Effects of tannic acid extract on performance and intestinal health of broiler chickens following coccidiosis vaccination and/or a mixed-species Eimeria challenge

Effects of tannic acid extract on performance and intestinal health of broiler chickens following... ABSTRACT Two experiments were conducted to investigate the effects of tannic acid extract (TAE) formulations on the performance and intestinal health of male Cobb × Cobb 500 broilers exposed to coccidiosis. In the first experiment, 320 broiler chicks were randomly assigned to 5 treatments with 8 replicates. Treatments included non-medicated, uninfected (NC); non-medicated, infected (PC); salinomycin (SAL, 66 mg/kg); tannic acid (TA, 0.5 g/kg) and TAE (TAE, 0.5 g/kg). On d 14, all groups (except NC) were orally inoculated with Eimeria acervulina, E. maxima and E. tenella oocysts. Intestinal lesion scores, fecal oocyst counts (OPG) and performance were evaluated on d 20. The PC had greater lesions and higher FCR than infected, supplemented groups. Only TAE reduced OPG compared to PC (P < 0.05). In the second experiment, 3,000 broiler chicks were vaccinated on day of hatch with live coccidial oocysts, then randomly assigned to 5 treatments with 15 replicates. Treatments included non-medicated (CNT); salinomycin (SAL, 66 mg/kg); robenidine (ROB, 33 mg/kg); TAE (0.5 g/kg) and TAE with Bacillus coagulans (TAE+BC, 0.5 g/kg). On d 29, a subset of pens (n = 20) were challenged with a mixed Eimeria spp. oral inoculum; performance, lesions and OPG were evaluated on d 35. An immune challenge was created in half the pens by issuing broilers feed without supplementation materials during the challenge. For the non-challenged pens (n = 55), performance was measured up to d 49. Performance of non-challenged, vaccinated-CNT birds was improved with all treatments at d 21 and d 49. Among the challenged birds, withdrawal of SAL or ROB resulted in FCR similar to the challenged CNT group (P > 0.05), whereas withdrawal of TAE or TAE+BC maintained improved FCR compared to challenged-CNT birds (P < 0.05). These findings indicate supplementation of TAE and TAE+BC with coccidiosis vaccination can be considered as a potential alternative strategy to address coccidiosis in broiler chickens. INTRODUCTION Coccidiosis is a ubiquitous disease in the commercial poultry industry and is estimated to cost poultry producers more than $800 million each year (Williams, 1998). In poultry, the causative agents of coccidiosis are apicomplexan protozoan parasites of the genus Eimeria. These parasites invade the intestinal epithelium in a site-specific manner, causing inflammation and necrosis of the mucosal barrier and underlying tissue (Vermeulen, 2001). Lesion formation in the intestinal epithelium results in nutrient malabsorption, diarrhea, reduced weight gain and a concomitant decrease in feed efficiency (Lillehoj and Trout, 1993; Williams, 2005). The reductions in performance due to subclinical Eimeria spp. infection attribute approximately 80% of the worldwide cost of coccidiosis (Williams, 1999). Of the seven species of Eimeria known to infect poultry, E. acervulina, E. maxima and E. tenella are the most frequently diagnosed. For the past five decades, chemotherapeutic agents have been used to control coccidiosis (Lillehoj and Lillehoj, 2000). Large-scale preventative use of these chemical anticoccidials and ionophoric feed additives is convenient for producers and has greatly enhanced the productivity of the commercial poultry industry (Chapman, 1997). However, long-term, wide-spread use of a limited pool of chemotherapeutics has resulted in decreased sensitivity of Eimeria to many anticoccidials used in commercial poultry production today (Long, 1982; Chapman, 1997). Growing public health concerns related to development of antimicrobial resistance and anxiety over potential drug residues in egg and meat products have further increased the pressure on commercial poultry producers to reduce, even eliminate, the use of antimicrobials and anticoccidials in poultry diets. Therefore, poultry producers are actively looking for alternative methods of medication to control coccidiosis, such as vaccinations or use of feed additives like plant extracts and probiotics. Live coccidia vaccines function by enhancing the natural immunity of the animal to coccidia, due to recycling of Eimeria oocysts through poultry litter (Chapman et al., 2002). In order to provide broad-spectrum immunity, vaccines typically contain attenuated or non-attenuated live oocysts of multiple Eimeria spp. (Lillehoj and Lillehoj, 2000; Dalloul and Lillehoj, 2005). While vaccines have been used for many years to control coccidiosis in broiler breeders (Chapman, 1999), implementation of vaccination for preventative coccidiosis control in meat-producing broilers has not been universally accepted by the U.S. poultry industry (Danforth, 1998). Producer hesitancy to utilize vaccination as a coccidiosis management practice is based on reports of reduced weight gain and feed efficiency in vaccinated broilers compared to non-vaccinated broilers (Danforth, 1998; Williams, 2002). Despite these concerns, commercial vaccination programs are playing a more prominent role in commercial broiler production, as using vaccines can potentially reestablish drug sensitivity of Eimeria to anticoccidial chemicals (Chapman, 2000; Chapman et al., 2002; Chapman and Jeffers, 2014). Feed additives, such as plant extracts and probiotics, have shown success in positively influencing the intestinal microbial balance of poultry. Numerous reports have suggested plant extracts, especially polyphenolic bioactive molecules, may have potential as alternative anticoccidial agents (Allen and Fetterer, 2002; Naidoo et al., 2008; Windisch et al., 2008; Abbas et al., 2012; Wunderlich et al., 2014). Plant bioactives such as tannins, saponins and flavonoids have well known antioxidant and anti-inflammatory activities. These functional activities provide potential modes of action in which bioactives may help protect the intestinal epithelium from oxidative damage (Kaur et al., 2008; Hamiza et al., 2012). Further, tannins have known anti-parasitic (Min and Hart, 2003) and antimicrobial activities due to their ability to complex with microbial enzymes and metal ions (Scalbert, 1991; Chung et al., 1998a). Although tannins are often considered undesirable in poultry diets because of their ability to precipitate proteins and inhibit digestive enzymes (Chung et al., 1998b), both the level of dietary tannin and the tannin structure impact the nutritive or anti-nutritive properties of the tannin (Mueller-Harvey, 2006). Tannins are a structurally diverse group of complex polyphenolic compounds, which are classified into two groups, hydrolysable and condensed tannins, based on molecular structure. Recent studies have suggested that tannins may be potential alternative growth promoters for poultry diets (Tosi et al., 2013; Redondo et al., 2014). Unlike antibiotic growth promoters, the development of bacterial resistance to tannins is postulated to be difficult due to the complex structure of the tannin molecules. In vitro (Ahn et al., 1998; Chung et al., 1998a; Elizondo et al., 2010; Redondo et al., 2015) and in vivo (Tosi et al. 2013) studies have shown several tannin sources to be effective at inhibiting growth of poultry pathogens, including C. perfringens, the causative agent of necrotic enteritis. Multiple studies have tested the efficacy of common tannin sources, chestnut (Castanea sativa; hydrolysable tannin) and quebracho (Schinopsis lorentzii; condensed tannin), to control Eimeria infections (McCann et al., 2006; Cejas et al., 2011; Hooge et al., 2012). However, there is a paucity of literature discussing the use of tannic acid, a model hydrolysable tannin, to control avian parasitic diseases like coccidiosis (Mansoori and Modirsanei, 2012; Kaleem et al., 2014). Probiotic supplementation in human and animal health is associated with improved intestinal microbial balance, enhanced immune activity and stronger gut defense against enteric disease (Huyghebaert, et al. 2011). According to Yang et al. (2009), probiotics can maintain beneficial microbial populations in the gut via competitive exclusion and immune modulation. An increasing number of studies have been reported, which evaluated the ability of probiotics to control parasitic diseases like coccidiosis (Lee et al., 2007; Stringfellow et al., 2011; Abdelrahman et al., 2014). Further, indirect effects of probiotics towards reducing Eimeria induced intestinal lesions has recently been suggested (Bozkurt et al., 2014). Multifaceted strategies towards modulating poultry gut health and immune development are necessary for controlling coccidiosis. Vaccination combined with dietary supplementation of tannins or probiotics could alleviate intestinal challenges associated with vaccination, thereby providing an effective alternative method of coccidiosis control and further mitigation of concurrent enteric diseases (Van Immerseel et al. 2004; Williams, 2005). The overall aim of the present study was thus to evaluate the protective effects of a gallnut tannic acid extract (TAE) to control coccidiosis under the experimental conditions. Three objectives were proposed: (1) to evaluate the ability of tannic acid products to reduce a mixed Eimeria spp. challenge; (2) to compare the effects of TAE, TAE combined with a probiotic and in-feed anticoccidials on broilers vaccinated at day of hatch with live oocysts; and (3) to assess the impact of the aforementioned materials on protective immunity development in vaccinated broilers infected with mixed Eimeria spp. challenge at 29 days of age. MATERIALS AND METHODS The two experiments were conducted at Southern Poultry Research in Athens, Georgia. In both experiments, Cobb × Cobb 500 broilers were obtained from the Cobb-Vantress hatchery in Cleveland, Georgia. Bird sexing and vaccination were completed at the hatchery without administration of any coccidia vaccine. For all experiments, age-appropriate supplemental heat was provided following breeder recommendations. Access to feed and water was provided ad libitum. Experiments were conducted in accordance with the principles and specific guidelines presented in the Federation of Animal Science Societies (FASS, 2010). Birds and housing facilities were monitored twice daily. Facilities were checked for general health status, feed and water supply, temperature, unexpected events and removal of dead birds. Daily mortality record was maintained and birds were not replaced. Coccidial oocysts used for challenge studies were field-strain Eimeria and were isolated from U.S. commercial production broiler houses. Coccidia strains were maintained as individual strains by periodic propagation and passage and were enumerated prior to challenge. Experiment 1 A total of 320 male Cobb-500 broiler chicks were randomly assigned to 5 dietary treatments, with 8 replicate cages of 8 birds per cage (0.63 ft2/bird). Broilers were housed in thermostatically controlled Petersime battery units (Petersime Incubator, Co., Gettysburg, OH) with the battery cage serving as the experimental unit. A commercial starter diet composition (Table 1) without additives, anticoccidial and growth promoter was formulated to meet NRC (1994) requirements. Treatment groups consisted of (1) negative control group (NC): commercial feed, untreated, unchallenged; (2) positive control group (PC): commercial feed, untreated, challenged with a mixed Eimeria spp.; (3) control diet supplemented with salinomycin (SAL): commercial feed with 66 mg salinomycin/kg of feed, challenged with a mixed Eimeria spp.; (4) control diet supplemented with tannic acid (TA): commercial feed with TA, challenged with a mixed Eimeria spp.; (5) control diet supplemented with tannic acid extract (TAE): commercial feed with TAE, challenged with a mixed Eimeria species. The products used in this experiment were: ionophore anticoccidial salinomcyin, Sacox 60 (Huvepharma, Inc., Peachtree City, GA) added at 66 g/MT to feed, a commercial food grade TA (Hubei Province, China) and a food grade TAE (Kemin Industries, Inc., Des Moines, IA). The TAE product is a proprietary formulation of hydrolysable tannins extracted from Rhus chinensis gallnuts. Both TA and TAE were mixed into finished feed at inclusion rates of 500 g/MT of feed, giving final concentrations of 100 mg TA per kg of feed and 100 mg TAE per kg of feed, respectively. All feeds were pelletized and were provided as crumbled pellets for the duration of the study. Table 1. Composition of the experimental starter diet and its nutrient profile (Experiment 1). Amount (% Item unless noted) Ingredient  Corn 57.10  Soybean meal dehulled 36.86  Fat 2.33  Dicalcium phosphate 1.41  Calcium carbonate 1.26  Sodium chloride 0.44  DL-Methionine 0.31  L-Lysine 0.10  L-Threonine (98.5%) 0.02  Ronozyme P-(ct) 0.02  Trace Mineral1 0.08  Vitamin premix2 0.07 Analyzed value3 (%)  DM 88.08  CP 23.44  Crude fiber 2.38  Crude fat 4.57  Calcium 0.90  Phosphorus (total) 0.60 Calculated value  ME (kcal/kg) 3067  Methionine 0.62  TSAA 0.90  Lysine 1.35  Threonine 0.95  Tryptophan 0.30  Sodium 0.21  Potassium 0.84  Chloride 0.28 Amount (% Item unless noted) Ingredient  Corn 57.10  Soybean meal dehulled 36.86  Fat 2.33  Dicalcium phosphate 1.41  Calcium carbonate 1.26  Sodium chloride 0.44  DL-Methionine 0.31  L-Lysine 0.10  L-Threonine (98.5%) 0.02  Ronozyme P-(ct) 0.02  Trace Mineral1 0.08  Vitamin premix2 0.07 Analyzed value3 (%)  DM 88.08  CP 23.44  Crude fiber 2.38  Crude fat 4.57  Calcium 0.90  Phosphorus (total) 0.60 Calculated value  ME (kcal/kg) 3067  Methionine 0.62  TSAA 0.90  Lysine 1.35  Threonine 0.95  Tryptophan 0.30  Sodium 0.21  Potassium 0.84  Chloride 0.28 1Trace mineral mix provided the following (per kg of diet): manganese (MnSO4•H2O), 60 mg; iron (FeSO4•7H2O), 30 mg; zinc (ZnO), 50 mg; copper (CuSO4•5H2O), 5 mg; iodine (ethylene diamine dihydroiodide), 0.15 mg; selenium (NaSe03), 0.3 mg. 2Vitamin mix provided the following (per kg of diet): Vitamin A, 12,345 IU; Vitamin D3, 3472 IU; 25-hydroxyvitamin D3, 96.6 μg; Vitamin E, 49 IU; vitamin B12 (cobalamin), 21.7 μg; Biotin, 0.23 mg; Menadione, 2.78 mg; Thiamine, 2.62 mg; Riboflavin, 10.8 mg; d-Panthothenic Acid, 18.5 mg; Vitamin B6, 4.6 mg; Niacin, 61.7 mg; Folic Acid, 1.5 mg. 3Analyzed values refer to basal (control) diet. View Large Table 1. Composition of the experimental starter diet and its nutrient profile (Experiment 1). Amount (% Item unless noted) Ingredient  Corn 57.10  Soybean meal dehulled 36.86  Fat 2.33  Dicalcium phosphate 1.41  Calcium carbonate 1.26  Sodium chloride 0.44  DL-Methionine 0.31  L-Lysine 0.10  L-Threonine (98.5%) 0.02  Ronozyme P-(ct) 0.02  Trace Mineral1 0.08  Vitamin premix2 0.07 Analyzed value3 (%)  DM 88.08  CP 23.44  Crude fiber 2.38  Crude fat 4.57  Calcium 0.90  Phosphorus (total) 0.60 Calculated value  ME (kcal/kg) 3067  Methionine 0.62  TSAA 0.90  Lysine 1.35  Threonine 0.95  Tryptophan 0.30  Sodium 0.21  Potassium 0.84  Chloride 0.28 Amount (% Item unless noted) Ingredient  Corn 57.10  Soybean meal dehulled 36.86  Fat 2.33  Dicalcium phosphate 1.41  Calcium carbonate 1.26  Sodium chloride 0.44  DL-Methionine 0.31  L-Lysine 0.10  L-Threonine (98.5%) 0.02  Ronozyme P-(ct) 0.02  Trace Mineral1 0.08  Vitamin premix2 0.07 Analyzed value3 (%)  DM 88.08  CP 23.44  Crude fiber 2.38  Crude fat 4.57  Calcium 0.90  Phosphorus (total) 0.60 Calculated value  ME (kcal/kg) 3067  Methionine 0.62  TSAA 0.90  Lysine 1.35  Threonine 0.95  Tryptophan 0.30  Sodium 0.21  Potassium 0.84  Chloride 0.28 1Trace mineral mix provided the following (per kg of diet): manganese (MnSO4•H2O), 60 mg; iron (FeSO4•7H2O), 30 mg; zinc (ZnO), 50 mg; copper (CuSO4•5H2O), 5 mg; iodine (ethylene diamine dihydroiodide), 0.15 mg; selenium (NaSe03), 0.3 mg. 2Vitamin mix provided the following (per kg of diet): Vitamin A, 12,345 IU; Vitamin D3, 3472 IU; 25-hydroxyvitamin D3, 96.6 μg; Vitamin E, 49 IU; vitamin B12 (cobalamin), 21.7 μg; Biotin, 0.23 mg; Menadione, 2.78 mg; Thiamine, 2.62 mg; Riboflavin, 10.8 mg; d-Panthothenic Acid, 18.5 mg; Vitamin B6, 4.6 mg; Niacin, 61.7 mg; Folic Acid, 1.5 mg. 3Analyzed values refer to basal (control) diet. View Large On the day of experimental diet placement, birds were randomly allotted to dietary treatments and bird weights by cage were recorded to ensure average initial cage weight was similar between groups. Live body weight (BW), feed intake (FI), body weight gain (BWG) and feed conversion ratio (FCR) were determined at d 14 and 20. FCR was calculated and corrected for BW of mortality. On d 14, all broilers, except those in NC treatment, were challenged with a 1 mL mixed-species Eimeria oral inoculum consisting of E. acervulina (5 × 105), E. maxima (8.5 × 104) and E. tenella (1.75 × 105) sporulated oocysts. NC birds were inoculated with 1 mL of saline solution. Samples of feces passed between 120 to 144 h post-challenge were collected from all cages for oocyst counting. Feces collected per cage