The effect of intra-amniotic and posthatch dietary synbiotic administration on the performance, intestinal histomorphology, cecal microbial population, and short-chain fatty acid composition of broiler chickens

The effect of intra-amniotic and posthatch dietary synbiotic administration on the performance, intestinal histomorphology, cecal microbial population, and short-chain fatty acid composition of broiler chickens Ali Calik,∗ Ahmet Ceylan,† Burcu Ekim,‡ Shahram Golzar Adabi,§ Furkan Dilber,∗ ¶ ¨ Alev Gurol Bayraktaroglu,† Turgay Tekinay,‡,# Do˘ gukan Ozen, and Pinar Sacakli∗,1 ∗

Department of Animal Nutrition & Nutritional Diseases, Faculty of Veterinary Medicine, Ankara University, Ankara, 06110, Turkey; † Department of Histology Embryology, Faculty of Veterinary Medicine, Ankara University, Ankara, 06110, Turkey; ‡ Life Sciences Research and Application Center, Gazi University, Ankara, 06830, Turkey; § Department of Animal Science, Faculty of Agriculture, Ankara University, Ankara, 06110, Turkey; # Medical Biology and Genetics, Faculty of Medicine, Gazi University, Ankara, 06500, Turkey; and ¶ Department of Biostatistics, Faculty of Veterinary Medicine, Ankara University, Ankara, 06110, Turkey

Key words: broiler, in ovo feeding, synbiotic, intestinal histomorphology, cecal microflora 2016 Poultry Science 0:1–15 http://dx.doi.org/10.3382/ps/pew218

INTRODUCTION

chicks, making the birds more susceptible to intestinal disorders (de Oliveira et al., 2014; Gong et al., 2008). Therefore, ensuring that the intestines of the hatchlings are quickly colonized by beneficial bacteria helps establish a favorable gut microflora balance that competitively excludes pathogens from the intestinal tract and promotes disease resistance (Bohorquez, 2007). Early intestinal colonization with beneficial bacteria not only prevents pathogenic bacteria-related intestinal disorders, but also improves intestinal maturation and integrity (Lan et al., 2013). Nutrient digestion and absorption, and therefore chick growth, are directly related to the functional capabilities of the intestine. Therefore, any improvement in early intestinal

In modern broiler production, newly hatched birds initially contact bacteria in the hatchery and house environment, rather than that of the hen or nest material. This arrangement leads to insufficient bacterial colonization of the gastrointestinal tract of the

 C 2016 Poultry Science Association Inc. Received December 20, 2015. Accepted June 1, 2016. 1 A portion of this manuscript was presented as part of a symposium at the 3rd International Poultry Meat Congress 22–26 April 2015. 2 Corresponding author: [email protected]

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eggs on d 17 of incubation did not affect the hatchability or hatching weight of the birds. However, intra-amniotic synbiotic inclusion had a positive effect on FCR at d 0 to 42 (P = 0.041) and d 22 to 42 (P = 0.036). There was no significant interaction effect on the growth performance of the birds between the intra-amniotic and dietary synbiotic treatment during different or entire experimental periods. Villus height and goblet and proliferating cell nuclear antigen (PCNA) positive cell counts were positively influenced by intra-amniotic and dietary synbiotic treatments. Our results also indicated that intra-amniotic synbiotic injection followed by dietary supplementation with a synbiotic significantly increased Lactobacillus colonization and decreased coliform population in the broiler cecum. Cecal butyric acid concentration increased proportionally to the cecal Lactobacillus count with dietary synbiotic supplementation on d 42. In summary, combined intra-amniotic and dietary synbiotic treatments improved broiler intestinal integrity and increased cecal beneficial bacteria populations.

ABSTRACT This study evaluated the effect of intraamniotic synbiotic inclusion and continued synbiotic supplementation in the diet on the performance, intestinal epithelium integrity, and cecal microflora of broiler chickens. In Experiment 1, 510 eggs containing viable embryos were divided into 3 groups of 170 eggs each. The first group was not injected and served as a negative control (NC). The next group was injected with 0.9% NaCl and was the positive control (PC). The synbiotic-injected group (S) was injected with a 0.5% inulin and 1 × 106 Enterococcus faecium solution. The non-injected and synbiotic injected groups were further divided into 2 sets for Experiment 2 and the birds were offered either a basal or synbiotic supplemented diet (1% inulin and 2 × 109 E. faecium cfu/kg feed). One hundred ninety-six broiler hatchlings were randomly allocated in a 2 × 2 factorial arrangement that included an intra-amniotic treatment (non-injected or synbiotic injected) and a dietary treatment (basal or synbiotic supplemented diet). The results showed that the administration of an intra-amniotic synbiotic to embryonated

