Composition comprising guanidinoacetic acid for use in the treatment and / or prophylaxis of a condition induced by coccidiosis in poultry

Description

1 / 17 COMPOSITION COMPRISING GUANIDINOACETIC ACID FOR USE IN THE TREATMENT AND / OR PROPHYLAXIS OF A CONDITION INDUCED BY COCCIDIOSIS IN POULTRY

[0001] Necrotic enteritis (NE) is one of the most relevant enteric diseases in poultry and entails high costs in the poultry industry worldwide (see Skinner et al., Avian Diseases 2010, 54(4): 1237-40). This results in annual losses of US$ 6 billion in global poultry production, and this controllable disease is increasing. One reason is the voluntary or legally required reduction of antibiotics in animal production. This trend is driven by the increasing occurrence of antimicrobial resistance, as well as consumer demand. Another reason is the reduction of ionophores which, in addition to their activity against coccidia, are also effective against clostridia. When live anticoccidial vaccines are used, the application of these ionophores is not possible, and cases of clostridia / necrotic enteritis increase (Williams, 2005).

[0002] Although this is a widespread problem in all birds, for broiler chickens in particular necrotic enteritis and coccidiosis are a significant health problem.

[0003] Necrotic enteritis is a multifactorial disease. Although its etiology is under investigation, the main theory is as follows (see Fathima et al., Microorganisms 2022, 10(10): 1958; Abd El-Hack et al., Poultry Science 2022,101(2): 101590): Factors such as coccidiosis or heat stress weaken intestinal integrity. Coccidiosis is an endemic disease in the commercial broiler industry and is caused by the development and reproduction of parasitic species of Eimeria, which can infect a specific area of ​​the intestine and cause tissue damage to intestinal epithelial cells. Disruption of intestinal epithelial cells leads to impaired nutrient absorption and increased intestinal permeability (see Teng et al., Poultry Science 2020, 99(9): 4203-4216), which can lead to... Petition 870250089342, dated 10 / 01 / 2025, page 14 / 33 2 / 17 to a high risk of bacterial infections. Additionally, the active immunosuppression used by the parasite to remain in the host makes the host more susceptible to secondary infections, for example, with avian-specific Clostridium perfringens leading to necrotic enteritis.

[0004] Since coccidiosis is often part of the disease's development, coccidiostats are used to control Eimeria infection (see Mesa-Pineda et al., Frontiers in Veterinary Science 2021, 8:87653) and antibiotics to control pathogenic bacteria, thus preventing the development of enteric diseases. Like antibiotics, chemical anticoccidials or coccidiostats induce the development of resistance in Eimeria species. Therefore, it is recommended to discontinue their use at least once a year throughout the broiler chicken's growth cycle. Instead, live vaccines containing attenuated strains of Eimeria species are used. However, vaccination often carries the risk of mild coccidiosis. With the ban on antimicrobial growth promoters and the emergence of the "totally antibiotic-free" movement, the impact of necrotic enteritis increases even further.Therefore, many recent studies on necrotizing enteritis have focused on finding different ways to control the disease and understand its pathogenesis. Premixes or technical products that can improve intestinal health or have a direct impact on infection, but are not antibiotics, may play an important role in controlling the disease and negative commercial effects.

[0005] Generally, necrotic enteritis occurs in broiler chickens aged 2 to 6 weeks. In subclinical forms, it is characterized by impaired digestion. Clinical forms cause serious problems and increase flock mortality in a very short period.

[0006] The clinical form of necrotic enteritis is characterized Petition 870250089342, dated 10 / 01 / 2025, page 15 / 33 3 / 17 due to acute, dark diarrhea, resulting in wet bedding and a sudden increase in flock mortality of up to 1% per day after the appearance of the first clinical signs (Ducatelle and Van Immerseel, 2010), sometimes reaching mortality rates of 50% (Van der Sluis, 2013). The birds have ruffled feathers, lethargy, and inappetence.