were homogenized and stored at 4°C until measurement of oocyst counts. Counts were determined via dilution and microscopic enumeration using a McMaster counting chamber and are reported as oocysts per gram of excreta. On d 20 (6 d post-challenge), all birds were euthanized and necropsied to determine the presence and degree of coccidiosis lesions in the upper, middle and cecal regions of the intestinal tract (Johnson and Reid, 1970). Experiment 2 A total of 3000 male Cobb-500 broiler chicks were randomly assigned to 5 dietary treatments, with 15 replicate pens of 40 birds per pen. The experimental house contained 75 pens of equal size, each having an area of 50 ft2, with built-up wood shavings as bedding with a thickness of approximately 4 inches. The built-up bedding was from 3 grow-out cycles. Each pen had 5 ft high side walls with 1.5 ft bottom solid wood to prevent bird migration. Water was provided ad libitum from one Plasson-type watering fount per pen. The initial stocking density, after subtracting out for equipment, was 1.16 ft2/bird. The pen served as the experimental unit. The growth period was divided into 3 phases: starter (d 0 to 21), grower (d 21 to 35) and finisher (d 35 to 49). Commercial feed (Table 2) without additives, anticoccidial and growth promoter was formulated to meet NRC guidelines (1994) and the requirements for the birds’ growing stage. All diets were pelletized, with the starter phase provided as crumbled pellets. Treatment groups consisted of (1) control group (CNT): commercial feed, cocci-vaccinated; (2) control supplemented with salinomycin (SAL): commercial feed with 66 mg salinomycin/kg of feed, cocci-vaccinated.; (3) control supplemented with robenidine (ROB): commercial feed with 33 mg robenidine/kg of feed, cocci-vaccinated; (4) control supplemented with tannic acid extract (TAE): commercial feed with TAE, cocci-vaccinated; (5) control supplemented with TAE and probiotic (TAE+BC): commercial feed with TAE and probiotic, cocci-vaccinated. The anticoccidial products used in this experiment were: ionophore anticoccidial salinomycin Bio-Cox 60 (Zoetis, Florham Park, NJ) added at 66 g/MT of feed and chemical anticoccidial robenidine Robenz (Zoetis, Florham Park, NJ) added at 33 g/MT in feed. The TAE product (Kemin Industries, Inc., Des Moines, IA) was mixed into finished feed at an inclusion rate of 500 g/MT of feed, giving a final concentration of 100 mg TAE per kg of feed. The TAE and probiotic combination product used TAE combined with a single-species Bacillus coagulans probiotic having a content of ≥ 1.0 × 107 spores per kg (VANNIX C, Kemin Industries, Inc., Des Moines, IA). The product was mixed into the finished feed at an inclusion rate of 500 g/MT of feed, giving a final concentration of 100 mg TAE per kg of feed and ≥1.0 × 104 spores of B. coagulans per kg of feed. Table 2. Composition of the experimental starter, grower and finisher diets and their nutrient profiles (Experiment 2). Diet Item Starter Grower Finisher Ingredient ———————-%————————  Corn 55.44 60.05 65.37  Soybean meal dehulled 35.71 31.10 26.38  Corn DDGS 4.00 4.00 4.00  Fat 1.26 1.73 1.59  Dicalcium phosphate 1.28 0.99 0.77  Calcium carbonate 1.15 1.11 0.92  Sodium chloride 0.44 0.42 0.43  DL-Methionine 0.35 0.26 0.22  L-Lysine 0.21 0.20 0.17  Ronozyme P-(ct) 0.02 0.02 0.02  Trace Mineral1 0.08 0.08 0.08  Vitamin premix2 0.07 0.07 0.07 Analyzed value3 (%)  DM 88.10 88.05 87.93  CP 22.93 21.01 19.12  Crude fiber 2.42 2.37 2.33  Crude fat 3.96 4.54 4.54  Calcium 0.92 0.83 0.70  Phosphorus (total) 0.64 0.57 0.51 Calculated value  ME (kcal/kg) 3000 3080 3130  Methionine 0.67 0.57 0.51  TSAA 0.94 0.82 0.75  Lysine 1.42 1.28 1.13  Threonine 0.93 0.85 0.77  Tryptophan 0.30 0.27 0.24  Sodium 0.21 0.20 0.20  Potassium 0.89 0.81 0.74  Chloride 0.31 0.29 0.30 Diet Item Starter Grower Finisher Ingredient ———————-%————————  Corn 55.44 60.05 65.37  Soybean meal dehulled 35.71 31.10 26.38  Corn DDGS 4.00 4.00 4.00  Fat 1.26 1.73 1.59  Dicalcium phosphate 1.28 0.99 0.77  Calcium carbonate 1.15 1.11 0.92  Sodium chloride 0.44 0.42 0.43  DL-Methionine 0.35 0.26 0.22  L-Lysine 0.21 0.20 0.17  Ronozyme P-(ct) 0.02 0.02 0.02  Trace Mineral1 0.08 0.08 0.08  Vitamin premix2 0.07 0.07 0.07 Analyzed value3 (%)  DM 88.10 88.05 87.93  CP 22.93 21.01 19.12  Crude fiber 2.42 2.37 2.33  Crude fat 3.96 4.54 4.54  Calcium 0.92 0.83 0.70  Phosphorus (total) 0.64 0.57 0.51 Calculated value  ME (kcal/kg) 3000 3080 3130  Methionine 0.67 0.57 0.51  TSAA 0.94 0.82 0.75  Lysine 1.42 1.28 1.13  Threonine 0.93 0.85 0.77  Tryptophan 0.30 0.27 0.24  Sodium 0.21 0.20 0.20  Potassium 0.89 0.81 0.74  Chloride 0.31 0.29 0.30 1Trace mineral mix provided the following (per kg of diet): manganese (MnSO4•H2O), 60 mg; iron (FeSO4•7H2O), 30 mg; zinc (ZnO), 50 mg; copper (CuSO4•5H2O), 5 mg; iodine (ethylene diamine dihydroiodide), 0.15 mg; selenium (NaSe03), 0.3 mg. 2Vitamin mix provided the following (per kg of diet): Vitamin A, 12,345 IU; Vitamin D3, 3472 IU; 25-hydroxyvitamin D3, 96.6 μg; Vitamin E, 49 IU; vitamin B12 (cobalamin), 21.7 μg; Biotin, 0.23 mg; Menadione, 2.78 mg; Thiamine, 2.62 mg; Riboflavin, 10.8 mg; d-Panthothenic Acid, 18.5 mg; Vitamin B6, 4.6 mg; Niacin, 61.7 mg; Folic Acid, 1.5 mg. 3Analyzed values refer to basal (control) diet. View Large Table 2. Composition of the experimental starter, grower and finisher diets and their nutrient profiles (Experiment 2). Diet Item Starter Grower Finisher Ingredient ———————-%————————  Corn 55.44 60.05 65.37  Soybean meal dehulled 35.71 31.10 26.38  Corn DDGS 4.00 4.00 4.00  Fat 1.26 1.73 1.59  Dicalcium phosphate 1.28 0.99 0.77  Calcium carbonate 1.15 1.11 0.92  Sodium chloride 0.44 0.42 0.43  DL-Methionine 0.35 0.26 0.22  L-Lysine 0.21 0.20 0.17  Ronozyme P-(ct) 0.02 0.02 0.02  Trace Mineral1 0.08 0.08 0.08  Vitamin premix2 0.07 0.07 0.07 Analyzed value3 (%)  DM 88.10 88.05 87.93  CP 22.93 21.01 19.12  Crude fiber 2.42 2.37 2.33  Crude fat 3.96 4.54 4.54  Calcium 0.92 0.83 0.70  Phosphorus (total) 0.64 0.57 0.51 Calculated value  ME (kcal/kg) 3000 3080 3130  Methionine 0.67 0.57 0.51  TSAA 0.94 0.82 0.75  Lysine 1.42 1.28 1.13  Threonine 0.93 0.85 0.77  Tryptophan 0.30 0.27 0.24  Sodium 0.21 0.20 0.20  Potassium 0.89 0.81 0.74  Chloride 0.31 0.29 0.30 Diet Item Starter Grower Finisher Ingredient ———————-%————————  Corn 55.44 60.05 65.37  Soybean meal dehulled 35.71 31.10 26.38  Corn DDGS 4.00 4.00 4.00  Fat 1.26 1.73 1.59  Dicalcium phosphate 1.28 0.99 0.77  Calcium carbonate 1.15 1.11 0.92  Sodium chloride 0.44 0.42 0.43  DL-Methionine 0.35 0.26 0.22  L-Lysine 0.21 0.20 0.17  Ronozyme P-(ct) 0.02 0.02 0.02  Trace Mineral1 0.08 0.08 0.08  Vitamin premix2 0.07 0.07 0.07 Analyzed value3 (%)  DM 88.10 88.05 87.93  CP 22.93 21.01 19.12  Crude fiber 2.42 2.37 2.33  Crude fat 3.96 4.54 4.54  Calcium 0.92 0.83 0.70  Phosphorus (total) 0.64 0.57 0.51 Calculated value  ME (kcal/kg) 3000 3080 3130  Methionine 0.67 0.57 0.51  TSAA 0.94 0.82 0.75  Lysine 1.42 1.28 1.13  Threonine 0.93 0.85 0.77  Tryptophan 0.30 0.27 0.24  Sodium 0.21 0.20 0.20  Potassium 0.89 0.81 0.74  Chloride 0.31 0.29 0.30 1Trace mineral mix provided the following (per kg of diet): manganese (MnSO4•H2O), 60 mg; iron (FeSO4•7H2O), 30 mg; zinc (ZnO), 50 mg; copper (CuSO4•5H2O), 5 mg; iodine (ethylene diamine dihydroiodide), 0.15 mg; selenium (NaSe03), 0.3 mg. 2Vitamin mix provided the following (per kg of diet): Vitamin A, 12,345 IU; Vitamin D3, 3472 IU; 25-hydroxyvitamin D3, 96.6 μg; Vitamin E, 49 IU; vitamin B12 (cobalamin), 21.7 μg; Biotin, 0.23 mg; Menadione, 2.78 mg; Thiamine, 2.62 mg; Riboflavin, 10.8 mg; d-Panthothenic Acid, 18.5 mg; Vitamin B6, 4.6 mg; Niacin, 61.7 mg; Folic Acid, 1.5 mg. 3Analyzed values refer to basal (control) diet. View Large Live Performance Upon arrival at the trial facility, all birds were vaccinated on day of hatch by spray cabinet with a commercial coccidia vaccine, Advent (Novus International, Inc., St. Louis, MO), which contains viable attenuated oocysts of E. acervulina, E. maxima and E. tenella. Birds were allowed to preen for 1 h to improve vaccine uptake prior to placement. Vaccinated birds were randomly allotted to dietary treatments and bird weights by pen were recorded to ensure average initial pen weight was similar between groups. On d 1, 21, 35, and 49, BW, BWG and FI were determined and FCR was calculated for d 21, 35, and 49 following correction for BW of mortality. On d 28 and 49, litter conditions were graded. A grading scale of 0–4, 0 being dry and 1, 2, 3, or 4 indicating increased wetness and unacceptability, developed by Abdelrahman et al. (2014) was used for litter quality grading. Coccidial Immunity Challenge On d 28, 4 pens per treatment (n = 20 pens) were allocated to a coccidial immunity challenge. Birds and feed were weighed by pen and treatment pens were divided into two subgroups, one of which was reissued commercial grower feed containing supplemented additives (group 1) and the other, which additives were withdrawn from the reissued feed (group 2). On d 29, all birds in the 20 pens were challenged with a mixed-species Eimeria oral inoculum consisting of E. acervulina (1 × 105), E. maxima (5 × 104) and E. tenella (7.5 × 104) sporulated oocysts. On d 35 (6 d post-challenge), feed was weighed back, pen weights were taken and BW, BWG, FI and mortality corrected FCR were determined for the challenge period (d 28 to 35). In addition, 20 broilers from each pen were euthanized and necropsied for scoring of coccidiosis lesions in the upper, middle and cecal regions of the intestinal tract (Johnson and Reid, 1970). Fresh fecal samples (10 per pen) were also collected on d 35, which were pooled and stored at 4°C until measurement of oocyst counts. Counts were determined via dilution and microscopic enumeration using a McMaster counting chamber and are reported as oocysts per gram of excreta. Statistical Analysis Experiment 1 Data were analyzed by ANOVA using the GLM procedure of SPSS (IMB SPSS 19, IMB Corp., Armonk, NY). Cage served as the experimental unit. Statistical significance was determined by one-way ANOVA and means were separated by least significant differences. The threshold for statistical significance was P ≤ 0.05. Experiment 2 BWG, FI and FCR for d 0 to 21 and d 0 to 49 were analyzed by ANOVA using the GLM procedure of SPSS (IMB SPSS 19, IMB Corp., Armonk, NY). Pen served as the experimental unit. Statistical significance was determined by one-way ANOVA and means were separated by least significant differences. Due to the experimental blocking scheme used in the coccidial immunity challenge, vaccinated, challenged control pens were averaged separately (n = 2 pens) rather than together (n = 4 pens) for analysis. For the coccidial challenge, data collected at d 35, including post-challenge BWG, FI, FCR, lesion scores and oocysts, were analyzed using one-way ANOVA. Means were separated by Duncan's multiple range test with differences deemed significant at P ≤ 0.05. RESULTS Experiment 1 Initial cage weight was not different (P = 0.2473) among groups. As anticipated, broilers challenged with mixed Eimeria spp. had reduced performance compared to unchallenged broilers (Table 3). During the challenge period (d 14 to 20), broilers challenged with Eimeria had reduced (P < 0.05) BWG and FI and significantly higher FCR than NC. No difference was observed in the BWG of SAL, TA and TAE from PC, whereas TA had reduced (P < 0.05) BWG compared to SAL. Reduced (P < 0.05) FI was observed with TA compared to PC and SAL, but was not different from TAE. FCR was improved (P < 0.05) with SAL and TAE compared to PC, while TA was similar to PC, SAL and TAE. Over d 1 to 20, FI was similar for all groups, whereas Eimeria challenge resulted in depressed (P < 0.05) BWG and worsened (P < 0.05) FCR compared to NC. No difference in BWG and FCR was observed between PC, SAL, TA and TAE. During both d 14 to 20 and d 1 to 20, FI, BWG and FCR were similar (P ≥ 0.05) for SAL and TAE, however, no supplemented challenged group recovered performance to that observed in the NC. Table 3. Body weight gain (BWG; g), feed intake (FI; g) and mortality-corrected feed conversion ratio (FCR) of broilers which were infected with a mixed-species Eimeria spp. inoculum on d 14 of age, when fed diets supplemented with anticoccidial ionophore, tannic acid and tannic acid extract (Experiment 1). d 14 to 20 d 1 to 20 Infection1 Item2 BWG FI FCR BWG FI FCR − NC 295a 398a 1.35c 530a 791 1.50b + PC 177b,c 349b 2.00a 416b 730 1.76a SAL 198b 352b 1.79b 451b 774 1.72a TA 173c 311c 1.86a,b 433b 730 1.71a TAE 179b,c 324b,c 1.82b 423b 726 1.72a SEM3 $$\phantom{>}$$8.1 $$\phantom{0}$$11.0 $$\phantom{>}$$0.05 14.6 18.0 0.03a P-value <0.001 <0.001 <0.001 <0.001 0.051 <0.001 d 14 to 20 d 1 to 20 Infection1 Item2 BWG FI FCR BWG FI FCR − NC 295a 398a 1.35c 530a 791 1.50b + PC 177b,c 349b 2.00a 416b 730 1.76a SAL 198b 352b 1.79b 451b 774 1.72a TA 173c 311c 1.86a,b 433b 730 1.71a TAE 179b,c 324b,c 1.82b 423b 726 1.72a SEM3 $$\phantom{>}$$8.1 $$\phantom{0}$$11.0 $$\phantom{>}$$0.05 14.6 18.0 0.03a P-value <0.001 <0.001 <0.001 <0.001 0.051 <0.001 a-cMeans within a column not sharing the same superscript differ significantly (P < 0.05). 1Mixed-species challenge contained Eimeria acervulina (5 × 105), Eimeria maxima (8.5 × 104) and Eimeria tenella (1.75 × 105) sporulated oocysts. 2NC = negative control. The broilers were fed a non-medicated diet containing no anticoccidial and growth promoter and were supplemented with anticoccidial ionophore salinomycin (SAL; 66 mg/kg of diet), tannic acid (TA; 100 mg/kg of diet) and tannic acid extract (TAE; 100 mg/kg of diet). 3Pooled standard error of the mean. View Large Table 3. Body weight gain (BWG; g), feed intake (FI; g) and mortality-corrected feed conversion ratio (FCR) of broilers which were infected with a mixed-species Eimeria spp. inoculum on d 14 of age, when fed diets supplemented with anticoccidial ionophore, tannic acid and tannic acid extract (Experiment 1). d 14 to 20 d 1 to 20 Infection1 Item2 BWG FI FCR BWG FI FCR − NC 295a 398a 1.35c 530a 791 1.50b + PC 177b,c 349b 2.00a 416b 730 1.76a SAL 198b 352b 1.79b 451b 774 1.72a TA 173c 311c 1.86a,b 433b 730 1.71a TAE 179b,c 324b,c 1.82b 423b 726 1.72a SEM3 $$\phantom{>}$$8.1 $$\phantom{0}$$11.0 $$\phantom{>}$$0.05 14.6 18.0 0.03a P-value <0.001 <0.001 <0.001 <0.001 0.051 <0.001 d 14 to 20 d 1 to 20 Infection1 Item2 BWG FI FCR BWG FI FCR − NC 295a 398a 1.35c 530a 791 1.50b + PC 177b,c 349b 2.00a 416b 730 1.76a SAL 198b 352b 1.79b 451b 774 1.72a TA 173c 311c 1.86a,b 433b 730 1.71a TAE 179b,c 324b,c 1.82b 423b 726 1.72a SEM3 $$\phantom{>}$$8.1 $$\phantom{0}$$11.0 $$\phantom{>}$$0.05 14.6 18.0 0.03a P-value <0.001 <0.001 <0.001 <0.001 0.051 <0.001 a-cMeans within a column not sharing the same superscript differ significantly (P < 0.05). 1Mixed-species challenge contained Eimeria acervulina (5 × 105), Eimeria maxima (8.5 × 104) and Eimeria tenella (1.75 × 105) sporulated oocysts. 2NC = negative control. The broilers were fed a non-medicated diet containing no anticoccidial and growth promoter and were supplemented with anticoccidial ionophore salinomycin (SAL; 66 mg/kg of diet), tannic acid (TA; 100 mg/kg of diet) and tannic acid extract (TAE; 100 mg/kg of diet). 