Effects of coccidial vaccination and dietary antimicrobial alternatives on the growth performance, internal organ development, and intestinal morphology of Eimeria-challenged male broilers

Xi Wang, E. David Peebles, Aaron S. Kiess, Kelley G. S. Wamsley, and Wei Zhai1 Department of Poultry Science, Mississippi State University, 39762 ABSTRACT Effects of the coccidial vaccination and dietary antimicrobial alternatives on growth performance, internal organ development, and intestinal morphology of male broilers subjected to an Eimeria challenge were determined. A total of 1,120 one d-old Ross × Ross 708 male broilers were randomly distributed to 80 floor pens (10 treatments, 8 replication pens/treatment, and 14 chicks/pen). A 2 × 5 factorial arrangement of treatments was used to determine the main and interaction effects of the coccidial vaccination (vaccinated or non-vaccinated) and the dietary additive [1) corn and soybean-meal basal diet, 2) basal diet + antimicrobials (bacitracin and salinomycin), 3) basal diet + probiotics (3 Bacillus subtilis strains), 4) basal diet + prebiotics (mannan-oligosaccharides and β -glucans), and 5) basal diet + probiotics + prebiotics]. To mimic the Eimeria challenge, all chicks were gavaged with a 20x dose of a different coccidial vaccine (live Eimeria oocysts) at Day 14. The coccidial vaccination decreased Day 0–14 and 29–42 BW gain

(BWG) and subsequently decreased Day 0–56 BWG. Broilers fed diets with antimicrobials exhibited the lowest feed conversion ratio (FCR) during the periods of Day 0–14 and 15–28, the shallowest ileal crypt depth on Day 28, and the lowest relative duodenum weight on Day 28 and 42. The Pre+Pro diets helped the broilers to reach a lower overall FCR than did the Pro alone diets and helped the broilers reach a FCR similar to that of the Anti diets. However, broilers fed diets supplemented with prebiotics and probiotics exhibited the deepest intestinal crypt depth on Day 28. There was no interaction between coccidial vaccination and dietary additive on growth performance or any carcass yield. In conclusion, antimicrobial additives might reduce the intestinal size of broilers; whereas prebiotic and B. subtilis-based probiotic additives might promote the growth of several digestive organs. Prebiotics can be safely used with B. subtilis-probiotics in broiler feed without compromising feed conversion ability.

Key words: antimicrobial alternative, broiler, coccidial vaccination, Eimeria challenge, growth performance 2019 Poultry Science 0:1–12 http://dx.doi.org/10.3382/ps/pey552

INTRODUCTION

public health. More broiler integrators have started to implement antibiotic-free programs. Important human antibiotics as well as non-human antibiotics, including ionophore anticoccidials, are being phased out of broiler feeds. In current antibiotic-free programs, the prevention of enteric diseases is the primary issue for broiler producing companies, due to a lack of efficient control of bacteria and coccidia. It has become a priority to develop dietary antibiotic alternatives in order to maintain intestinal microbial balance while also improve the growth of broilers. Coccidiosis is a common and financially devastating enteric disease for broilers (Williams, 2005). It is caused by the apicomplexan protozoan Eimeria. Among the various species of Eimeria, E. acervulina, E. maxima, and E. tenella contribute significantly to coccidiosis infection in chickens (De Gussem, 2007). In the poultry industry, global economic loss due to coccidiosis is approximately $750 million annually (Shirley et al., 2007). This loss is attributed to clinical

Antibiotics have been used in broiler production for the treatment and prevention of bacterial infections and subsequent growth promotion. According to Food and Drug Administration statistics, 80% of antibacterial drugs used in the United States in 2011 were used in animal production (FDA, 2011, 2012; Done et al., 2015). Although how dietary antibiotics contribute to the overall antibiotic resistance is not clear, some scientists are concerned about the overuse of antibiotics in animal production (Mathew et al., 2007; Landers et al., 2012). This concern stems from a potential acceleration of the selection of antibiotic-resistant bacteria in food supplies which may subsequently threaten

 C 2018 Poultry Science Association Inc. Received November 3, 2017. Accepted November 27, 2018. 1 Corresponding author: [email protected]

1

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Effects of coccidial vaccination and dietary antimicrobial alternatives on the growth performance, internal organ development, and intestinal morphology of Eimeria-challenged male broilers

2

WANG ET AL.

MATERIALS AND METHODS Treatments and Bird Management The Institutional Animal Care and Use Committee of Mississippi State University have approved all bird husbandry and handling methods in this study (IACUC# 15–002). A total of 1120 one-day-old Ross × Ross 708 male broilers were obtained from a commercial hatchery. All birds received Newcastle and infectious bronchitis vaccines on the day of hatch by a spray. Half of the birds (560) were also spray-vaccinated with a commercial coccidiosis vaccine at recommended levels in a commercial hatchery. The other 560 chicks did not receive the coccidiosis vaccine. Chicks were kept in the delivery baskets for 5 h before being placed in experimental floor pens. The coccidiosis vaccine contained live oocytes of E. maxima, E. acervulina, and E. tenella. Broilers were fed 1 of the 5 diets from Day 0 to 56 (crumbled diets from Day 0 to 14, pelleted after Day 14). The 5 diets were as followed: 1) a corn and soybean-meal control diet (Con), 2) an antibioticsupplemented diet (Anti, Con + 50 g of bacitracin and 40 g of salinomycin sodium/ton of finished feed), 3) a prebiotic-supplemented diet (Pre, Con + 170 g of mannan-oligosaccharides and 250 g of β -glucans/ton of finished feed), 4) a probiotic-supplemented diet (Pro, Con + 2084, LSSAOl, and 15A-P4 of Bacillus subtilis; 280,000 CFU/g of finished feed), and 5) a prebiotic and probiotic combination-supplemented diet (Pre+Pro, products as described above). An anticoccidial additive (salinomycin) was not included in Anti diet from Day 0 to 14 to allow for the full efficacy of the coccidiosis vaccination. All additives are commercially available. Bacillus subtilis counts in the finished feed were confirmed in a blind test by a commercial laboratory. All major ingredients (corn, soybean meal, and meat and bone meal) were analyzed using near-infrared spectroscopy (NIR system, model: XDS-XM-1100 series, FOSS, Sweden) and a commercial database (Precise Nutrition Evaluation, Adisseo, Alpharetta, GA) for determination of digestible AA and AME values. These values were used to formulate the Con diet that met all requirements of male broilers exhibiting standard performance (Rostagno et al., 2011, Table 1). Antibiotics, as well as prebiotics and probiotics, were incorporated into the diets with the equivalent amount of sand in the Con diet. Eighty floor pens (0.91 × 1.22 meter) were divided into 8 blocks that were equally spaced in an environmentally controlled facility. Ten pens, each assigned a particular treatment group (2 vaccination status × 5 diets), were randomly assigned to each block. Fourteen broiler chicks were randomly distributed among the 80 floor pens (0.0796m2 /bird). Each floor pen was equipped with 3 nipple drinkers, 1 hanging feeder, 3 rubber dividers, and top-dressed with fresh woodshavings litter. Chicks had ad libitum access to water and feed. Light programs of 24L:0D and 20L:4D were

