) for acclimatization. The (experimental) animals were housed individually in 10L tanks and fed the same diet as during the first 18 days. The tanks were filled with artificial seawater with a salinity of 20 g / L and equipped with mechanical / biological filters to ensure water quality. The water temperature was maintained at 27°C, and the room containing the tanks was illuminated for 12 hours daily. Acclimatization was allowed for 3 days.

[0161] Each treatment (control group or treatment group) was replicated in 3 blocks, with 10 shrimp per block per treatment. Shrimp were hand-fed four times daily (9:00 AM, 11:30 AM, 2:00 PM, and 4:30 PM), with additional feedings at 8:00 PM and 12:00 PM on the day of AHPND inoculation. Clinical signs of disease and mortality were monitored twice daily.

[0162] This challenge was for acute hepatopancreatic necrosis disease (AHPND). The bacteria used were *Vibrio parahaemolyticus* isolated from shrimp infected with AHPND. The bacterial samples were preserved at -70°C. After thawing, the preserved samples were aseptically inoculated into culture medium and grown under standard conditions. The optical density of the resulting bacterial suspension was determined spectrophotometrically. These data were used to determine the bacterial concentration per milliliter of colony-forming units (CFU) in the suspension. Throughout the experiment, shrimp were inoculated using a fixed amount of bacterial suspension via immersion. The same dose of bacteria was used for each tank.

[0163] Mortality rates were measured, and the cumulative mortality rate (in percentage) 14 days after infection was calculated. The results are shown in Table 7 below.

[0164] Table 7. Mortality rate of shrimp infected with Vibrio parahaemolyticus.

[0165] Cumulative mortality rate (%) 14 days after infection control group 59.5％ Processing group 50％ Effects (%) of the treatment group versus the control group -16％

[0166] Adding licorice leaves to the shrimp diet significantly reduced mortality due to Vibrio parahaemolyticus.

[0167] Example 5

[0168] The effects of dried Glycyrrhiza glabra leaves on the production performance of broiler chickens were studied.

[0169] Licorice glaze leaves are cultivated. Cultivation and drying follow Good Agricultural and Collecting Practices. The licorice glaze leaves are ground into particles <0.3 mm and fed as a powder according to the age of the chickens: starter feed from day 0 to day 14, growing feed from day 15 to day 28, and finishing feed from day 29 to day 42. The basic diet composition is described in Table 8 below.

[0170] Table 8. Composition of basic starter feed, growing feed and finishing feed.

[0171] Raw materials, in kg / 100kg Open-end material Growth feed Fattening feed cereals 57.02 58.49 58.189 soybean meal 29.693 23.505 18.654 Dried lees and solubles 6.00 10.00 15.000 meat meal and bone meal 4.00 4.00 4.000 soybean oil 1.105 2.213 2.833 limestone 0.333 0.309 0.212 dicalcium phosphate 0.567 0.236 Salt 0.433 0.391 0.342 choline 0.062 0.064 0.054 Lysine 0.238 0.277 0.275 Methionine 0.285 0.260 0.213 threonine 0.110 0.103 0.074 A mixture that supplies other minerals and vitamins 0.154 0.152 0.154

[0172] The experimental treatments were conducted as follows. The control group received basal starter feed, basal growing feed, and basal finishing feed. The treatment group received basal feed supplemented with 55g of dried and pulverized licorice leaves per metric ton of feed.

[0173] Each experimental treatment was divided into 12 enclosures per treatment. Each enclosure (4'×5') contained one drinker and one feeder. Chickens were raised on fresh wood chips. Thirty-four day 0 (1-day-old) Ross 308 chicks were assigned to each enclosure. Chicks were evenly distributed into 24 study enclosures based on weight. Continuous lighting was provided from day 0 to day 42.

[0174] When the chickens were 0, 14, 28, and 42 days old, all broilers were weighed by pen. Feed was added as needed by weighing, and any remaining feed was weighed when the chickens were weighed. The following data were collected during the study: broiler body weight on day 0 and day 42 (to calculate weight gain), feed intake on day 42, feed conversion ratio on day 42, and mortality rate. Based on these results, feed conversion ratio and adjusted feed conversion ratio were calculated.

[0175] Calculate the feed conversion ratio for a single pen by dividing (the sum of the total weight of all chickens in the pen + the sum of the total weight of all dead broilers in the pen) by the total feed consumption.