3Pooled standard error of the mean. View Large Oocyst shedding was reduced by 58% in TAE compared to PC, while SAL reduced oocysts by 21% compared to PC, albeit all Eimeria challenged groups had increased (P < 0.05) oocyst shedding compared to NC (Figure 1). A 51% reduction in oocysts was additionally observed with TAE compared to TA. Overall, significant reduction in oocyst shedding was observed with TAE compared to PC and TA, whereas SAL was not different from PC, TA and TAE. Lesion scores were recorded separately for the upper, middle, and cecal intestinal regions (Figure 2). No lesions were observed in any region of the intestine in NC. Eimeria challenged broilers supplemented with SAL, TA and TAE had reduced (P < 0.05) lesions in the upper intestine, middle intestine and ceca compared to PC. Lesion scores in the upper intestine and ceca were not different between SAL, TAE and TA, whereas TAE had significantly fewer middle intestine lesions than SAL. While performance and lesion reductions were similar for TA and TAE, TAE showed significantly improved reduction in oocyst shedding compared to TA and was therefore selected for use in Experiment 2. Figure 1. View largeDownload slide Total oocysts per gram (OPG) of excreta in broilers infected with Eimeria spp. on d 14 of age in Experiment 1. Excreta samples were collected from broilers 6 d after mixed Eimeria spp. challenge. Means represent 8 replicates per treatment (1 sample/cage). NC = negative control (non-medicated feed, uninfected); PC = positive control (non-medicated feed, infected); SAL = salinomycin supplemented feed, infected (Sacox 60, 66 mg/kg of diet); TA = tannic acid supplemented feed, infected (100 mg/kg of diet); TAE = tannic acid extract supplemented feed, infected (TAE, 100 mg/kg of diet). Means with different letters (a-c) differ significantly (P < 0.01). Figure 1. View largeDownload slide Total oocysts per gram (OPG) of excreta in broilers infected with Eimeria spp. on d 14 of age in Experiment 1. Excreta samples were collected from broilers 6 d after mixed Eimeria spp. challenge. Means represent 8 replicates per treatment (1 sample/cage). NC = negative control (non-medicated feed, uninfected); PC = positive control (non-medicated feed, infected); SAL = salinomycin supplemented feed, infected (Sacox 60, 66 mg/kg of diet); TA = tannic acid supplemented feed, infected (100 mg/kg of diet); TAE = tannic acid extract supplemented feed, infected (TAE, 100 mg/kg of diet). Means with different letters (a-c) differ significantly (P < 0.01). Figure 2. View largeDownload slide Intestinal lesion scores (LS) observed in broilers infected with Eimeria spp. on d 14 of age in Experiment 1. Lesion scores were assessed 6 d after mixed Eimeria spp. challenge. A lesion score was assigned from 0 (no gross lesions) to 4 (extensive lesions) according to the system of Johnson and Reid (1970). Means represent 8 replicates per treatment (8 samples/cage). NC = negative control (non-medicated feed, uninfected); PC = positive control (non-medicated feed, infected); SAL = salinomycin supplemented feed, infected (Sacox 60, 66 mg/kg of diet); TA = tannic acid supplemented feed, infected (100 mg/kg of diet); TAE = tannic acid extract supplemented feed, infected (TAE, 100 mg/kg of diet). Within each intestinal region, means with different letters (a-d) differ significantly (P < 0.01). Figure 2. View largeDownload slide Intestinal lesion scores (LS) observed in broilers infected with Eimeria spp. on d 14 of age in Experiment 1. Lesion scores were assessed 6 d after mixed Eimeria spp. challenge. A lesion score was assigned from 0 (no gross lesions) to 4 (extensive lesions) according to the system of Johnson and Reid (1970). Means represent 8 replicates per treatment (8 samples/cage). NC = negative control (non-medicated feed, uninfected); PC = positive control (non-medicated feed, infected); SAL = salinomycin supplemented feed, infected (Sacox 60, 66 mg/kg of diet); TA = tannic acid supplemented feed, infected (100 mg/kg of diet); TAE = tannic acid extract supplemented feed, infected (TAE, 100 mg/kg of diet). Within each intestinal region, means with different letters (a-d) differ significantly (P < 0.01). Experiment 2 Live Performance Initial chick BW was 40.60 ± 0.55 g and was similar between all groups (P = 0.3360). During the starter phase (d 1 to 21), FCR was improved (P < 0.05) with supplementation of SAL, ROB, TAE and TAE+BC compared to CNT and the improvement in FCR was similar (P > 0.05) between the supplemented groups (Table 4). Increased (P < 0.05) BWG during d 1 to 21 was observed in SAL, ROB and TAE compared to TAE+BC and CNT. Similar to d 1 to 21, from d 1 to 49, vaccinated broiler FCR was significantly improved with SAL, ROB, TAE and TAE+BC compared to CNT, with SAL and ROB having lower (P < 0.05) FCR than TAE and TAE+BC over the full trial. Increased (P < 0.05) BWG was observed in SAL and ROB compared to CNT, while BWG of TAE was similar (P > 0.05) to all groups. No significant difference in BWG was observed between TAE+BC, TAE and ROB, however, BWG of TAE+BC was reduced (P < 0.05) compared to SAL. Both mortality and FI were similar (P ≥ 0.05) for all groups during the experiment. Litter scores at d 28 and 49 were not different between CNT, SAL, ROB, TAE and TAE+BC groups (Figure 3). Overall, live performance of vaccinated broilers was enhanced with supplementation of all treatments, and differences between TAE and TAE+BC supplemented broilers were minimal over d 1 to 49. Figure 3. View largeDownload slide Litter scores on d 28 and 49 of Experiment 2. No significant differences (P > 0.05) were observed between treatments on both days. CNT = non-medicated control; SAL = salinomycin (Bio-Cox, 66 mg/kg of diet) supplemented; ROB = robenidine (Robenz, 33 mg/kg of diet) supplemented; TAE = tannic acid extract (100 mg/kg of diet) supplemented; TAE+BC = tannic acid extract with Bacillus coagulans (110 mg/kg of diet). Figure 3. View largeDownload slide Litter scores on d 28 and 49 of Experiment 2. No significant differences (P > 0.05) were observed between treatments on both days. CNT = non-medicated control; SAL = salinomycin (Bio-Cox, 66 mg/kg of diet) supplemented; ROB = robenidine (Robenz, 33 mg/kg of diet) supplemented; TAE = tannic acid extract (100 mg/kg of diet) supplemented; TAE+BC = tannic acid extract with Bacillus coagulans (110 mg/kg of diet). Table 4. Body weight gain (BWG; g), feed intake (FI; g), mortality-corrected feed conversion ratio (FCR) and mortality of broilers cocci-vaccinated with live oocysts1 on d 1 of age when fed diets supplemented with anticoccidial chemical, anticoccidial ionophore, tannic acid extract and tannic acid extract combined with Bacillus coagulans (Experiment 2). d 1 to 21 d 1 to 49 Diet2 BWG FI FCR BWG FI FCR Mort. CNT 597b 985 1.55a 2,795c 5197 1.87a 4.6 SAL 637a 1003 1.49b 2,991a 5345 1.79c 3.9 ROB 630a 993 1.48b 2,945a,b 5322 1.79c 3.6 TAE 624a 996 1.50b 2,869a,b,c 5260 1.83b 3.6 TAE+BC 557b 962 1.51b 2,844b,c 5183 1.84b 5.2 SEM3 $$\phantom{00}$$8.8 58.1 0.01 $$\phantom{00,}$$51.4 80.1 0.01 1.1 P-value $$\phantom{00}$$0.003 0.125 0.007 $$\phantom{0,}$$>0.040 0.495 <0.001 0.821 d 1 to 21 d 1 to 49 Diet2 BWG FI FCR BWG FI FCR Mort. CNT 597b 985 1.55a 2,795c 5197 1.87a 4.6 SAL 637a 1003 1.49b 2,991a 5345 1.79c 3.9 ROB 630a 993 1.48b 2,945a,b 5322 1.79c 3.6 TAE 624a 996 1.50b 2,869a,b,c 5260 1.83b 3.6 TAE+BC 557b 962 1.51b 2,844b,c 5183 1.84b 5.2 SEM3 $$\phantom{00}$$8.8 58.1 0.01 $$\phantom{00,}$$51.4 80.1 0.01 1.1 P-value $$\phantom{00}$$0.003 0.125 0.007 $$\phantom{0,}$$>0.040 0.495 <0.001 0.821 a-cMeans within a column not sharing the same superscript differ significantly (P < 0.05). 1Cocci-vaccinated with Advent (Novus International, Inc., St. Louis, MO) at 1 day of age via a spray cabinet. 2The broilers were fed a non-medicated control (CNT) diet containing no anticoccidial and growth promoter and were supplemented with anticoccidial ionophore salinomycin (SAL; 66 mg/kg of diet), anticoccidial chemical robenidine (ROB; 33 mg/kg of diet), tannic acid extract (TAE; 100 mg/kg of diet) and TAE combined with probiotic Bacillus coagulans (TAE+BC; 110 mg/kg of diet). 3Pooled standard error of the mean. View Large Table 4. Body weight gain (BWG; g), feed intake (FI; g), mortality-corrected feed conversion ratio (FCR) and mortality of broilers cocci-vaccinated with live oocysts1 on d 1 of age when fed diets supplemented with anticoccidial chemical, anticoccidial ionophore, tannic acid extract and tannic acid extract combined with Bacillus coagulans (Experiment 2). d 1 to 21 d 1 to 49 Diet2 BWG FI FCR BWG FI FCR Mort. CNT 597b 985 1.55a 2,795c 5197 1.87a 4.6 SAL 637a 1003 1.49b 2,991a 5345 1.79c 3.9 ROB 630a 993 1.48b 2,945a,b 5322 1.79c 3.6 TAE 624a 996 1.50b 2,869a,b,c 5260 1.83b 3.6 TAE+BC 557b 962 1.51b 2,844b,c 5183 1.84b 5.2 SEM3 $$\phantom{00}$$8.8 58.1 0.01 $$\phantom{00,}$$51.4 80.1 0.01 1.1 P-value $$\phantom{00}$$0.003 0.125 0.007 $$\phantom{0,}$$>0.040 0.495 <0.001 0.821 d 1 to 21 d 1 to 49 Diet2 BWG FI FCR BWG FI FCR Mort. CNT 597b 985 1.55a 2,795c 5197 1.87a 4.6 SAL 637a 1003 1.49b 2,991a 5345 1.79c 3.9 ROB 630a 993 1.48b 2,945a,b 5322 1.79c 3.6 TAE 624a 996 1.50b 2,869a,b,c 5260 1.83b 3.6 TAE+BC 557b 962 1.51b 2,844b,c 5183 1.84b 5.2 SEM3 $$\phantom{00}$$8.8 58.1 0.01 $$\phantom{00,}$$51.4 80.1 0.01 1.1 P-value $$\phantom{00}$$0.003 0.125 0.007 $$\phantom{0,}$$>0.040 0.495 <0.001 0.821 a-cMeans within a column not sharing the same superscript differ significantly (P < 0.05). 1Cocci-vaccinated with Advent (Novus International, Inc., St. Louis, MO) at 1 day of age via a spray cabinet. 2The broilers were fed a non-medicated control (CNT) diet containing no anticoccidial and growth promoter and were supplemented with anticoccidial ionophore salinomycin (SAL; 66 mg/kg of diet), anticoccidial chemical robenidine (ROB; 33 mg/kg of diet), tannic acid extract (TAE; 100 mg/kg of diet) and TAE combined with probiotic Bacillus coagulans (TAE+BC; 110 mg/kg of diet). 3Pooled standard error of the mean. View Large Coccidial Immunity Challenge Similar (P ≥ 0.05) BWG, FI and FCR were observed for the CNT groups following mixed Eimeria spp. challenge (Table 5). In group 1, challenged broilers supplemented with SAL, ROB, TAE and TAE+BC were observed to have increased (P < 0.05) BWG compared to CNT. Supplementation of ROB, TAE and TAE+BC provided similar improvement in BWG, while SAL had higher (P < 0.05) BWG than ROB and TAE+BC but was not different (P ≥ 0.05) from TAE. Similarly, FI was not different (P > 0.05) between CNT and birds supplemented with SAL, ROB, TAE and TAE+BC during the Eimeria challenge. Compared to challenged CNT birds, a significant improvement in FCR was observed upon supplementation with SAL, ROB and TAE, with no difference between FCR of broilers fed the supplemented products (P ≥ 0.05). Supplementation with TAE+BC during challenge resulted in similar FCR to ROB and TAE, however, improvement in FCR was not significantly different from CNT. Table 5. Body weight gain (BWG; g), feed intake (FI; g) and mortality-corrected feed conversion ratio (FCR) of cocci-vaccinated1 broilers d 6 after mixed Eimeria spp. challenge2 at d 29, when fed diets supplemented with anticoccidial chemical, anticoccidial ionophore, tannic acid extract and tannic acid extract combined with Bacillus coagulans (Experiment 2). d 28 to 35 Diet3 Supplemented feed4 BWG FI FCR CNT NA 469c,d 825a,b 1.80a,b SAL + 623a 868a 1.41d ROB 564b 769a,b,c 1.55c,d TAE 561a,b 868a 1.58c,d TAE+BC 552b 904a 1.67a,b,c CNT NA 458c,d 841a,b 1.84a SAL - 469c,d 796a,b 1.80a,b ROB 381e 710b,c 1.86a TAE 444d,e 618c 1.55c,d TAE+BC 516b,c 806a,b 1.61b,c,d SEM5 $$\phantom{0}$$20.7 $$\phantom{0}$$48.1 0.07 P-value $$\phantom{.}$$<0.001 $$\phantom{00}$$0.047 0.009 d 28 to 35 Diet3 Supplemented feed4 BWG FI FCR CNT NA 469c,d 825a,b 1.80a,b SAL + 623a 868a 1.41d ROB 564b 769a,b,c 1.55c,d TAE 561a,b 868a 1.58c,d TAE+BC 552b 904a 1.67a,b,c CNT NA 458c,d 841a,b 1.84a SAL - 469c,d 796a,b 1.80a,b ROB 381e 710b,c 1.86a TAE 444d,e 618c 1.55c,d TAE+BC 516b,c 806a,b 1.61b,c,d SEM5 $$\phantom{0}$$20.7 $$\phantom{0}$$48.1 0.07 P-value $$\phantom{.}$$<0.001 $$\phantom{00}$$0.047 0.009 a-eMeans within a column not sharing the same superscript differ significantly (P < 0.05). 1Cocci-vaccinated with Advent (Novus International, Inc., St. Louis, MO) at day 1 of age via a spray cabinet. 2Mixed-species challenge contained Eimeria acervulina (1 × 105), Eimeria maxima (5 × 104) and Eimeria tenella (7.5 × 104) sporulated oocysts. 3The broilers were fed a non-medicated control (CNT) diet containing no anticoccidial and growth promoter and were supplemented with anticoccidial ionophore salinomycin (SAL; 66 mg/kg of diet), anticoccidial chemical robenidine (ROB; 33 mg/kg of diet), tannic acid extract (TAE; 100 mg/kg of diet) and TAE combined with probiotic Bacillus coagulans (TAE+BC; 110 mg/kg of diet). 4On d 28, broilers were issued commercial grower feed containing supplemental treatment materials (+) or CNT grower feed with the previously added supplemental treatment materials withdrawn (−). Feed issued to CNT treatment during the challenge was the same for all challenged control pens (NA) as it never contained supplemental materials. 5Pooled standard error of the mean. View Large Table 5. Body weight gain (BWG; g), feed intake (FI; g) and mortality-corrected feed conversion ratio (FCR) of cocci-vaccinated1 broilers d 6 after mixed Eimeria spp. challenge2 at d 29, when fed diets supplemented with anticoccidial chemical, anticoccidial ionophore, tannic acid extract and tannic acid extract combined with Bacillus coagulans (Experiment 2). d 28 to 35 Diet3 Supplemented feed4 BWG FI FCR CNT NA 469c,d 825a,b 1.80a,b SAL + 623a 868a 1.41d ROB 564b 769a,b,c 1.55c,d TAE 561a,b 868a 1.58c,d TAE+BC 552b 904a 1.67a,b,c CNT NA 458c,d 841a,b 1.84a SAL - 469c,d 796a,b 1.80a,b ROB 381e 710b,c 1.86a TAE 444d,e 618c 1.55c,d TAE+BC 516b,c 806a,b 1.61b,c,d SEM5 $$\phantom{0}$$20.7 $$\phantom{0}$$48.1 0.07 P-value $$\phantom{.}$$<0.001 $$\phantom{00}$$0.047 0.009 d 28 to 35 Diet3 Supplemented feed4 BWG FI FCR CNT NA 469c,d 825a,b 1.80a,b SAL + 623a 868a 1.41d ROB 564b 769a,b,c 1.55c,d TAE 561a,b 868a 1.58c,d TAE+BC 552b 904a 1.67a,b,c CNT NA 458c,d 841a,b 1.84a SAL - 469c,d 796a,b 1.80a,b ROB 381e 710b,c 1.86a TAE 444d,e 618c 1.55c,d TAE+BC 516b,c 806a,b 1.61b,c,d SEM5 $$\phantom{0}$$20.7 $$\phantom{0}$$48.1 0.07 P-value $$\phantom{.}$$<0.001 $$\phantom{00}$$0.047 0.009 a-eMeans within a column not sharing the same superscript differ significantly (P < 0.05). 1Cocci-vaccinated with Advent (Novus International, Inc., St. Louis, MO) at day 1 of age via a spray cabinet. 2Mixed-species challenge contained Eimeria acervulina (1 × 105), Eimeria maxima (5 × 104) and Eimeria tenella (7.5 × 104) sporulated oocysts. 3The broilers were fed a non-medicated control (CNT) diet containing no anticoccidial and growth promoter and were supplemented with anticoccidial ionophore salinomycin (SAL; 66 mg/kg of diet), anticoccidial chemical robenidine (ROB; 33 mg/kg of diet), tannic acid extract (TAE; 100 mg/kg of diet) and TAE combined with probiotic Bacillus coagulans (TAE+BC; 110 mg/kg of diet). 4On d 28, broilers were issued commercial grower feed containing supplemental treatment materials (+) or CNT grower feed with the previously added supplemental treatment materials withdrawn (−). Feed issued to CNT treatment during the challenge was the same for all challenged control pens (NA) as it never contained supplemental materials. 5Pooled standard error of the mean. View Large An immunity challenge was created in group 2 by issuing previously supplemented, vaccinated broilers feed without supplemental materials during the Eimeria spp. challenge. Withdrawal of ROB resulted in significantly reduced broiler BWG compared to CNT and broilers supplemented with ROB (group 1). Similar BWG to CNT was observed after withdrawal of TAE, SAL and TAE+BC, however, when the materials were supplemented (group 1), broilers had increased (P < 0.05) BWG as compared to vaccinated, challenged broilers during the challenge. Withdrawal of SAL and TAE+BC did not impact FI compared to CNT birds, however, FI was reduced (P < 0.05) following withdrawal of TAE and ROB during the challenge. Withdrawal of ROB and SAL resulted in FCR similar to CNT, whereas supplementation with ROB and SAL during challenge was found to significantly improve FCR of vaccinated, challenged broilers. In contrast, withdrawal of TAE and TAE+BC did not negatively impact broiler FCR during the immunity challenge. Broilers previously treated with TAE and TAE+BC maintained significantly improved FCR compared to the vaccinated, challenged CNT birds. Overall, withdrawal of ROB and SAL resulted in a > 0.30 increase in broiler FCR, whereas withdrawal of TAE and TAE+BC minimally impacted vaccinated, Eimeria challenged broilers. Following Eimeria challenge, lesions were observed in the challenged, supplemented; challenged, non-supplemented; and CNT groups (Table 6). Lesions in the ceca, indicative of E. tenella, were increased compared to those due to E. acervulina and E. maxima in the upper and middle intestine, respectively; however, lesions did not differ (P ≥ 0.05) among challenged groups. Withdrawal of SAL, ROB, TAE and TAE+BC resulted in ≥ 2-fold increase in oocyst shedding compared to when supplemented in-feed following Eimeria challenge. Although oocyst counts were found to be consistently reduced in CNT and TAE+BC groups, the differences were not significant (P ≥ 0.05). Table 6. Intestinal lesion scores1 and total oocysts per gram (OPG) of excreta of cocci-vaccinated2 broilers d 6 after mixed Eimeria spp. challenge3 at d 29, when fed diets supplemented with anticoccidial chemical, anticoccidial ionophore, tannic acid extract and tannic acid extract combined with Bacillus coagulans (Experiment 2). Diet4 Supplemented feed5 Upper Middle Ceca Total OPG CNT NA 0.58 0.43 1.70 1830 SAL + 0.95 0.60 1.05 1970 ROB 0.45 0.30 2.30 2340 TAE 0.75 0.30 1.45 2070 TAE+BC 0.33 0.23 0.38 230 CNT NA 0.95 0.50 1.07 270 SAL - 1.50 0.78 1.00 21,180 ROB 1.38 0.68 1.00 8940 TAE 1.08 1.13 1.83 4370 TAE+BC 0.73 0.60 1.15 570 SEM6 0.23 0.21 0.34 6310 P-value 0.061 0.223 0.128 $$\phantom{0}$$0.470 Diet4 Supplemented feed5 Upper Middle Ceca Total OPG CNT NA 0.58 0.43 1.70 1830 SAL + 0.95 0.60 1.05 1970 ROB 0.45 0.30 2.30 2340 TAE 0.75 0.30 1.45 2070 TAE+BC 0.33 0.23 0.38 230 CNT NA 0.95 0.50 1.07 270 SAL - 1.50 0.78 1.00 21,180 ROB 1.38 0.68 1.00 8940 TAE 1.08 1.13 1.83 4370 TAE+BC 0.73 0.60 1.15 570 SEM6 0.23 0.21 0.34 6310 P-value 0.061 0.223 0.128 $$\phantom{0}$$0.470 1Lesion scores were assigned from 0 (no gross lesions) to 4 (extensive lesions) according to the system of Johnson and Reid (1970). 2Cocci-vaccinated with Advent (Novus International, Inc., St. Louis, MO) at day 1 of age via a spray cabinet. 