[0007] Necropsy usually shows swollen small intestines with a rough mucosal surface, lesions, and brownish (diphtheritic) pseudomembranes. There is a lot of watery, brown fluid with blood spots, and an unpleasant odor during the postmortem examination. The liver is dark, swollen, and misshapen, and the gallbladder is distended (Hofacre et al., 2018).

[0008] In the case of hyperacute necrotic enteritis, birds may die without showing any preliminary signs.

[0009] When birds suffer from the subclinical form, chronic damage to the intestinal mucosa and an increase in the amount of mucus in the small intestine lead to impaired digestion and absorption of nutrients, resulting in poor growth performance.

[0010] The worsening of feed conversion and the consequent reduction in performance become particularly noticeable around the 35th day of age. As feed contributes approximately 65 to 70% of the input cost to produce a broiler chicken, poor feed conversion increases production costs and significantly impacts profitability. Often, due to the lack of clear symptoms, this subclinical disease remains untreated and permanently affects production efficiency.

[0011] Consequently, there was still a need for a way to treat any type of coccidiosis-induced condition in birds. This problem was found to be solved by a composition comprising guanidinoacetic acid, a guanidinoacetic acid salt, or a mixture of either of these.

[0012] One objective of the present invention is, therefore, a Petition 870250089342, dated 10 / 01 / 2025, p. 16 / 33 4 / 17 composition for use in the treatment and / or prophylaxis of a coccidiosis-induced condition in poultry, wherein the composition comprises guanidinoacetic acid, a guanidinoacetic acid salt, or a mixture of either.

[0013] The aforementioned composition is administered to birds with a condition induced by coccidiosis.

[0014] The term "birds" is used in the context of the present invention to denote any type of domesticated bird, raised in captivity for its intended use. Examples of birds are domestic fowl, including chickens (or broilers and laying hens), turkeys, geese, quails, and ducks, raised for meat or eggs. Preferably, the term "birds" refers to chickens or broilers in the context of the present invention.

[0015] Supplementation with guanidinoacetic acid leads to greater creatine availability in all tissue cells, not just muscle tissue, and therefore to greater cellular energy availability in all cells. Furthermore, phosphorylated creatine acts as an energy reservoir under conditions of cellular stress and diverts energy production from glycolysis to the more efficient oxidative phosphorylation pathway. Additionally, it leads to increased expression of creatine kinase. Therefore, guanidinoacetic acid supplementation improves energy digestibility, minimizing energy loss, increasing energy availability for the immune response in immune cells, and leading to a more favorable metabolic profile in response to Eimeria infection or necrotic enteritis.

[0016] Coccidiosis is caused by the development and reproduction of parasitic species of Eimeria, which can infect a specific area of ​​the intestine and cause tissue damage to intestinal epithelial cells. Furthermore, the active immune suppression used by the parasite to remain in the host makes the host more susceptible to secondary infections, for example, with specific Clostridium perfringens. Petition 870250089342, dated 10 / 01 / 2025, page 17 / 33 5 / 17 of birds leading to necrotic enteritis. However, the administration of guanidinoacetic acid aids in the growth of birds by improving their energy efficiency, which can be impaired due to parasitic infection or induced secondary inflammation. Specifically, the administration of guanidinoacetic acid improves limited mitochondrial energy, which is highly beneficial for bird welfare. Therefore, the administration of guanidinoacetic acid in particular improves the growth and resistance of broiler chickens to a multifactorial necrotic enteritis challenge involving Eimeria spp. and Clostridium perfringens pathogens.

[0017] In one embodiment of the composition for use according to the present invention, coccidiosis is caused by Eimeria spp. and / or Clostridium perfringens.

[0018] In another embodiment of the composition for use according to the present invention, the coccidiosis-induced condition is one or more of bacterial enteritis, necrotic enteritis, diarrhea, and lesion.

