Use of CHD–FA in broiler production

Abstract

Carbohydrate derived fulvic acid (CHD-FA) provides a novel feed additive in broiler production. The CHD-FA is used in the prophylactic and / or therapeutic treatment of broilers to mitigate the risk of infection during the early stages of broiler production. The feed additive comprising CHD-FA accelerates stabilization of the treated broilers' microbiota, which is significant in reducing the susceptibility of chickens to different infections at a much earlier stage in their growth cycle, increasing their chances of survival and viability.

Description

[0001] USE OF CHD–FA IN BROILER PRODUCTION BACKGROUND OF THE INVENTION THIS invention relates to carbohydrate derived fulvic acid (CHD-FA) and its use as a novel feed additive in broiler production. The invention further relates to the use of CHD-FA in the prophylactic and / or therapeutic treatment of broilers to mitigate the risk of infection during the early stages of broiler production. Antibiotics have traditionally been used in poultry farming with a view to prophylactically reducing the incidence and frequency of diseases, as well as to assist with improved growth performance and enhance feeding efficacy. This has been driven by the ever-increasing tendency towards intensive poultry production. The increase in intensive production has had a negative effect on disease and stress resistance making disease control more difficult. Intensive production has also led to an increase in ammonia production and this in combination with lowered disease resistance results in respiratory diseases that negatively impact production (Tang et al., 2023). The overuse of antibiotics in industry has only made disease control more challenging as antibiotic resistance increases, making the use of antibiotics less effective and negatively affecting production. In some cases, countries have begun to ban the use of antibiotics in the poultry industry. As a consequence, alternative additives to antibiotics are needed as well as additives that can combat the decline in immunity, productivity and feed efficiency caused by high stocking densities, intensive feeding, and antibiotic resistance (Feng et al., 2022). Fulvic acid (FA) is one of three categories (categories are based on solubility) that fall under the group humic substances, which are organic acids formed through the decomposition and transformation of microbial materials, plants, and animal residues. Fulvic acid is soluble in both alkaline and acidic solutions (Mao, 2019). More specifically fulvic acid is a weak acid mixture that can be extracted from various sources. It has a low molecular weight and many biologically active molecules (Feng et al., 2022). This is owing to the numerous functional groups found within the fulvic acid, such as aliphatic and phenolic hydroxyl (-OH), primary amine (-NH2), secondary amine (-NH-), and tertiary amine (-N=), carbonyl (C=O) and carboxyl (-COOH) groups (Xiao et al., 2022). The difficulties faced by scientists to characterise the molecular structure of fulvic acid (Abbt-Braun et al., 2004; Samios et al., 2007; Yang et al., 1993) are related to the abundance of the polar functional groups in its structure (Dixon & Larive, 1997) and demonstrated by Day & Hansen, 2007 who isolated more than 4,000 distinct compounds from only one Suwannee River fulvic acid sample. Characterising the molecular structure of fulvic acid is further complicated by significant variations in the location of it within a geographical area and in the depth of the vertical soil layers from where fulvic acid is sourced. Carbohydrate derived fulvic acid (CHD-FA) on the other hand is produced by a controlled wet oxidation process in a GMP facility that yields a uniform and well- characterised product, which is devoid of the contaminants typically found in environmentally derived forms. Carbohydrate derived fulvic acid is a well- characterised mixture of aliphatic and aromatic carboxylic acids that are the products obtained from the wet oxidation of sucrose using only heat, pressure, pure oxygen and reverse osmosis water. This CHD-FA is then further processed into a 5000Da fraction for non-pharmaceutical applications and a 400Da fraction for use as a medicine. Traditionally fulvic acid has been used to treat digestive tract diseases and has also been known to treat ulcerative carbuncles. It has been shown to have antioxidant, anti-inflammatory, immunomodulatory, antiviral and antidiabetic properties. Carbohydrate derived fulvic acid has been demonstrated to be effective in treating rat paw oedema, as well as having antimicrobial efficacy, showing favourable minimum inhibitory concentration against a number of pathogens including Clostridium, Salmonella, E.coli, MRSA, Zygomycetes, Fusaria, Scedosporium and Candida sp. Studies have suggested that the addition of fulvic acid could improve growth rate, feed conversion ratio and immunity. Fulvic acid has the potential to regulate productive performance through its regulation of immune activity and influence on gut microbiota and mechanisms that underly production performance (Feng et al., 2022). Fulvic acid has been seen to have a chelating effect on metals (Xiao et al., 2022). For environmentally derived fulvic acids, this is a problem because they tend to adsorb and bind heavy metals from the soil or water sources from which they are isolated. These heavy metals are in turn toxic to the humans or animals dosed with the environmental fulvic acid. Carbohydrate derived fulvic acid on the other hand is free from such heavy metals because it is produced under controlled, heavy metal- free conditions. Once ingested, fulvic acid increases the uptake of copper thus reducing the potential of copper toxicity as well as prompting the secretion of digestive enzymes such as lysozyme, protease and acid-alkaline phosphate which promotes intestinal absorption and production performance (Tang et al., 2023). The increase in minerals due to fulvic acid can influence the maintenance of homeostasis, pH, activation of enzymes, vitamin uptake and blood enzyme activity. Fulvic acid has also been seen to have an effect on blood metabolites, such as glucose, protein, triglyceride, high density lipoproteins (HDL), low density lipoproteins (LDL) and, total cholesterol, which influences animal health and meat quality. Colour, water holding capacity (WHC) and pH are important for meat quality and have been seen to be affected by the addition of fulvic acid (Ozturk et al., 2012). There is evidence that fulvic acid has the potential to improve meat quality through alteration of the total protein and fat content owing to the lower oxidation and higher antioxidant activity (Hudák et al., 2021). Notwithstanding the above, research into the direct effects of fulvic acid are lacking, the use of fulvic acid as a feed additive is relatively new and there is a lack of knowledge regarding the optimum inclusion levels. There is also much debate due to variations in results of studies on the use of fulvic acid specifically in the poultry industry (Feng et al., 2022). Salmonella is a genus of gram-negative rod bacteria comprised of over 2500 serovars. The majority are pathogenic to humans. Food borne Salmonella is mainly non-typhoidal and can cause fever, abdominal pain, and inflammatory diarrhoea. It has been estimated that this type of Salmonella causes 420000 deaths globally per annum (Vinueza-Burgos et al., 2019). Hence Salmonella is considered one of the most global foodborne zoonotic pathogens in industry. Poultry and poultry products are among the most common causes of human Salmonella infections and so pose a risk to human health. The risk of Salmonella in broilers is often high due to the systems used to raise broilers (high densities, multiple inputs) (Pieskus et al., 2008). In the USA poultry has led to 40% of the 1.4 million non-typhoidal salmonellosis cases. Even though regulations have been put in place to lower the presence of Salmonella in end products, these levels still pose a threat to consumers (Liljebjelke et al., 2005). The rise of antimicrobial resistant Salmonella poses a serious challenge and concern in the poultry industry. Resistance has developed due to the misuse of antibiotics and thus an alternative need to be found to replace their use (Vinueza-Burgos et al., 2019). A study in KwaZulu Natal using Cobb 500 broilers showed that 32.1% of samples taken were contaminated with Salmonella with 30.9% on farm, 0.6% at the abattoir and 0.6% during house decontamination (Ramtahal et al., 2022). A systematic review of Salmonella serovars in South Africa showed that the pooled prevalence estimates of Salmonella detection were 79.6% for human, 61.6% for environment, 56.5% for animal and 43.2% for environment / animal samples (Ramatla et al., 2022). Salmonella leads to a loss in production. Chicks are more severely affected and can suffer from high temperatures, diarrhoea leading to fluid loss and death. While adults don’t die from Salmonella, they can experience weakness and diarrhoea, weight loss and decreased growth rate (Heinzl & Nutrition, 2021). The effects of fulvic acid on gut microbiota is even less well documented and the direct effects on pathogenic bacteria such as Salmonella, E.coli and Campylcobacter are non-existent. Due to the wide variation of findings and inclusions used as well as a lack of research the benefits and inclusion levels of fulvic acid also need further investigation (Tang et al., 2023). This is even more so in respect of (CHD-FA). SUMMARY OF THE INVENTION According to a first aspect of the invention there is provided a feed additive comprising a prophylactically and / or therapeutically effective amount of a carbohydrate derived fulvic acid (CHD-FA) for use in broiler production. In exemplary embodiments of the invention, the amount of CHD-FA in the feed additive is from about 20 mg / kg / day, or from about 40 mg / kg / day, or from about 60 mg / kg / day, or from about 80 mg / kg / day, or from about 90 mg / kg / day CHD-FA to about 500 mg / kg / day, or about 350 mg / kg / day, or about 300 mg / kg / day, or about 250 mg / kg / day, or about 200 mg / kg / day, or about 150 mg / kg / day, in particular about 100 mg / kg / day, CHD-FA. In certain preferred embodiments of the invention, the amount of CHD-FA in the feed additive is greater than about 20 mg / kg / day, or greater than about 30 mg / kg / day, or greater than about 40 mg / kg / day, or greater than about 50 mg / kg / day, or greater than about 60 mg / kg / day, or greater than about 70 mg / kg / day, or greater than about 80 mg / kg / day or greater than about 90 mg / kg / day CHD-FA and less than about 250 mg / kg / day, or less than about 200 mg / kg / day, or less than about 150 mg / kg / day, or less than about 140 mg / kg / day, or less than about 130 mg / kg / day, or less than about 120 mg / kg / day, or less than about 110 mg / kg / day CHD-FA, and in a particularly preferred embodiments of the invention the amount of CHD-FA in the feed additive is about 100 mg / kg / day CHD-FA. By “x mg / kg / day” is meant the amount of dry weight CHD-FA measured in milligrams per kilogram body weight of each broiler per day. By “a prophylactically and / or therapeutically effective amount of CHD-FA” is meant an amount of CHD-FA that is sufficient to stabilize the gut microbiota of the treated broiler preferably as determined by a reduction of the E.coli and / or Salmonella and / or Campylobacter sufficient for broiler survival and below acceptable limits for human consumption. In some preferred embodiments of the invention the E.coli count of broilers treated with the CHD-FA of the invention stabilised around 2 log to 2.1 log (about 100 CFU / swab to about 128 CFU / swab) between 14 and 21 days. In some preferred embodiments of the invention, the CHD-FA feed additive of the invention accelerates the stabilization of the treated broilers microbiota, as determined by the log reduction of E.coli, to between 14 and 21 days. The accelerated stabilization of the treated broilers’ microbiota is surprising, and far superior to the comparative control and antibiotic containing feed additive, which only achieved stabilization between 21 and 28 days of treatment. This accelerated stabilization is significant in reducing the susceptibility of chickens to different infections at a much earlier stage in their growth cycle, increasing their chances of survival and viability.