[0176] The feed conversion ratio after final BW adjustment on day 42 is calculated as follows: Feed conversion ratio + (2.22kg - actual body weight) / 7.

[0177] Table 9. Production performance of broiler chickens at 42 days.

[0178] Production results control group Processing group Effect,% Feed intake (kg) 4.11 4.16 +1.2％ Weight gain (kg) 2.37 2.44 +2.9％ Adjusted FCR 1.69 1.65 -2.4％

[0179] The results shown in Table 9 demonstrate that adding licorice root leaves to the diet of broiler chickens improved production performance: it increased final body weight by 2.9%. The addition of licorice root leaves reduced feed conversion ratio by 2.4%.

[0180] Example 6

[0181] The effects of dried Glycyrrhiza glabra leaves on the production performance of salmon were studied.

[0182] Licorice glaze leaves are cultivated. Cultivation and drying follow Good Agricultural and Collecting Practices. The licorice glaze leaves are ground to a particle size of <0.3 mm. A basic diet is formulated to meet the nutritional requirements of Atlantic salmon at levels of approximately 41% crude protein, 28% crude fat, 7% moisture, 24 kJ / g energy, and 5% ash.

[0183] The experimental treatments were conducted as follows. Control group salmon received a basal diet. Treatment group salmon received a basal diet supplemented with 130g of dried, ground licorice root leaves per metric ton of feed instead of wheat. All macro and micro components (including the dried, ground licorice root leaf component) were dry-mixed before extruding the complete mixture to produce 4mm pellets.

[0184] This experiment involved two treatments (control group and treatment group) using Atlantic salmon with an average weight of 420g. Each treatment was replicated in three places using 1 cubic meter tanks with 40 fish per tank. The water temperature was maintained at 12°C at a salinity of 31 ppt. The salmon were fed three times a day. The lighting program was 24 hours of light. The experiment lasted 54 days. The salmon were weighed on the first day (and the average initial weight, in g) and on the last day of the experiment (which provides the average final weight, in g). The feeding days were 54. Other results were measured: final biomass (in g), total weight of dead fish (in g), and total feed intake (in g). Specific growth rate (SGR) and feed intake (both expressed as a percentage of daily intake) were calculated as follows.

[0185] SGR = (Average final weight / Average initial weight) (1 / Number of feeding days) -1 ×100

[0186] Feed intake = SGR × Total feed consumed / (Final biomass - Average initial weight + Total dead fish weight)

[0187] Table 10 shows the results of the experiment.

[0188] Table 10. Production performance of salmon fed with Glycyrrhiza glabra leaves

[0189] control group Processing group Effect(%) Feed intake, % / day 0.527 0.566 7.4％ <![CDATA[RGR 1 ]]> 0.906 0.934 3.1％ SGR 1.169 1.206 3.2％

[0190] 1 RGR stands for Relative Growth Rate.

[0191] The addition of 130 ppm of Glycyrrhiza glabra leaf increased feed intake by 7.4% and improved specific growth rate by 3.2%.

[0192] Example 7

[0193] In this embodiment, the effect of Glycyrrhiza glabra leaf on alleviating the effects of aflatoxin B1 on hepatocytes was studied.

[0194] Alpha mouse liver 12 cells (AML12) were grown and differentiated in DMEM / F12 medium supplemented with 10% FBS, 1% insulin-transferrin-selenium, 100 IU / mL penicillin, 100 μg / mL streptomycin, and 40 ng / mL dexamethasone at 37°C under a humid atmosphere with 5% CO2. The cell line was maintained by continuous passage.

[0195] 0.1 g of dried licorice root leaves were suspended in 500 μL of DMSO, DMSO aqueous solution (DMSO:H2O = 1:1), or sterile water to obtain a stock solution. After vigorous stirring to ensure complete dissolution, the suspension was filtered through a 0.22 μm filter membrane and stored at -20°C. Purified aflatoxin B1 was dissolved in basal medium or DMSO stored at -20°C before dilution in cell culture medium. Control samples were treated with culture medium.

[0196] The cytotoxic effects of aflatoxin B1 and various concentrations of PE solution on AML-12 cells were assessed using the Cell Counting Kit-8 (CCK-8) reagent. Briefly, after treatment with 8 mg / L aflatoxin B1 (with or without licorice root leaf extract solution, GG; added at 10 μg / mL, 15 μg / mL, and 20 μg / mL) for 24 h, 10 μL of CCK-8 solution was added according to the manufacturer's instructions. A control group without aflatoxin B1 and without GG was used. Absorbance at 450 nm was determined by microplate reader at the indicated time.