3Mixed-species challenge contained Eimeria acervulina (1 × 105), Eimeria maxima (5 × 104) and Eimeria tenella (7.5 × 104) sporulated oocysts. 4The broilers were fed a non-medicated control (CNT) diet containing no anticoccidial and growth promoter and were supplemented with anticoccidial ionophore salinomycin (SAL; 66 mg/kg of diet), anticoccidial chemical robenidine (ROB; 33 mg/kg of diet), tannic acid extract (TAE; 100 mg/kg of diet) and TAE combined with probiotic Bacillus coagulans (TAE+BC; 110 mg/kg of diet). 5On d 28, broilers were issued commercial grower feed containing supplemental treatment materials (+) or CNT grower feed with the previously added supplemental treatment materials withdrawn (−). 6Pooled standard error of the mean. View Large Table 6. Intestinal lesion scores1 and total oocysts per gram (OPG) of excreta of cocci-vaccinated2 broilers d 6 after mixed Eimeria spp. challenge3 at d 29, when fed diets supplemented with anticoccidial chemical, anticoccidial ionophore, tannic acid extract and tannic acid extract combined with Bacillus coagulans (Experiment 2). Diet4 Supplemented feed5 Upper Middle Ceca Total OPG CNT NA 0.58 0.43 1.70 1830 SAL + 0.95 0.60 1.05 1970 ROB 0.45 0.30 2.30 2340 TAE 0.75 0.30 1.45 2070 TAE+BC 0.33 0.23 0.38 230 CNT NA 0.95 0.50 1.07 270 SAL - 1.50 0.78 1.00 21,180 ROB 1.38 0.68 1.00 8940 TAE 1.08 1.13 1.83 4370 TAE+BC 0.73 0.60 1.15 570 SEM6 0.23 0.21 0.34 6310 P-value 0.061 0.223 0.128 $$\phantom{0}$$0.470 Diet4 Supplemented feed5 Upper Middle Ceca Total OPG CNT NA 0.58 0.43 1.70 1830 SAL + 0.95 0.60 1.05 1970 ROB 0.45 0.30 2.30 2340 TAE 0.75 0.30 1.45 2070 TAE+BC 0.33 0.23 0.38 230 CNT NA 0.95 0.50 1.07 270 SAL - 1.50 0.78 1.00 21,180 ROB 1.38 0.68 1.00 8940 TAE 1.08 1.13 1.83 4370 TAE+BC 0.73 0.60 1.15 570 SEM6 0.23 0.21 0.34 6310 P-value 0.061 0.223 0.128 $$\phantom{0}$$0.470 1Lesion scores were assigned from 0 (no gross lesions) to 4 (extensive lesions) according to the system of Johnson and Reid (1970). 2Cocci-vaccinated with Advent (Novus International, Inc., St. Louis, MO) at day 1 of age via a spray cabinet. 3Mixed-species challenge contained Eimeria acervulina (1 × 105), Eimeria maxima (5 × 104) and Eimeria tenella (7.5 × 104) sporulated oocysts. 4The broilers were fed a non-medicated control (CNT) diet containing no anticoccidial and growth promoter and were supplemented with anticoccidial ionophore salinomycin (SAL; 66 mg/kg of diet), anticoccidial chemical robenidine (ROB; 33 mg/kg of diet), tannic acid extract (TAE; 100 mg/kg of diet) and TAE combined with probiotic Bacillus coagulans (TAE+BC; 110 mg/kg of diet). 5On d 28, broilers were issued commercial grower feed containing supplemental treatment materials (+) or CNT grower feed with the previously added supplemental treatment materials withdrawn (−). 6Pooled standard error of the mean. View Large DISCUSSION The current work investigated the protective effect of tannic acid based feed additives to control coccidiosis in broiler chickens in two experiments. In the first experiment, the ability of commercial TA and the proprietary TAE to alleviate coccidiosis in broilers challenged with mixed Eimeria species was assessed. The second experiment then evaluated the ability of TAE formulations and commercial anticoccidials to impact performance of broilers vaccinated for coccidiosis and to influence the development of protective immunity in vaccinated broilers challenged with mixed Eimeria species. Mixed results on the efficacy of diets containing tannins to reduce the negative impact of coccidiosis in broilers have previously been observed. Some authors report improved performance, reduced lesions and fewer oocysts upon tannin supplementation (Lee et al., 2012; Kaleem et al., 2014), while others observed mixed effects (Cejas et al., 2011) or no impact of tannins on coccidiosis (Mansoori and Modirsanei, 2012). The results of our first experiment align with those of McCann et al. (2006), who observed only small improvements in performance of Eimeria challenged broilers fed tannins, but found that tannin-supplemented diets were able to lessen the detrimental effects of coccidiosis by reducing intestinal lesions. Gross intestinal lesions, due to replication of Eimeria parasites within host intestinal epithelia cells, diminish intestinal barrier integrity, absorptive capacity and create high levels of oxidative and inflammatory stress within the intestine. Tannins are potent antioxidants, which have been reported to reduce oxidative and inflammatory stress in macrophages (Kaur et al., 2008), as well as colon tissue (Hamiza et al., 2012). It is possible that TAE and TA lessened intestinal lesions caused by Eimeria via reducing oxidative stress and/or inflammation within the intestine, leading to a more intact intestinal barrier. Critical indicators of a protection against coccidiosis infection are increased BWG and reduced oocyst shedding. While BWG was not different between challenged treatments, birds supplemented with TAE were observed to have reduced (P < 0.05) oocyst shedding compared to the challenged control (Figure 1). Tannins are known to have anti-protozoal activity; thus, it is possible that TAE had a direct activity towards Eimeria parasites. Mechanisms such as inhibition of extracellular parasitic enzymes or tannin complexation with metals, such as iron, have been suggested as antimicrobial methods by which tannins prevent parasite growth and proliferation (Scalbert, 1991; Chung et al., 1998a, 1998b). Additionally, Min and Hart (2003) reported that tannins may form complexes with nutrients and inhibit their availability to the parasite for normal growth, development and motility and thus, decrease the metabolism of parasite directly through inhibition of oxidative phosphorylation or electron transport. Along with other phytobiotic feed additives, the specific chemical and physical properties of tannins are highly variable (Windisch et al., 2008; Huyghebaert et al., 2011). In our study, the proprietary TAE was found to reduce (P < 0.05) oocyst shedding by 58% and 51% for PC birds and TA treated birds, respectively (Figure 1). Kaleem et al. (2014) previously reported that tannin extracts from Emblica officinalis also had improved efficacy in reducing the impact of coccidiosis challenge, as compared to commercial TA. These results, and those of others (Mueller-Harvey, 2006; Schiavone et al., 2008; Elizondo et al., 2010), provide additional support for the need to characterize tannins based on individual assessment, as opposed to an assumption that all tannin extracts and commercial TA products have equivalent biological efficacy. Along with their anti-inflammatory and anti-parasitic functions, tannins may be involved in modulation of the interaction between the microbiota and gastrointestinal tract (Redondo et al., 2014). Thus, dietary tannin supplementation may help in alleviating the intestinal challenges associated with vaccine-induced Eimeria oocyst replication, thereby improving performance of coccidiosis vaccinated broilers. Thus far, few studies have been reported which assess the compatibility of tannin feed additives with coccidiosis vaccination. In the live performance portion of Experiment 2, supplementation of TAE was found to improve (P < 0.05) the FCR of broilers vaccinated with a live, non-attenuated anti-coccidial vaccine (Table 4). These significant improvements in FCR of vaccinated birds supplemented with TAE were consistent, even though the flock was split at d 28 to accommodate the coccidial immunity challenge. Our results align with Hooge et al. (2012), who observed improvements in d 0 to 21 FCR of vaccinated broilers supplemented with sweet chestnut tannins and contrast the negative impact of commercial TA on vaccine effectiveness observed by Mansoori and Modirsanei (2012). Combination strategies towards controlling coccidia and preventing intestinal dysbacteriosis are likely to be most effective methods for controlling concurrent enteric diseases such as coccidiosis and necrotic enteritis in broilers (Van Immerseel et al., 2004; Williams 2005). Bacillus coagulans has previously been shown to have immune modulating activities in vitro (Jensen et al., 2010) and in vivo (Lin et al., 2012). Previous work has further shown supplementation of B. coagulans can improve survivability of Clostridium difficile-infected mice (Fitzpatrick et al., 2011) and enhance growth performance of broilers (Lin et al., 2011). By combining the probiotic functions of B. coagulans with the antioxidant and anti-inflammatory activities of the proprietary TAE (TAE+BC), the combination would provide dual modes of action by which synergistic benefits towards broiler live performance may be observed as compared to TAE alone. During the vaccination study, broilers supplemented with TAE+BC were found to have improved FCR compared to CNT birds, but live performance of TAE+BC was similar to, rather than synergistic to, TAE alone (Table 4). Probiotics are known to support intestinal integrity by enhancing immune activity and maintaining intestinal microflora balance (Huyghebaert et al., 2011). Under the conditions of this experiment, it is possible that coccidiosis vaccination alone did not provide a sufficient enteric challenge in which enhanced growth promoting effects of B. coagulans could be observed over TAE. Future studies should utilize enteric pathogen challenges, such as C. perfringens infection, to create a more robust enteric challenge by which the beneficial probiotic activities of B. coagulans should enhance the benefits of TAE+BC beyond the anti-parasitic and antioxidant effects observed with TAE. Reduced disease susceptibility in vaccinated broilers is based on the acquisition of protective immunity, a result of recycling the vaccine parasites through the litter. Previous studies have shown either additive benefits (Oviedo-Rondón et al., 2006; Stringfellow et al., 2011; Mathis et al., 2014) or detrimental effects (Küçükyilmaz et al., 2012; Mansoori and Modirsanei, 2012) of feed additive supplementation upon the efficacy of coccidiosis vaccination to mitigate the impact of enteric challenges in later growth phases. Commercial TA was previously reported to hamper coccidial vaccine oocyst cycling, resulting in insufficient disease protection following a secondary Eimeria challenge in broilers (Mansoori and Modirsanei, 2012). Experiment 2 was therefore designed to assess whether the proprietary TAE and TAE+BC combinations would impact efficacy of coccidiosis vaccination using a mixed Eimeria challenge during d 29 to 35. The goal of the coccidial immunity challenge portion of Experiment 2 was twofold. The first objective was to assess whether supplementation of TAE or TAE+BC in vaccinated broiler diets would enhance recovery following Eimeria challenge as compared to vaccination alone (group 1). The second, more important objective, was to evaluate any potential impact of TAE and TAE+BC on altering the effectiveness of vaccination to induce protective immunity development in broilers (group 2). To accomplish these purposes, vaccinated broilers were split into subgroups, which were either provided feed supplemented with TAE, TAE+BC, SAL, or ROB (group 1) during the mixed Eimeria challenge, or were provided feed in which additives were withdrawn (group 2). This design allowed for collection of challenge recovery, as well as vaccine compatibility, information to be gathered within the study, albeit due to reduced replication, the chance for pen to pen variability to impact detection of significant differences among treatments was increased. Based on previous reports, the mixed Eimeria challenge was expected to cause increased lesions, higher oocyst shedding, reduced BWG and higher FCR in Eimeria challenged broilers treated with additives that interfered with vaccine efficacy. The results of interfering additives in this study were therefore expected to be similar to results previously seen in non-vaccinated, Eimeria challenged broilers, as compared to vaccinated, challenged broilers (Oviedo-Rondón et al., 2006; Lee et al., 2011; Mansoori and Modirsanei, 2012; Mathis et al., 2014). Due to the design of Experiment 2, a comparison of the effects of Eimeria challenge in vaccinated versus non-vaccinated broilers was unable to be drawn, but assessment of the impact of treatments on enhancing broiler recovery following the mixed Eimeria challenge was evaluated. In group 1, neither intestinal lesions nor oocyst shedding were altered by Eimeria challenge (Table 6), however, vaccination combined with TAE or TAE+BC supplementation was found to enhance broiler performance following Eimeria challenge as compared to vaccination alone (Table 5). This result indicates that TAE and TAE+BC enhanced disease resistance in vaccinated broilers, which is in contrast with the results of Mansoori and Modirsanei (2012). Differences in tannin source (tannin extract versus commercial TA) and tannin inclusion in feed (100 mg/kg TAE versus 10 g/kg commercial TA) may explain the opposing results of these vaccination studies. According to Chapman (1999), insufficient immunity development following vaccination is indicated by increased fecal oocyst shedding and reduced performance after withdrawal of supplemental medication or feed additive from broiler diets. In group 2, oocyst shedding was numerically increased in broilers in which ROB and SAL were withdrawn from feed (Table 6). Broiler performance was additionally found to be significantly reduced when ROB and SAL were withdrawn from feed, as compared to when they were kept in feed during the Eimeria challenge (Table 5). ROB is known to markedly suppress oocyst production of drug-sensitive Eimeria strains while ionophores, like SAL, can indirectly inhibit parasite replication (Chapman, 1999). The results of our coccidial immunity challenge support the idea that concomitant use of some anticoccidial drugs and vaccination is not advisable during the initial weeks following vaccination due to the risk of negatively impacting vaccine efficacy (Chapman, 1999; Vermeulen et al., 2001; Williams, 2005). In contrast to the negative impact of anticoccidial withdrawal on broiler FCR, withdrawal of TAE and TAE+BC did not depress Eimeria challenged broiler performance. Broiler FCR was found to be equivalent (P > 0.05) whether TAE and TAE+BC were supplemented or withdrawn during Eimeria challenge (Table 5). In addition, broilers that had TAE or TAE+BC withdrawn during the challenge had significantly improved FCR (P < 0.05) compared to vaccinated, challenged control birds. These results indicate that TAE and TAE+BC, unlike ROB and SAL, allowed protective immunity to develop following coccidiosis vaccination. Unlike TAE withdrawal, which resulted in lower BWG and FI as compared to when it was supplemented, withdrawal of TAE+BC did not significantly impact these variables during the coccidial immunity challenge. Further, broilers supplemented with TAE+BC consistently showed numerically reduced intestinal lesions and oocyst shedding compared to birds supplemented with TAE during the Eimeria challenge in both groups 1 and 2. Increased disease prevention, altered intestinal microflora composition and enhancement of host immune response have been observed from oral feeding of probiotics (Dalloul and Lillehoj, 2005; Lee et al., 2010; Lin et al., 2011; Stringfellow et al., 2011; Lin et al., 2012; Abdelrahman et al., 2014; Bozkurt et al., 2014). Bacillus coagulans has also been observed to modulate the composition of intestinal microflora and improve non-specific immunity (Lin et al., 2011; Lin et al., 2012). The differences observed between TAE and TAE+BC after withdrawal therefore may have been the result of B. coagulans exerting an immune enhancing effect, either following the initial coccidiosis vaccination or during the Eimeria challenge itself, which could not be achieved with TAE alone. To conclude, the data from these experiments provide additional evidence supporting the need to individually characterize plant bioactive feed additives for use in poultry nutrition. Supplementation of broiler diets with TAE reduced the negative effects of coccidiosis in Eimeria challenged broilers. Both TAE alone and in combination with the probiotic B. coagulans (TAE+BC) provided additive FCR benefits in coccidiosis vaccinated broilers and more importantly, were found to permit protective immunity development in vaccinated broilers. Further observations indicate TAE+BC may have improved biological activity compared to TAE alone in Eimeria infected broilers, which warrants additional research. 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J. 65 : 97 – 114 . Google Scholar CrossRef Search ADS © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Effects of tannic acid extract on performance and intestinal health of broiler chickens following coccidiosis vaccination and/or a mixed-species Eimeria challenge

Poultry Science , Volume 97 (9) – Sep 1, 2018

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© 2018 Poultry Science Association Inc.