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treatment cost and subclinical broiler performance depression (poor feed conversion and slow-growth). Two rotational methods are used to prevent coccidiosis in the poultry industry: dietary anticoccidials (ionophore and chemical) and vaccination. Most chemical anticoccidials are considered less effective than ionophores in controlling coccidiosis due to the rapid build-up of resistance by coccidia (De Gussem, 2007). Therefore, coccidial vaccination becomes more important for the prevention of coccidiosis in current antibiotic-free production programs. The early exposure of live oocytes boosts specific immunity and protects chicks from coccidiosis challenges in the later phases of growth. Nevertheless, this initial exposure may compromise early BW gain (BWG) and feed conversion efficiency of broiler chicks (Lee et al., 2011). Previous research has shown that probiotic administration in drinking water has alleviated depressing effects of coccidial vaccination on the early growth of chicks (Ritzi et al., 2016). One common probiotic product, Bacillus subtilis spores, benefits broilers by competitively excluding pathogenic bacteria such as Salmonella, Clostridium, and E. coli O78: K80 (La Ragione et al., 2001; La Ragione and Woodward, 2003). Spores of B. subtilis are widely applied in current poultry feed due to their heat-resistance during feed pelleting and their high recovery rate in the GI tract (Latorre et al., 2014). Bacillus subtilis spores added to broiler feed have the ability to lower pathogenic bacteria counts in gastrointestinal tract, improve intestinal integrity and apparent nutrient retention, and subsequently improve feed conversion efficiency of broilers (Molnar et al., 2011; Sen et al., 2012; Jeong and Kim, 2014). The previous study from our lab, feeding B. subtilis spores alone with beta-glucans and mannan-oligosaccharides to broilers improved their growth rate and enabled them to reach their target weights sooner (Wang et al., 2016). Beta-glucans and mannan-oligosaccharides served as prebiotics. In an in vitro study, it was shown that betaglucans enhanced the growth, viability, and colonization of the probiotic organism (Russo et al., 2012). Furthermore, in another in vivo study, it was shown that feeding beta-glucans and manna-oligosaccharides to broilers enhanced the adhesion of beneficial bacteria (Lactobacillus) to the intestinal mucosa (Wang et al., 2016). Manno-oligosaccharides are also able to lower levels of pathogenic bacteria by offering to them competitive binding sites (Fernandez et al., 2000; Biggs et al., 2007). Based on these findings, it is hypothesized that the combined use of B. subtilis-based probiotics and prebiotics in the feed may alleviate the negative effects of Eimeria vaccination on chickens. Therefore, in this study, effects of coccidial vaccination and dietary antibiotic alternatives on growth, internal organ development, and intestinal integrity of broilers subjected to an Eimeria challenge were evaluated.

3

0.060 0.484

Coccidial vaccination is given to one-day-old chicks by spraying a commercial vaccine containing live oocytes of Eimeria maxima, E. acervulina, and E. tenella. Experiment diets included Con (control, corn and soybean-meal basal diet), Anti (antimicrobial, basal diet supplemented with 50 g of bacitracin and 40 g of salinomycin sodium/ton of finished feed), Pre (prebiotics, basal diet supplemented with 170 g of mannan-oligosaccharides and 250 g of β -glucans/ton of finished feed), Pro (probiotics, basal diet supplemented with 3 Bacillus subtilis strains of 2084, LSSAOl, and 15A-P4 at equal amounts, 300,000 CFU/g of finished feed) and Pre+Pro (basal diet supplemented with prebiotic and probiotics products above). a,b Means in a column not sharing a common superscript are different (P ≤ 0.05). Means of non-significant interaction are not listed. 2

1

0.504 0.179 0.734 0.765 0.030 0.912 0.002 0.567 0.582 0.356 0.568 0.383 0.210 0.497 0.441 0.719 0.838 0.659 0.001 0.415 0.689 0.272 0.264 0.605 0.268 0.138 0.650 0.021 0.249 0.949 0.021 0.353 0.791 0.237 0.159 0.705 0.676 0.562

< .0001 0.007

0.613 0.007 0.175 0.017 0.614 0.610 0.142 0.014 0.212 0.012 0.208 0.917

Con Anti Pre Pro Pre+Pro SEM

0.087 0.448 0.380

1.388 1.400 0.0056 1.400a 1.366b 1.399a 1.397a 1.408a 0.0087 0.445a 0.437b 0.0025 0.434 0.446 0.440 0.441 0.445 0.0046 0.617 0.608 0.0033 0.607 0.609 0.616 0.613 0.619 0.0052

0.022 0.274 0.756

7.50 9.02 1.59 7.14 3.13 9.82 10.93 10.27 2.515 1.919 1.923 0.0093 1.920a,b 1.890b 1.919a,b 1.961a 1.912b 0.0142 4.600a 4.511b 0.0266 4.542 4.603 4.540 4.517 4.621 0.0413 8.536a 8.335b 0.0444 8.446 8.396 8.360 8.465 8.512 0.0690 3.423 2.089 1.0128 4.278 3.125 0.893 3.660 1.823 1.6013 2.159 2.106 0.0278 2.150 2.092 2.109 2.192 2.118 0.0439 1.530 1.519 0.0282 1.512 1.524 1.508 1.521 1.558 0.0343 3.286a 3.148b 0.0304 3.242 3.174 3.175 3.210 3.283 0.0494 3.326 1.788 0.9790 1.610 0.000 4.201 2.773 4.200 1.5479 1.901 1.926 0.0151 1.910 1.920 1.902 1.962 1.874 0.0288 1.486a 1.434b 0.0154 1.478 1.438 1.458 1.428 1.498 0.0243 2.820a 2.769b 0.0150 2.817 2.752 2.793 2.808 2.802 0.0238 2.516 4.277 1.0424 2.491 0.000 4.848 4.637 5.008 1.6482 1.588 1.592 0.0084 1.595a 1.556b 1.594a 1.595a 1.611a 0.0116 1.140 1.145 0.0113 1.118b 1.196a 1.134b 1.140b 1.123b 0.0140 1.813 1.820 0.0125 1.791b 1.861a 1.807b 1.815b 1.808b 0.0144

FCR FCR FCR

0.896b 2.858a 0.5636 1.339 1.339 1.786 3.126 1.786 0.8911

FCR BWG (kg) Mort (%)

FI (kg)

Day 43 to 56

Mort (%)

FI (kg)

Day 0 to 56

Mort (%)

P-value Coccidial vaccination Diet Vaccination × Diet

Experimental diets were provided to the broilers in accordance with the following growth phases: starter (Day 0 to 14), grower (Day 15 to 28), finisher (Day 29 to 42), and withdrawal (Day 43 to 56). Mortality, dead

None Vaccinated SEM

Growth Performance and Carcass Yield

Diet2

provided from Days 0 to 7 and 8 to 56, respectively. To develop clinical coccidiosis, a 20x dose of a different commercial vaccine (different from cocci vaccine used in the hatchery) was administered to each broiler by oral gavage on Day 14. The vaccine used for challenge contained live oocysts of E. acervulina, E. mivati, E. maxima, and E. tenella. Eimeria challenge was confirmed by intestinal lesion observation at Days 28, 41, and 56.