[0002] BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described in more detail, by way of example only, with reference to the accompanying drawings in which: Figure 1 is a graphical representation of the Mean (± standard deviation) of the European production efficiency factor (EPEF) for broilers receiving a feed additive comprising 100 mg / kg / day of carbohydrate derived fulvic acid (CHD-FA), indicated as Neg100FA, when compared to a negative control (NegCon, Tap water with no additive), positive control (PosCon, commercial antibiotic in the feed), and acidified water with 0.3M citric acid to pH4 and having a commercial antibiotic in the feed (PosCit), Figure 2 is a graphical representation of the Mean (± standard deviation) of the average daily gain (ADG) for broilers receiving a feed additive comprising 100 mg / kg / day of carbohydrate derived fulvic acid (CHD- FA), indicated as Neg100FA, when compared to a negative control (NegCon, Tap water with no additive), positive control (PosCon, commercial antibiotic in the feed), and acidified water with 0.3M citric acid to pH4 and having a commercial antibiotic in the feed (PosCit); Figure 3 is a graphical representation of the Mean (± standard deviation) of the feed conversion ratio (FCR) for broilers receiving a feed additive comprising 100 mg / kg / day of carbohydrate derived fulvic acid (CHD- FA), indicated as Neg100FA, when compared to a negative control (NegCon, Tap water with no additive), positive control (PosCon, commercial antibiotic in the feed), and acidified water with 0.3M citric acid to pH4 and having a commercial antibiotic in the feed (PosCit); Figure 4 is a graphical representation of the Mean (± standard deviation) of the protein efficiency ratio (PER) for broilers receiving a feed additive comprising 100 mg / kg / day of carbohydrate derived fulvic acid (CHD- FA), indicated as Neg100FA, when compared to a negative control (NegCon, Tap water with no additive), positive control (PosCon, commercial antibiotic in the feed), and acidified water with 0.3M citric acid to pH4 and having a commercial antibiotic in the feed (PosCit); and Figure 5 is a graphical representation of the log reduction in E.coli count for broilers receiving a feed additive comprising 100 mg / kg / day of carbohydrate derived fulvic acid (CHD-FA), indicated as Neg100FA, when compared to a negative control (NegCon, Tap water with no additive), positive control (PosCon, commercial antibiotic in the feed), and acidified water with 0.3M citric acid to pH4 and having a commercial antibiotic in the feed (PosCit). DESCRIPTION OF PREFERRED EMBODIMENTS Carbohydrate derived fulvic acid was tested as a novel feed additive in drinking water in a broiler health, growth and performance trial. The trial consisted of 10 treatments with eight repetitions per treatment. Treatments were according to the inclusion of CHD-FA with three different inclusion levels, a control without acid and an acidified treatment with citric acid. Each treatment was divided into two groups with one group receiving in-feed antibiotics while the others did not. Production parameters, carcass characteristics, meat quality characteristics, organ data and bone mineralization were measured and calculated. Health parameters including a microbiological assessment of common enteric pathogens and a survey of the microbiome were also examined. The results show a clear difference between the treatments and the negative control for production parameters with the treatments showing similar results to that of the positive control indicating that the use of CHD-FA has the ability to replace antibiotics in the feed of broilers. On the other hand, the use of the CHD-FA did not have any effect on carcass and meat quality parameters, which indicates that the use of this product will not negatively influence the yields expected at the abattoir and processing plant post slaughter. In addition to the commercially valuable characteristics, it is noted that organ weights, ratios and gizzard characteristics were also not influenced by the use of CHD-FA indicating that the use of the product can be deemed safe. Importantly, the results indicate that the highest inclusion level of CHD-FA did not actually yield the most notable effects. In order to establish the safety and value of this novel feed additive in broiler production, bioassays were conducted. These included a gizzard erosion study, a production parameter study terminating in analysis of carcass and organ characteristics, meat quality and bone mineralisation. These assessments shed light on possible damage that the additive could cause to the intestine, effects that it has on production parameters, carcass characteristics and meat quality as well as calcium metabolism and subsequent skeletal disorders. The novel feed additive was also shown to benefit the health of broilers by positively affecting the microbiome and, for example reducing the incidence of typical faecal pathogens such as Salmonella and Escherichia coli. Materials and Methods For this trial Cobb 500 broilers as hatched (expected a 50:50 male:female) were used for all experiments. Birds were sexed at slaughter. In the broiler industry birds are grown as hatched (Vantress, 2008) and a normal distribution is accepted and at end point sex differences are small. The trial consisted of a sufficient number of birds and repetitions to raise them as hatched. In the event of outliers, these were excluded using statistical methods (3). Analytical and mathematical methodologies The base feed i.e., starter, grower and finisher were analysed to confirm composition. Dry matter (DM) was determined according to the Official Method 934.01 (A.O.A.C. 2002). The subsamples retained from the DM analysis were used for the determination of ash content using Official Method 942.05 (A.O.A.C. 2002) and crude fibre (CF) content were determined using Official Method 962.09 (A.O.A.C. 2002). Ether extract (EE) was determined using the Foss Tecator Soxtec HT 1043 Extraction Unit, Höganäs, Sweden while fat was extracted using diethyl ether using Official Method 920.39 (A.O.A.C. 2002). Crude protein (CP) contents were determined by measuring the total N content according to Official Method 4.2.07 (A.O.A.C. 2002) in the LECO FP-528, Protein / Nitrogen Determinator, (LECO corporation, 3000 Lakeview Avenue, St Joseph, MI 49085-2396) and crude protein value was then obtained by multiplying the N content by 6.25. Mineral composition was determined using the combustion method as described by Method no. 6.1.1 for feeds and plants (AgriLASA, 2007). Using this method, the minerals P, K, Ca, Mg, Na, Cu, Mn, Fe, Al, Zn and B were determined. Gizzard erosion study The gizzard erosion study consisted of two sections, the first was based on an assay where birds were kept for the purpose of gizzard scoring only, while the second formed part of the organ data. The first section was a separate assay which was done on young birds and assessed the ability of this additive to cause gizzard erosion. For this section 60 broiler chicks, as hatched were used thus allowing 20 birds per treatment. The chicks were vaccinated, at hatch, against Newcastle disease and Infectious bronchitis. Chicks were maintained in a bio-assay unit comprising a temperature-controlled room equipped with wire cages. Each cage was equipped with a feeder and a drinker. Artificial lighting was provided at a pattern of 18 hours light and 6 hours of darkness. Ventilation was set to a minimum of six air changes per hour. Chicks had ad libitum access to feed and water during the duration of the period. During the first seven days chicks were maintained on a commercial starter diet formulated to produce marketable chickens weighing 1.9kg at 35 days of age (Cobb International. 2008). Hereafter the chicks received, for a period of seven days, water containing either 40 mg / kg / day (i.e., twice the lowest dose used in the production parameter assessment) or 500 mg / kg / day (i.e., twice the highest dose used in the production parameter assessment) CHD-FA or the control (no additive). Body weight of broilers was determined at arrival and weekly thereafter until slaughter. At the end of the period birds were sacrificed by cervical dislocation and the gizzards removed for scoring. Gizzards were scored on an ordinal scale 1 to 5 (Table 1). Table 1 Gizzard erosion scoring description. Production parameters This section produced data on the growth performance of birds given this novel additive while the slaughter of the birds at the end of the trial produced additional data on bone mineralisation and possible organ damage. All trials were conducted at the poultry section, Mariendahl Experimental Farm of Stellenbosch University. For the purpose of the trial 800-day old broiler chicks, as hatched and vaccinated at the hatchery against infectious bronchitis and Newcastle disease were used. This design allowed for eight replicates per treatment and 10 birds per replicate. Birds were maintained in a temperature-controlled house with environmental conditions maintained as prescribed by the primary breeder. Cages measure 900mm X 600mm X 600mm and contained two nipple drinkers and a tube feeder. During the starter phase additional chick feeders and chick founts were supplied. Chicks had ad libitum access to feed and water for the duration of the trial. A three-phase diet consisting of a starter, grower, and finisher, was used according to the minimum nutrient specifications as supplied by the primary breeder. All birds received the same feed, and treatments were supplied via water. Feed allocation was 900g per bird during the starter phase, 1200g per bird during the grower phase and finisher feed was supplied until slaughter at day 33. Water was supplied via chick founts and nipple drinkers for the first six days whereafter the chick founts were removed, and the nipple drinkers were the only water supply. Treatments were added to the water using a manual dosing system. Water for the chick founts was taken from the corresponding dosing system to ensure similar treatment. The pH of the water in the water lines was measured every morning at 08h00 using a calibrated (standard buffers pH 4.0 and 7.0 at 25°C) portable Crison pH25 meter (Alella, Barcelona) by first flushing the water line into a bucket and then inserting the pH electrode into the centre of the bucket. At the same time the electric conductivity (EC) of the water was measured using a handheld EC meter. Water tanks were drained and refilled with freshly dosed water on weighing days. Before the start of the trial, the water system was flushed using undiluted 8% CHD-FA. The water system was drained and then filled with the 8% CHD-FA, maintained in this manner for 48 hours and then drained, rinsed with fresh tap water, and then filled according to treatment. This was to ensure removal of any biofouling which could have been present. The CHD-FA solutions and the citric acid solutions were prepared according to standard operating procedures. Body weight of broilers was determined on a per pen basis at arrival and weekly thereafter until slaughter. Feed was supplied ad libitum and weekly intake was determined by weighing the initial amount of feed supplied, adding the amount added during the week and subtracting the amount remaining in the feeder at the end of the week. Mortalities and morbidities were recorded twice daily. All dead or culled birds removed from the pen were weighed immediately upon removal. Data obtained was used for the determination of growth rate, feed conversion ratio (FCR) (efficiency with which feed is converted to body weight), protein efficiency ratio (PER) (efficiency with which supplied protein is converted to body weight) and European production efficiency factor (EPEF) (factor considering liveability, FCR, growth rate and age). Table 2 Treatment numbers, names and descriptions for the evaluation of the inclusion of carbohydrate derived fulvic acid (CHD-FA) in the diets of broiler chicks from hatch until day of slaughter at 33 days of age. Selection of broiler chicks for carcass, meat quality and organ data collection Two