[0019] Guanidinoacetic acid is soluble in water, but compared to some amino acids such as glycine or arginine, it has a relatively low solubility in water. However, the solubility of guanidinoacetic acid in water can be increased by transferring this compound to an acid addition salt. Suitable acid addition salts of guanidinoacetic acid can be formed with hydrogen chloride, sulfuric acid, and phosphoric acid.

[0020] In a further embodiment of the composition for use according to the present invention, the guanidinoacetic acid salt is an acid addition salt.

[0021] In a preferred embodiment of the composition for use according to the present invention, the guanidinoacetic acid salt is guanidinoacetic acid hydrogen chloride, guanidinoacetic acid hydrogen sulfate, hydrogen phosphate of Petition 870250089342, dated 10 / 01 / 2025, page 18 / 33 6 / 17 guanidinoacetic acid or a mixture of any of these.

[0022] In principle, the composition according to the present invention is not subject to any limitation as to the content of guanidinoacetic acid. Instead, the content of guanidinoacetic acid in the composition according to the present invention is more or less determined by national or regional regulations for the registration of food items.

[0023] In one embodiment, the composition for use according to the present invention comprises up to 1,500 ppm of guanidinoacetic acid, a guanidinoacetic acid salt, or a mixture of either. Preferably, the composition according to the present invention comprises up to 1,200 ppm of guanidinoacetic acid, a guanidinoacetic acid salt, or a mixture of either.

[0024] In addition to guanidinoacetic acid, the composition according to the present invention may comprise other components, in particular, components that are beneficial to the health of birds. Efficient animal production is a balancing act between nutrition, health, and animal welfare, especially when reducing or eliminating the use of antibiotics. In this context, ensuring intestinal health is absolutely essential. Challenges such as disease, environmental factors, and the quality of feed ingredients can negatively impact the intestinal microbial balance, leading to slower growth and reduced performance, which in turn results in economic losses. Microbiota imbalance (dysbiosis), as well as the overgrowth of Clostridium perfringens, allows the growth of opportunistic bacteria, such as Escherichia coli, which can result in various disorders. These disorders, such as diarrhea, wet litter, necrotic enteritis, leaky gut (impaired intestinal barrier function), intestinal inflammation (colitis), and low immune status, ultimately impair feed efficiency, growth, and animal welfare. Petition 870250089342, dated 10 / 01 / 2025, page 19 / 33 7 / 17 Therefore, it is beneficial that the composition according to the present invention also includes a probiotic.

[0025] In another embodiment, the composition for use according to the present invention further comprises a probiotic.

[0026] It has been found that a probiotic further enhances the benefits of the composition according to the present invention. In particular, it has been found that guanidinoacetic acid and an additional probiotic synergistically improve the growth of birds infected with coccidiosis and resistance to a multifactorial necrotic enteritis challenge involving Eimeria spp. and Clostridium perfringens pathogens.

[0027] In a preferred embodiment of the composition for use according to the present invention, the probiotic comprises Bacillus spp. spores.

[0028] A probiotic comprising Bacillus subtilis spores, such as Evonik® GutCare, is considered a direct inhibitor of the bacterial component, namely Clostridium perfringens, while stabilizing the microbiome and reducing leaky gut-induced bacterial translocation.

[0029] A probiotic comprising Bacillus amyloliquefaciens spores, such as Evonik® EcoBiol, improves animal health and production conditions and helps producers improve product quality, business profitability, and address sustainability challenges. In detail, feed supplemented with a probiotic containing Bacillus amyloliquefaciens spores, such as Evonik® EcoBiol, helps balance the gut microbiome. This probiotic helps reduce production costs by improving feed conversion and reducing time to slaughter.

[0030] The composition according to the present invention may further comprise one of the two types of probiotics mentioned above, alone or in combination.

[0031] In an additional preferred embodiment of composition Petition 870250089342, dated 10 / 01 / 2025, page 20 / 33 8 / 17 for use in accordance with the present invention, the probiotic comprises spores of Bacillus amyloliquefaciens and / or Bacillus subtilis.