Day 29 to 42

1 Nutrient contents of corn, soybean meal, and meat and bone meal were analyzed by near-infrared spectroscopy. 2 Premix provided the following ingredients per kilogram of finished feed: 2.654 μ g retinyl acetate, 110 μ g cholecalciferol, 9.9 mg DL-α tocopherol acetate, 0.9 mg menadione, 0.01 mg vitamin B12, 0.6 μ g folic acid, 379 mg choline, 8.8 mg D-pantothenic acid, 5.0 mg riboflavin, 33 mg niacin, 1.0 mg thiamine, 0.1 mg D-biotin, 0.9 mg pyridoxine, 28 mg ethoxyquin, 55 mg manganese, 50 mg zinc, 28 mg iron, 4 mg copper, 0.5 mg iodine, and 0.1 mg selenium. 3 The phytase product contained 10,000 FTY/g phytase. One unit (FYT) of the phytase can liberate 1 mM of inorganic phosphate per min from sodium phytate at pH 5.5 and 37◦ C. 4 The Xylanase product contained 160,000 BXU/g xylanase. One unit (BXU) of the xylanase can liberate 0.06 mM of reducing sugars from birch xylan per min at pH 5.3 and 50◦ C. 5 Experimental feed additives [commercial prebiotics (170 g of mannan-oligosaccharides and 250 g of β -glucans/ton of finished feed), probiotics (300,000 CFU of Bacillus subtilis/g of finished feed, Bacillus product consisted of 3 strains, 2084, LSSAOl, and 15A-P4 at equal amounts), or antibiotics (50 g of bacitracin and 40 g of salinomycin sodium/ton of finished feed)] replaced the equivalent amount of sand in diet. 6 Nutrient contents were calculated on a dry matter basis.

BWG (kg)

3208 17.30 0.60 0.26 0.20 0.43 0.68 0.94 0.61

FI (kg)

3153 18.00 0.64 0.30 0.20 0.48 0.74 1.01 0.66

Mort (%)

3010 20.80 0.82 0.39 0.22 0.57 0.87 1.17 0.77

Day 15 to 28

2955 22.20 0.92 0.47 0.22 0.66 0.96 1.31 0.86

BWG (kg)

74.37 19.95 2.77 0.50 0.51 0.22 0.59 0.19 0.28 0.08 0.25 0.02 0.01 0.06 0.20

FI (kg)

71.33 22.50 3.09 0.50 0.51 0.13 0.75 0.23 0.31 0.10 0.25 0.02 0.01 0.07 0.20

Mort (%)

62.79 31.47 1.22 0.50 0.56 0.64 1.58 0.29 0.26 0.11 0.25 0.02 0.01 0.11 0.20

Day 0 to 14

58.70 34.55 1.79 0.50 0.57 0.55 1.88 0.37 0.36 0.16 0.25 0.02 0.01 0.10 0.20

BWG (kg)

D 43 to 57

FI (kg)

D 29 to 42

Coccidial vaccination1

Nutrient content6 ME (Kcal/kg) CP, % Ca, % Available P, % Na, % Digestible methionine, % Digestible TSAA, % Digestible lysine, % Digestible threonine, %

D 15 to 28

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Ingredient1 , % Corn Soybean meal Meat and bone meal Poultry fat Salt Calcium carbonate Dicalcium phosphate DL-Methionine L-Lysine hydrochloride L-Threonine Premix2 Phytase3 Xylanase4 Choline chloride Sand5

D 0 to 14

Table 2. Effects of coccidial vaccination and dietary additive on the growth performance of male broilers.

Items

BWG (kg)

Table 1. Feed ingredient composition and calculated nutrient contents of basal diets.

FCR

COCCIDIAL VACCINATION AND FEED ADDITIVES

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WANG ET AL.

Table 3. Effects of coccidial vaccination and dietary additive on absolute weights of carcass and parts of male broilers at 42 D of age (g). Coccidial vaccination1

Diet2

Con Anti Pre Pro Pre+Pro SEM P-value Coccidial vaccination Diet Vaccination × Diet

3209 3227 21.2 3198 3217 3203 3235 3238 33.0 0.546 0.879 0.325

Carcass 2270 2268 15.9 2226 2280 2275 2290 2275 25.2 0.918 0.419 0.554

Wing 246 245 1.8 244 247 244 248 244 2.8 0.855 0.726 0.471

Breast

Drumstick

635 622 6.9 612 630 630 629 641 11.0

281 284 2.3 282 284 282 286 279 3.7

0.174 0.477 0.591

0.316 0.677 0.727

Thigh 385 384 3.9 378 383 383 388 389 6.2 0.880 0.712 0.677

Tender 129 126 1.7 128 127 127 127 129 2.7 0.280 0.983 0.921

1 Coccidial vaccination is given to 1-day-old chicks by spraying a commercial vaccine containing live oocytes of Eimeria maxima, E. acervulina, and E. tenella. 2 Experiment diets included Con (control, corn and soybean-meal basal diet), Anti (antimicrobials, basal diet supplemented with 50 g of bacitracin and 40 g of salinomycin sodium/ton of finished feed), Pre (prebiotics, basal diet supplemented with 170 g of mannan-oligosaccharides and 250 g of β -glucans/ton of finished feed), Pro (probiotics, basal diet supplemented with 3 Bacillus subtilis strains of 2084, LSSAOl, and 15A-P4 at equal amounts, 300,000 CFU/g of finished feed), and Pre+Pro (basal diet supplemented with both prebiotic and probiotics products above). Means of non-significant interaction are not listed.