chicks per replicate were slaughtered at day 33. The one chick was used for the determination of carcass and meat quality characteristics while the other was used for collecting organ data including gizzard erosion, microbiome data, histology, and bone mineralisation. The two chicks most closely representing the middle weight group were selected. Chicks were sexed at slaughter. For clarity, a total of 16 chicks per treatment were slaughtered for analysis. Of these, 8 were assessed for carcass and meat quality and 8 were assessed for gut and organ data. Gut and organ data Organs were removed (gizzard, heart, liver, spleen, and bursa of Fabriscious) and weighed. Liver colour, an indicator of possible oxidative stresses associated with toxicity, was determined using the BYK- Gardner Colour Guide with colour described according to the CIElab system (Commition International de L’Eclairage, 1976) with three measurements; L* (lightness), a* (redness) and b* (yellowness). Organ weights were used to calculate various ratios used as indicators of immune status of the birds, which in turn indicate the presence of stressors. After the removal of the organs and histology samples the pH of the gut was measured inside the gizzard, proventriculus, duodenum (on the gizzard side of the duodenum at the start of the pancreas), jejunum (approximately centre), ileum (5mm from Meckel’s diverticulum to the ileocecal junction) and caecum. The pH was measured using a calibrated (standard buffers pH 4.0 and 7.0 at 25°C) portable Crison pH25 meter (Alella, Barcelona) by inserting the pH electrode into the centre of the area of the digestive tract to be measured. The probe was thoroughly rinsed with distilled water between readings and stored in buffered solution. Microbiology Microbiome samples were taken from the jejunum (5cm segment length samples at 4-5cm from the distal end) prior to any other treatment or measurements to ensure the natural flora is not disturbed. This was done aseptically by placing the jejunum biopsy samples into individual DNA / RNA Shield faecal collection tubes (Zymo, Cat #R1101). The samples were collected from the same birds used for the gut and organ data; three birds per treatment were randomly selected and sampled for subsequent Amplicon Sequencing & Taxonomic Profiling of the entire variable region (V1–V9) of the bacterial 16S rRNA gene. The samples were refrigerated immediately after sampling and stored at 4°C in the DNA / RNA Shield faecal collection tubes until further processing (DNA extraction) and sequencing. Faecal samples were collected on days 1, 15, 22 and 29 of the trial. These were in the form of a cloacal swab. Samples were randomly taken from five birds per treatment. Swabs were stored in 10% PBS at 4°C until further processing and isolation of bacteria could commence. Sample collection Sterile cotton swabs in 10mL buffered peptone water (Lasec, South Africa) were used to swab the area of the cloaca of the broiler birds on Day 0, 14, 21 and 28 of the trial. The swabs were sealed in leakproof containers and frozen at -20°C until further processing and isolation of bacteria could commence. Isolation of Escherichia coli The samples (swabs) were allowed to thaw at room temperature (20°C) prior to analysis. The swabs were incubated at 37°C for 24 hours (± 2 hours) in 10mL buffered peptone water (BPW; Oxoid, Hampshire, United Kingdom) before preparing a serial dilution using physiological saline solution (PSS) in 9mL units. A spread plate technique was then used to spread 0.1mL of inoculated peptone water onto Tryptone Bile X-glucuronide (TBX) agar (Oxoid, Hampshire, United Kingdom) and incubated at 37°C for 24 hours to determine the number of colony forming units (CFU) per swab sample. Escherichia coli colonies were identified by their vibrant blue-green appearance on the TBX-agar, whereafter the number of colonies per dilution were counted and recorded. Single colonies of E. coli were then also further streaked onto nutrient agar (Oxoid, Hampshire, United Kingdom) in preparation for stock cultures and safekeeping. Isolation of Salmonella spp. The standard ISO 6579-1:2017 protocol for isolation of Salmonella spp. in animal faeces was followed. All sample swabs were thawed to room temperature (20°C) before being placed into 10mL of BPW, and incubated at 37°C for 24 hours, whereafter 0.1mL of the inoculated BPW was transferred to 10mL Rappaport- Vasiliadis soy peptone (RVS) broth (Oxoid, Hampshire, United Kingdom) and incubated at 41.5°C (± 0.5°C) for 18 to 24 hours. Xylose lysine deoxycholate (XLD) agar (Oxoid, Hampshire, United Kingdom) plates were used to spread a loop full of the inoculated RVS broth onto the plates and were incubated overnight at 37°C. Salmonella spp. was identified as black colonies with a translucent red halo on XLD agar. Histology For histological analysis, samples were taken from the midpoint of the jejunum after slaughter. The samples were taken using the intestinal strip method and were 2 to 3cm in length. Samples were rinsed with a phosphate buffer of pH 7.4 and then fixed in a 10% neutral buffered formalin until a decision would be made about further analysis. Carcass and meat quality characteristics Slaughter characteristics At and after slaughter the following data was collected: ● Live weight at slaughter. ● Carcass weight at slaughter (warm carcass weight). ● Cold carcass weight after 24 h of chilling. ● Carcass drip loss. ● Dressing percentage. Cut yield of the carcasses were obtained as follows • The carcass was halved, and the right side of the carcass cut up into breast, thigh, leg, wing and back. Each portion was weighed and expressed as a percentage of the total. • Skin and fat, and bone were removed from the breast and weights determined. Meat quality characteristics ● pH of breast muscle at slaughter (pHi) and 24 h after chilling (pHu) ● Colour of the breast muscle was determined on the breast that was tissue yield breast after blooming for 30 min. Colour was determined using the BYK- Gardner Colour Guide with colour described according to the CIElab system (Commition International de L’Eclairage, 1976) with three measurements; L* (lightness), a* (redness) and b* (yellowness). Bone mineralisation ● The tibia of both legs was removed at slaughter, all tissue was removed, and the bones vacuum packed and frozen. Eight samples per treatment were taken and six of these were subjected to analysis. ● Before analysis the bones were defrosted over-night. ● After defrosting the bones were dried at 60°C and subsequently defatted and then incinerated. The remaining ash was used for mineral analysis. Statistical analysis Statistical analysis included analysis of variance (ANOVA) with Fisher’s least significant difference (l.s.d.) post hoc test were used for comparing treatments; differences were accepted as significant at p<0.05. The ANOVA allowed differences between the means of the treatments to be analysed. This was done using the statistical software Statistica™. For data points that are measured throughout the trial at different times, a repeated measures analysis of variance was done. For the repeated measures it was investigated if a growth curve can be fitted to the data, dependent on the distribution of the data. In this report four p values are presented, p Interaction, p Treatment, p Antibiotic and p Overall. The p Treatment is related to the means of the five water treatments (water, citric acid, 20 mg / kg / day CHD-FA, 100 mg / kg / day CHD-FA and 250 mg / kg / day CHD-FA), the p Antibiotic is related to the means of the two antibiotic levels either being antibiotic positive or negative, the p Interaction is related to the dependence between the five water treatments and the two antibiotic levels and the p Overall is related to the means of the 10 individual treatments (the combination of water treatment and antibiotic). The p Interaction, p Treatment and p Antibiotic give an indication as to what component of the overall treatment has an effect and causes significant differences. If only the p Treatment is significant then only the water treatment had a significant effect likewise if only the p Antibiotic is significant only the presence or absence thereof had an effect. If the p Interaction is significant the effect of the 10 individual treatments and differences between the 10 individual treatments (the combination of the water treatment and the presence or absence of antibiotics) needs to be investigated through the p Overall. In cases where only the p Treatment or p Antibiotic were significant the means of these groups have been pooled and presented in a separate table. An asterisk denotes where winsorised values have been used due to an outlier present in the measurement. Results and discussion For the purpose of this study, unless specifically indicated otherwise or in terms of the specific context, the following abbreviations apply to the respective treatments. NegCon: Negative control: Tap water with no additive PosCon: Positive control: Commercial antibiotic in feed NegCit: Acidified water with 0.3M citric acid to pH 4 PosCit: Water acidified with 0.3M citric acid to pH 4 and commercial antibiotic in feed Neg20FA: Water with 20 mg / kg / day 8% CHD-FA Pos20FA: Water with 20 mg / kg / day 8% CHD-FA and commercial antibiotic in feed Neg100FA: Water with 100 mg / kg / day 8% CHD-FA Pos100FA: Water with 100 mg / kg / day 8% CHD-FA and commercial antibiotic in feed Neg250FA: Water with 250 mg / kg / day 8% CHD-FA Pos250FA: Water with 250 mg / kg / day 8% CHD-FA and commercial antibiotic in feed For convenience, the respective treatments utilized “8% CHD-FA”. By “8% CHD-FA” is meant 8g dry weight of pure CHD-FA per 100mL of pure water (i.e. w / v). However, it is important to note that dosages were calculated in terms of dry weight CHD-FA. Note that for the purposes of the trial, the dose was adjusted every 7 days from Day 0 to Day 33 to ensure the specified dose was given for an average of the body weights of the growing birds over each 7-day period. Temperature and humidity Temperature (°C) and relative humidity (%) data was collected using Logtag data loggers. Two loggers were placed in the house, one in the front and one in the back. This was to ensure maintenance of the temperature-controlled environment conditions for the study. Feed and feeding A three-phase diet was obtained from a commercial feed mill and fed to the birds at a rate of 900g starter and 1200g grower feed allocated per bird. Finisher feed was fed until slaughter. The proximate composition of the respective feeds is shown in Table 3 and the ingredient composition in Table 4. Table 3 Proximate composition of starter, grower and finisher feed of the three- phase diet fed to broiler chickens. Table 4 Ingredient composition of starter, grower and finisher feed of the three- phase diet fed to broiler chickens. Gizzard erosion study The short-term gizzard erosion study served to determine whether a high concentration of CHD-FA over a short period would have any negative effect on gizzard health. Gizzard erosion can be caused by several factors such as mycotoxins, the presence of harmful compounds and stress (Džaja et al., 1996). Differences in gizzard scores (Table 5) between treatments were not significant (p=0.14). Overall, the scores remained low with no scores of five and majority of scores being twos or threes, showing that high dosages of CHD-FA over a short period did not present any negative effects. Table 5 Number of observations per treatment of the short-term gizzard erosion scores recorded from broilers receiving different carbohydrate derived-fulvic acid (CHD-FA) treatments. The long-term effects of the treatments at commercial inclusion levels (Table 6) presented similar results to that of the short-term gizzard erosion study with no significant differences between treatments (p=0.43) and very few scores above three. The addition of CHD-FA had no effect on gizzard erosion and showed no negative effects. Table 6 Number of observations per treatment of the long-term gizzard erosion scores recorded from broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments.