[0197] The results are shown in Table 11 below.

[0198] Table 11. Cell viability (%)

[0199] deal with Cell viability (%) [1] Control group 100 [2] = [1] + 8 mg / L aflatoxin B1 51.8 [3] = [2] + 10 ug / mL of GG 55.9 [4] = [2] + 15 ug / mL of GG 64.7 [5] = [2] + 20 ug / mL of GG 74.0

[0200] It can be inferred that the leaves of Glycyrrhiza glabra alleviate the effect of aflatoxin B1 on cells.

[0201] Example 8

[0202] Newly hatched chicks (from Longnecker's hatchery in Elizabeth, PA) were placed in Petersime brooders with free access to feed and water. All chicks were weighed and assigned to cages. Each treatment consisted of 60 chicks (5 chicks / cage = 12 replicates). All treatments had the same initial weight.

[0203] For E. maxima infection, chicks were infected with a newly propagated strain of E. maxima (ARS strain). On day 14 post-hatching, chicks were infected via gavage with 10,000 E. maxima oocysts.

[0204] The experimental treatments were as follows: [1] = negative control group (NC), uninfected and untreated chicks; [2] = positive control group (PC) = [1] + infected; [3] = [2] + monensin (MO) at 90 mg / kg feed; [4] = [2] + 55 ppm dried licorice leaves; [5] = [2] + 160 ppm dried licorice leaves. Monensin is currently the standard product used to control coccidiosis.

[0205] Chicks were weighed individually on days 7, 14, 18, 20, and 23 to calculate their weight gain.

[0206] Oocyst shedding: Feces were collected from each group on days 6–8 post-coccidiosis infection (dpi). To count coccidia oocysts, various dilutions were first performed to determine the optimal dilution for counting oocysts in each sample. Oocysts were counted under a microscope using a McMaster counting chamber via sodium chloride flotation. The total number of oocysts shed per chick was calculated using the following formula: Total oocysts / chicks = (Number of oocysts × Dilution factor × Fecal sample volume / Counting chamber volume) / Number of chicks per cage.

[0207] result

[0208] The production performance results are shown in Table 12 below.

[0209] Table 12. Production performance data of chicks: Uninfected with Eimeria giantiformis (NC) or infected with Eimeria giantiformis (PC, MO, GG) (lacking further treatment (PC) or treated with monensin (MO), treated with 55 ppm Glycyrrhiza glabra leaf (55 ppm GG) or treated with 160 ppm Glycyrrhiza glabra leaf (160 ppm GG)).

[0210]

[0211] DPI refers to days post-infection; BW refers to body weight; ADG refers to average daily gain; ADFI refers to average daily feed intake; and FCR refers to feed conversion ratio.

[0212] Monensin has demonstrated remarkable effectiveness in maintaining the growth performance of chicks infected with Eimeria giantiformis. Studies have also found that supplementing the diet with dried licorice root leaves improved the growth performance of chicks infected with Eimeria giantiformis.

[0213] Figure 2 The results of fecal oocyst shedding are shown. The study found that monensin reduced lesion scores and the number of oocysts, corresponding to growth outcomes. Licorice root leaves also significantly reduced the number of oocysts. When licorice root leaves were added to the feed at a concentration of 160 ppm, the number of oocysts was even lower than when monensin was added.

[0214] Example 9

[0215] The effects of dietary supplementation with Glycyrrhiza glabra leaves on the response of shrimp to viral diseases (white spot disease or white spot syndrome) were investigated.

[0216] Licorice root leaves are cultivated. Cultivation and drying follow Good Agricultural and Collecting Practices. Licorice root leaves are ground into particles <0.3 mm in size. The basic dietary components are presented in Table 13.

[0217] Table 13. Basic dietary components of extruded shrimp feed.

[0218] raw material Addition level (in kg / 100kg) Soy products / by-products 35-40 Wheat products / by-products 35-40 Fish meal / byproducts 8-15 Major minerals 4-6 Soybean phospholipids / oil 1.5-2.5 Fish oil NA 1.5-2 Crustacean attractants 1.5-3 Hydrolysis products 1.5-3 amino acid mixture 0.5-1.2 Vitamin-mineral premix 0.3-0.5

[0219] The diet was made into extruded pellets of 1.4 ± 0.1 mm.