[0032] For example, the probiotic comprises a selected strain of B. subtilis (e.g., DSM 32315 or DSM 32540), B. amyloliquefaciens (e.g., CECT 5940) and / or a mixture of any of these.

[0033] In principle, the composition according to the present invention can be administered to birds at any stage, in an infinite number of stages, or in all stages of the birds' feeding, that is, at any stage, in an infinite number of stages, or in all stages of their life. The lifespan of laying hens can be divided into a pre-laying phase and three production phases or three production periods, namely, the initial production phase, the juvenile production phase, and the nesting phase, sometimes also called the pre-laying phase and phases I to III. The lifespan of birds raised in captivity for meat production can be divided into three phases: initial phase, growth phase, and finishing phase. For example, the entire lifespan of a hen can reach 39 days, of which the days (d) from d-0 to d-10 are called the initial phase, the days from d-10 to d-21 are called the growth phase, and the days from d-21 to d-39 are called the finishing phase.Birds raised for egg production also have different growth and feeding periods. In principle, the composition according to the present invention, when administered to birds with a coccidiosis-induced condition, for example, birds infected with coccidiosis, is not limited to any specific phase or period in the birds' lives. Therefore, the composition can be administered to birds with a coccidiosis-induced condition, for example, birds infected with coccidiosis, at any conceivable point in time in or during any of the phases, i.e., in or during the initial, growth and / or finishing phases. However, it is preferable. Petition 870250089342, dated 10 / 01 / 2025, p. 21 / 33 9 / 17 administer the composition to birds with coccidiosis-induced conditions, for example, birds infected with coccidiosis, in or during the finishing phase.

[0034] In one embodiment, the composition for use according to the present invention is administered to birds at any stage, in an infinite number of stages, or in all stages of their life.

[0035] It was observed that the daily weight gain of birds infected with coccidiosis that received the composition according to the present invention was significantly greater than that of birds infected with coccidiosis that received an antibiotic through the drinking water during the challenge period from day 16 to day 22 and, overall, from day 0 to day 42.

[0036] In another embodiment, the composition for use according to the present invention is administered to birds from the beginning of the initial phase until slaughter or from the beginning of the growth phase until slaughter or any other phase of life.

[0037] During the growth phase, birds infected with coccidiosis that received the composition according to the present invention gained approximately 9 g / day more than the negative control and, overall, gained approximately 2 g / day more.

[0038] In a further embodiment, the composition for use according to the present invention is administered to birds in the growth phase.

[0039] In principle, the composition according to the present invention is not subject to a specific state of aggregation or matrix.

[0040] In one embodiment, the composition for use according to the present invention is a liquid or solid composition.

[0041] When the composition according to the present invention is a solid composition, it is preferable that it be an effervescent tablet. In this case, the composition comprises a carbon dioxide-producing compound and a gas-releasing compound. Preferably, the equivalent ratio of the gas-releasing compound to Petition 870250089342, dated 10 / 01 / 2025, page 22 / 33 The ratio of gas to the carbon dioxide-producing compound is 1:1 or greater, and the molar ratio of the carbon dioxide-producing compound to guanidinoacetic acid is 1:1 or greater.

[0042] This composition is particularly suitable for water supply applications, preferably drinking water, because the carbon dioxide-producing compound reacts with the gas-releasing compound in the presence of water to release carbon dioxide. As a result, the dissolution process of guanidinoacetic acid is greatly accelerated.

[0043] In order to supply or produce carbon dioxide, it is preferable that the carbon dioxide-producing compound be a carbonic acid salt.

[0044] In one embodiment of the composition according to the present invention, the carbon dioxide-producing compound is therefore an alkaline carbonate, an alkaline-earth carbonate, an ammonium carbonate, an alkaline hydrogen carbonate, an alkaline-earth hydrogen carbonate, an ammonium hydrogen carbonate or a mixture of any of these.