[0003] Production parameters Liveability is an indication of how well a treatment survived, it is expressed as the percentage of birds surviving until slaughter. The European production efficiency factor (EPEF) is an overall indicator of broiler performance that takes into account a number of factors such as liveability, live weight gain, age and feed conversion ratio (FCR). The EPEF allows for comparison of performance between different flocks. When liveability and live weight gain are maximized and age and FCR are minimized a greater EPEF is seen. A higher EPEF indicates higher efficiency. A higher EPEF points to a flock that has a higher survival rate and greater gain in weight over time, while converting feed to live weight more efficiently i.e. using less feed (Mavromati et al., 2018). The feed conversion ratio (FCR) shows a ratio of the amount of feed needed in order for a broiler to gain 1kg of live weight. It is a measure of how efficiently feed is converted to live weight. A lower FCR shows a greater efficiency. Average daily gain (ADG) is the average gain in live weight per day over the lifespan of the broiler. The protein efficiency ratio (PER) is calculated by dividing weight gain by protein intake. A higher ratio shows more effective use of protein as weight gain increases. Liveability, EPEF, ADG, FCR and PER results are captured in Table 7. There was no significant difference in liveability between treatments (p=0.94). A comparison between the antibiotic fed and non-antibiotic fed birds also showed no significance (p=0.42) with a nonsignificant interaction between CHD-FA inclusion level and the inclusion of antibiotics (p=0.11). Overall differences in liveability were not significant (p=0.42). When looking at EPEF, the effect of the interaction (p<0.01), antibiotic (p<0.01) and the overall treatment (p<0.01) were highly significant while the effect of antibiotic (p=0.18) was not. The NegCon had the lowest EPEF and was no different from the NegCit and Pos250FA and in turn these two were no different from the Pos20FA. The PosCit had the highest EPEF and was the same as the PosCon and Pos100FA. The Neg20FA, Pos20FA, Neg100FA, Pos100FA and Neg250FA were the same as the PosCon and the Pos100FA was the same as the Neg20FA, Pos20FA, Neg100FA, Neg250FA and Pos250FA. For ADG the effect of the interaction (p<0.01), the treatment (p<0.01) and overall treatment (p<0.01) was significant but not antibiotics (p=0.41). The PosCon, PosCit and Neg100FA had the highest ADG and were similar. The Neg20FA, Pos100FA and Neg250FA were the same and no different from the PosCon, PosCit and Neg100FA. The NegCit had one of the lowest ADG and was the same as the Pos20FA and Pos250FA. The NegCon had the lowest ADG and was the same as the Pos250FA. Interaction, antibiotics and overall treatment had an effect on FCR, but water treatment alone did not (p=0.34). The PosCit, Neg100FA and Pos100FA were the same and had the lowest FCR and were no different from the PosCon, Neg20FA and Neg250FA which were the same as each other. The NegCit and Pos20FA had higher FCRs and were the same, they were not significantly different from the PosCon, Neg20FA and Neg250FA. The Pos250FA had the second highest FCR and was the same as the NegCit and Pos20FA. The NegCon had the highest FCR and was the same statistically as the Pos250FA. Interaction, antibiotics, and overall treatment had significant (p<0.01) effect on PER, but water treatment alone did not (p=0.46). The PosCit, Neg100FA and Pos100FA had the highest PER and were the same these were no different form the PosCon, Neg20FA and Neg250FA (these were the same as each other). The NegCon had the lowest PER and was the same as the NegCit and the Pos250FA. Industry norms regarding the measurement in Table 7 are almost non-existent. Annual reports that do contain these factors often report them as a percentage increase or decrease of the previous year’s performance and do not give an exact value. A case study done in 2005 on poultry farms in Botswana reported a liveability of 96%, EPEF values ranging from 240,83 to 266,90; an ADG of 50g and FCR values ranging from 1.67 to 1.85 (Kelebemang, 2005). Table 7 Mean (± standard deviation) of liveability (%) and European production efficiency factor (EPEF), average daily gain (ADG), feed conversion ratio (FCR) and, protein efficiency ratio (PER) for broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. a,b,c,d,eMeans within columns with different superscripts differ significantly (p<0.05) Referring to Figures 1 to 4 of the accompanying drawings it can be seen that the CHD-FA feed additive of the invention (neg100FA) outperformed the water only control and compared favourably on all counts with the treatment additive comprising a commercial antibiotic (respectively, PosCon and PosCit). The average weight per bird (Table 8) per week was significantly different between treatments (p=0.02). The inclusion of antibiotics to the feed had a significant effect (p=0.02) while a highly significant (p<0.01) interaction between treatment and antibiotic inclusion was also observed. Differences were only significant from day 7 onwards. On day 7 (p=0.03) the PosCit resulted in the heaviest weights followed by the Pos20FA while the Neg100FA resulted in the lightest weights. All other treatments were very similar and fell in between these treatments. On day 14 (p=0.01) the PosCit and Pos20FA were the same and resulted in the heaviest weights and the NegCit and Neg20FA in the lightest weights. The PosCit and Pos20FA were the same as the PosCon, Pos100FA, Neg250FA and Pos250FA. The NegCit and Neg20FA were the same as the NegCon, Neg100FA, Neg250FA and Pos250FA. On day 21 (p<0.01) the PosCon and PosCit were the same having the heaviest weights followed by the Pos100FA. All these were heavier than the NegCon, NegCit and the same as the Pos20FA and the Neg100FA. The NegCon had the lightest live weight and was the same as the NegCit, Pos250FA and the Neg250FA. On Day 28 (p<0.01) the NegCon was the lightest and was the same as the NegCon and Pos20FA, Pos250FA. The PosCit had the heaviest weight and was the same as the PosCon, Pos100FA and Neg100FA. The Neg20FA and the Neg250FA were the same and were both significantly lighter than the PosCit and significantly heavier than the NegCon. When looking at day 33 (p<0.01) the PosCit, PosCon, Pos100FA and Neg100FA were the same and had the heaviest weights and were not different from the Neg20FA and the Neg250FA. The Pos250FA had the lightest weight and was the same as the NegCon and NegCit which were the same as each other. The Pos20FA was an intermediate treatment being lighter than the PosCit, PosCon, Pos100FA and Neg100FA and heavier than the Pos250FA. Table 8 Mean (± standard deviation) of average weekly bird weight of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33.a,b,c,d,eMeans within columns with different superscripts differ significantly (p<0.05) Table 9 shows the average weekly feed intake of the birds which had a significant interaction (p<0.01) and was highly significantly different on days 14, 28 and 33 (p<0.01). Treatment and antibiotics alone also had a significant effect (p=0.02). On day 14 the NegCon had the highest intake and was the same as the Neg20FA. The NegCit, Neg100FA and Neg250FA had the second highest intake and were the same as the PosCit, PosCon and Pos20FA, which were similar. The Pos100FA had the lowest intake and was the same as the Pos250FA. For day 28 the PosCit had the highest intake and was the same as the PosCon, Pos100FA, Neg100FA and the Pos250FA, which were the same as each other. This was followed by the Pos20FA and the Neg20FA which were the same as each other. The Pos250FA had the lowest intake and was the same as the NegCon and the NegCit. On day 33 the PosCit, PosCon, Pos100, Neg20FA, Neg100FA and Neg250FA were all the same and had the highest intake. This was followed by the Pos20FA and then lastly the lowest intake was the Pos250. Both the Pos20FA and the Pos250FA were the same as the NegCon and the NegCit. Table 9 Mean (± standard deviation) of average weekly feed intake per bird per week (g) of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33.a,b,c,d,e,fMeans within columns with different superscripts differ significantly (p<0.05) Treatment had a significant effect (p=0.02) on the average cumulative feed intake of the birds (Table 10) as did the interaction (p<0.01) between treatment and antibiotics but the inclusion of antibiotics per se (p=0.36) showed no significant difference. Significant differences were observed from day 14 onwards. On day 14 (p<0.01) the NegCon was the highest and the same as the Neg20FA and the Neg100FA and was higher than the other treatments. The Neg100FA was the lowest but was the same as the PosCit, PosCon, Pos20FA and Neg250FA. On day 21 (p=0.01) the NegCon and PosCit were the same and significantly higher than the Pos100FA, Neg250FA and the Pos250FA and both the NegCon and PosCit were the same as all other treatments. The Pos250FA was the lowest and was the same as the PosCon, Pos20FA, Pos100FA, Neg100FA and the Neg250FA. On day 28 (p<0.01) the PosCit was the highest and was the same as the PosCon, Neg20FA, Neg100FA and the Neg250FA, and higher than the NegCon, Pos20FA and Pos100FA. The Pos250FA had the lowest intake but was the same as the NegCit. On day 33 (p<0.01) the PosCit, PosCon and Neg100FA were the same and were the highest intake and were not different from the Pos100FA, Neg20FA and the Neg250FA. The NegCon and NegCit were the same and were the second lowest and not different from the Pos20FA and Pos100FA. The Pos250 had the lowest intake. Table 10 Mean (± standard deviation) of average cumulative feed intake per bird per week (g) of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. a,b,c,dMeans within columns with