Table 4. Effects of dietary additives and coccidial vaccination on relative weights of carcass and parts of male broilers at 42 days of age (%). Coccidial vaccination1

Diet2

None Vaccinated SEM Con Anti Pre Pro Pre+Pro SEM P-value Coccidial vaccination Diet Vaccination × Diet vaccination

Carcass/BW

Wing/carcass

Breast/carcass

Drumstick/carcass

Thigh/carcass

Tender/carcass

70.50 70.20 0.165 69.86 70.70 70.37 70.57 70.27 0.265

10.81 10.81 0.058 10.94 10.84 10.75 10.82 10.70 0.095

27.84a 27.20b 0.184 27.36 27.67 27.42 27.37 27.78 0.287

12.37a 12.64b 0.075 12.70 12.48 12.54 12.50 12.28 0.116

16.89 17.04 0.141 16.96 17.00 16.81 16.95 17.10 0.229

5.67 5.55 0.059 5.76 5.47 5.59 5.63 5.62 0.096

0.202 0.200 0.918

0.969 0.404 0.554

0.026 0.780 0.663

0.011 0.167 0.959

0.472 0.925 0.730

0.168 0.344 0.544

1 Coccidial vaccination is given to one-day-old chicks by spraying a commercial vaccine containing live oocytes of Eimeria maxima, E. acervulina, and E. tenella. 2 Experiment diets included Con (control, corn and soybean-meal basal diet), Anti (antimicrobials, basal diet supplemented with 50 g of bacitracin and 40 g of salinomycin sodium/ton of finished feed), Pre (prebiotics, basal diet supplemented with 170 g of mannan-oligosaccharides and 250 g of β -glucans/ton of finished feed), Pro (probiotics, basal diet supplemented with 3 Bacillus subtilis strains of 2084, LSSAOl, and 15A-P4 at equal amounts, 300,000 CFU/g of finished feed), and Pre+Pro (basal diet supplemented with both prebiotic and probiotics products above). a,b Means in a column not sharing a common superscript are different (P ≤ 0.05). Means of non-significant interaction are not listed.

bird BW, weights of feed and birds on a pen basis were recorded and used to calculate bird BWG, feed intake, and mortality corrected feed conversion ratio (FCR) within each growing phase. Birds used for sampling and processing were not considered as mortality. Mortality was calculated by dividing the number of live birds on the last day of the each period by the number of live birds on the first day of the period. Three broilers per pen on Day 42 were randomly selected and weighed individually for processing. Feed and water were withdrawn from the birds for 16 h before processing. Birds were automatically processed, chilled in an ice-water slurry for 4 h, and manually deboned. Carcasses were weighed at the time of processing. Wings, drumsticks, thighs, boneless and skin-

less breasts, and tenders were weighed at the time of deboning.

Intestine Sampling and Morphology Exam On Day 14, 28, 42, and 56, one bird per pen was randomly selected for sampling of internal organs. Each broiler was weighed individually, euthanized by CO2 asphyxiation, and dissected. Lengths of each segment of the small intestine (duodenum, jejunum, and ileum) were measured as described by Wang et al. (2015). The pH of the contents of gizzard and ileum were measured using a portable pH meter (Accumet AP110, Thermo Fisher Scientific, Waltham, MA). Weights of the

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None Vaccinated SEM

Individual BW

5

COCCIDIAL VACCINATION AND FEED ADDITIVES

Pancreas/BW at 14 days of age (%) 0.500 0.450

a abc

c

bc

0.400 0.350

a abc

ab abc

bc

0.300 None

0.250

Vaccinated

0.200

P = 0.038

0.150 0.100 0.050 0.000 Control

Antibiotics Prebiotics Probiotics

Pre+Pro

Figure 1. Interaction between coccidial vaccination and dietary additive on pancreas/BW of broilers at 14 D of age. common superscript are different.

a–c

Means not sharing a

pancreas, spleen, empty gizzard, proventriculus, duodenum, jejunum, and ileum were recorded. On Day 28, 42, and 56, ileal digesta were collected, stored on ice, and then centrifuged at 7200 × g for 10 min to collect the supernatant. The viscosity of the ileal digesta supernatant was read at 20 rpm and 25◦ C using a Brookfield Programmable Viscometer (LVDV-II + Pro, Brookfield Engineering Laboratories, Stoughton, MA). On Day 28, the intestinal morphology of vaccinated broilers was examed. A 2.5 cm intestine section from the middle of each intestine segment (duodenum, jejunum, and ileum) was obtained for histological analysis. Histological samples were processed as described by Wang et al. (2015). Villus length, width, crypt depth, muscle thickness, and goblet cell size were measured (Fasina et al., 2010).

dietary additive were tested only if there was no significant interaction. Mortality data were arc sin transformed before ANOVA analysis. Results of mortality were transformed back to original unit in Table 2. Results of intestinal morphology were generated by a oneway ANOVA analysis of the 5 dietary treatments. The PROC GLM procedure (SAS version 9.2, SAS Institute, 2010) was used to determine the main effect of dietary additive with dietary additive as a fixed effect and block as a random effect. Fisher’s least significant difference test was conducted if there were significant differences among treatments. All significant levels were set at α = 0.05.

Experimental Design and Data Analysis

Growth Performance and Carcass Yield

A randomized complete block design was applied in this study. A two-way factorial arrangement of treatments was used to test the main effects of dietary additives and coccidial vaccination as well as their interaction effects. Each of the 8 blocks in the housing facility served as a unit of replication. Each pen in each block was randomly assigned one of the 10 treatments. Main and interaction treatment effects on growth performance, carcass yield, and gut development were tested by two-way ANOVA analysis (2 coccidial vaccination treatments × 5 diets). An unrestricted-mixed model in the PROC GLM procedure (SAS version 9.2, SAS Institute, 2010) was used with coccidial vaccination, dietary additive, and their interaction as fixed effects and with a block as a random effect. Significant differences among fixed factors were determined in a hierarchical order. Main effects of coccidial vaccination and

Growth performance was not affected by a coccidial vaccination and dietary additive treatment interaction (P > 0.05, Table 2). Coccidial vaccination on day of hatch decreased feed intake from Days 29 to 42 (P = 0.021) and Day 43 to 56 (P = 0.001), and subsequently decreased overall feed intake from Day 0 to 56 (P = 0.002). This coccidial vaccination also decreased BWG from Days 0 to 14 (P = 0.012) and Day 29 to 42 (P = 0.021), and subsequently decreased overall BWG from Day 0 to 56 (P = 0.022). In addition, the coccidial vaccination increased early mortality from Day 0 to 14 (P = 0.017) without affecting later mortalities or overall mortality from Day 0 to 56. Broilers fed Anti diets exhibited the lowest FCR from Day 0 to 14 (P = 0.014) and from Day 15 to 28 (P = 0.007), and likewise exhibited the highest feed intake (P = 0.007) and BWG (P = <0.001) from Day 15 to 28. Broilers fed

RESULTS

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abc

< 0.001 0.230 0.229 0.164 0.722 0.854 0.080 0.173 0.455 0.034 0.901 0.248 0.797 0.798 0.862 0.727 0.424 0.579 0.960 0.870 0.436 0.652 0.612 0.290 0.685 0.542 0.766 0.601 0.234 0.038 0.120 0.018 0.569 0.367 0.001 0.420