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0032-5791
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Abstract

ABSTRACT Two experiments were conducted to investigate the effects of tannic acid extract (TAE) formulations on the performance and intestinal health of male Cobb × Cobb 500 broilers exposed to coccidiosis. In the first experiment, 320 broiler chicks were randomly assigned to 5 treatments with 8 replicates. Treatments included non-medicated, uninfected (NC); non-medicated, infected (PC); salinomycin (SAL, 66 mg/kg); tannic acid (TA, 0.5 g/kg) and TAE (TAE, 0.5 g/kg). On d 14, all groups (except NC) were orally inoculated with Eimeria acervulina, E. maxima and E. tenella oocysts. Intestinal lesion scores, fecal oocyst counts (OPG) and performance were evaluated on d 20. The PC had greater lesions and higher FCR than infected, supplemented groups. Only TAE reduced OPG compared to PC (P < 0.05). In the second experiment, 3,000 broiler chicks were vaccinated on day of hatch with live coccidial oocysts, then randomly assigned to 5 treatments with 15 replicates. Treatments included non-medicated (CNT); salinomycin (SAL, 66 mg/kg); robenidine (ROB, 33 mg/kg); TAE (0.5 g/kg) and TAE with Bacillus coagulans (TAE+BC, 0.5 g/kg). On d 29, a subset of pens (n = 20) were challenged with a mixed Eimeria spp. oral inoculum; performance, lesions and OPG were evaluated on d 35. An immune challenge was created in half the pens by issuing broilers feed without supplementation materials during the challenge. For the non-challenged pens (n = 55), performance was measured up to d 49. Performance of non-challenged, vaccinated-CNT birds was improved with all treatments at d 21 and d 49. Among the challenged birds, withdrawal of SAL or ROB resulted in FCR similar to the challenged CNT group (P > 0.05), whereas withdrawal of TAE or TAE+BC maintained improved FCR compared to challenged-CNT birds (P < 0.05). These findings indicate supplementation of TAE and TAE+BC with coccidiosis vaccination can be considered as a potential alternative strategy to address coccidiosis in broiler chickens. INTRODUCTION Coccidiosis is a ubiquitous disease in the commercial poultry industry and is estimated to cost poultry producers more than $800 million each year (Williams, 1998). In poultry, the causative agents of coccidiosis are apicomplexan protozoan parasites of the genus Eimeria. These parasites invade the intestinal epithelium in a site-specific manner, causing inflammation and necrosis of the mucosal barrier and underlying tissue (Vermeulen, 2001). Lesion formation in the intestinal epithelium results in nutrient malabsorption, diarrhea, reduced weight gain and a concomitant decrease in feed efficiency (Lillehoj and Trout, 1993; Williams, 2005). The reductions in performance due to subclinical Eimeria spp. infection attribute approximately 80% of the worldwide cost of coccidiosis (Williams, 1999). Of the seven species of Eimeria known to infect poultry, E. acervulina, E. maxima and E. tenella are the most frequently diagnosed. For the past five decades, chemotherapeutic agents have been used to control coccidiosis (Lillehoj and Lillehoj, 2000). Large-scale preventative use of these chemical anticoccidials and ionophoric feed additives is convenient for producers and has greatly enhanced the productivity of the commercial poultry industry (Chapman, 1997). However, long-term, wide-spread use of a limited pool of chemotherapeutics has resulted in decreased sensitivity of Eimeria to many anticoccidials used in commercial poultry production today (Long, 1982; Chapman, 1997). Growing public health concerns related to development of antimicrobial resistance and anxiety over potential drug residues in egg and meat products have further increased the pressure on commercial poultry producers to reduce, even eliminate, the use of antimicrobials and anticoccidials in poultry diets. Therefore, poultry producers are actively looking for alternative methods of medication to control coccidiosis, such as vaccinations or use of feed additives like plant extracts and probiotics. Live coccidia vaccines function by enhancing the natural immunity of the animal to coccidia, due to recycling of Eimeria oocysts through poultry litter (Chapman et al., 2002). In order to provide broad-spectrum immunity, vaccines typically contain attenuated or non-attenuated live oocysts of multiple Eimeria spp. (Lillehoj and Lillehoj, 2000; Dalloul and Lillehoj, 2005). While vaccines have been used for many years to control coccidiosis in broiler breeders (Chapman, 1999), implementation of vaccination for preventative coccidiosis control in meat-producing broilers has not been universally accepted by the U.S. poultry industry (Danforth, 1998). Producer hesitancy to utilize vaccination as a coccidiosis management practice is based on reports of reduced weight gain and feed efficiency in vaccinated broilers compared to non-vaccinated broilers (Danforth, 1998; Williams, 2002). Despite these concerns, commercial vaccination programs are playing a more prominent role in commercial broiler production, as using vaccines can potentially reestablish drug sensitivity of Eimeria to anticoccidial chemicals (Chapman, 2000; Chapman et al., 2002; Chapman and Jeffers, 2014). Feed additives, such as plant extracts and probiotics, have shown success in positively influencing the intestinal microbial balance of poultry. Numerous reports have suggested plant extracts, especially polyphenolic bioactive molecules, may have potential as alternative anticoccidial agents (Allen and Fetterer, 2002; Naidoo et al., 2008; Windisch et al., 2008; Abbas et al., 2012; Wunderlich et al., 2014). Plant bioactives such as tannins, saponins and flavonoids have well known antioxidant and anti-inflammatory activities. These functional activities provide potential modes of action in which bioactives may help protect the intestinal epithelium from oxidative damage (Kaur et al., 2008; Hamiza et al., 2012). Further, tannins have known anti-parasitic (Min and Hart, 2003) and antimicrobial activities due to their ability to complex with microbial enzymes and metal ions (Scalbert, 1991; Chung et al., 1998a). Although tannins are often considered undesirable in poultry diets because of their ability to precipitate proteins and inhibit digestive enzymes (Chung et al., 1998b), both the level of dietary tannin and the tannin structure impact the nutritive or anti-nutritive properties of the tannin (Mueller-Harvey, 2006). Tannins are a structurally diverse group of complex polyphenolic compounds, which are classified into two groups, hydrolysable and condensed tannins, based on molecular structure. Recent studies have suggested that tannins may be potential alternative growth promoters for poultry diets (Tosi et al., 2013; Redondo et al., 2014). Unlike antibiotic growth promoters, the development of bacterial resistance to tannins is postulated to be difficult due to the complex structure of the tannin molecules. In vitro (Ahn et al., 1998; Chung et al., 1998a; Elizondo et al., 2010; Redondo et al., 2015) and in vivo (Tosi et al. 2013) studies have shown several tannin sources to be effective at inhibiting growth of poultry pathogens, including C. perfringens, the causative agent of necrotic enteritis. Multiple studies have tested the efficacy of common tannin sources, chestnut (Castanea sativa; hydrolysable tannin) and quebracho (Schinopsis lorentzii; condensed tannin), to control Eimeria infections (McCann et al., 2006; Cejas et al., 2011; Hooge et al., 2012). However, there is a paucity of literature discussing the use of tannic acid, a model hydrolysable tannin, to control avian parasitic diseases like coccidiosis (Mansoori and Modirsanei, 2012; Kaleem et al., 2014). Probiotic supplementation in human and animal health is associated with improved intestinal microbial balance, enhanced immune activity and stronger gut defense against enteric disease (Huyghebaert, et al. 2011). According to Yang et al. (2009), probiotics can maintain beneficial microbial populations in the gut via competitive exclusion and immune modulation. An increasing number of studies have been reported, which evaluated the ability of probiotics to control parasitic diseases like coccidiosis (Lee et al., 2007; Stringfellow et al., 2011; Abdelrahman et al., 2014). Further, indirect effects of probiotics towards reducing Eimeria induced intestinal lesions has recently been suggested (Bozkurt et al., 2014). Multifaceted strategies towards modulating poultry gut health and immune development are necessary for controlling coccidiosis. Vaccination combined with dietary supplementation of tannins or probiotics could alleviate intestinal challenges associated with vaccination, thereby providing an effective alternative method of coccidiosis control and further mitigation of concurrent enteric diseases (Van Immerseel et al. 2004; Williams, 2005). The overall aim of the present study was thus to evaluate the protective effects of a gallnut tannic acid extract (TAE) to control coccidiosis under the experimental conditions. Three objectives were proposed: (1) to evaluate the ability of tannic acid products to reduce a mixed Eimeria spp. challenge; (2) to compare the effects of TAE, TAE combined with a probiotic and in-feed anticoccidials on broilers vaccinated at day of hatch with live oocysts; and (3) to assess the impact of the aforementioned materials on protective immunity development in vaccinated broilers infected with mixed Eimeria spp. challenge at 29 days of age. MATERIALS AND METHODS The two experiments were conducted at Southern Poultry Research in Athens, Georgia. In both experiments, Cobb × Cobb 500 broilers were obtained from the Cobb-Vantress hatchery in Cleveland, Georgia. Bird sexing and vaccination were completed at the hatchery without administration of any coccidia vaccine. For all experiments, age-appropriate supplemental heat was provided following breeder recommendations. Access to feed and water was provided ad libitum. Experiments were conducted in accordance with the principles and specific guidelines presented in the Federation of Animal Science Societies (FASS, 2010). Birds and housing facilities were monitored twice daily. Facilities were checked for general health status, feed and water supply, temperature, unexpected events and removal of dead birds. Daily mortality record was maintained and birds were not replaced. Coccidial oocysts used for challenge studies were field-strain Eimeria and were isolated from U.S. commercial production broiler houses. Coccidia strains were maintained as individual strains by periodic propagation and passage and were enumerated prior to challenge. Experiment 1 A total of 320 male Cobb-500 broiler chicks were randomly assigned to 5 dietary treatments, with 8 replicate cages of 8 birds per cage (0.63 ft2/bird). Broilers were housed in thermostatically controlled Petersime battery units (Petersime Incubator, Co., Gettysburg, OH) with the battery cage serving as the experimental unit. A commercial starter diet composition (Table 1) without additives, anticoccidial and growth promoter was formulated to meet NRC (1994) requirements. Treatment groups consisted of (1) negative control group (NC): commercial feed, untreated, unchallenged; (2) positive control group (PC): commercial feed, untreated, challenged with a mixed Eimeria spp.; (3) control diet supplemented with salinomycin (SAL): commercial feed with 66 mg salinomycin/kg of feed, challenged with a mixed Eimeria spp.; (4) control diet supplemented with tannic acid (TA): commercial feed with TA, challenged with a mixed Eimeria spp.; (5) control diet supplemented with tannic acid extract (TAE): commercial feed with TAE, challenged with a mixed Eimeria species. The products used in this experiment were: ionophore anticoccidial salinomcyin, Sacox 60 (Huvepharma, Inc., Peachtree City, GA) added at 66 g/MT to feed, a commercial food grade TA (Hubei Province, China) and a food grade TAE (Kemin Industries, Inc., Des Moines, IA). The TAE product is a proprietary formulation of hydrolysable tannins extracted from Rhus chinensis gallnuts. Both TA and TAE were mixed into finished feed at inclusion rates of 500 g/MT of feed, giving final concentrations of 100 mg TA per kg of feed and 100 mg TAE per kg of feed, respectively. All feeds were pelletized and were provided as crumbled pellets for the duration of the study. Table 1. Composition of the experimental starter diet and its nutrient profile (Experiment 1). Amount (% Item unless noted) Ingredient  Corn 57.10  Soybean meal dehulled 36.86  Fat 2.33  Dicalcium phosphate 1.41  Calcium carbonate 1.26  Sodium chloride 0.44  DL-Methionine 0.31  L-Lysine 0.10  L-Threonine (98.5%) 0.02  Ronozyme P-(ct) 0.02  Trace Mineral1 0.08  Vitamin premix2 0.07 Analyzed value3 (%)  DM 88.08  CP 23.44  Crude fiber 2.38  Crude fat 4.57  Calcium 0.90  Phosphorus (total) 0.60 Calculated value  ME (kcal/kg) 3067  Methionine 0.62  TSAA 0.90  Lysine 1.35  Threonine 0.95  Tryptophan 0.30  Sodium 0.21  Potassium 0.84  Chloride 0.28 Amount (% Item unless noted) Ingredient  Corn 57.10  Soybean meal dehulled 36.86  Fat 2.33  Dicalcium phosphate 1.41  Calcium carbonate 1.26  Sodium chloride 0.44  DL-Methionine 0.31  L-Lysine 0.10  L-Threonine (98.5%) 0.02  Ronozyme P-(ct) 0.02  Trace Mineral1 0.08  Vitamin premix2 0.07 Analyzed value3 (%)  DM 88.08  CP 23.44  Crude fiber 2.38  Crude fat 4.57  Calcium 0.90  Phosphorus (total) 0.60 Calculated value  ME (kcal/kg) 3067  Methionine 0.62  TSAA 0.90  Lysine 1.35  Threonine 0.95  Tryptophan 0.30  Sodium 0.21  Potassium 0.84  Chloride 0.28 1Trace mineral mix provided the following (per kg of diet): manganese (MnSO4•H2O), 60 mg; iron (FeSO4•7H2O), 30 mg; zinc (ZnO), 50 mg; copper (CuSO4•5H2O), 5 mg; iodine (ethylene diamine dihydroiodide), 0.15 mg; selenium (NaSe03), 0.3 mg. 2Vitamin mix provided the following (per kg of diet): Vitamin A, 12,345 IU; Vitamin D3, 3472 IU; 25-hydroxyvitamin D3, 96.6 μg; Vitamin E, 49 IU; vitamin B12 (cobalamin), 21.7 μg; Biotin, 0.23 mg; Menadione, 2.78 mg; Thiamine, 2.62 mg; Riboflavin, 10.8 mg; d-Panthothenic Acid, 18.5 mg; Vitamin B6, 4.6 mg; Niacin, 61.7 mg; Folic Acid, 1.5 mg. 3Analyzed values refer to basal (control) diet. View Large Table 1. Composition of the experimental starter diet and its nutrient profile (Experiment 1). Amount (% Item unless noted) Ingredient  Corn 57.10  Soybean meal dehulled 36.86  Fat 2.33  Dicalcium phosphate 1.41  Calcium carbonate 1.26  Sodium chloride 0.44  DL-Methionine 0.31  L-Lysine 0.10  L-Threonine (98.5%) 0.02  Ronozyme P-(ct) 0.02  Trace Mineral1 0.08  Vitamin premix2 0.07 Analyzed value3 (%)  DM 88.08  CP 23.44  Crude fiber 2.38  Crude fat 4.57  Calcium 0.90  Phosphorus (total) 0.60 Calculated value  ME (kcal/kg) 3067  Methionine 0.62  TSAA 0.90  Lysine 1.35  Threonine 0.95  Tryptophan 0.30  Sodium 0.21  Potassium 0.84  Chloride 0.28 Amount (% Item unless noted) Ingredient  Corn 57.10  Soybean meal dehulled 36.86  Fat 2.33  Dicalcium phosphate 1.41  Calcium carbonate 1.26  Sodium chloride 0.44  DL-Methionine 0.31  L-Lysine 0.10  L-Threonine (98.5%) 0.02  Ronozyme P-(ct) 0.02  Trace Mineral1 0.08  Vitamin premix2 0.07 Analyzed value3 (%)  DM 88.08  CP 23.44  Crude fiber 2.38  Crude fat 4.57  Calcium 0.90  Phosphorus (total) 0.60 Calculated value  ME (kcal/kg) 3067  Methionine 0.62  TSAA 0.90  Lysine 1.35  Threonine 0.95  Tryptophan 0.30  Sodium 0.21  Potassium 0.84  Chloride 0.28 1Trace mineral mix provided the following (per kg of diet): manganese (MnSO4•H2O), 60 mg; iron (FeSO4•7H2O), 30 mg; zinc (ZnO), 50 mg; copper (CuSO4•5H2O), 5 mg; iodine (ethylene diamine dihydroiodide), 0.15 mg; selenium (NaSe03), 0.3 mg. 2Vitamin mix provided the following (per kg of diet): Vitamin A, 12,345 IU; Vitamin D3, 3472 IU; 25-hydroxyvitamin D3, 96.6 μg; Vitamin E, 49 IU; vitamin B12 (cobalamin), 21.7 μg; Biotin, 0.23 mg; Menadione, 2.78 mg; Thiamine, 2.62 mg; Riboflavin, 10.8 mg; d-Panthothenic Acid, 18.5 mg; Vitamin B6, 4.6 mg; Niacin, 61.7 mg; Folic Acid, 1.5 mg. 3Analyzed values refer to basal (control) diet. View Large On the day of experimental diet placement, birds were randomly allotted to dietary treatments and bird weights by cage were recorded to ensure average initial cage weight was similar between groups. Live body weight (BW), feed intake (FI), body weight gain (BWG) and feed conversion ratio (FCR) were determined at d 14 and 20. FCR was calculated and corrected for BW of mortality. On d 14, all broilers, except those in NC treatment, were challenged with a 1 mL mixed-species Eimeria oral inoculum consisting of E. acervulina (5 × 105), E. maxima (8.5 × 104) and E. tenella (1.75 × 105) sporulated oocysts. NC birds were inoculated with 1 mL of saline solution. Samples of feces passed between 120 to 144 h post-challenge were collected from all cages for oocyst counting. Feces collected per cage were homogenized and stored at 