different superscripts differ significantly (p<0.05) In terms of average weekly gain per bird (Table 11) there was a significant difference across all days due to the interaction (p<0.01) between antibiotics and treatments, and within the treatments alone (p<0.01). The major differences on day 7 (p=0.03) was the PosCit having the highest gain and being significantly higher than the NegCon, NegCit, Pos100FA and Neg100FA. The Neg100FA had the lowest gain but was only significantly lower than the PosCit, PosCon and Pos20FA. On day 14 (p=0.02) the main differences were between treatment Pos20FA which had the highest gain and treatment Neg20FA that had the lowest gain. Treatment Pos20FA was also higher than the NegCon and NegCit that were the same as the Neg20FA. The PosCon and PosCit were also higher than the NegCit and Neg20FA. On day 21 (p<0.01) the PosCon was the highest and significantly higher than the NegCit, Pos20FA, Pos100FA, Pos250FA and the Neg250FA. The NegCon was the lowest but was not different from the NegCit, Pos250FA and Neg250FA. On day 28 (p<0.01) the PosCit, PosCon, Pos100FA, Neg20FA and Neg100FA were the highest and were not different from the Neg250FA nor was the Neg250FA different from the NegCit and Pos20FA. The NegCon and Pos250FA were the lowest and not different from the NegCit and Pos20FA. On day 33 (p<0.01) the Neg100FA had the highest gain and was significantly higher than the NegCon, Pos20FA and Pos100FA. The NegCon was significantly lower than the PosCit, Neg20FA, Neg100FA and the Neg250FA. The Pos250FA had the lowest gain overall. Table 11 Mean (± standard deviation) of average weekly gain per bird per week (g) of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. a,b,c,d,e,fMeans within columns with different superscripts differ significantly (p<0.05) For average cumulative weight gain, the effect of the interaction (p<0.01), treatment (p=0.01) and antibiotics (p=0.02) were all significant (Table 12). On day 7 (p=0.05) the PosCit had the highest gain but was only significantly higher than the NegCon, NegCit, Pos100FA and Neg100FA. The NegCit and Neg100FA were the lowest but only significantly lower than the PosCit, PosCon and Pos20FA. On day 14 (p<0.01) The PosCit and Pos20FA were the highest but only significantly higher than the NegCon, NegCit, Neg20FA and the Neg100FA. The NegCit and Neg20FA had the lowest gain but were no different from the Pos250FA, NegCon and Neg100FA. On day 21 (p<0.01) the PosCon and PosCit were the highest but the same as the Pos20FA and the Neg100FA). The NegCon was the lowest but was not different from the NegCit, Pos250FA and Neg250FA. On day 28 (p<0.01) the PosCit was the highest and was the same as the PosCon, Pos100FA and Neg100FA. The NegCon was the lowest and no different from the NegCit, Pos20FA and the Pos250FA. The Neg20FA and Neg250FA were the same and fell between the PosCit and NegCon. On day 33 (p<0.01) the NegCit, PosCon, Pos100FA and Neg100FA were all significantly the same and higher than the other treatments except for the Neg20FA and Neg250FA which were the same as the previous treatments and each other. The Pos250FA was the lowest treatment but was not different from the NegCon and NegCit. Table 12 Mean (± standard deviation) of average cumulative gain per bird per week (g) of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. a,b,c,d,eMeans within columns with different superscripts differ significantly (p<0.05) The protein intake per bird (Table 13) had a highly significant interaction (p<0.01) between antibiotics and water treatment, this was seen in the overall treatment on day 14 (p=0.02), day 28 (p<0.01) and day 33 (p<0.01) and followed a similar pattern as total feed intake. On day 14 the Neg250FA had the highest intake and was the same as the NegCon, Neg20FA and Neg100FA. The Pos100FA had the lowest intake and was the same as the PosCit, Pos20FA and Pos250FA. The PosCon and NegCit were the same as each other and every other treatment. On day 28 the Neg250FA had the highest intake and was the same as the PosCon, PosCit and Neg100FA. The Pos250FA was the lowest but was the same as the NegCon and NegCit. On day 33 the PosCon, PosCit, Neg100FA and Neg250FA were the same and the highest and not significantly different from the Pos100FA which itself was the same as the Pos20FA. The NegCon, NegCit and Pos250FA were the same and the lowest intake but not different form the Pos20FA. Table 13 Mean (± standard deviation) of average crude protein intake per bird per week (g) of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. a,b,c,d,e,fMeans within columns with different superscripts differ significantly (p<0.05) The average cumulative crude protein intake is shown in (Table 14). There was a highly significant interaction (p<0.01) and treatment effect (p=0.03). Significant overall treatment differences were seen from day 14 onwards but followed a similar pattern to feed intake. On day 14 (p<0.01) the NegCon was the highest but no different from the Neg20FA and the Neg100FA. The Pos250FA had the lowest intake but was only significantly lower than the NegCon, NegCit, Neg20FA and Neg100FA. On day 21 (p=0.01) the NegCon and NegCit were the highest and the same as the NegCit and Neg20FA. The Pos250FA was the lowest but not different from the Pos100FA and the Neg250FA. The PosCon, Pos20FA and Neg100FA were all the same as each other and no different from any other treatment. On day 28 (p<0.01) the PosCit was the highest and was the same as the PosCon, Neg20FA, Neg100FA and Neg250FA. The Pos250FA was the lowest and was the same as the NegCit. The NegCon, Pos20FA and Pos100FA were the same and fell in between the PosCit and the Pos250FA. On day 33 (p<0.01) the PosCon, PosCit and Neg100FA were the same and the highest and were no different form the Pos100FA, Neg20FA and Neg250FA. The NegCon and NegCit were the same and the second lowest but not different from the Pos20FA and Pos100FA. The Pos250FA was the lowest overall. Table 14 Mean (± standard deviation) of average cumulative crude protein intake per bird per week (g) of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. a,b,c,dMeans within columns with different superscripts differ significantly (p<0.05) Gut and organ data The pH (Table 15) of the proventriculus and gizzard were affected by the addition of antibiotics but there were no differences owing to the treatments. The Ileum was the only gut component to show significant difference between treatment (p=0.01) and to show a significant interaction between the treatment and antibiotics (p=0.04). When looking at the 10 individual treatments they were highly significantly different (p<0.01). The NegCit and the Pos100FA were the same and the lowest, they were not different from the NegCon, PosCit, Pos20FA or Neg20FA treatments. The Neg100FA had the highest pH and was not different from Neg250FA and Pos250FA treatments, the Neg20FA or the PosCon. The Neg250FA and Pos250FA treatments were higher than the PosCit and Pos20FA. The PosCit, Pos20FA, Neg20FA, PosCon and NegCon were not significantly different. While the Jejunum pH did not have a significant Interaction (p=0.46), antibiotics did (p=0.02) and treatment (p=0.08) showed a trend towards significance. The Neg100FA had the highest pH while the Pos20FA had the lowest while the other treatments fell in between. Table 15 Mean (± standard deviation of the gut pH of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33.a,b,c,dMeans within columns with different superscripts differ significantly (p<0.05) Antibiotics (Table 16) had a significant effect on proventriculus pH (p=0.03) which pulled through to the overall significance (p=0.04), and then on gizzard pH (p=0.05). For the proventriculus the negative group had a higher pH than the positive group whereas with the gizzard pH the negative group had the lower pH than the positive group. Table 16 Mean (± standard deviation) of the proventriculus and gizzard pH of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. Organ weights for the different treatments are shown in Table 17. The only significant difference observed was for the effect of CHD-FA (p<0.01) on heart weight with the control and the citric acid treatments having heavier hearts than Pos100FA, Neg100FA treatments and the Pos250FA treatment but were the same as the remaining treatments. The PosCit, NegCit and the Neg20FA treatment were the same and heavier than the Pos100FA, Pos250FA and Neg250FA treatments. The Neg20FA, Neg100Fa, Pos250FA and Neg250FA were the same. Table 17 Mean (± standard deviation) of organ weights of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. a,b,cMeans within columns with different superscripts differ significantly (p<0.05) When looking at the organ to liveweight ratios (Table 18), the only significant effects are of treatment on heart weight ratio (p<0.01) and antibiotics (p=0.02) on bursa weight ratio. The NegCon, PosCon, NegCit, Pos20FA, Pos250FA and Neg250FA were the same and were greater than the Pos100FA treatment. The NegCon was greater than the PosCit, Neg20FA, Pos100FA, Neg100FA, Pos250FA and Neg250FA treatments. The NegCit was greater than PosCit, Neg100FA and Pos100FA but the same as the Pos250FA and Neg250FA treatments. The PosCon and Pos250FA treatment were the same as each other and greater than PosCit, Pos100FA and Neg100FA treatments which were in turn no different from each other. The Pos20FA treatment and the Neg250FA treatment were the same as each other and greater than the Pos100FA treatment, which was no different from the PosCit, Neg20FA treatment and the Neg100FA treatment. When looking at the bursa weight ratio (Table 19) the antibiotic negative group had a higher ratio than the antibiotic positive group. Table 18 Mean (± standard deviation) of the organ to live-weight ratios (%) of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. a,b,c,d,eMeans within columns with different superscripts differ significantly (p<0.05) Table 19 Mean (± standard deviation) of the bursa weight ratio of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. From Table 20 it can be seen that the heart to liver ratio (p=0.03) and heart to spleen ratio (p=0.03) was significantly different between treatments. With regards to heart to liver ratio the NegCon had the highest ratio but was no different from the PosCon, NegCit, Pos20FA, Pos250FA and Neg250FA. The Pos100FA (0.21 ± 0.029) had the lowest ratio but was no different from the PosCon, NegCit, Pos250FA, Neg20FA and Neg100FA. With regards to heart to spleen ratio the NegCon and PosCon were the same and had the highest ratio and were no different from the NegCit, Pos250FA, Neg20FA and Neg250FA. The PosCit had the lowest ratio but was no different from all treatments except the NegCon, PosCon and Neg20FA. Antibiotics (Table 21) had a significant effect on spleen bursa ratio (p=0.05), the negative group having a lower ratio than the positive group. Table 20 Mean (± standard deviation) of the organ weight ratios of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. a,b,c,dMeans within columns with different superscripts differ significantly (p<0.05) Table 21 Mean (± standard deviation) of the spleen to bursa ratio of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. There was a significant interaction (p=0.03) between the treatment and antibiotics for the L* colour value (Table 22), meaning that those two factors were potentially dependent on each other, and the combination may have had an effect on the means. But when looking at the effects of antibiotics (p=0.62) and treatment (p=0.42) these were non-significant. The effect of the overall treatment was also not significant (p=0.11). It can then be concluded due to the sensitive nature of the statistical testing that the significant interaction was a false positive. Therefore, there were no differences seen in terms of liver colour between the treatments. Table 22 Mean (± standard deviation) of liver colour measurements (CIE-Lab) for broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. Carcass and meat quality characteristics Carcass and meat quality characteristics are reported in Table 23. No significant differences were observed for any parameter, treatment, or interaction. Thus, neither acidification, the presence of CHD-FA, the presence of antibiotics or combinations thereof had any effect on carcass and meat quality characteristics. Table 23 Mean % (± standard deviation) of dressing percentage and carcass cut yields (% of cold carcass weight) of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. There were significant interactions between the treatments and antibiotics with regards to live weight (p=0.01), warm carcass weight (p<0.01) and cold carcass weight (p<0.01), which lead to significant differences overall (p<0.01) between treatments (Table 24). The PosCon and PosCit had the heaviest liveweight, followed by the NegCon. The PosCon, PosCit and NegCon were the same as the Neg20FA, Pos100FA, Neg100FA and Neg250FA. The NegCon was the same as the NegCit and Pos20FA. The Pos250FA had the lightest weight and was the same as the NegCit and Pos20FA. The warm carcass weight of the PosCon, PosCit and NegCit was heavier than that of the NegCon, NegCit, Pos20FA and Pos250FA treatment. The Neg20FA was heavier than that of the NegCit, Pos20FA and Pos250FA which were the same as each other. The Pos250FA was significantly lower than the Pos100FA, Neg100FA and the Neg250FA treatments. The cold carcass weights of the PosCon, PosCit and NegCit were the same and heavier than the NegCon, NegCit, Pos20FA and Pos250FA treatments. The Neg20FA was heavier than the NegCit and Pos250FA treatments. The Pos100FA, Neg100FA and the Neg250FA were heavier than the Pos250FA. Since the effect of treatment was seen on liveweight and treatment had no effect on dressing percentage the effect on carcass weight is expected. Table 24 Mean (± standard deviation) live weights and carcass weights of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33.a,b,c,d,Means within columns with different superscripts differ significantly (p < 0.05) In terms of breast muscle components (Table 25) there was a significant (p<0.01) interaction between the treatment and antibiotics which lead to a significant effect overall (p<0.01). The PosCon and PosCit were the same, and both heavier than the NegCon, NegCit, Pos20FA and the Pos250FA treatments when looking at muscle. The Neg250FA was heavier than the NegCit as well as Pos20FA and the Pos250FA. The Pos100FA and Neg100FA treatments were heavier than the Pos250FA. Skin and fat had a significant interaction (p=0.03) and a trend towards significance overall (p=0.07). The Neg20FA had the heaviest weight but was not different from the PosCon, Pos100FA and Neg250FA. The NegCon and the Pos20FA were the lightest but were not different from the PosCit, PosCon, NegCit, Pos100FA, Neg100FA and the Pos250FA. Table 25 Mean (± standard deviation) absolute weights of breast muscle components of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33.a,b,c,dMeans within columns with different superscripts differ significantly (p < 0.05) These observed differences were, however, not repeated in the tissue yields expressed as a percentage of the cut (Table 26). For the percentage skin and fat only the water treatment had an effect (p=0.03), and this was seen again in the overall treatment effect (p=0.04). When looking at the effect of water treatment on percentage skin and fat (Table 27), the 20FA and the 250FA had the highest percentage and the Con had the lowest. While the 20FA and 250FA were different from the Con none of these treatments were any different from the other treatments. Table 26 Mean % (± standard deviation) muscle, skin and fat and bone of the breast relative to the breast of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. Table 27 Mean (± standard deviation) of percentage skin and fat of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. a,bMeans within columns with different superscripts differ significantly (p<0.05) Con: water with or without antibiotics Cit: Citric acid with or without antibiotics 20FA: 20 mg / kg / day CHD-FA with or without antibiotics 100FA: 100 mg / kg / day CHD-FA with or without antibiotics 250FA: 250 mg / kg / day CHD-FA with or without antibiotics The initial temperature (Tempi) (Table 28) of the carcasses showed significant differences (p<0.01) (Tempi) for treatment as well as a significant interaction (p<0.01) between treatment and the antibiotics and for overall treatment (p<0.01). The NegCon had the lowest temperature overall but was the same as the NegCit. The Pos100FA and Neg100FA had the highest temperature but were not different from the Neg20FA and the Neg100FA treatment. The Neg100FA was higher than both the NegCit and PosCit as well as both Pos20FA, Pos250FA and the Neg250FA treatments. The PosCon and Neg20FA were the same and higher than NegCit and PosCit treatments and the Pos20FA. The NegCit was lower than both Pos250FA and the Neg250FA which were not different from each other or the PosCit and Pos20FA treatment. Table 28 Mean (± standard deviation) temperature (°C) and pH measurements of the breast muscles of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. a,b,c,d,e,fMeans within columns with different superscripts differ significantly (p<0.05) Breast colour was also not influenced by treatment (Table 29). Table 29 Mean (± standard deviation) of breast colour measurements (CIE-Lab) of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. Table 30 shows the mineral percentages of the tibial bones of the broilers. Here it is seen that only the potassium and magnesium percentage were highly significantly (p<0.01) different between the 10 treatments. The overall significant difference observed for calcium and phosphorous is due to the water treatment and not the other factors and thus Table 33 should be consulted for differences in calcium and phosphorous. When looking at potassium percentages the Neg20FA, Pos250FA and Neg250FA had the highest levels and were the same as the NegCon, Pos100FA and Neg100FA. The Pos20FA had the lowest levels and was the same as the NegCit and PosCit. The PosCon was an intermediate treatment and was the same as the NegCon, PosCit, NegCit, Pos100FA and Neg100FA. Magnesium percentages followed a similar pattern. NegCon, Neg20FA, Pos100FA and Pos250FA were the same and the highest and were the same as the Neg100FA and Neg250FA. Table 30 Mean (± standard deviation) of tibial bone mineral percentages of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33.a,b,c,d,eMeans within columns with different superscripts differ significantly (p<0.05) Only the iron content of the bones (Table 31) was highly significantly (p<0.01) different with the Neg100FA having the highest content and being the same as the PosCit. The NegCon, PosCon, NegCit and Pos20FA were the same and had the lowest iron content and were the same as the Neg20FA, Pos100FA, Pos250FA and the Neg250FA which were all statistically the same as each other. The overall significant difference observed for sodium and zinc is due to the water treatment and not the other factors and thus Table 34 should be consulted for differences in sodium and zinc. Table 31 Mean (± standard deviation) of tibial bone mineral contents of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. a,b,c,Means within columns with different superscripts differ significantly (p<0.05) The manganese and boron (Table 32) contents of the bones were not significantly different. The overall significant difference observed for aluminium is due to the water treatment and not the other factors and thus Table 