Coccidial vaccination is given to one-day-old chicks by spraying a commercial vaccine containing live oocytes of Eimeria maxima, E. acervulina, and E. tenella. Experiment diets included Con (control, corn and soybean-meal basal diet), Anti (antimicrobials, basal diet supplemented with 50 g of bacitracin and 40 g of salinomycin sodium/ton of finished feed), Pre (prebiotics, basal diet supplemented with 170 g of mannan-oligosaccharides and 250 g of β -glucans/ton of finished feed), Pro (probiotics, basal diet supplemented with 3 Bacillus subtilis strains of 2084, LSSAOl, and 15A-P4 at equal amounts, 300,000 CFU/g of finished feed), and Pre+Pro (basal diet supplemented with both prebiotic and probiotics products above). 3 Interaction on relative pancreas weight is presented in Figure 1. Means of non-significant interaction are not listed. a–c Means in a column not sharing a common superscript are different (P ≤ 0.05). 2

1

None Vaccinated SEM

P-value Coccidial vaccination Diet Vaccination × Diet

Con Anti Pre Pro Pre+Pro SEM

0.456 0.163 0.228

6.878a 5.999b 0.1441 6.364 6.878 6.254 6.542 6.152 0.1776 2.097 2.173 0.0382 2.133 2.111 2.174 2.181 2.076 0.0605 1.441 1.381 0.0238 1.395 1.385 1.365 1.416 1.494 0.0398 451 456 5.0 468 456 455 444 443 7.8

0.695 0.712 0.0136 0.693b,c 0.647c 0.708b 0.782a 0.688b,c 0.0219

3.072 2.938 0.0609 2.883b 2.973b 3.253a 2.829b 3.087a,b 0.0975

0.396 0.390 0.0083 0.376 0.377 0.401 0.412 0.401 0.0138

0.100 0.980 0.0038 0.091 0.102 0.100 0.104 0.097 0.0064

20.31 20.13 0.292 20.75 20.09 19.88 19.91 20.47 0.461

49.85 49.79 0.823 50.16 49.22 50.41 50.45 48.88 1.300

47.91 48.41 1.001 45.56 48.47 48.47 48.49 49.81 1.578

1.121 1.110 0.0283 1.108 1.120 1.094 1.094 1.164 0.0449

2.087a 1.969b 0.0391 2.004 2.051 2.070 2.019 1.995 0.0631

Ileal pH Ileum/BW (%) Jejunum/ BW (%) Ileum (cm) Jejunum (cm) Duodenum (cm) Spleen/ BW (%) Pancreas/ BW (%)3 Gizzard/BW (%) Proventriculus/ BW(%) BW (g) Diet2

Table 5. Effects of coccidial vaccination and dietary additive on the internal organs of broilers at 14 D of age.

At Day 14, there was an interaction effect between coccidial vaccination and dietary additive on the relative pancreas weight (P = 0.038, Figure 1). For nonvaccinated broilers, feeding Pre diets increased relative pancreas weights as compared to feeding Anti diets. For vaccinated broilers, feeding Pro diets increased relative pancreas weights as compared to feeding Con or Pre diets. The coccidial vaccination decreased relative jejunum weight (Table 5, P = 0.034) and ileal pH (P < 0.001). Broilers fed Pro diets exhibited a higher relative proventriculus weight than those fed Con, Anti, Pre, and Pre+Pro diets (P = 0.009). Broilers fed Pre diets exhibited a higher relative proventriculus weight than those fed Anti diets. In addition, broilers fed Pre diets exhibited the highest relative gizzard weight (P = 0.018). At Day 28, there was no interaction between coccidial vaccination and dietary additive on any internal organ sizes, or on ileal pH or viscosity (Table 6). However, coccidial vaccination decreased relative gizzard weight (P = 0.002). Broilers fed Anti diets exhibited the lowest relative duodenum (P = 0.021), jejunum (P = 0.039), and ileum weights (P = 0.003). Conversely, broilers fed Pre+Pro diets exhibited the highest relative duodenum and jejunum weights. Also, broilers fed Pro diets exhibited a longer ileum than those fed Con, Anti, and Pre+Pro diets (P = 0.046). At Day 42, there was no interaction between coccidial vaccination and dietary additive on any internal organ sizes or any other measured variables (Table 7). However, coccidial vaccination decreased relative duodenum (P = 0.006) and jejunum weights (P = 0.044). In addition, broilers fed Anti diets exhibited lower relative duodenum weights than those fed Con, Pro, and Pre+Pro diets (P = 0.025). At Day 56, there was a significant (P = 0.026) interaction between coccidial vaccination and dietary additive on gizzard pH (Figure 2). In broilers that did not receive the coccidial vaccination, the Pro diet decreased their gizzard pH in comparison to the Con diet; and in broilers that received a coccidial vaccination, the Pre diet decreased their gizzard pH in comparison to the Con and Pro diets. Nevertheless, coccidial vaccination increased the length (P = 0.038, Table 8) and relative weight (P = 0.030) of the duodenum.

Duodenum/ BW (%)

Internal Organs

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Pre+Pro diets as well as those fed Anti diets exhibited a lower overall FCR from Day 0 to 56 than broilers fed Pro diets (P = 0.030). There were no main or interaction effects of coccidial vaccination and dietary additive on the absolute weights of carcass and parts on Day 42 (Table 3). Coccidial vaccination decreased relative breast (P = 0.026, Table 4) and drumstick weights on Day 42 (P = 0.011). Dietary additive did not affect any relative weights of carcass on Day 42.

Gizzard pH

WANG ET AL.

Coccidial vaccination1

6

Con Anti Pre Pro Pre+Pro SEM

Diet2

0.094 0.307 0.698

1578 1526 21.6 1510 1611 1540 1557 1543 34.2

BW (g)

0.644 0.600 0.451

0.431 0.424 0.0096 0.430 0.433 0.426 0.439 0.405 0.0158 0.002 0.300 0.564

1.748a 1.591b 0.0338 1.756 1.595 1.663 1.645 1.687 0.0545

Gizzard/BW (%)

0.361 0.462 0.996

0.244 0.251 0.0056 0.256 0.240 0.254 0.239 0.251 0.0089 0.849 0.768 0.617

0.119 0.118 0.0046 0.113 0.119 0.115 0.124 0.123 0.0069 0.095 0.701 0.360

26.96 25.98 0.406 26.81 25.88 26.09 26.55 27.00 0.654

Pancreas/ Spleen/BW Duodenum BW (%) (%) (cm)

0.074 0.729 0.917

67.97 70.87 1.112 67.69 68.88 69.91 71.36 69.27 1.775

Jejunum (cm)

0.505 0.046 0.713

69.05 69.95 1.267 67.63b 68.34b 70.94a,b 72.83a 67.63b 1.692 0.720 0.021 0.705

0.761 0.752 0.0174 0.785a 0.680b 0.771a 0.741a,b 0.805a 0.0281 0.537 0.039 0.848

1.049 1.519 0.0318 1.556a 1.372b 1.500a,b 1.535a 1.565a 0.0475

Duodenum/ Jejunum/ Ileum (cm) BW (%) BW (%)