[0220] The feed production process follows standard guidelines for shrimp feed extrusion. After extrusion, the pellets are dried until the moisture content is below 10%. The final feed is then packaged and stored at 4°C.

[0221] The experimental treatments were conducted as follows: The "control group" shrimp received a basal diet. "Treatment group 1" shrimp received a basal diet supplemented with 1040 g of dried and ground licorice root leaves per metric ton of feed (1040 ppm). "Treatment group 2" shrimp received a basal diet supplemented with 1040 g of dried and ground licorice root leaves per metric ton of feed and 150 g of capsicum oleoresin per metric ton (1040 ppm).

[0222] Shrimp were individually housed in 10L glass tanks filled with artificial seawater, prepared by adding a commercial salt mixture at 20g / L to purified water. Each tank was equipped with an independent mechanical / biological filter. Water flow between tanks was strictly controlled. Water quality was maintained through filtration, aeration, and regular water changes. The water temperature was kept at 27°C. The room was provided with 12 hours of light per day. Shrimp were fed three times daily. Clinical signs of disease and mortality were monitored twice daily.

[0223] Each treatment was repeated in 3 blocks, with 10 shrimp per block per treatment. The blocks were placed at different locations within the challenge facility.

[0224] Three days prior to the start of disease challenge, shrimp were transferred to the disease challenge facility. Shrimp were fed different treatment diets. White spot syndrome virus (WSSV) was used for disease challenge in this experiment. Virus samples were preserved at -70°C in the facility. Solid WSSV inoculum were prepared and used in oral infection experiments. The assessment parameter was mortality (%). Results are shown in Table 14 below.

[0225] Table 14

[0226] deal with Final mortality rate (%) The effect compared to the control group, % control group 46.7 - Processing Group 1 40.0 -14％ Processing Group 2 33.3 -29％

[0227] The addition of licorice root leaf extract improved shrimp survival rates against WSSV virus infection. The addition of products containing capsaicin further improved resistance to this virus.

[0228] Example 10

[0229] This experiment aimed to test the efficacy of dried licorice root leaves in peripartum dairy cows, with the first dose being 260 mg / head / day.

[0230] Thirty multiparous (2+ litters) dairy cows were divided according to their birth weight (BW), previous miltage (MY), and expected calving date, and assigned to one of the following treatments during lactation from week -3 to week +6 (n = 15 / trt): 1) control group; 2) 260 mg / head / day of dried licorice root leaves.

[0231] The measurement indicators throughout the study were: i) individual feed intake (daily); individual milk production (daily); milk composition (weekly; fat, protein, lactose); and individual body weight (weekly).

[0232] Daily data were processed using two-way ANOVA. Weekly data were submitted for one-way ANOVA with repeated measures.

[0233] Results and Conclusions

[0234] The results are shown in Table 15 below.

[0235] Table 15. Production performance parameters of dairy cows.

[0236]

[0237] Studies have found that supplementing the feed of dairy cows with dried licorice leaves during the peripartum period increases the percentage of milk fat in milk fat (p = 0.05) and increases the weight of the cows (p = 0.05).

[0238] Example 11

[0239] Target

[0240] The overall objective of this project is to determine, using a transgenic mouse infection model, the efficacy of a composition comprising cinnamon oil, tea extract, and pomegranate extract (CO / TE / PE; 1:1:1) in reducing parasite load and shedding, slowing disease progression, inhibiting pathogenesis, and increasing protective innate mucosal immunity in animal hosts during Cryptosporidium parvum infection.

[0241] Materials and methods

[0242] Twenty-four (24) 8-week-old male interferon-gamma knockout (IFN-γKO) mice (B6.129S7-IfngTM1Ts / J) were purchased and quarantined and acclimatized for one week prior to the start of the experiment. There were three treatment groups, with eight mice in each group housed individually. The treatments consisted of the following: Group 1, uninfected – untreated; Group 2, Cryptosporidium microsporidium infection – untreated (control group, only reconstituted DMSO); Group 3, infected – dried licorice leaves. On day 3 after the start of the experiment, mice were administered 10 mg of licorice root extract orally via gavage. 4 Each mouse from the infected group was infected with a Cryptosporidium microsporidium AUCP-1 isolated oocyst.