4°C until measurement of oocyst counts. Counts were determined via dilution and microscopic enumeration using a McMaster counting chamber and are reported as oocysts per gram of excreta. On d 20 (6 d post-challenge), all birds were euthanized and necropsied to determine the presence and degree of coccidiosis lesions in the upper, middle and cecal regions of the intestinal tract (Johnson and Reid, 1970). Experiment 2 A total of 3000 male Cobb-500 broiler chicks were randomly assigned to 5 dietary treatments, with 15 replicate pens of 40 birds per pen. The experimental house contained 75 pens of equal size, each having an area of 50 ft2, with built-up wood shavings as bedding with a thickness of approximately 4 inches. The built-up bedding was from 3 grow-out cycles. Each pen had 5 ft high side walls with 1.5 ft bottom solid wood to prevent bird migration. Water was provided ad libitum from one Plasson-type watering fount per pen. The initial stocking density, after subtracting out for equipment, was 1.16 ft2/bird. The pen served as the experimental unit. The growth period was divided into 3 phases: starter (d 0 to 21), grower (d 21 to 35) and finisher (d 35 to 49). Commercial feed (Table 2) without additives, anticoccidial and growth promoter was formulated to meet NRC guidelines (1994) and the requirements for the birds’ growing stage. All diets were pelletized, with the starter phase provided as crumbled pellets. Treatment groups consisted of (1) control group (CNT): commercial feed, cocci-vaccinated; (2) control supplemented with salinomycin (SAL): commercial feed with 66 mg salinomycin/kg of feed, cocci-vaccinated.; (3) control supplemented with robenidine (ROB): commercial feed with 33 mg robenidine/kg of feed, cocci-vaccinated; (4) control supplemented with tannic acid extract (TAE): commercial feed with TAE, cocci-vaccinated; (5) control supplemented with TAE and probiotic (TAE+BC): commercial feed with TAE and probiotic, cocci-vaccinated. The anticoccidial products used in this experiment were: ionophore anticoccidial salinomycin Bio-Cox 60 (Zoetis, Florham Park, NJ) added at 66 g/MT of feed and chemical anticoccidial robenidine Robenz (Zoetis, Florham Park, NJ) added at 33 g/MT in feed. The TAE product (Kemin Industries, Inc., Des Moines, IA) was mixed into finished feed at an inclusion rate of 500 g/MT of feed, giving a final concentration of 100 mg TAE per kg of feed. The TAE and probiotic combination product used TAE combined with a single-species Bacillus coagulans probiotic having a content of ≥ 1.0 × 107 spores per kg (VANNIX C, Kemin Industries, Inc., Des Moines, IA). The product was mixed into the finished feed at an inclusion rate of 500 g/MT of feed, giving a final concentration of 100 mg TAE per kg of feed and ≥1.0 × 104 spores of B. coagulans per kg of feed. Table 2. Composition of the experimental starter, grower and finisher diets and their nutrient profiles (Experiment 2). Diet Item Starter Grower Finisher Ingredient ———————-%————————  Corn 55.44 60.05 65.37  Soybean meal dehulled 35.71 31.10 26.38  Corn DDGS 4.00 4.00 4.00  Fat 1.26 1.73 1.59  Dicalcium phosphate 1.28 0.99 0.77  Calcium carbonate 1.15 1.11 0.92  Sodium chloride 0.44 0.42 0.43  DL-Methionine 0.35 0.26 0.22  L-Lysine 0.21 0.20 0.17  Ronozyme P-(ct) 0.02 0.02 0.02  Trace Mineral1 0.08 0.08 0.08  Vitamin premix2 0.07 0.07 0.07 Analyzed value3 (%)  DM 88.10 88.05 87.93  CP 22.93 21.01 19.12  Crude fiber 2.42 2.37 2.33  Crude fat 3.96 4.54 4.54  Calcium 0.92 0.83 0.70  Phosphorus (total) 0.64 0.57 0.51 Calculated value  ME (kcal/kg) 3000 3080 3130  Methionine 0.67 0.57 0.51  TSAA 0.94 0.82 0.75  Lysine 1.42 1.28 1.13  Threonine 0.93 0.85 0.77  Tryptophan 0.30 0.27 0.24  Sodium 0.21 0.20 0.20  Potassium 0.89 0.81 0.74  Chloride 0.31 0.29 0.30 Diet Item Starter Grower Finisher Ingredient ———————-%————————  Corn 55.44 60.05 65.37  Soybean meal dehulled 35.71 31.10 26.38  Corn DDGS 4.00 4.00 4.00  Fat 1.26 1.73 1.59  Dicalcium phosphate 1.28 0.99 0.77  Calcium carbonate 1.15 1.11 0.92  Sodium chloride 0.44 0.42 0.43  DL-Methionine 0.35 0.26 0.22  L-Lysine 0.21 0.20 0.17  Ronozyme P-(ct) 0.02 0.02 0.02  Trace Mineral1 0.08 0.08 0.08  Vitamin premix2 0.07 0.07 0.07 Analyzed value3 (%)  DM 88.10 88.05 87.93  CP 22.93 21.01 19.12  Crude fiber 2.42 2.37 2.33  Crude fat 3.96 4.54 4.54  Calcium 0.92 0.83 0.70  Phosphorus (total) 0.64 0.57 0.51 Calculated value  ME (kcal/kg) 3000 3080 3130  Methionine 0.67 0.57 0.51  TSAA 0.94 0.82 0.75  Lysine 1.42 1.28 1.13  Threonine 0.93 0.85 0.77  Tryptophan 0.30 0.27 0.24  Sodium 0.21 0.20 0.20  Potassium 0.89 0.81 0.74  Chloride 0.31 0.29 0.30 1Trace mineral mix provided the following (per kg of diet): manganese (MnSO4•H2O), 60 mg; iron (FeSO4•7H2O), 30 mg; zinc (ZnO), 50 mg; copper (CuSO4•5H2O), 5 mg; iodine (ethylene diamine dihydroiodide), 0.15 mg; selenium (NaSe03), 0.3 mg. 2Vitamin mix provided the following (per kg of diet): Vitamin A, 12,345 IU; Vitamin D3, 3472 IU; 25-hydroxyvitamin D3, 96.6 μg; Vitamin E, 49 IU; vitamin B12 (cobalamin), 21.7 μg; Biotin, 0.23 mg; Menadione, 2.78 mg; Thiamine, 2.62 mg; Riboflavin, 10.8 mg; d-Panthothenic Acid, 18.5 mg; Vitamin B6, 4.6 mg; Niacin, 61.7 mg; Folic Acid, 1.5 mg. 3Analyzed values refer to basal (control) diet. View Large Table 2. Composition of the experimental starter, grower and finisher diets and their nutrient profiles (Experiment 2). Diet Item Starter Grower Finisher Ingredient ———————-%————————  Corn 55.44 60.05 65.37  Soybean meal dehulled 35.71 31.10 26.38  Corn DDGS 4.00 4.00 4.00  Fat 1.26 1.73 1.59  Dicalcium phosphate 1.28 0.99 0.77  Calcium carbonate 1.15 1.11 0.92  Sodium chloride 0.44 0.42 0.43  DL-Methionine 0.35 0.26 0.22  L-Lysine 0.21 0.20 0.17  Ronozyme P-(ct) 0.02 0.02 0.02  Trace Mineral1 0.08 0.08 0.08  Vitamin premix2 0.07 0.07 0.07 Analyzed value3 (%)  DM 88.10 88.05 87.93  CP 22.93 21.01 19.12  Crude fiber 2.42 2.37 2.33  Crude fat 3.96 4.54 4.54  Calcium 0.92 0.83 0.70  Phosphorus (total) 0.64 0.57 0.51 Calculated value  ME (kcal/kg) 3000 3080 3130  Methionine 0.67 0.57 0.51  TSAA 0.94 0.82 0.75  Lysine 1.42 1.28 1.13  Threonine 0.93 0.85 0.77  Tryptophan 0.30 0.27 0.24  Sodium 0.21 0.20 0.20  Potassium 0.89 0.81 0.74  Chloride 0.31 0.29 0.30 Diet Item Starter Grower Finisher Ingredient ———————-%————————  Corn 55.44 60.05 65.37  Soybean meal dehulled 35.71 31.10 26.38  Corn DDGS 4.00 4.00 4.00  Fat 1.26 1.73 1.59  Dicalcium phosphate 1.28 0.99 0.77  Calcium carbonate 1.15 1.11 0.92  Sodium chloride 0.44 0.42 0.43  DL-Methionine 0.35 0.26 0.22  L-Lysine 0.21 0.20 0.17  Ronozyme P-(ct) 0.02 0.02 0.02  Trace Mineral1 0.08 0.08 0.08  Vitamin premix2 0.07 0.07 0.07 Analyzed value3 (%)  DM 88.10 88.05 87.93  CP 22.93 21.01 19.12  Crude fiber 2.42 2.37 2.33  Crude fat 3.96 4.54 4.54  Calcium 0.92 0.83 0.70  Phosphorus (total) 0.64 0.57 0.51 Calculated value  ME (kcal/kg) 3000 3080 3130  Methionine 0.67 0.57 0.51  TSAA 0.94 0.82 0.75  Lysine 1.42 1.28 1.13  Threonine 0.93 0.85 0.77  Tryptophan 0.30 0.27 0.24  Sodium 0.21 0.20 0.20  Potassium 0.89 0.81 0.74  Chloride 0.31 0.29 0.30 1Trace mineral mix provided the following (per kg of diet): manganese (MnSO4•H2O), 60 mg; iron (FeSO4•7H2O), 30 mg; zinc (ZnO), 50 mg; copper (CuSO4•5H2O), 5 mg; iodine (ethylene diamine dihydroiodide), 0.15 mg; selenium (NaSe03), 0.3 mg. 2Vitamin mix provided the following (per kg of diet): Vitamin A, 12,345 IU; Vitamin D3, 3472 IU; 25-hydroxyvitamin D3, 96.6 μg; Vitamin E, 49 IU; vitamin B12 (cobalamin), 21.7 μg; Biotin, 0.23 mg; Menadione, 2.78 mg; Thiamine, 2.62 mg; Riboflavin, 10.8 mg; d-Panthothenic Acid, 18.5 mg; Vitamin B6, 4.6 mg; Niacin, 61.7 mg; Folic Acid, 1.5 mg. 3Analyzed values refer to basal (control) diet. View Large Live Performance Upon arrival at the trial facility, all birds were vaccinated on day of hatch by spray cabinet with a commercial coccidia vaccine, Advent (Novus International, Inc., St. Louis, MO), which contains viable attenuated oocysts of E. acervulina, E. maxima and E. tenella. Birds were allowed to preen for 1 h to improve vaccine uptake prior to placement. Vaccinated birds were randomly allotted to dietary treatments and bird weights by pen were recorded to ensure average initial pen weight was similar between groups. On d 1, 21, 35, and 49, BW, BWG and FI were determined and FCR was calculated for d 21, 35, and 49 following correction for BW of mortality. On d 28 and 49, litter conditions were graded. A grading scale of 0–4, 0 being dry and 1, 2, 3, or 4 indicating increased wetness and unacceptability, developed by Abdelrahman et al. (2014) was used for litter quality grading. Coccidial Immunity Challenge On d 28, 4 pens per treatment (n = 20 pens) were allocated to a coccidial immunity challenge. Birds and feed were weighed by pen and treatment pens were divided into two subgroups, one of which was reissued commercial grower feed containing supplemented additives (group 1) and the other, which additives were withdrawn from the reissued feed (group 2). On d 29, all birds in the 20 pens were challenged with a mixed-species Eimeria oral inoculum consisting of E. acervulina (1 × 105), E. maxima (5 × 104) and E. tenella (7.5 × 104) sporulated oocysts. On d 35 (6 d post-challenge), feed was weighed back, pen weights were taken and BW, BWG, FI and mortality corrected FCR were determined for the challenge period (d 28 to 35). In addition, 20 broilers from each pen were euthanized and necropsied for scoring of coccidiosis lesions in the upper, middle and cecal regions of the intestinal tract (Johnson and Reid, 1970). Fresh fecal samples (10 per pen) were also collected on d 35, which were pooled and stored at 4°C until measurement of oocyst counts. Counts were determined via dilution and microscopic enumeration using a McMaster counting chamber and are reported as oocysts per gram of excreta. Statistical Analysis Experiment 1 Data were analyzed by ANOVA using the GLM procedure of SPSS (IMB SPSS 19, IMB Corp., Armonk, NY). Cage served as the experimental unit. Statistical significance was determined by one-way ANOVA and means were separated by least significant differences. The threshold for statistical significance was P ≤ 0.05. Experiment 2 BWG, FI and FCR for d 0 to 21 and d 0 to 49 were analyzed by ANOVA using the GLM procedure of SPSS (IMB SPSS 19, IMB Corp., Armonk, NY). Pen served as the experimental unit. Statistical significance was determined by one-way ANOVA and means were separated by least significant differences. Due to the experimental blocking scheme used in the coccidial immunity challenge, vaccinated, challenged control pens were averaged separately (n = 2 pens) rather than together (n = 4 pens) for analysis. For the coccidial challenge, data collected at d 35, including post-challenge BWG, FI, FCR, lesion scores and oocysts, were analyzed using one-way ANOVA. Means were separated by Duncan's multiple range test with differences deemed significant at P ≤ 0.05. RESULTS Experiment 1 Initial cage weight was not different (P = 0.2473) among groups. As anticipated, broilers challenged with mixed Eimeria spp. had reduced performance compared to unchallenged broilers (Table 3). During the challenge period (d 14 to 20), broilers challenged with Eimeria had reduced (P < 0.05) BWG and FI and significantly higher FCR than NC. No difference was observed in the BWG of SAL, TA and TAE from PC, whereas TA had reduced (P < 0.05) BWG compared to SAL. Reduced (P < 0.05) FI was observed with TA compared to PC and SAL, but was not different from TAE. FCR was improved (P < 0.05) with SAL and TAE compared to PC, while TA was similar to PC, SAL and TAE. Over d 1 to 20, FI was similar for all groups, whereas Eimeria challenge resulted in depressed (P < 0.05) BWG and worsened (P < 0.05) FCR compared to NC. No difference in BWG and FCR was observed between PC, SAL, TA and TAE. During both d 14 to 20 and d 1 to 20, FI, BWG and FCR were similar (P ≥ 0.05) for SAL and TAE, however, no supplemented challenged group recovered performance to that observed in the NC. Table 3. Body weight gain (BWG; g), feed intake (FI; g) and mortality-corrected feed conversion ratio (FCR) of broilers which were infected with a mixed-species Eimeria spp. inoculum on d 14 of age, when fed diets supplemented with anticoccidial ionophore, tannic acid and tannic acid extract (Experiment 1). d 14 to 20 d 1 to 20 Infection1 Item2 BWG FI FCR BWG FI FCR − NC 295a 398a 1.35c 530a 791 1.50b + PC 177b,c 349b 2.00a 416b 730 1.76a SAL 198b 352b 1.79b 451b 774 1.72a TA 173c 311c 1.86a,b 433b 730 1.71a TAE 179b,c 324b,c 1.82b 423b 726 1.72a SEM3 $$\phantom{>}$$8.1 $$\phantom{0}$$11.0 $$\phantom{>}$$0.05 14.6 18.0 0.03a P-value <0.001 <0.001 <0.001 <0.001 0.051 <0.001 d 14 to 20 d 1 to 20 Infection1 Item2 BWG FI FCR BWG FI FCR − NC 295a 398a 1.35c 530a 791 1.50b + PC 177b,c 349b 2.00a 416b 730 1.76a SAL 198b 352b 1.79b 451b 774 1.72a TA 173c 311c 1.86a,b 433b 730 1.71a TAE 179b,c 324b,c 1.82b 423b 726 1.72a SEM3 $$\phantom{>}$$8.1 $$\phantom{0}$$11.0 $$\phantom{>}$$0.05 14.6 18.0 0.03a P-value <0.001 <0.001 <0.001 <0.001 0.051 <0.001 a-cMeans within a column not sharing the same superscript differ significantly (P < 0.05). 1Mixed-species challenge contained Eimeria acervulina (5 × 105), Eimeria maxima (8.5 × 104) and Eimeria tenella (1.75 × 105) sporulated oocysts. 2NC = negative control. The broilers were fed a non-medicated diet containing no anticoccidial and growth promoter and were supplemented with anticoccidial ionophore salinomycin (SAL; 66 mg/kg of diet), tannic acid (TA; 100 mg/kg of diet) and tannic acid extract (TAE; 100 mg/kg of diet). 3Pooled standard error of the mean. View Large Table 3. Body weight gain (BWG; g), feed intake (FI; g) and mortality-corrected feed conversion ratio (FCR) of broilers which were infected with a mixed-species Eimeria spp. inoculum on d 14 of age, when fed diets supplemented with anticoccidial ionophore, tannic acid and tannic acid extract (Experiment 1). d 14 to 20 d 1 to 20 Infection1 Item2 BWG FI FCR BWG FI FCR − NC 295a 398a 1.35c 530a 791 1.50b + PC 177b,c 349b 2.00a 416b 730 1.76a SAL 198b 352b 1.79b 451b 774 1.72a TA 173c 311c 1.86a,b 433b 730 1.71a TAE 179b,c 324b,c 1.82b 423b 726 1.72a SEM3 $$\phantom{>}$$8.1 $$\phantom{0}$$11.0 $$\phantom{>}$$0.05 14.6 18.0 0.03a P-value <0.001 <0.001 <0.001 <0.001 0.051 <0.001 d 14 to 20 d 1 to 20 Infection1 Item2 BWG FI FCR BWG FI FCR − NC 295a 398a 1.35c 530a 791 1.50b + PC 177b,c 349b 2.00a 416b 730 1.76a SAL 198b 352b 1.79b 451b 774 1.72a TA 173c 311c 1.86a,b 433b 730 1.71a TAE 179b,c 324b,c 1.82b 423b 726 1.72a SEM3 $$\phantom{>}$$8.1 $$\phantom{0}$$11.0 $$\phantom{>}$$0.05 14.6 18.0 0.03a P-value <0.001 <0.001 <0.001 <0.001 0.051 <0.001 a-cMeans within a column not sharing the same superscript differ significantly (P < 0.05). 1Mixed-species challenge contained Eimeria acervulina (5 × 105), Eimeria maxima (8.5 × 104) and Eimeria tenella (1.75 × 105) sporulated oocysts. 2NC = negative control. The broilers were fed a non-medicated diet containing no anticoccidial and growth promoter and were supplemented with anticoccidial ionophore salinomycin (SAL; 66 mg/kg of diet), tannic acid (TA; 100 mg/kg of diet) and tannic acid extract (TAE; 100 mg/kg of diet). 