0.916 0.003 0.515

1.046 1.044 0.0184 1.128a 0.963c 1.067a,b 1.034b,c 1.034b,c 0.0275 0.189 0.812 0.343

2.695 2.552 0.0760 2.531 2.725 2.631 2.571 2.661 0.1199

Ileum/BW (%) Gizzard pH

Ileal Viscosity (cP) 2.70 2.76 0.115 2.87 2.71 2.59 2.68 2.79 0.172 0.769 0.863 0.195

Ileal pH 6.11 6.04 0.087 6.00 6.34 5.83 6.21 6.00 0.138 0.550 0.119 0.976

Con Anti Pre Pro Pre+Pro SEM

Diet2

0.368 0.257 0.755

2981 3022 37.2 2902 3030 3048 3013 3014 48.6

BW (g)

0.083 0.289 0.357

0.321 0.304 0.0065 0.335 0.308 0.302 0.309 0.310 0.0101 0.196 0.292 0.197

1.591 1.538 0.0286 1.631 1.554 1.602 1.516 1.520 0.0443

Gizzard/BW (%)

0.716 0.245 0.543

0.175 0.178 0.0351 0.187 0.168 0.173 0.178 0.175 0.0567

Pancreas/ BW (%)

0.730 0.678 0.709

0.126 0.124 0.0495 0.118 0.128 0.119 0.132 0.127 0.0076

Spleen/ BW (%)

0.560 0.864 0.119

30.12 29.73 0.444 29.94 29.53 30.15 30.39 29.62 0.677

Duodenum (cm)

0.797 0.180 0.337

75.65 74.93 2.310 71.56 72.88 78.66 74.68 78.68 2.901

Jejunum (cm)

0.269 0.074 0.993

79.47 81.84 2.127 79.02 75.97 84.70 80.65 82.94 2.560

0.006 0.025 0.611

0.574 0.527b 0.0116 0.586a 0.509b 0.545a,b 0.577a 0.534a 0.0184

a

0.044 0.104 0.229

1.053 0.997b 0.0188 1.071 0.958 1.036 1.037 1.025 0.0308

a

Duodenum/ Jejunum/ Ileum (cm) BW (%) BW (%)

0.368 0.109 0.254

0.743 0.722 0.0156 0.759 0.673 0.738 0.735 0.759 0.0243

0.351 0.148 0.766

2.92 2.80 0.090 2.65 2.75 2.97 3.06 2.89 0.126

Ileum/BW (%) Gizzard pH

Ileal viscosity (cP) 2.95 2.96 0.123 3.00 3.07 2.92 2.90 2.89 0.158 0.796 0.809 0.882

Ileal pH 6.07 6.29 0.148 6.19 6.46 6.12 6.26 5.86 0.213 0.244 0.344 0.378

2

1

a–c

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Means in a column not sharing a common superscript are different (P ≤ 0.05). Means of non-significant interaction are not listed. Coccidial vaccination is given to one-day-old chicks by spraying a commercial vaccine containing live oocytes of Eimeria maxima, E. acervulina, and E. tenella. Experiment diets included Con (control, corn and soybean-meal basal diet), Anti (antimicrobials, basal diet supplemented with 50 g of bacitracin and 40 g of salinomycin sodium/ton of finished feed), Pre (prebiotics, basal diet supplemented with 170 g of mannan-oligosaccharides and 250 g of β -glucans/ton of finished feed), Pro (probiotics, basal diet supplemented with 3 Bacillus subtilis strains of 2084, LSSAOl, and 15A-P4 at equal amounts, 300,000 CFU/g of finished feed), and Pre+Pro (basal diet supplemented with both prebiotic and probiotics products above).

P-value Coccidial vaccination Diet Vaccination × Diet

None Vaccinated SEM

Coccidial vaccination1

Proventriculus/ BW(%)

Table 7. Effects of coccidial vaccination and dietary additive on the internal organs of broilers at 42 D of age.

2

Coccidial vaccination is given to one-day-old chicks by spraying a commercial vaccine containing live oocytes of Eimeria maxima, E. acervulina, and E. tenella. Experiment diets included Con (control, corn and soybean-meal basal diet), Anti (antimicrobials, basal diet supplemented with 50 g of bacitracin and 40 g of salinomycin sodium/ton of finished feed), Pre (prebiotics, basal diet supplemented with 170 g of mannan-oligosaccharides and 250 g of β -glucans/ton of finished feed), Pro (probiotics, basal diet supplemented with 3 Bacillus subtilis strains of 2084, LSSAOl, and 15A-P4 at equal amounts, 300,000 CFU/g of finished feed), and Pre+Pro (basal diet supplemented with both prebiotic and probiotics products above). a–c Means in a column not sharing a common superscript are different (P ≤ 0.05). Means of non-significant interaction are not listed.

1

P-value Coccidial vaccination Diet Vaccination × Diet

None Vaccinated SEM

Coccidial vaccination1

Proventriculus/ BW (%)

Table 6. Effects of coccidial vaccination and dietary additive on the internal organs of broilers at 28 D of age.

COCCIDIAL VACCINATION AND FEED ADDITIVES

7

8

WANG ET AL.

Gizzard pH at 56 days of age 4.00

a a

3.00

a abc

ab c

ab abc

bc

2.50 None

2.00

Vaccinated 1.50

P = 0.026

1.00 0.50 0.00 Control

Antibiotics Prebiotics Probiotics

Pre+Pro

Figure 2. Interaction between coccidial vaccination and dietary additive on gizzard pH at 56 D of age. superscript are different.

Intestinal Morphology Dietary additive did not affect duodenum or jejunum morphology in Eimeria-challenged broilers on Day 28 (Table 9). However, broilers fed Pre+Pro diets exhibited a higher ileal crypt depth than those fed Con and Anti diets (P = 0.041). In addition, broilers fed Pre diets or Pro diets exhibited a higher crypt depth than those fed Anti diets.