[0243] 1. Parasites

[0244] Cryptosporidium microsporidium AUCP-1 isolates were maintained and propagated in male Holstein calves. Cryptosporidium microsporidium oocysts were extracted and purified from freshly collected calf feces using sequential sieving, sheather's sugar flotation, and discontinuous sucrose density gradient centrifugation. The purified oocysts were washed and stored in phosphate-buffered saline (PBS) at 4°C within 3 months to ensure maximum viability as determined by exocystization.

[0245] 2. Animals

[0246] Seven-week-old male IFN-γ knockout mice (B6.129S7-Ifngtm1Ts / J) were obtained from The Jackson Laboratory in the United States and housed under biosafety level 2 conditions. They were provided with free access to commercial mouse food and water. The animals were allowed to acclimatize to the environment for four days before the start of the experiment.

[0247] 3. Preparation of Glycyrrhiza glabra

[0248] The powdered dried licorice leaves were reconstituted in molecular-grade dimethyl sulfoxide (DMSO), then sterilized by filtration using a 0.22 μm filter, and stored at room temperature away from light until use.

[0249] 4. Processing, infection, sample collection, and determination of physiological parameters.

[0250] The mice were divided into three groups of eight mice each. Within each group, each mouse was housed in a separate cage. The groups are as follows:

[0251] • Uninfected and Untreated Group (UU)

[0252] • DMSO Infection Treatment Group (DMSO)

[0253] • Infection GG-treatment Group

[0254] Three days prior to infection with Cryptosporidium parvum, mice in each treatment group began daily oral gavage administration of either the following combination or DMSO: GG = 55 mg / kg; DMSO = 0.05 ml DMSO; UU = 0.05 ml DMSO. On day three after the start of treatment, mice in the infection groups (DMSO, GG) each received 10 mg / kg of PBS via oral gavage. 4 Suspensions of Cryptosporidium oocysts were prepared, and each group underwent daily treatment for 14 days post-infection. Mice were provided with free access to standard commercial food and water during treatment. Fecal samples were collected daily throughout the treatment period, and fecal physical consistency was determined. The animals' physiological status and activity, body weight, and appetite were also assessed. Furthermore, mice were euthanized, and their distal small intestine was harvested for histopathological examination.

[0255] result

[0256] 1. Physiological parameters:

[0257] Overall, uninfected, untreated (UU) mice were active, healthy, had a good appetite throughout, and gained weight. The observed weight gain and activity are typical of healthy mice of this age (7 weeks), as they are still in the growth phase. DMSO-treated mice exhibited typical cryptosporidiosis progression, including diarrhea, weight loss, poor condition, and reduced activity. Overall, although they were still growing, they experienced a decrease in experimental body weight during the experimental phase.

[0258] Mice infected with GG showed better physiological outcomes than the DMSO-treated group. They did not develop diarrhea, remained active and healthy throughout the course of the infection, and gained weight, although not as much as the uninfected mice. At necropsy, they had outwardly healthy intestines without bleeding. These observations clearly demonstrate that GG treatment alleviates cryptosporidium infection and improves the health of infected animals.

[0259] 2. Effect of combined treatment on Cryptosporidium microsporidium oocyst load in feces of infected mice:

[0260] As expected, mice infected with DMSO-treated control mice showed a gradual increase in oocyst shedding, reaching approximately 2 × 10⁻⁶ oocysts at 10 days post-infection. 8 The first peak in oocyst load per gram of feces (OPG) was observed, and 2.2 × 10⁻⁶ was observed at 15 days post-infection. 8 The second wave of higher peak values ​​for oocysts. Treatment with GG showed significantly lower oocyst loads (P<0.05) throughout the treatment phase compared to DMSO-infected mice. These results confirm the physiological parameters indicating that GG treatment mitigates disease progression and outcomes.

[0261] 3. Improvement of histopathological damage:

[0262] At the end of treatment, the mice were euthanized and their distal small intestines were used for histopathological examination. As expected, the uninfected control mice showed healthy intestinal mucosa with prominent villi. On the other hand, the DMSO-infected mice exhibited intestinal damage characterized by mucosal erosion, villi atrophy, crypt hypertrophy, and accumulation of inflammatory cells. The GG-treated mice showed even better results than the DMSO-infected mice, displaying essentially intact mucosa with prominent villi. Although the GG-treated mice showed accumulation of inflammatory cells in the mucosa, there was no significant hypertrophy of the intestinal crypts, indicating that these mice still possessed the ability to replenish their intestinal epithelial cells from intact crypts. These findings are consistent with the significantly reduced number of oocysts detected in the GG-treated mice and therefore suggest that this treatment alleviates parasite proliferation and intestinal pathological symptoms in infected mice.