3Pooled standard error of the mean. View Large Oocyst shedding was reduced by 58% in TAE compared to PC, while SAL reduced oocysts by 21% compared to PC, albeit all Eimeria challenged groups had increased (P < 0.05) oocyst shedding compared to NC (Figure 1). A 51% reduction in oocysts was additionally observed with TAE compared to TA. Overall, significant reduction in oocyst shedding was observed with TAE compared to PC and TA, whereas SAL was not different from PC, TA and TAE. Lesion scores were recorded separately for the upper, middle, and cecal intestinal regions (Figure 2). No lesions were observed in any region of the intestine in NC. Eimeria challenged broilers supplemented with SAL, TA and TAE had reduced (P < 0.05) lesions in the upper intestine, middle intestine and ceca compared to PC. Lesion scores in the upper intestine and ceca were not different between SAL, TAE and TA, whereas TAE had significantly fewer middle intestine lesions than SAL. While performance and lesion reductions were similar for TA and TAE, TAE showed significantly improved reduction in oocyst shedding compared to TA and was therefore selected for use in Experiment 2. Figure 1. View largeDownload slide Total oocysts per gram (OPG) of excreta in broilers infected with Eimeria spp. on d 14 of age in Experiment 1. Excreta samples were collected from broilers 6 d after mixed Eimeria spp. challenge. Means represent 8 replicates per treatment (1 sample/cage). NC = negative control (non-medicated feed, uninfected); PC = positive control (non-medicated feed, infected); SAL = salinomycin supplemented feed, infected (Sacox 60, 66 mg/kg of diet); TA = tannic acid supplemented feed, infected (100 mg/kg of diet); TAE = tannic acid extract supplemented feed, infected (TAE, 100 mg/kg of diet). Means with different letters (a-c) differ significantly (P < 0.01). Figure 1. View largeDownload slide Total oocysts per gram (OPG) of excreta in broilers infected with Eimeria spp. on d 14 of age in Experiment 1. Excreta samples were collected from broilers 6 d after mixed Eimeria spp. challenge. Means represent 8 replicates per treatment (1 sample/cage). NC = negative control (non-medicated feed, uninfected); PC = positive control (non-medicated feed, infected); SAL = salinomycin supplemented feed, infected (Sacox 60, 66 mg/kg of diet); TA = tannic acid supplemented feed, infected (100 mg/kg of diet); TAE = tannic acid extract supplemented feed, infected (TAE, 100 mg/kg of diet). Means with different letters (a-c) differ significantly (P < 0.01). Figure 2. View largeDownload slide Intestinal lesion scores (LS) observed in broilers infected with Eimeria spp. on d 14 of age in Experiment 1. Lesion scores were assessed 6 d after mixed Eimeria spp. challenge. A lesion score was assigned from 0 (no gross lesions) to 4 (extensive lesions) according to the system of Johnson and Reid (1970). Means represent 8 replicates per treatment (8 samples/cage). NC = negative control (non-medicated feed, uninfected); PC = positive control (non-medicated feed, infected); SAL = salinomycin supplemented feed, infected (Sacox 60, 66 mg/kg of diet); TA = tannic acid supplemented feed, infected (100 mg/kg of diet); TAE = tannic acid extract supplemented feed, infected (TAE, 100 mg/kg of diet). Within each intestinal region, means with different letters (a-d) differ significantly (P < 0.01). Figure 2. View largeDownload slide Intestinal lesion scores (LS) observed in broilers infected with Eimeria spp. on d 14 of age in Experiment 1. Lesion scores were assessed 6 d after mixed Eimeria spp. challenge. A lesion score was assigned from 0 (no gross lesions) to 4 (extensive lesions) according to the system of Johnson and Reid (1970). Means represent 8 replicates per treatment (8 samples/cage). NC = negative control (non-medicated feed, uninfected); PC = positive control (non-medicated feed, infected); SAL = salinomycin supplemented feed, infected (Sacox 60, 66 mg/kg of diet); TA = tannic acid supplemented feed, infected (100 mg/kg of diet); TAE = tannic acid extract supplemented feed, infected (TAE, 100 mg/kg of diet). Within each intestinal region, means with different letters (a-d) differ significantly (P < 0.01). Experiment 2 Live Performance Initial chick BW was 40.60 ± 0.55 g and was similar between all groups (P = 0.3360). During the starter phase (d 1 to 21), FCR was improved (P < 0.05) with supplementation of SAL, ROB, TAE and TAE+BC compared to CNT and the improvement in FCR was similar (P > 0.05) between the supplemented groups (Table 4). Increased (P < 0.05) BWG during d 1 to 21 was observed in SAL, ROB and TAE compared to TAE+BC and CNT. Similar to d 1 to 21, from d 1 to 49, vaccinated broiler FCR was significantly improved with SAL, ROB, TAE and TAE+BC compared to CNT, with SAL and ROB having lower (P < 0.05) FCR than TAE and TAE+BC over the full trial. Increased (P < 0.05) BWG was observed in SAL and ROB compared to CNT, while BWG of TAE was similar (P > 0.05) to all groups. No significant difference in BWG was observed between TAE+BC, TAE and ROB, however, BWG of TAE+BC was reduced (P < 0.05) compared to SAL. Both mortality and FI were similar (P ≥ 0.05) for all groups during the experiment. Litter scores at d 28 and 49 were not different between CNT, SAL, ROB, TAE and TAE+BC groups (Figure 3). Overall, live performance of vaccinated broilers was enhanced with supplementation of all treatments, and differences between TAE and TAE+BC supplemented broilers were minimal over d 1 to 49. Figure 3. View largeDownload slide Litter scores on d 28 and 49 of Experiment 2. No significant differences (P > 0.05) were observed between treatments on both days. CNT = non-medicated control; SAL = salinomycin (Bio-Cox, 66 mg/kg of diet) supplemented; ROB = robenidine (Robenz, 33 mg/kg of diet) supplemented; TAE = tannic acid extract (100 mg/kg of diet) supplemented; TAE+BC = tannic acid extract with Bacillus coagulans (110 mg/kg of diet). Figure 3. View largeDownload slide Litter scores on d 28 and 49 of Experiment 2. No significant differences (P > 0.05) were observed between treatments on both days. CNT = non-medicated control; SAL = salinomycin (Bio-Cox, 66 mg/kg of diet) supplemented; ROB = robenidine (Robenz, 33 mg/kg of diet) supplemented; TAE = tannic acid extract (100 mg/kg of diet) supplemented; TAE+BC = tannic acid extract with Bacillus coagulans (110 mg/kg of diet). Table 4. Body weight gain (BWG; g), feed intake (FI; g), mortality-corrected feed conversion ratio (FCR) and mortality of broilers cocci-vaccinated with live oocysts1 on d 1 of age when fed diets supplemented with anticoccidial chemical, anticoccidial ionophore, tannic acid extract and tannic acid extract combined with Bacillus coagulans (Experiment 2). d 1 to 21 d 1 to 49 Diet2 BWG FI FCR BWG FI FCR Mort. CNT 597b 985 1.55a 2,795c 5197 1.87a 4.6 SAL 637a 1003 1.49b 2,991a 5345 1.79c 3.9 ROB 630a 993 1.48b 2,945a,b 5322 1.79c 3.6 TAE 624a 996 1.50b 2,869a,b,c 5260 1.83b 3.6 TAE+BC 557b 962 1.51b 2,844b,c 5183 1.84b 5.2 SEM3 $$\phantom{00}$$8.8 58.1 0.01 $$\phantom{00,}$$51.4 80.1 0.01 1.1 P-value $$\phantom{00}$$0.003 0.125 0.007 $$\phantom{0,}$$>0.040 0.495 <0.001 0.821 d 1 to 21 d 1 to 49 Diet2 BWG FI FCR BWG FI FCR Mort. CNT 597b 985 1.55a 2,795c 5197 1.87a 4.6 SAL 637a 1003 1.49b 2,991a 5345 1.79c 3.9 ROB 630a 993 1.48b 2,945a,b 5322 1.79c 3.6 TAE 624a 996 1.50b 2,869a,b,c 5260 1.83b 3.6 TAE+BC 557b 962 1.51b 2,844b,c 5183 1.84b 5.2 SEM3 $$\phantom{00}$$8.8 58.1 0.01 $$\phantom{00,}$$51.4 80.1 0.01 1.1 P-value $$\phantom{00}$$0.003 0.125 0.007 $$\phantom{0,}$$>0.040 0.495 <0.001 0.821 a-cMeans within a column not sharing the same superscript differ significantly (P < 0.05). 1Cocci-vaccinated with Advent (Novus International, Inc., St. Louis, MO) at 1 day of age via a spray cabinet. 2The broilers were fed a non-medicated control (CNT) diet containing no anticoccidial and growth promoter and were supplemented with anticoccidial ionophore salinomycin (SAL; 66 mg/kg of diet), anticoccidial chemical robenidine (ROB; 33 mg/kg of diet), tannic acid extract (TAE; 100 mg/kg of diet) and TAE combined with probiotic Bacillus coagulans (TAE+BC; 110 mg/kg of diet). 3Pooled standard error of the mean. View Large Table 4. Body weight gain (BWG; g), feed intake (FI; g), mortality-corrected feed conversion ratio (FCR) and mortality of broilers cocci-vaccinated with live oocysts1 on d 1 of age when fed diets supplemented with anticoccidial chemical, anticoccidial ionophore, tannic acid extract and tannic acid extract combined with Bacillus coagulans (Experiment 2). d 1 to 21 d 1 to 49 Diet2 BWG FI FCR BWG FI FCR Mort. CNT 597b 985 1.55a 2,795c 5197 1.87a 4.6 SAL 637a 1003 1.49b 2,991a 5345 1.79c 3.9 ROB 630a 993 1.48b 2,945a,b 5322 1.79c 3.6 TAE 624a 996 1.50b 2,869a,b,c 5260 1.83b 3.6 TAE+BC 557b 962 1.51b 2,844b,c 5183 1.84b 5.2 SEM3 $$\phantom{00}$$8.8 58.1 0.01 $$\phantom{00,}$$51.4 80.1 0.01 1.1 P-value $$\phantom{00}$$0.003 0.125 0.007 $$\phantom{0,}$$>0.040 0.495 <0.001 0.821 d 1 to 21 d 1 to 49 Diet2 BWG FI FCR BWG FI FCR Mort. CNT 597b 985 1.55a 2,795c 5197 1.87a 4.6 SAL 637a 1003 1.49b 2,991a 5345 1.79c 3.9 ROB 630a 993 1.48b 2,945a,b 5322 1.79c 3.6 TAE 624a 996 1.50b 2,869a,b,c 5260 1.83b 3.6 TAE+BC 557b 962 1.51b 2,844b,c 5183 1.84b 5.2 SEM3 $$\phantom{00}$$8.8 58.1 0.01 $$\phantom{00,}$$51.4 80.1 0.01 1.1 P-value $$\phantom{00}$$0.003 0.125 0.007 $$\phantom{0,}$$>0.040 0.495 <0.001 0.821 a-cMeans within a column not sharing the same superscript differ significantly (P < 0.05). 1Cocci-vaccinated with Advent (Novus International, Inc., St. Louis, MO) at 1 day of age via a spray cabinet. 2The broilers were fed a non-medicated control (CNT) diet containing no anticoccidial and growth promoter and were supplemented with anticoccidial ionophore salinomycin (SAL; 66 mg/kg of diet), anticoccidial chemical robenidine (ROB; 33 mg/kg of diet), tannic acid extract (TAE; 100 mg/kg of diet) and TAE combined with probiotic Bacillus coagulans (TAE+BC; 110 mg/kg of diet). 3Pooled standard error of the mean. View Large Coccidial Immunity Challenge Similar (P ≥ 0.05) BWG, FI and FCR were observed for the CNT groups following mixed Eimeria spp. challenge (Table 5). In group 1, challenged broilers supplemented with SAL, ROB, TAE and TAE+BC were observed to have increased (P < 0.05) BWG compared to CNT. Supplementation of ROB, TAE and TAE+BC provided similar improvement in BWG, while SAL had higher (P < 0.05) BWG than ROB and TAE+BC but was not different (P ≥ 0.05) from TAE. Similarly, FI was not different (P > 0.05) between CNT and birds supplemented with SAL, ROB, TAE and TAE+BC during the Eimeria challenge. Compared to challenged CNT birds, a significant improvement in FCR was observed upon supplementation with SAL, ROB and TAE, with no difference between FCR of broilers fed the supplemented products (P ≥ 0.05). Supplementation with TAE+BC during challenge resulted in similar FCR to ROB and TAE, however, improvement in FCR was not significantly different from CNT. Table 5. Body weight gain (BWG; g), feed intake (FI; g) and mortality-corrected feed conversion ratio (FCR) of cocci-vaccinated1 broilers d 6 after mixed Eimeria spp. challenge2 at d 29, when fed diets supplemented with anticoccidial chemical, anticoccidial ionophore, tannic acid extract and tannic acid extract combined with Bacillus coagulans (Experiment 2). d 28 to 35 Diet3 Supplemented feed4 BWG FI FCR CNT NA 469c,d 825a,b 1.80a,b SAL + 623a 868a 1.41d ROB 564b 769a,b,c 1.55c,d TAE 561a,b 868a 1.58c,d TAE+BC 552b 904a 1.67a,b,c CNT NA 458c,d 841a,b 1.84a SAL - 469c,d 796a,b 1.80a,b ROB 381e 710b,c 1.86a TAE 444d,e 618c 1.55c,d TAE+BC 516b,c 806a,b 1.61b,c,d SEM5 $$\phantom{0}$$20.7 $$\phantom{0}$$48.1 0.07 P-value $$\phantom{.}$$<0.001 $$\phantom{00}$$0.047 0.009 d 28 to 35 Diet3 Supplemented feed4 BWG FI FCR CNT NA 469c,d 825a,b 1.80a,b SAL + 623a 868a 1.41d ROB 564b 769a,b,c 1.55c,d TAE 561a,b 868a 1.58c,d TAE+BC 552b 904a 1.67a,b,c CNT NA 458c,d 841a,b 1.84a SAL - 469c,d 796a,b 1.80a,b ROB 381e 710b,c 1.86a TAE 444d,e 618c 1.55c,d TAE+BC 516b,c 806a,b 1.61b,c,d SEM5 $$\phantom{0}$$20.7 $$\phantom{0}$$48.1 0.07 P-value $$\phantom{.}$$<0.001 $$\phantom{00}$$0.047 0.009 a-eMeans within a column not sharing the same superscript differ significantly (P < 0.05). 1Cocci-vaccinated with Advent (Novus International, Inc., St. Louis, MO) at day 1 of age via a spray cabinet. 2Mixed-species challenge contained Eimeria acervulina (1 × 105), Eimeria maxima (5 × 104) and Eimeria tenella (7.5 × 104) sporulated oocysts. 3The broilers were fed a non-medicated control (CNT) diet containing no anticoccidial and growth promoter and were supplemented with anticoccidial ionophore salinomycin (SAL; 66 mg/kg of diet), anticoccidial chemical robenidine (ROB; 33 mg/kg of diet), tannic acid extract (TAE; 100 mg/kg of diet) and TAE combined with probiotic Bacillus coagulans (TAE+BC; 110 mg/kg of diet). 4On d 28, broilers were issued commercial grower feed containing supplemental treatment materials (+) or CNT grower feed with the previously added supplemental treatment materials withdrawn (−). Feed issued to CNT treatment during the challenge was the same for all challenged control pens (NA) as it never contained supplemental materials. 5Pooled standard error of the mean. View Large Table 5. Body weight gain (BWG; g), feed intake (FI; g) and mortality-corrected feed conversion ratio (FCR) of cocci-vaccinated1 broilers d 6 after mixed Eimeria spp. challenge2 at d 29, when fed diets supplemented with anticoccidial chemical, anticoccidial ionophore, tannic acid extract and tannic acid extract combined with Bacillus coagulans (Experiment 2). d 28 to 35 Diet3 Supplemented feed4 BWG FI FCR CNT NA 469c,d 825a,b 1.80a,b SAL + 623a 868a 1.41d ROB 564b 769a,b,c 1.55c,d TAE 561a,b 868a 1.58c,d TAE+BC 552b 904a 1.67a,b,c CNT NA 458c,d 841a,b 1.84a SAL - 469c,d 796a,b 1.80a,b ROB 381e 710b,c 1.86a TAE 444d,e 618c 1.55c,d TAE+BC 516b,c 806a,b 1.61b,c,d SEM5 $$\phantom{0}$$20.7 $$\phantom{0}$$48.1 0.07 P-value $$\phantom{.}$$<0.001 $$\phantom{00}$$0.047 0.009 d 28 to 35 Diet3 Supplemented feed4 BWG FI FCR CNT NA 469c,d 825a,b 1.80a,b SAL + 623a 868a 1.41d ROB 564b 769a,b,c 1.55c,d TAE 561a,b 868a 1.58c,d TAE+BC 552b 904a 1.67a,b,c CNT NA 458c,d 841a,b 1.84a SAL - 469c,d 796a,b 1.80a,b ROB 381e 710b,c 1.86a TAE 444d,e 618c 1.55c,d TAE+BC 516b,c 806a,b 1.61b,c,d SEM5 $$\phantom{0}$$20.7 $$\phantom{0}$$48.1 0.07 P-value $$\phantom{.}$$<0.001 $$\phantom{00}$$0.047 0.009 a-eMeans within a column not sharing the same superscript differ significantly (P < 0.05). 1Cocci-vaccinated with Advent (Novus International, Inc., St. Louis, MO) at day 1 of age via a spray cabinet. 2Mixed-species challenge contained Eimeria acervulina (1 × 105), Eimeria maxima (5 × 104) and Eimeria tenella (7.5 × 104) sporulated oocysts. 3The broilers were fed a non-medicated control (CNT) diet containing no anticoccidial and growth promoter and were supplemented with anticoccidial ionophore salinomycin (SAL; 66 mg/kg of diet), anticoccidial chemical robenidine (ROB; 33 mg/kg of diet), tannic acid extract (TAE; 100 mg/kg of diet) and TAE combined with probiotic Bacillus coagulans (TAE+BC; 110 mg/kg of diet). 4On d 28, broilers were issued commercial grower feed containing supplemental treatment materials (+) or CNT grower feed with the previously added supplemental treatment materials withdrawn (−). Feed issued to CNT treatment during the challenge was the same for all challenged control pens (NA) as it never contained supplemental materials. 5Pooled standard error of the mean. View Large An immunity challenge was created in group 2 by issuing previously supplemented, vaccinated broilers feed without supplemental materials during the Eimeria spp. challenge. Withdrawal of ROB resulted in significantly reduced broiler BWG compared to CNT and broilers supplemented with ROB (group 1). Similar BWG to CNT was observed after withdrawal of TAE, SAL and TAE+BC, however, when the materials were supplemented (group 1), broilers had increased (P < 0.05) BWG as compared to vaccinated, challenged broilers during the challenge. Withdrawal of SAL and TAE+BC did not impact FI compared to CNT birds, however, FI was reduced (P < 0.05) following withdrawal of TAE and ROB during the challenge. Withdrawal of ROB and SAL resulted in FCR similar to CNT, whereas supplementation with ROB and SAL during challenge was found to significantly improve FCR of vaccinated, challenged broilers. In contrast, withdrawal of TAE and TAE+BC did not negatively impact broiler FCR during the immunity challenge. Broilers previously treated with TAE and TAE+BC maintained significantly improved FCR compared to the vaccinated, challenged CNT birds. Overall, withdrawal of ROB and SAL resulted in a > 0.30 increase in broiler FCR, whereas withdrawal of TAE and TAE+BC minimally impacted vaccinated, Eimeria challenged broilers. Following Eimeria challenge, lesions were observed in the challenged, supplemented; challenged, non-supplemented; and CNT groups (Table 6). Lesions in the ceca, indicative of E. tenella, were increased compared to those due to E. acervulina and E. maxima in the upper and middle intestine, respectively; however, lesions did not differ (P ≥ 0.05) among challenged groups. Withdrawal of SAL, ROB, TAE and TAE+BC resulted in ≥ 2-fold increase in oocyst shedding compared to when supplemented in-feed following Eimeria challenge. Although oocyst counts were found to be consistently reduced in CNT and TAE+BC groups, the differences were not significant (P ≥ 0.05). Table 6. Intestinal lesion scores1 and total oocysts per gram (OPG) of excreta of cocci-vaccinated2 broilers d 6 after mixed Eimeria spp. challenge3 at d 29, when fed diets supplemented with anticoccidial chemical, anticoccidial ionophore, tannic acid extract and tannic acid extract combined with Bacillus coagulans (Experiment 2). Diet4 Supplemented feed5 Upper Middle Ceca Total OPG CNT NA 0.58 0.43 1.70 1830 SAL + 0.95 0.60 1.05 1970 ROB 0.45 0.30 2.30 2340 TAE 0.75 0.30 1.45 2070 TAE+BC 0.33 0.23 0.38 230 CNT NA 0.95 0.50 1.07 270 SAL - 1.50 0.78 1.00 21,180 ROB 1.38 0.68 1.00 8940 TAE 1.08 1.13 1.83 4370 TAE+BC 0.73 0.60 1.15 570 SEM6 0.23 0.21 0.34 6310 P-value 0.061 0.223 0.128 $$\phantom{0}$$0.470 Diet4 Supplemented feed5 Upper Middle Ceca Total OPG CNT NA 0.58 0.43 1.70 1830 SAL + 0.95 0.60 1.05 1970 ROB 0.45 0.30 2.30 2340 TAE 0.75 0.30 1.45 2070 TAE+BC 0.33 0.23 0.38 230 CNT NA 0.95 0.50 1.07 270 SAL - 1.50 0.78 1.00 21,180 ROB 1.38 0.68 1.00 8940 TAE 1.08 1.13 1.83 4370 TAE+BC 0.73 0.60 1.15 570 SEM6 0.23 0.21 0.34 6310 P-value 0.061 0.223 0.128 $$\phantom{0}$$0.470 1Lesion scores were assigned from 0 (no gross lesions) to 4 (extensive lesions) according to the system of Johnson and Reid (1970). 