[0045] In a preferred embodiment of the composition according to the present invention, the carbon dioxide-producing compound is a sodium bicarbonate, a potassium bicarbonate, an ammonium bicarbonate, or a mixture of any of these.

[0046] In the case of an effervescent tablet, the gas-releasing compound reacts with the carbon dioxide-producing compound in the presence of water to form carbon dioxide. In principle, the underlying reaction is therefore an acid-base reaction; the hydronium ions (HaO+) supplied by the gas-releasing compound react with carbonate (COs2-) and / or bicarbonate ions (HCCh-) to form carbonic acid (H2CO3). However, the carbonic acid thus released is not thermodynamically stable and readily decays into carbon dioxide and water, leading to the foaming effect and therefore, Petition 870250089342, dated 10 / 01 / 2025, page 23 / 33 11 / 17 to the accelerated dissolution of guanidinoacetic acid.

[0047] To allow the best possible release of carbon dioxide, it is preferable that the gas-releasing compound be a stronger (or even much stronger) acid in terms of pKa value(s) than the carbon dioxide-producing compound, in particular, the carbonic acid salt. This condition is normally met when the gas-releasing compound in the composition is an acid. In comparison, the carbonate in the carbonic acid salt is a base. Carbonic acid has pKa values ​​pKa1 = 6.35 and pKa2 = 10.33. Therefore, any gas-releasing compound suitable for use in the composition according to the present invention must have pKa values ​​lower than those of carbonic acid.

[0048] In one embodiment of the composition according to the present invention, the gas-releasing compound is an acid, for example, an inorganic acid, an organic acid, or a mixture of either.

[0049] Preferably, the acid has one or more pKa values ​​less than 6.35. This requirement is met by a variety of organic acids, such as citric acid (pKa1 = 3.13, pKa2 = 4.76 and pKa3 = 6.4), tartaric acid (pKa1 = 2.98 and pKa2 = 4.34) and malic acid (pKa1 = 3.46 and pKa2 = 5.10).

[0050] The use of a solid organic acid makes it possible to provide the composition according to the present invention in solid form. A solid form of the composition according to the present invention is the most concentrated form of said composition and therefore requires much less space than the liquid form, i.e., a solution of said composition. Therefore, it is preferable that the composition according to the present invention be a solid composition.

[0051] In a preferred embodiment of the composition according to the present invention, the gas-releasing compound is therefore a solid organic acid.

[0052] Preferably, the gas-releasing compound is an acid Petition 870250089342, dated 10 / 01 / 2025, p. 24 / 33 12 / 17 citric acid, tartaric acid, malic acid, or a mixture of any of these.

[0053] In the context of the present invention, the term equivalent ratio therefore denotes the ratio between the gas-releasing compound and the carbon dioxide-producing compound that is required to give one (or more) molecule(s) of carbon dioxide. For example, where the gas-releasing compound is citric acid with 3 carboxylic acid groups and the carbon dioxide-producing compound is sodium bicarbonate (NaHCO3), 3 equivalents of citric acid react with 3 equivalents of sodium bicarbonate to give 3 equivalents of carbon dioxide. Here, the equivalent ratio of the gas-releasing compound to the carbon dioxide-producing compound is 1:1, i.e., 1. In another example, where the gas-releasing compound is citric acid with 3 carboxylic acid groups and the carbon dioxide-producing compound is sodium carbonate (Na2CO3), 6 equivalents of citric acid react with 3 equivalents of sodium carbonate to give 3 equivalents of carbon dioxide.Here, the equivalent ratio of the gas-releasing compound to the carbon dioxide-producing compound is 2:1, or 2.

[0054] Preferably, the equivalent ratio of the gas-releasing compound to the carbon dioxide-producing compound ranges from 1:1 to 3:1 in the composition according to the present invention.

[0055] To ensure that carbon dioxide is produced or supplied as completely as possible, it is preferable that the equivalent ratio between the gas release and the gas-producing compound be at least 1.05:1.