DISCUSSION The prevention of coccidiosis in poultry has relied upon dietary anticoccidials and vaccination administration programs. The current trend toward the use of antimicrobial-free poultry diets and the increased drugresistance of Eimeria strains have forced companies to rely more on vaccination administration for the control of coccidiosis (De Gussem, 2007). Previous research has indicated that coccidial vaccination may depress the growth of broilers at a young age (Lee et al., 2011). In the current study, coccidial vaccination at the day of hatch resulted in compromised growth performance in broilers (lowered feed intake and BWG). On Day 14, a clinical-coccidiosis was induced to chicks. The gut hemorrhage and a sudden increase in mortality were observed. One possible explanation is broiler chicks may have not developed a fully mature immune system at Day 14. They might not be able to protect themselves against a severe Eimeria challenge. Protective immunity from a vaccination relies on Eimeria oocytes that are recycled through litter, which may take 2 to 3 life cycles (Long et al., 1986). Eimeria has a complex life cycle, including intracellular, extracellular, asexual, and

a–c

Means not sharing a common

sexual stages, which leads to a complex host immune response (Yun et al., 2000). Parasite-specific antibodies produced by intestinal B cells, and cytokines produced by intestinal T lymphocytes, are responsible for a specific immunity to coccidiosis (Lillehoj and Lillehoj, 2000). In addition, this specific immunity has been found to be strain specific, especially with regard to the E. maxima (Fizt-Coy, 1992; Martin et al., 1997). In the current study, the coccidial vaccination (E. maxima, E. acervulina, and E. tenella) given to chicks on the day of hatch was different from the one used for the Eimeria challenge (E. acervulina, E. mivati, E. maxima, and E. tenella). Then the vaccination failed to provide the coverage against the challenge. As a consequence, the vaccination has aggravated the insult imposed by Eimeria challenge and thereby irreversibly depressed growth performance. In addition to slowing down the growth rate of broilers, coccidiosis oocyst vaccination may also depress the growth of the small intestines of broilers at an early age. Chapman (2014) summarized that Eimeria spp. infection can result in the malabsorption of nutrients (E. acervulina and E. mitis), epithelial inflammation (E. maxima), and villi destruction (E. tenella). Eimeria sporozoites and merozoites can secrete proteins that can form a moving junction at the parasite-host cell membrane (Lal et al., 2009; Cowper et al., 2012). This moving junction allows sporozoites and merozoites to invade host epithelial cells and to absorb nutrients competitively. The inadequate absorption of nutrients including L-arginine, threonine (Wils-Plotz et al., 2013) and vitamin E (Allen and Fetterer, 2002) by epithelial cells may ultimately lead to their damage to death. In this study, mucosa morphology was not significantly

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3.50

Coccidial vaccination is given to one-day-old chicks by spraying a commercial vaccine containing live oocytes of Eimeria maxima, Eimeria acervulina, and Eimeria tenella. Experiment diets included Con (control, corn and soybean-meal basal diet), Anti (antibiotics, basal diet supplemented with 50 g of bacitracin and 40 g of salinomycin sodium/ton of finished feed), Pre (prebiotics, basal diet supplemented with 170 g of mannan-oligosaccharides and 250 g of β -glucans/ton of finished feed), Pro (probiotics, basal diet supplemented with 3 Bacillus subtilis strains of 2084, LSSAOl, and 15A-P4 at equal amounts, 300,000 CFU/g of finished feed), and Pre+Pro (basal diet supplemented with both prebiotic and probiotics products above). 3 Interaction on gizzard pH is summarized in Figure 2. Means of non-significant interaction are not listed. a,b Means in a column not sharing a common superscript are different (P ≤ 0.05). 2

1

0.567 0.209 0.111 0.094 0.310 0.910 P-value Coccidial vaccination Diet Vaccination × Diet

Con Anti Pre Pro Pre+Pro SEM

0.502 0.067 0.438

0.487 0.258 0.658

0.429 0.504 0.215

0.158 0.603 0.492

0.940 0.661 0.184

0.038 0.676 0.791

0.670 0.501 0.354

0.916 0.671 0.267

0.030 0.452 0.190

0.651 0.616 0.502

0.879 0.323 0.900

0.844 0.208 0.026

2.38 2.33 0.085 2.17 2.56 2.34 2.33 2.40 0.123 6.366 6.119 0.1025 6.010 6.464 6.193 6.171 6.376 0.1630 82.62 83.68 1.306 82.50 85.31 84.54 82.41 80.97 2.131 0.134 0.141 0.0035 0.131 0.136 0.143 0.141 0.136 0.0056 0.972 0.995 0.0020 1.007 0.968 1.020 0.974 0.950 0.0318 0.238 0.231 0.0060 0.239 0.216 0.237 0.234 0.247 0.0093 4681 4622 62.4 4736 4778 4610 4410 4714 97.2 None Vaccinated SEM

Coccidial vaccination1

Diet2

BW (g)

0.097 0.096 0.0032 0.096 0.100 0.101 0.093 0.092 0.0051

31.87b 33.01a 0.383 33.09 32.72 32.33 32.07 32.00 0.596

89.65 90.01 1.481 88.75 92.16 91.10 89.33 87.81 1.453

0.428b 0.457a 0.0088 0.439 0.428 0.464 0.434 0.448 0.0139

0.818 0.833 0.0216 0.808 0.804 0.867 0.807 0.843 0.0342

0.612 0.616 0.0181 0.598 0.588 0.668 0.605 0.609 0.0284

3.134 3.112 0.0805 3.367 3.063 2.939 3.099 3.146 0.1272

Ileal pH Gizzard pH3 Ileum/BW (%) Duodenum/ Jejunum/ Ileum (cm) BW (%) BW (%) Jejunum (cm) Duodenum (cm) Spleen/ BW (%) Pancreas/ BW (%) Gizzard/BW (%) Proventriculus/ BW (%)

Table 8. Effects of dietary additive and coccidial vaccination on the internal organs of broilers at 56 D of age.

9

affected by the coccidial vaccination. However, vaccinated broilers exhibited a lower relative ileum weight on Day 14, and lower relative of the duodenum and jejunum on Day 42. This would indicate that vaccinated broilers had poor intestinal development. In this study, the combined use of bacitracin and salinomycin in a corn and soybean-meal basal diet served as a practical control in a conventional feeding program. Consistent with a similar study conducted by our lab (Wang et al., 2016), this antimicrobial diet was shown to improve the growth of broilers at an early age. In that study, the use of bacitracin in combination with other anticoccidials (narasin and nicarbazin) improved broiler BWG from Day 15 to 27 and breast weight on Day 42. In the current study, the combined use of bacitracin and salinomycin improved broiler FCR from Day 0 to 14 and Day 15 to 28. In addition, no deaths occurred when broilers were fed the antimicrobial diet from Day 15 to 42. In previous studies, in which no Eimeria challenge was administrated, intestinal size (lengths and weights) was reduced in broilers fed diets containing antimicrobials (Miles et al., 2006; Wang et al., 2016). Shorter intestinal lengths in fast-growing chickens have been associated with efficient nutrient absorption (Dibner and Richards, 2005). In the current study, in which an Eimeria challenge was employed, the inclusion of antimicrobials in the diet also decreased the relative weight of the small intestine. In addition, the use of dietary antimicrobials reduced the relative weight of the proventriculus of broilers without affecting their BW. More energy may have been saved and partitioned towards growth rather than towards organ maintenance. The primary mechanism behind the growth promoting effects of antimicrobials may be through their inhibition of bacterial growth. When compared to conventional animals, germfree animals have thinner intestinal mucosa and shallower crypt (Thompson and Trexler, 1971). These morphology features in germ-free intestines are considered to be the result of a lack of immunological stimulus by bacteria (Thompson and Trexler, 1971). In this study, a shallower ileal crypt was also observed in antibiotictreated broilers. Neither of the prebiotic or probiotic treatment improved the growth or carcass yield of broilers. This is contradictory to other probiotic studies, in which a mixture of different bacteria species used (Bozkurt et al., 2014; Ritzi et al., 2016). A mixture of Enterococcus, Bifidobacterium, Pediococcus, and Lactobacillus alleviated the negative effects of the coccidial challenge on growth (Ritzi et al., 2016). A mixture of L. acidophilus, L. casei, Enterococcus faecium, and B. bifidum improved the BWG of Eimeria-infected broilers (Bozkurt et al., 2014). However, 3 different strains of B. subtilis, which were included as the probiotic treatment in the current study, failed to improve the growth of Eimeria-challenged broilers. The failure to improve the growth of broilers fed prebiotic or probiotic diets may be a result of the severity of challenge.