[0263] Example 12

[0264] The objective of this experiment is to determine, using a calf infection model, the efficacy of Glycyrrhiza glabra leaves in reducing parasite load and shedding, inhibiting disease development and pathogenesis, and enhancing the protective innate mucosal immunity of animal hosts during Cryptosporidium infection.

[0265] Experimental methods

[0266] Determination of efficacy in alleviating cryptosporidiosis

[0267] Three days prior to infection, calf vegetatively administered GG (dried and powdered licorice leaves, reconstituted in molecular-grade dimethyl sulfoxide (DMSO), and stored at room temperature protected from light) once daily via oral gavage, and continued for 14 days after Cryptosporidium infection. Calves were also provided with free access to milk replacer and water. Throughout the treatment period, fecal samples were collected daily for measuring Cryptosporidium oocyst shedding by real-time quantitative PCR and for determining fecal physical consistency. Animal physiological status and activity, weight, feed intake, body temperature, coat condition, and body posture were also assessed.

[0268] This experiment consists of the following 3 groups, each with 5 calves:

[0269] • Five calves infected with Cryptosporidium microsporidium were treated with GG (13.75 g / kg BW).

[0270] • Five untreated calves infected with Cryptosporidium microsporidium

[0271] · 5 uninfected and untreated calves

[0272] Results and Conclusions

[0273] Generally, uninfected, untreated calves were active, healthy, had a good appetite throughout, and gained weight. The observed weight gain and activity are typical characteristics of healthy calves of this age, as they are still in a rapid growth phase. DMSO-treated calves exhibited typical cryptosporidiosis progression, including diarrhea, weight loss, poor condition, and reduced activity. Over time, their condition gradually worsened, and two calves died from the disease on days 10 and 12, respectively, post-infection. Overall, despite being in a rapid growth phase, all calves in this group experienced weight loss during the experimental period.

[0274] Infected calves treated with GG showed better physiological outcomes than the untreated group. Initially, two animals in this group developed diarrhea on day 3 post-infection, but recovered within 2–3 days, although one calf remained somewhat lethargic for a few days. The remaining calves in the GG group were active throughout the course of infection, and they gained weight, although not as much as the untreated calves. These observations clearly demonstrate that GG treatment alleviates cryptosporidiosis and improves the health of infected animals.

[0275] As expected, DMSO-infected control calves showed a gradual increase in oocyst shedding, reaching approximately 2.5 × 10⁻⁶ oocysts by day 9 post-infection. 8 The peak oocyst load per gram of feces (OPG) was observed in GG treatment. GG treatment consistently showed significantly (P<0.05) lower oocyst loads than DMSO-infected calves throughout the treatment phase, with a peak load of 6.5 × 10⁻⁶ OPG observed on day 8 post-infection. 4 The first peak was observed at 5.5 × 10⁵ oocysts per gram of feces, coinciding with a second wave of oocyst shedding on day 11 post-infection. 4 The peak value of each ovum per gram of feces. These results confirm the physiological parameters that indicate GG treatment reduces disease progression and outcomes.

[0276] At the end of treatment, calves were euthanized and their distal small intestines were used for histopathological examination. As expected, uninfected control calves showed healthy intestinal mucosa with prominent villi. On the other hand, DMSO-infected calves exhibited intestinal lesions characterized by mucosal erosion. Notably, GG-treated calves showed better results than DMSO-infected calves, displaying essentially intact mucosa with prominent villi. On average, uninfected calves, DMSO-infected calves, and GG-infected calves had villi of 300 μm, 260 μm, and 285 μm, respectively. These findings are consistent with a significant reduction in the number of oocysts detected in GG-treated calves and therefore suggest that GG treatment alleviates parasite proliferation and intestinal pathological symptoms in infected calves.

[0277] Example 13

[0278] The objective of this experiment was to observe whether treatment with dried licorice root would have a beneficial effect on enterocytozoon hepatocellular carcinoma (EHP) infection in shrimp. EHP is one of the most prevalent pathogens and poses a major challenge to shrimp farming worldwide. It is an intracellular parasitic disease associated with slow growth and development, large size variations in farmed shrimp, and significant economic losses for shrimp farmers.