34 should be consulted for differences in aluminium. Table 32 Mean (± standard deviation) of tibial bone manganese, boron and aluminium contents of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. When looking at the phosphorus percentage (Table 33) of the bones there was a highly significant difference (p<0.01) with the 20FA having a higher levelthe 100FA and 250FA which were the same as each other. The Cit was significantly lower than all treatments. Con was significantly higher than the Cit and lower than the 20FA but the same as both the 100FA and 250FA. The calcium percentages also differed significantly (p<0.01) The Con, 20FA, 100FA and 250FA were statistically the same and higher than the Cit. Table 33 Mean (± standard deviation) of tibial bone phosphorus and calcium percentage of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. a,b,cMeans within columns with different superscripts differ significantly (p<0.05) Con: water with or without antibiotics Cit: Citric acid with or without antibiotics 20FA: 20 mg / kg / day CHD-FA with or without antibiotics 100FA: 100 mg / kg / day CHD-FA with or without antibiotics 250FA: 250 mg / kg / day CHD-FA with or without antibiotics The sodium contents (Table 34) of the different water treatments were significantly different with the 250FA having the highest levels and being the same as the Con and 20FA. The Cit had the lowest levels while the 100FA was intermediate being lower than 250FA, higher than the Cit and the same as the Con and 20FA. In terms of copper content, the Con, 20FA and 250FA were the same and higher than the Cit. The Cit once again had the lowest levels while the 100FA was not different from any other treatment. The Con had the highest levels of zinc and was the same as the 20FA and 250FA. The 100FA had the lowest levels and was the same as the Cit. The Cit was also the same as the 20FA and the 250FA. The aluminium content of the 20FA was the highest and the same as the Cit which was no different from any other treatment. The Con, 100FA and 250FA were the same. As seen in Table 34, the standard deviation of the 20FA group was very high. This indicates a large variation within the treatment group. Table 34 Mean (± standard deviation) of tibial bone sodium, copper, zinc and aluminium of broilers receiving different carbohydrate derived fulvic acid (CHD-FA) treatments, grown from hatch to day 33. a,b,cMeans within columns with different superscripts differ significantly (p<0.05) Microbiology Information of treatment groups to which the animals were allocated during the trial was shared with the authors of this discussion, in order to visualise relevant trends in the data. On each sampling day (Day 0, 14, 21, 28) five samples were taken from each of the 10 treatment groups. All samples plated on the TBX media were identified as containing E. coli. At Day 0, all positive E. coli samples contained less than 10 CFU / swab, and no Salmonella spp. were detected. At Day 14, all samples were still negative for Salmonella spp. Samples from the NegCon, NegCit and PosCit treatment groups indicated the highest counts for E. coli, averaging more than 4.5 log (36,000 CFU / swab). Furthermore, samples from the treatment groups Neg100FA, Pos100FA and Neg250FA indicated the lowest counts, averaging less than 3.9 log (8,900 CFU / swab) E. coli. Similar to the previous sampling days, no Salmonella spp. was detected for Day 21 and again the samples from treatment groups NegCon, NegCit, and PosCit presented with the highest counts of E.coli, averaging at more than 4 log (33,000 CFU / swab). Staying consistent with the trend on Day 14, samples from the treatment groups Neg100FA, Pos100FA and Pos20FA indicated the lowest (2.1 log (128 CFU / swab)) E. coli present. It appears as though CHD-FA inhibits the growth of E. coli up to 21 days. Following on the previous assumption, results from Day 28 are not consistent with the previous sampling days. All samples were yet again negative for Salmonella spp., but the addition of CHD-FA does not appear to have additional benefit at Day 28. All the samples from the 10 different treatment groups were in the region of 2 log (100 CFU / swab) E. coli, except for treatment Neg250FA which indicates a slightly higher (444 CFU / swab) count. In conclusion, a general 1.7 log reduction in E. coli counts can be observed from day 14 to 28. The internal gut microbiota seems to stabilise between day 21 and 28 for all treatment groups except Pos20FA, Neg100FA and Pos100FA where the gut microbiome appears to stabilise earlier, between day 14 and 21. The intestinal tract of commercially hatched chickens is gradually colonised from environmental sources only; however they can be colonised by microorganisms from the very first days of life and become resistant to infections with pathogenic E. coli or Salmonella spp. The interactions of chickens and their microbiota is an extremely interesting area mainly due to the fact that commercial hatching completely separates chickens from contact with adult hens. This makes newly hatched chicks highly susceptible to different infections. The gastrointestinal tract of chickens is characterised by a relatively long, bifurcated caecum and short colon (in comparison to humans or pigs). Digestion and nutrient resorption in the small intestine are continuous but resemble batch cultivation in the caecum. Faecal droppings can therefore be of ileal or caecal origin, the former being enriched for Lactobacillaceae while the latter being enriched for Bacteroidaceae and gram-negative bacteria, like Enterobactereacea. Most natural environments harbour a diverse collection of microbial species. Within these communities, bacteria compete with their neighbours for space and resources. Competitive exclusion works by the beneficial bacteria in the gut of the chicken being at such a level that the more harmful bacteria are denied the environment they need to survive and multiply. This could also be the case for when certain feed supplements or additives are used. In this trial, it can be concluded that the ideal microflora was reached by day 28, thus creating a stabilised environment within the gastrointestinal tract of the animals. While most treatment groups reached stabilisation between 21 - 28 days, some of the CHD-FA treatments (Neg100FA, Pos100FA, and Pos20FA) appear to have stabilised earlier and consequently may have influenced the E. coli burden of the birds. Referring to Figure 5 of the accompanying drawings, it can be seen that the preferred feed additive of the invention (Neg100FA) outperformed all of the other treatments, obtaining a stabilised microbiota between 14 to 21 as opposed to the greater than 21 days stabilisation achieved by the control (NegCon) and the antibiotic containing feed additives (PosCon and PosCit, respectively). When looking at average live weight (Table 8), average weekly gain (Table 11) and average cumulative gain (Table 12) on days 21 and 28 a loose link can be seen between treatments and E. coli burden. The live weight of the NegCon, NegCit and PosCit was lower than the Neg20FA, Neg100FA and Pos100FA on day 21, while on day 28 the Neg20FA was the same as the NegCit. This was also true for the average gain and average cumulative gain, respectively. So, it appears that there could be a link between E. coli burden and live weight, average gain and average cumulative gain and the effect of CHD-FA. Conclusion The results show a clear difference between the treatments and the negative control for production parameters with the treatments, specifically the 20FA and 100FA groups, showing similar results to that of the positive control (PosCon) indicating that the use of CHD-FA has the ability to replace antibiotics in the feed of broilers. The CHD-FA had no negative impact on the health of the broilers in term of gizzard erosion even when included at a level double the highest inclusion used in the trial (500 mg / kg / day). Equally the use of the CHD-FA did not have any negative effect on carcass and meat quality parameters which indicates that the use of this product will not negatively influence the yields that are expected at the abattoir and processing plant post slaughter. Besides these commercially valuable characteristics, it was noted that organ weights, ratios and gizzard characteristics were also not influenced by the use of CHD-FA indicating that the use of the product can be deemed safe. The results indicate that the highest inclusion level did not actually yield the most positive effects. In many cases, the lower CHD-FA inclusion levels often had similar or better results. Significant differences were seen in the EPEF, ADG, FCR, PER, average weekly weight, average weekly feed intake, average cumulative feed intake, average weekly gain, average cumulative gain, average crude protein intake, average cumulative crude protein intake, ileum and jejunum pH, heart weight, heart to live weight ratio, heart to liver ratio, heart to spleen ratio, live weight, warm carcass weight, cold carcass weight, breast muscle weight, breast skin and fat weight, skin and fat percentage, initial carcass temperature and tibial bone mineral content. When looking at microbiology between 21 - 28 days, the CHD-FA treatments (Neg100FA, Pos100FA, and Pos20FA) are believed to have accelerated gut microbiome stabilisation and influenced the E. coli burden of the birds. Further genome studies of the chicken gut microbiota during this time may create a clearer picture on whether the decreased E. coli burden on the flock can be fully attributed to the addition of CHD-FA in the diets of these birds. Overall, it can be concluded that the use of CHD-FA may be a potential replacement for antibiotics in the feed of broilers and will add value to broiler production without any negative effect on the product.