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Ileal Viscosity (cP)

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10

WANG ET AL.

Table 9. Effects of dietary additive on intestinal morphology of vaccinated broilers at 28 D of age.1 Villus width (mm)

Con Anti Pre Pro Pre+Pro SEM P-value

3.328 3.242 3.439 3.132 2.902 0.2479 0.695

0.344 0.322 0.306 0.298 0.341 0.0290 0.764

Con Anti Pre Pro Pre+Pro SEM P-value

3.277 2.960 2.890 2.902 2.876 0.2185 0.729

0.276 0.306 0.296 0.299 0.333 0.0298 0.699

Con Anti Pre Pro Pre+Pro SEM P-value

1.740 1.576 1.903 1.968 1.510 0.1593 0.225

0.277 0.301 0.295 0.271 0.372 0.0292 0.288

Crypt depth (mm) Duodenum 0.341 0.346 0.351 0.340 0.439 0.0404 0.711 Jejunum 0.371 0.304 0.348 0.375 0.474 0.0363 0.102 Ileum 0.252b,c 0.225c 0.324a,b 0.321a,b 0.346a 0.0290 0.041

Muscle thickness (mm)

Goblet cell size (μ m2 )

0.242 0.256 0.214 0.244 0.231 0.0229 0.763

107.6 106.6 93.9 110.8 106.3 13.21 0.883

0.243 0.247 0.206 0.236 0.254 0.0325 0.869

121.0 112.7 131.4 85.8 107.5 15.26 0.276

0.212 0.166 0.216 0.234 0.170 0.0197 0.058

94.53 84.9 141.6 86.3 86.5 23.66 0.339

Means in a column not sharing a common superscript are different (P ≤ 0.05). Intestine samples were taken from vaccinated birds (8 replications/diet treatment, n = 40). Experiment diets included Con (control, corn and soybean-meal basal diet), Anti (antibiotics, basal diet supplemented with 50 g of bacitracin and 40 g of salinomycin sodium/ton of finished feed), Pre (prebiotics, basal diet supplemented with 170 g of mannan-oligosaccharides and 250 g of β -glucans/ton of finished feed), Pro (probiotics, basal diet supplemented with 3 Bacillus subtilis strains of 2084, LSSAOl, and 15A-P4 at equal amounts, 300,000 CFU/g of finished feed), and Pre+Pro (basal diet supplemented with both prebiotic and probiotics products above. a–c 1 2

In this study, specific coccidiosis lesions, intestinal hemorrhaging, necrotic lesions, and high mortality have been observed in broilers after an Eimeria challenge. To control this heavy coccidiosis, the inclusion of probiotics or prebiotics as feed additives alone is not enough. Even though the prebiotic and B. subtilis-based probiotics treatments applied in this study did not improve the growth or meat yield of broilers, both of them promoted the growth of the digestive organs of the broilers. On Day 14, broilers fed diets containing prebiotics (mannan oligosaccharides and β -glucans) exhibited the highest gizzard weights. Another mannan oligosaccharides prebiotic product (TechnoMos, Biochem, German) increased the gizzard size of broilers when they were not subjected to a coccidiosis challenge (Sojoudi et al., 2012). Further studies need to be conducted to understand better how prebiotics promote gizzard growth. In addition, broilers fed diets containing B. subtilis-based probiotics exhibited the highest proventriculus relative weight on Day 14. Up to 90% of B. subtilis spores can germinate into active vegetable cells in the crop (Latorre et al., 2014). The mechanism by which active B. subtilis cells stimulate the growth of the proventriculus is not clear. However, it is suggested that it may be related to the enrichment of acid bacteria, such as Lactobacillus spp., which can stimulate the growth of proventriculus (Jeong and Kim, 2014). In addition, the inclusion of B. subtilis cells enhances innate immunity in the proventriculus of broilers

(Monammed et al., 2015), which may indirectly promote their growth. A synergistic activity of B. subtilis and prebiotics was observed in the current study. The Pre+Pro diets helped the broilers to reach a lower overall FCR than did the Pro diets and helped the broilers reach an FCR similar to that by the Anti diets. Mannanoligosaccharides are reported to lower pathogenic bacterial growth (White et al., 2002), which may expose more sites for Lactobacillus to bind to the mucosal layer (Wang et al., 2016). Using mannan-oligosaccharides might also help the growth of B. subtilis by lowering the numbers of competitive bacteria. In conclusion, antimicrobial administration might reduce the intestinal size of broilers; whereas prebiotic and B. subtilis-based probiotic additives have an ability to promote the growth of several digestive organs. Even though the combined use of prebiotics and B. subtilisprobiotics did not improve the meat production of broilers subjected to Eimeria challenge, the combined use may help broilers to reach an overall FCR similar to that in the traditional program with antimicrobials.

ACKNOWLEDGMENTS This publication is a contribution of the Mississippi Agricultural and Forestry Experiment Station. This material is based upon work that is supported by the National Institute of Food and Agriculture, U. S.

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Villus length (mm)

Diet2

COCCIDIAL VACCINATION AND FEED ADDITIVES

Department of Agriculture, Hatch projects under accession numbers of MIS-322280 and MIS-701180.

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REFERENCES

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WANG ET AL. barrier function by dietary threonine and purified fiber during a coccidiosis challenge in broiler chicks. Poult. Sci. 92:735–745. White, L. A., M. C. Newman, G. L. Cromwell, and M. D. Lindemann. 2002. Brewers dried yeast as a source of mannan oligosaccharides for weanling pigs. J. Anim. Sci. 80:2619–2628. Yun, C. H., H. S. Lillehoj, and E. P. Lillehoj. 2000. Intestinal immune responses to coccidiosis. Dev. Comp. Immunol. 24:303–324.

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and resident Lactobacillus of male broilers. Poult. Sci. 95:1332– 1340. Williams, R. B. 2005. Intercurrent coccidiosis and necrotic enteritis of chickens: rational, integrated disease management by maintenance of gut integrity. Avian Pathol. 34:159–180. Wils-Plotz, E. L., M. C. Jenkins, and R. N. Dilger. 2013. Modulation of the intestinal environment, innate immune response, and