[0056] When the diet according to the present invention is a solid matrix, it is preferable that said diet be a poultry diet.

[0057] Another object of the present invention is a diet for use in the treatment and / or prophylaxis of a condition induced by Petition 870250089342, dated 10 / 01 / 2025, page 25 / 33 13 / 17 coccidiosis in poultry, wherein the diet comprises the composition according to the present invention. EXAMPLES

[0058] 0 The in vivo study aimed to test the beneficial effects of GAA against necrotic enteritis (NE). The general procedures and details of a floor pen study to determine the efficacy of GAA in preventing and assisting experimentally induced NE in broiler chickens are described below.

[0059] The experimental design consisted of 3 treatments: the first consisted of a basal diet as a negative control (IUC), the second was a positive control with the same basal diet, but the birds received an antibiotic through the drinking water (20 mg / kg BW / day administered for 5 days) (ITC), and the last was the same basal diet with supplemental guanidinoacetic acid (GAA) at 0.12% (1.2 g / kg), but without antibiotics in the drinking water. The feeding treatments were applied in all rearing phases (starter phase from day 0 to day 16, grower phase from day 16 to day 23, and finisher phase from day 23 to day 42). Table 1 below summarizes the formulation and nutritional composition of the basal diets. Item (%, as supplied) Start (d0-dl6) Growth (dl6-d23) Finish (d23-d42) Wheat 39.29 44.19 46.15 Soybean meal, 48% 33.62 -24.54 Corn 20.00 -20.00 Soybean oil 3.38 10.00 5.98 Animal fat (lard) -6.61 Rye -7.50 Wheat bran -5.00 Whole soybeans -5.00 Fish meal, 70% -30.00 Limestone (CaCO3) 1.37 -1.19 Monocalcium phosphate Ca(H2PO4)2 0.66 -0.35 Salt (NaCl) 0.20 -0.22 Sodium bicarbonate NaHCO3 0.26 0.19 0.50 Vitamin and mineral premix 0.50 0.50 0.50 Petition 870250089342, dated 10 / 01 / 2025, page 26 / 33 14 / 17 Choline chloride, 60% 0.02 - 0.01 Phytase (500 FTU) 0.01 0.005 0.01 DL-Met (99%) 0.29 - 0.24 L-Lys * HCl 0.25 - 0.22 L-Thr (98.5%) 0.09 - 0.08 L-Val 0.06 - 0.02 Formulated Nutrients Starter (d0-d16) Grower (d16-d23) Finisher (d23-d42) Crude protein 22.72 29.38 19.87 ME, kcal / kg 3000 3257 3200 SID Met + Cys 0.88 0.97 0.76 SID Lys 1.21 1.71 1.00 SID Thr 0.77 0.95 0.65 SID Arg 1.34 1.53 1.10 SID Val 0.96 1.20 0.80 Table 1: Formulation and nutritional composition of basal diets in the starter, grower and finisher phases.

[0060] The NE challenge was implemented in the grower phase. All birds in this phase were challenged. The feed in the initial and finishing phases was a typical Northern European corn-soybean-wheat diet, which is easily digestible and does not contain many antinutritional factors. The growth diet included a large proportion of fishmeal, wheat bran, and rye, resulting in a diet high in crude protein. These conditions and crude ingredients are known to be detrimental to intestinal health. Additionally, on days 14 and 16, the broilers received a 10-fold overdose of live Eimeria Paracox-8 vaccine by oral administration. On each of days 18 to 21, the broilers also received Clostridium perfringens inoculations.

[0061] Each treatment was replicated in 12 pens with 18 male Ross 308 broiler chickens each, in a completely randomized block design. The feed formulation of the diets was based on the energy, amino acid, and macromineral requirements of Ross 308 broiler chickens. The diets were provided as meal for the starter phase and as pellets for the grower and finisher phases. The chickens were fed ad libitum and had free access to water throughout the experiment. The birds were vaccinated at the hatchery against Petition 870250089342, dated 10 / 01 / 2025, page 27 / 33 Newcastle disease (15 / 17). No other commercial vaccines were administered during the study.