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Claims

AMENDED CLAIMS received by the International Bureau on 04 August 2025 (04.08.2025)1. A feed additive comprising a carbohydrate derived fulvic acid (CHD-FA) for use in broiler production, the feed additive comprising from about 20 mg / kg / day to about 500 mg / kg / day CHD-FA, measured as the dry weight CHD-FA in milligrams per kilogram body weight of each broiler per day.

2. A feed additive according to claim 1 , comprising from about 40 mg / kg / day to about 350 mg / kg / day CHD-FA.

3. A feed additive according to claim 1 or claim 2, comprising from about 80 mg / kg / day to about 150 mg / kg / day CHD-FA.

4. A feed additive according to any one of claims 1 to 3, comprising about 100 mg / kg / day CHD-FA.

5. A method of stabilizing the gut microbiota of a broiler chick, comprising administering to the chick a feed additive according to any one of claims 1 to 4, thereby to enhance the viability of the broiler chic.

6. A method according to claim 5, wherein the gut microbiota of the broiler chick is stabilized in 21 days.

7. A method according to claim 6 or claim 7, wherein the gut microbiota of the broiler chic is stabilized by a reduction of the E.coli and / or Salmonella and / or Campylcobacter sufficient for broiler survival and below acceptable limits for human consumption.

8. A method according to claim 7, wherein the E.coli count in the gut microbiota of treated broilers is stabilized around 2 log to 2.1 log (about 100CFU / swab to about 128 CFU / swab) between 14 and 21 days.

9. A method of preventing and / or treating a disease in a broiler chick, comprising administering to the broiler chick from about 20 mg / kg / day to about 500 mg / kg / day CHD-FA, measured as the dry weight CHD-FA, in milligrams per kilogram body weight of each broiler per day.

10. A composition comprising CHD-FA for use in a method of preventing and / or treating a disease in a broiler chick the composition comprising from about 20 mg / kg / day to about 500 mg / kg / day CHD-FA, measured as the dry weight CHD-FA, in milligrams per kilogram body weight of each broiler per day, the method comprising administering the composition to the broiler chick during broiler production.[0001][0002]Statement under Article 19(1)[0003](i) Claim 1 has been amended to incorporate the feature of original claim 2, which was found by ISA to be both novel and to involve an inventive step.[0004](ii) Claim 2 has been deleted as a consequence of the amendment to claim 1.[0005](iii) Original claim 6 (now claim 5) has been amended to specify that stabilization is of the gut microbiota. Basis for this amendment can be found, for example, on p5 final paragraph, and p49 final paragraph, of the specification.[0006](iv) Similarly original claims 7 to 9 (now claims 6 to 8) have been amended to reference gut microbiota for consistency of claiming.[0007](v) Original claims 10 and 11 (now claims 9 and 10, respectively) have been amended to include the feature of original claim 12, which was also found by the ISA to be novel and to involve an inventive step.[0008](vi) Original claim 12 has been deleted as a consequence of the amendment to original claims 10 and 11 (now claims 9 and 10, respectively).[0009](vii) Original claims 1 , 3 - 5, 10 and 11 have further been amended for clarity purposes.

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