2Cocci-vaccinated with Advent (Novus International, Inc., St. Louis, MO) at day 1 of age via a spray cabinet. 3Mixed-species challenge contained Eimeria acervulina (1 × 105), Eimeria maxima (5 × 104) and Eimeria tenella (7.5 × 104) sporulated oocysts. 4The broilers were fed a non-medicated control (CNT) diet containing no anticoccidial and growth promoter and were supplemented with anticoccidial ionophore salinomycin (SAL; 66 mg/kg of diet), anticoccidial chemical robenidine (ROB; 33 mg/kg of diet), tannic acid extract (TAE; 100 mg/kg of diet) and TAE combined with probiotic Bacillus coagulans (TAE+BC; 110 mg/kg of diet). 5On d 28, broilers were issued commercial grower feed containing supplemental treatment materials (+) or CNT grower feed with the previously added supplemental treatment materials withdrawn (−). 6Pooled standard error of the mean. View Large Table 6. Intestinal lesion scores1 and total oocysts per gram (OPG) of excreta of cocci-vaccinated2 broilers d 6 after mixed Eimeria spp. challenge3 at d 29, when fed diets supplemented with anticoccidial chemical, anticoccidial ionophore, tannic acid extract and tannic acid extract combined with Bacillus coagulans (Experiment 2). Diet4 Supplemented feed5 Upper Middle Ceca Total OPG CNT NA 0.58 0.43 1.70 1830 SAL + 0.95 0.60 1.05 1970 ROB 0.45 0.30 2.30 2340 TAE 0.75 0.30 1.45 2070 TAE+BC 0.33 0.23 0.38 230 CNT NA 0.95 0.50 1.07 270 SAL - 1.50 0.78 1.00 21,180 ROB 1.38 0.68 1.00 8940 TAE 1.08 1.13 1.83 4370 TAE+BC 0.73 0.60 1.15 570 SEM6 0.23 0.21 0.34 6310 P-value 0.061 0.223 0.128 $$\phantom{0}$$0.470 Diet4 Supplemented feed5 Upper Middle Ceca Total OPG CNT NA 0.58 0.43 1.70 1830 SAL + 0.95 0.60 1.05 1970 ROB 0.45 0.30 2.30 2340 TAE 0.75 0.30 1.45 2070 TAE+BC 0.33 0.23 0.38 230 CNT NA 0.95 0.50 1.07 270 SAL - 1.50 0.78 1.00 21,180 ROB 1.38 0.68 1.00 8940 TAE 1.08 1.13 1.83 4370 TAE+BC 0.73 0.60 1.15 570 SEM6 0.23 0.21 0.34 6310 P-value 0.061 0.223 0.128 $$\phantom{0}$$0.470 1Lesion scores were assigned from 0 (no gross lesions) to 4 (extensive lesions) according to the system of Johnson and Reid (1970). 2Cocci-vaccinated with Advent (Novus International, Inc., St. Louis, MO) at day 1 of age via a spray cabinet. 3Mixed-species challenge contained Eimeria acervulina (1 × 105), Eimeria maxima (5 × 104) and Eimeria tenella (7.5 × 104) sporulated oocysts. 4The broilers were fed a non-medicated control (CNT) diet containing no anticoccidial and growth promoter and were supplemented with anticoccidial ionophore salinomycin (SAL; 66 mg/kg of diet), anticoccidial chemical robenidine (ROB; 33 mg/kg of diet), tannic acid extract (TAE; 100 mg/kg of diet) and TAE combined with probiotic Bacillus coagulans (TAE+BC; 110 mg/kg of diet). 5On d 28, broilers were issued commercial grower feed containing supplemental treatment materials (+) or CNT grower feed with the previously added supplemental treatment materials withdrawn (−). 6Pooled standard error of the mean. View Large DISCUSSION The current work investigated the protective effect of tannic acid based feed additives to control coccidiosis in broiler chickens in two experiments. In the first experiment, the ability of commercial TA and the proprietary TAE to alleviate coccidiosis in broilers challenged with mixed Eimeria species was assessed. The second experiment then evaluated the ability of TAE formulations and commercial anticoccidials to impact performance of broilers vaccinated for coccidiosis and to influence the development of protective immunity in vaccinated broilers challenged with mixed Eimeria species. Mixed results on the efficacy of diets containing tannins to reduce the negative impact of coccidiosis in broilers have previously been observed. Some authors report improved performance, reduced lesions and fewer oocysts upon tannin supplementation (Lee et al., 2012; Kaleem et al., 2014), while others observed mixed effects (Cejas et al., 2011) or no impact of tannins on coccidiosis (Mansoori and Modirsanei, 2012). The results of our first experiment align with those of McCann et al. (2006), who observed only small improvements in performance of Eimeria challenged broilers fed tannins, but found that tannin-supplemented diets were able to lessen the detrimental effects of coccidiosis by reducing intestinal lesions. Gross intestinal lesions, due to replication of Eimeria parasites within host intestinal epithelia cells, diminish intestinal barrier integrity, absorptive capacity and create high levels of oxidative and inflammatory stress within the intestine. Tannins are potent antioxidants, which have been reported to reduce oxidative and inflammatory stress in macrophages (Kaur et al., 2008), as well as colon tissue (Hamiza et al., 2012). It is possible that TAE and TA lessened intestinal lesions caused by Eimeria via reducing oxidative stress and/or inflammation within the intestine, leading to a more intact intestinal barrier. Critical indicators of a protection against coccidiosis infection are increased BWG and reduced oocyst shedding. While BWG was not different between challenged treatments, birds supplemented with TAE were observed to have reduced (P < 0.05) oocyst shedding compared to the challenged control (Figure 1). Tannins are known to have anti-protozoal activity; thus, it is possible that TAE had a direct activity towards Eimeria parasites. Mechanisms such as inhibition of extracellular parasitic enzymes or tannin complexation with metals, such as iron, have been suggested as antimicrobial methods by which tannins prevent parasite growth and proliferation (Scalbert, 1991; Chung et al., 1998a, 1998b). Additionally, Min and Hart (2003) reported that tannins may form complexes with nutrients and inhibit their availability to the parasite for normal growth, development and motility and thus, decrease the metabolism of parasite directly through inhibition of oxidative phosphorylation or electron transport. Along with other phytobiotic feed additives, the specific chemical and physical properties of tannins are highly variable (Windisch et al., 2008; Huyghebaert et al., 2011). In our study, the proprietary TAE was found to reduce (P < 0.05) oocyst shedding by 58% and 51% for PC birds and TA treated birds, respectively (Figure 1). Kaleem et al. (2014) previously reported that tannin extracts from Emblica officinalis also had improved efficacy in reducing the impact of coccidiosis challenge, as compared to commercial TA. These results, and those of others (Mueller-Harvey, 2006; Schiavone et al., 2008; Elizondo et al., 2010), provide additional support for the need to characterize tannins based on individual assessment, as opposed to an assumption that all tannin extracts and commercial TA products have equivalent biological efficacy. Along with their anti-inflammatory and anti-parasitic functions, tannins may be involved in modulation of the interaction between the microbiota and gastrointestinal tract (Redondo et al., 2014). Thus, dietary tannin supplementation may help in alleviating the intestinal challenges associated with vaccine-induced Eimeria oocyst replication, thereby improving performance of coccidiosis vaccinated broilers. Thus far, few studies have been reported which assess the compatibility of tannin feed additives with coccidiosis vaccination. In the live performance portion of Experiment 2, supplementation of TAE was found to improve (P < 0.05) the FCR of broilers vaccinated with a live, non-attenuated anti-coccidial vaccine (Table 4). These significant improvements in FCR of vaccinated birds supplemented with TAE were consistent, even though the flock was split at d 28 to accommodate the coccidial immunity challenge. Our results align with Hooge et al. (2012), who observed improvements in d 0 to 21 FCR of vaccinated broilers supplemented with sweet chestnut tannins and contrast the negative impact of commercial TA on vaccine effectiveness observed by Mansoori and Modirsanei (2012). Combination strategies towards controlling coccidia and preventing intestinal dysbacteriosis are likely to be most effective methods for controlling concurrent enteric diseases such as coccidiosis and necrotic enteritis in broilers (Van Immerseel et al., 2004; Williams 2005). Bacillus coagulans has previously been shown to have immune modulating activities in vitro (Jensen et al., 2010) and in vivo (Lin et al., 2012). Previous work has further shown supplementation of B. coagulans can improve survivability of Clostridium difficile-infected mice (Fitzpatrick et al., 2011) and enhance growth performance of broilers (Lin et al., 2011). By combining the probiotic functions of B. coagulans with the antioxidant and anti-inflammatory activities of the proprietary TAE (TAE+BC), the combination would provide dual modes of action by which synergistic benefits towards broiler live performance may be observed as compared to TAE alone. During the vaccination study, broilers supplemented with TAE+BC were found to have improved FCR compared to CNT birds, but live performance of TAE+BC was similar to, rather than synergistic to, TAE alone (Table 4). Probiotics are known to support intestinal integrity by enhancing immune activity and maintaining intestinal microflora balance (Huyghebaert et al., 2011). Under the conditions of this experiment, it is possible that coccidiosis vaccination alone did not provide a sufficient enteric challenge in which enhanced growth promoting effects of B. coagulans could be observed over TAE. Future studies should utilize enteric pathogen challenges, such as C. perfringens infection, to create a more robust enteric challenge by which the beneficial probiotic activities of B. coagulans should enhance the benefits of TAE+BC beyond the anti-parasitic and antioxidant effects observed with TAE. Reduced disease susceptibility in vaccinated broilers is based on the acquisition of protective immunity, a result of recycling the vaccine parasites through the litter. Previous studies have shown either additive benefits (Oviedo-Rondón et al., 2006; Stringfellow et al., 2011; Mathis et al., 2014) or detrimental effects (Küçükyilmaz et al., 2012; Mansoori and Modirsanei, 2012) of feed additive supplementation upon the efficacy of coccidiosis vaccination to mitigate the impact of enteric challenges in later growth phases. Commercial TA was previously reported to hamper coccidial vaccine oocyst cycling, resulting in insufficient disease protection following a secondary Eimeria challenge in broilers (Mansoori and Modirsanei, 2012). Experiment 2 was therefore designed to assess whether the proprietary TAE and TAE+BC combinations would impact efficacy of coccidiosis vaccination using a mixed Eimeria challenge during d 29 to 35. The goal of the coccidial immunity challenge portion of Experiment 2 was twofold. The first objective was to assess whether supplementation of TAE or TAE+BC in vaccinated broiler diets would enhance recovery following Eimeria challenge as compared to vaccination alone (group 1). The second, more important objective, was to evaluate any potential impact of TAE and TAE+BC on altering the effectiveness of vaccination to induce protective immunity development in broilers (group 2). To accomplish these purposes, vaccinated broilers were split into subgroups, which were either provided feed supplemented with TAE, TAE+BC, SAL, or ROB (group 1) during the mixed Eimeria challenge, or were provided feed in which additives were withdrawn (group 2). This design allowed for collection of challenge recovery, as well as vaccine compatibility, information to be gathered within the study, albeit due to reduced replication, the chance for pen to pen variability to impact detection of significant differences among treatments was increased. Based on previous reports, the mixed Eimeria challenge was expected to cause increased lesions, higher oocyst shedding, reduced BWG and higher FCR in Eimeria challenged broilers treated with additives that interfered with vaccine efficacy. The results of interfering additives in this study were therefore expected to be similar to results previously seen in non-vaccinated, Eimeria challenged broilers, as compared to vaccinated, challenged broilers (Oviedo-Rondón et al., 2006; Lee et al., 2011; Mansoori and Modirsanei, 2012; Mathis et al., 2014). Due to the design of Experiment 2, a comparison of the effects of Eimeria challenge in vaccinated versus non-vaccinated broilers was unable to be drawn, but assessment of the impact of treatments on enhancing broiler recovery following the mixed Eimeria challenge was evaluated. In group 1, neither intestinal lesions nor oocyst shedding were altered by Eimeria challenge (Table 6), however, vaccination combined with TAE or TAE+BC supplementation was found to enhance broiler performance following Eimeria challenge as compared to vaccination alone (Table 5). This result indicates that TAE and TAE+BC enhanced disease resistance in vaccinated broilers, which is in contrast with the results of Mansoori and Modirsanei (2012). Differences in tannin source (tannin extract versus commercial TA) and tannin inclusion in feed (100 mg/kg TAE versus 10 g/kg commercial TA) may explain the opposing results of these vaccination studies. According to Chapman (1999), insufficient immunity development following vaccination is indicated by increased fecal oocyst shedding and reduced performance after withdrawal of supplemental medication or feed additive from broiler diets. In group 2, oocyst shedding was numerically increased in broilers in which ROB and SAL were withdrawn from feed (Table 6). Broiler performance was additionally found to be significantly reduced when ROB and SAL were withdrawn from feed, as compared to when they were kept in feed during the Eimeria challenge (Table 5). ROB is known to markedly suppress oocyst production of drug-sensitive Eimeria strains while ionophores, like SAL, can indirectly inhibit parasite replication (Chapman, 1999). The results of our coccidial immunity challenge support the idea that concomitant use of some anticoccidial drugs and vaccination is not advisable during the initial weeks following vaccination due to the risk of negatively impacting vaccine efficacy (Chapman, 1999; Vermeulen et al., 2001; Williams, 2005). In contrast to the negative impact of anticoccidial withdrawal on broiler FCR, withdrawal of TAE and TAE+BC did not depress Eimeria challenged broiler performance. Broiler FCR was found to be equivalent (P > 0.05) whether TAE and TAE+BC were supplemented or withdrawn during Eimeria challenge (Table 5). In addition, broilers that had TAE or TAE+BC withdrawn during the challenge had significantly improved FCR (P < 0.05) compared to vaccinated, challenged control birds. These results indicate that TAE and TAE+BC, unlike ROB and SAL, allowed protective immunity to develop following coccidiosis vaccination. Unlike TAE withdrawal, which resulted in lower BWG and FI as compared to when it was supplemented, withdrawal of TAE+BC did not significantly impact these variables during the coccidial immunity challenge. Further, broilers supplemented with TAE+BC consistently showed numerically reduced intestinal lesions and oocyst shedding compared to birds supplemented with TAE during the Eimeria challenge in both groups 1 and 2. Increased disease prevention, altered intestinal microflora composition and enhancement of host immune response have been observed from oral feeding of probiotics (Dalloul and Lillehoj, 2005; Lee et al., 2010; Lin et al., 2011; Stringfellow et al., 2011; Lin et al., 2012; Abdelrahman et al., 2014; Bozkurt et al., 2014). Bacillus coagulans has also been observed to modulate the composition of intestinal microflora and improve non-specific immunity (Lin et al., 2011; Lin et al., 2012). The differences observed between TAE and TAE+BC after withdrawal therefore may have been the result of B. coagulans exerting an immune enhancing effect, either following the initial coccidiosis vaccination or during the Eimeria challenge itself, which could not be achieved with TAE alone. To conclude, the data from these experiments provide additional evidence supporting the need to individually characterize plant bioactive feed additives for use in poultry nutrition. Supplementation of broiler diets with TAE reduced the negative effects of coccidiosis in Eimeria challenged broilers. Both TAE alone and in combination with the probiotic B. coagulans (TAE+BC) provided additive FCR benefits in coccidiosis vaccinated broilers and more importantly, were found to permit protective immunity development in vaccinated broilers. Further observations indicate TAE+BC may have improved biological activity compared to TAE alone in Eimeria infected broilers, which warrants additional research. 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Poultry ScienceOxford University Press

Published: Sep 1, 2018

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