[0062] On days 21 and 22, four chickens per pen were sacrificed for intestinal lesion scoring (adapted from Timbermont et al., Avian Pathology 2010, 39: 117-221). The same chickens were also evaluated for typical coccidiosis lesions, according to the Johnson and Reid method (Johnson and Reid, Experimental Parasitology 1970, 28(1): 30-36) with a score of 0 (no lesions) to 4 (severe lesions) for the species relevant to broiler chickens described in this scoring system. The chickens in this study were evaluated for E. acervuline and E. maxima).

[0063] For statistical evaluations, a Dunnett test was used, comparing antibiotic and GAA treatments with an untreated challenged control (UTC). The tables below summarize the resulting means and p-values ​​for GAA treatment. Bold text highlights significant differences from IUC, also for antibiotic treatment. Performance parameters for growth IUC ITC GAA p-value for GAA BW D0 47 47 46 0.441 BW D16 725 714 711 0.141 BW D2 2 1139 1312 1161 0.5962 BW D34 2467 2745 2463 0.992 BW D4 2 3376 3714 3332 0.753 DWG D0-D16 42.2 41.4 41.0 0.072 DWG D16-D22 63.4 100.6 72.2 0.057 DWG D22-D34 72.1 82.2 75.3 0.499 DWG D34-D42 110.7 114.5 108.7 .929 DWG D0-D42 60.3 70.0 62.6 .187 DFI D0-D16 47.3 46.6 45.9 .045 DFI D16-D22 81.5 100.2 84.4 .444 DFI D22-D34 115.8 125.0 117.3 0.847 DFI D34-D42 199.2 209.5 191.0 0.370 DFI D0-D42 113.7 115.9 112.1 0.233 FCR D0-D16 1.12 1.13 1.12 0.951 FCR D16-D22 1.31 1.00 1.18 0.017 FCR D22-D34 1.63 1.52 1.56 0.418 FCR D34-D42 1.82 1.88 1.77 0.885 FCR D0-42 1.90 1.66 1.80 0.067 Mortality related to EN 13.33 0 3.33 0.044 Petition 870250089342, dated 10 / 01 / 2025, pp. 28 / 33 16 / 17 Table 1: Summary of growth performance parameters for all treatments and corresponding p-values.

[0064] Table 1 summarizes the growth performance parameters of broiler chickens for all treatments and all rearing phases, including p-values. In general, infected birds treated with antibiotics (ITC) performed better than untreated infected birds (UTC). Large differences in body weight (BW), daily weight gain (DWG), and feed conversion ratio (FCR) show that antibiotics are an effective and efficient treatment for NE in broiler chickens. The daily weight gain (DWG) of birds supplemented with GAA was significantly higher than that of IUC birds during the challenge period from day 16 to day 22. In the growing phase, birds supplemented with GAA gained approximately 9 g / day more than the negative control and, overall, gained approximately 2 g / day more.There was no significant effect on daily feed intake (DFI) (p>0.05) at any time point for birds treated with GAA compared to untreated birds, except for a small effect in the pre-challenge period. Since DWG was higher and DFI was similar, the FCR of birds treated with GAA was significantly lower than that of untreated birds during the challenge period and overall. Finally, overall, the percentage of mortality related to NE was strongly reduced with GAA supplementation and comparable to the antibiotic-treated control. Daily Intestinal Health Scores IUC ITC GAA p-value for GAA NE Score 21 3.42 0.40 3.52 0.906 E. acervulina 21 0.15 0.02 0.08 0.536 E. maxima 21 0.81 0.38 0.52 0.038 TMLS 21 0.96 0.40 0.60 0.0268 Percentage of NE 21 88 13 85 0.957 NE Score 22 3.25 0.52 2.33 0.001 E. acervulina 22 0.17 0.06 0.06 0.270 E. maxima 22 0.85 0.79 0.88 0.975 TMLS 22 1.02 0.85 0.94 0.769 Percentage of NE 22 90 23 73 0.081 Table 2: Summary of gut health scores for Petition 870250089342, dated 10 / 01 / 2025, pp. 29 / 33 17 / 17 all treatments and corresponding p-values.