[0279] Materials and methods

[0280] The experimental design consisted of three groups: a negative control, a positive control, and treatment with dried licorice root leaves (“GG treatment”). The experiment was set up as a completely randomized design (CRD), with 50 shrimp per box. The experiment lasted for 63 days: including 14 days of pre-challenge, 7 days of EHP co-challenge, and the following 42 days of post-challenge.

[0281] During the experiment, shrimp were fed four times daily, according to their individual feed intake levels. The following feeding schedule was used: 5% biomass (BW) in the early stage of challenge, 3% BW during EHP infection challenge, and 5% BW in the late stage of challenge. Feed amounts were adjusted based on estimated biomass and shrimp feeding behavior in each tank.

[0282] The EHP-infected shrimp used for challenge (reserve inoculum) contained 10 [units of something] per gram of EHP-infected shrimp. 8 -10 9 The average EHP load / density was measured using qPCR. GG-treated and positive control boxes were challenged using a co-habitation method, with 10 EHP-infected shrimp per box. The negative control box was co-cultured with 10 specific pathogen-free (SPF) shrimp. The co-habitation challenge phase lasted 7 days. Then, the inoculum (EHP-infected shrimp) and SPF shrimp were removed from the boxes. The SPF and infected shrimp were separated by a mesh that allowed free water exchange. The co-habitation challenge method was designed as follows: a 40-liter rectangular mesh cage was installed in each 350L plastic box. During challenge, 10 EHP-infected shrimp were placed inside the suspended mesh, while the experimental shrimp were placed outside the mesh simultaneously. Additionally, 20L of water from the EHP-infected inoculum box was supplied to the positive control and GG-treated boxes to facilitate challenge.

[0283] To ensure optimal growth and survival of the shrimp throughout the experiment, good water quality was maintained during the study.

[0284] result

[0285] The survival rate of the shrimp is shown in Table 16 below.

[0286] Table 16. Shrimp survival rate at the end of the experiment

[0287]

[0288] The study found that shrimp treated with GG had the highest survival rate, which was significantly higher than that of the positive control (P<0.05).

[0289] At the end of the experiment, the shrimp were harvested and weighed for the calculation of the growth performance index. The growth performance parameters of the shrimp on the day of termination are described in Table 17.

[0290] Table 17. Growth performance parameters after 63 days of culture.

[0291]

[0292] EHP infection clearly affected the growth performance of shrimp (“positive control group”). Final mean body weight, mean body weight gain, mean daily weight gain, specific growth rate (SGR), feed consumption, and feed conversion ratio (FCR) were all negatively affected by EHP infection. Treatment with GG improved growth performance parameters compared to the positive control group.

[0293] The study found that at the end of the experiment, the positive control group had 6.6 × 10⁶ cells / day. 8 The average EHP load is 4.73 × 10⁻⁶. 5 The average EHP load in the control group was significantly lower than that in the positive control group. Therefore, based on EHP load as a measure of efficacy, GG treatment reduced the EHP load in infected shrimp.

[0294] In summary, the treatment with Glycyrrhiza glabra leaves improved the effectiveness of EHP infection in terms of growth performance and mortality.

Claims

1. An animal feed comprising the aboveground portion of Glycyrrhiza glabra or an extract thereof, wherein the aboveground portion of Glycyrrhiza glabra or an equivalent amount of the extract is included in an amount of about 1 mg / kg to about 10 g per kg of feed, wherein the aboveground portion of Glycyrrhiza glabra is preferably Glycyrrhiza glabra leaves.

2. The animal feed according to claim 1, wherein the above-ground portion of licorice root is dried and optionally pulverized, and the above-ground portion of licorice root is preferably licorice root leaves.

3. The animal feed according to any one of claims 1 or 2, wherein the extract is selected from the group consisting of: aqueous extract, ethanol extract, methanol extract, isopropanol extract, ethyl acetate extract, propanol extract, hexane extract or supercritical CO2 extract, or any mixture thereof.

4. Use of the above-ground parts of Glycyrrhiza glabra or its extracts as feed additive 5, wherein the feed additive is provided with written instructions to include the above-ground parts of Glycyrrhiza glabra or an equivalent amount of its extracts in animal feed at an amount of about 1 mg / kg to about 10,000 mg / kg of feed, wherein the above-ground parts of Glycyrrhiza glabra are preferably Glycyrrhiza glabra leaves.