[0065] Table 2 summarizes the effect of treatment on intestinal health scores. The effect of antibiotic treatment is evident and strong in improving NE scores, E. maxima lesions, and overall coccidiosis lesions (TMLS) compared to the untreated control on day 21. Birds supplemented with GAA also showed lower amounts of E. maxima lesions and coccidiosis on day 21, lower NE scores, and a lower percentage of birds affected by NE on day 22, similar to antibiotic treatment.

[0066] Based on these results, it can be concluded that GAA supplementation helps birds use feed energy more effectively to combat NE infection, potentially sustaining the energy-demanding immune response more effectively. In particular, this leads to a reduction in NE mortality, which also increases overall profitability under real-world conditions. Additionally, body weight gain and feed conversion rate are improved, while feed intake is not affected. In conclusion, GAA supplementation promotes the health of broiler chickens during an NE challenge and is likely a cost-effective and animal welfare-supporting solution, particularly in commercial farms that are completely antibiotic-free. Petition 870250089342, dated 10 / 01 / 2025, pages 30 / 33

Claims

1 / 2 CLAIMS 1. Composition for use in the treatment and / or prophylaxis of a coccidiosis-induced condition in poultry, characterized in that the composition comprises guanidinoacetic acid, a guanidinoacetic acid salt, or a mixture of either.

2. Composition for use, according to claim 1, characterized in that coccidiosis is caused by Eimeria spp. and / or Clostridium perfringens.

3. Composition for use, according to claim 1 or 2, characterized in that the condition induced by coccidiosis is one or more of bacterial enteritis, necrotic enteritis, diarrhea, and lesion.

4. Composition for use, according to any one of claims 1 to 3, characterized in that the guanidinoacetic acid salt is an acid addition salt.

5. Composition for use, according to any one of the salts of claims 1 to 4, characterized in that guanidinoacetic acid is hydrogen chloride of guanidinoacetic acid, hydrogen sulfate of guanidinoacetic acid, hydrogen phosphate of guanidinoacetic acid or a mixture of any of these.

6. Composition for use, according to any one of claims 1 to 5, characterized in that the composition comprises up to 1,500 ppm of guanidinoacetic acid, a salt of guanidinoacetic acid or a mixture of any of these.

7. Composition for use, according to any one of claims 1 to 6, characterized in that it further comprises a probiotic.

8. Composition for use, according to claim 7, characterized in that the probiotic comprises spores of Bacillus spp.

9. Composition for use, according to claim 7 or Petition 870250089342, dated 10 / 01 / 2025, page 31 / 33 2 / 2 8, characterized in that the probiotic comprises spores of Bacillus amyloliquefaciens and / or Bacillus subtilis.

10. Composition for use, according to any one of claims 1 to 9, characterized in that the composition is administered to birds at any stage, in an infinite number of stages, or in all stages of their life.

11. Composition for use, according to any one of claims 1 to 10, characterized in that the composition is administered to poultry from the beginning of the starter phase until slaughter or from the beginning of the growth phase until slaughter or any other phase of life.

12. Composition for use, according to any one of claims 1 to 11, characterized in that the composition is administered to birds in the growth phase.

13. Composition for use, according to any one of claims 1 to 12, characterized in that the composition is either a liquid or a solid composition.

14. Diet for use in the treatment and / or prophylaxis of a coccidiosis-induced condition in poultry, characterized in that the diet comprises the composition as defined in any one of claims 1 to 13. Petition 870250089342, dated 10 / 01 / 2025, pp. 32 / 33