5. Use of the above-ground parts of Glycyrrhiza glabra or its extracts as antibacterial agents, antiviral agents, antifungal agents, antiyeast agents, antiparasitic agents or feed preservatives, wherein the above-ground parts of Glycyrrhiza glabra are preferably Glycyrrhiza glabra leaves.

6. The use according to claim 4 or 5, wherein the feed additive, antibacterial agent, antiviral agent, antifungal agent, antiyeast agent, antiparasitic agent, or feed preservative is included in the premix.

7. A method of feeding an animal, the method comprising the step of administering animal feed according to any one of claims 1 to 3 to the animal.

8. The use of the above-ground portion of Glycyrrhiza glabra or its extract, or in animal feed according to any one of claims 1 to 3, for increasing the production performance of animals, wherein the above-ground portion of Glycyrrhiza glabra is preferably Glycyrrhiza glabra leaves.

9. The use of the above-ground portion of Glycyrrhiza glabra or its extracts or animal feed according to any one of claims 1 to 3 for increasing feed intake, increasing average daily weight gain, increasing feed efficiency (i.e., reducing feed conversion ratio), increasing relative growth rate, increasing specific growth rate and / or increasing milk fat production, wherein the above-ground portion of Glycyrrhiza glabra is preferably Glycyrrhiza glabra leaves.

10. A composition for use in reducing mortality, comprising the aboveground portion of Glycyrrhiza glabra or an extract thereof or animal feed according to any one of claims 1 to 3, wherein the aboveground portion of Glycyrrhiza glabra is preferably Glycyrrhiza glabra leaves.

11. A composition for use in preventing, mitigating, and / or reducing microbial infections, comprising the above-ground portion of Glycyrrhiza glabra or an extract thereof, or animal feed according to any one of claims 1 to 3, wherein the above-ground portion of Glycyrrhiza glabra is preferably Glycyrrhiza glabra leaves.

12. The composition for use according to claim 11, wherein the microbial infection is caused by a microorganism selected from the group consisting of bacteria, viruses, fungi, yeasts, or parasites.

13. The composition for use according to any one of claims 11 or 12, wherein the microbial infection is caused by Gram-negative bacteria, such as Gram-negative bacteria of the genus Vibrio, such as Gram-negative bacteria of the species Vibrio parahaemolyticus.

14. The composition for use according to any one of claims 11 or 12, wherein the microbial infection is caused by a virus, for example, vitiligo syndrome virus.

15. The composition for use according to any one of claims 11 or 12, wherein the microbial infection is caused by a parasite, for example, a parasite of the genus Eimeria, a parasite of the genus Cryptosporidium, or a parasite of the genus Enterocytozoa.

16. A composition for preventing hepatotoxicity or liver injury due to mycotoxin exposure, reducing the severity of hepatotoxicity or liver injury due to mycotoxin exposure, and / or treating hepatotoxicity or liver injury due to mycotoxin exposure, comprising the above-ground portion of Glycyrrhiza glabra or an extract thereof, or animal feed according to any one of claims 1 to 34, wherein the above-ground portion of Glycyrrhiza glabra is preferably Glycyrrhiza glabra leaves, and the mycotoxin exposure is, for example, exposure to aflatoxin B1.

17. The use according to any one of claims 4 to 6, or the composition for use according to any one of claims 10 to 16, wherein the extract is selected from the group consisting of: aqueous extracts, ethanol extracts, methanol extracts, isopropanol extracts, ethyl acetate extracts, propanol extracts, hexane extracts, or mixtures of these solvents, or supercritical CO2 extracts, or any mixtures thereof.

18. The use according to any one of claims 4 to 6, or the composition for use according to any one of claims 10 to 17, wherein the feed additive is intended to be included in the feed for farmed animals or companion animals.

19. The use according to claim 18, wherein the farmed animal is selected from the group consisting of: poultry; pigs; ruminants, such as beef cattle and dairy cattle; fish, such as salmon, trout, sea bream, sea bass, tilapia and tuna; and crustaceans, such as shrimp.

20. The use according to claim 18, wherein the companion animal is selected from the group consisting of: ornamental fish, cats, dogs, horses, rabbits, guinea pigs and hamsters.

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