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CALIK ET AL.

potentially synbiotic effect by improving the survival of probiotic strains throughout the upper intestinal tract. Previous studies have reported the superior benefits of synbiotics, as compared to probiotics alone, on broiler growth performance, intestinal microflora population, cecal volatile fatty acid concentration, and intestinal histomorphological parameters (Awad et al., 2009; Mookiah et al., 2014). The importance of establishing beneficial microflora in the chicken intestine early in production and its positive effects on intestinal health has long been known. Based on findings that also suggest the benefits of intra-amniotic pro- and/or prebiotic administrations, the current study hypothesized that intra-amniotic synbiotic inclusion and subsequent synbiotic supplementation in the diet may be an effective method to maintain intestinal epithelium integrity and cecal microflora of broiler chickens.

MATERIALS AND METHODS Animal Care and Use All experimental procedures were approved by The Animal Ethics Committee of Ankara University (20135-38).

Experiment 1 Incubation Procedures and In-Ovo Administration. Nine hundred eggs (Ross 308) were obtained from a 33-week-old maternal flock in a commercial hatchery (Beypili¸c A.S ¸ ., Bolu, Turkey). On arrival, all eggs were individually weighed, and 573 eggs with an average weight of 59.48 ± 2.54 g (mean ± SD) were selected and placed in the incubator under standard conditions. On d 17 of incubation (E17), the eggs were candled, and those that were unfertilized or had dead embryos were discarded; subsequently, 510 eggs containing viable embryos were divided into 3 groups of 170 eggs each. These eggs were marked to indicate the location of the amnion during the candling process on the day of injection (E17). The site of injection was disinfected with ethyl alcohol and then a sterile 21-gauge needle was used to punch a hole in the shell on the side of air-cell chamber. The first group was not injected and served as a negative control (NC). The second group was the positive control (PC) and was injected with 0.9% NaCl. The last group was injected with the synbiotic solution (S), which consisted of 0.5% inulin (wt/vol) (Orafti IPS, Beneo, Oreye, Belgium) and 1 × 106 E. faecium NCIMB 10415 (Cylactin ME20, DSM Nutritional Products, Basel, Switzerland). Intra-amniotic administration was performed by injecting the eggs with 0.6 mL of the test solution using selfrefilling syringes (Socorex, Ecublens, Switzerland), in accordance with the method described by Tako et al. (2004). The amount of time that the eggs were out of the incubator during the in ovo injection procedure was

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maturation and digestive capacity has a positive influence on chick growth and performance (Tako et al., 2004; Cheled-Shoval et al., 2011). Advances in intraamniotic administration techniques have made it possible to incorporate several nutrients and active compounds in late-term embryos, and these substances are subsequently swallowed, digested, and absorbed before hatching (Uni et al., 2005; Zhai et al., 2011; de Oliveira et al., 2014). Previous studies have shown that many nutrients, such as carbohydrates (Tako et al., 2004; Smirnov et al., 2006; Zhai et al., 2011), vitamins, and minerals (Yair and Uni, 2011), can be added to the amnion during the last quarter of the incubation period. More recent studies have demonstrated that probiotics, prebiotics, and synbiotics can be administered to chick eggs that contain viable embryos without any detrimental effect (Maiorano et al., 2012; de Oliveira et al., 2014; Madej et al., 2015; Pruszynska-Oszmalek et al., 2015). Enterococcus faecium, a lactic acid bacterium and normal inhabitant of the gut, is a probiotic that may be useful in animal health (Cao et al., 2013). Dietary supplementation with E. faecium alone (Samli et al., 2007) or in combination with several prebiotics (Awad et al., 2008) has marked effects on broiler performance and intestinal histomorphology. Furthermore, intra-amniotic E. faecium inoculation of embryonated eggs and the presence and viability of these bacteria in the cecum of chicks was demonstrated by de Oliveira et al. (2014). In addition, de Oliveira et al. (2014) revealed that Salmonella Enteritidis decreased with intra-amniotic inoculation of E. faecium and subsequent supplementation of these bacteria in the diet. Inulin is a mixture of linear polymers and oligomers of fructose linked by a β (2-1) glycosidic linkage (Rebole et al., 2010), a configuration that prevents inulin from being hydrolyzed by digestive enzymes and allows it to selectively stimulate the growth and activity of one or more bacteria in the intestine (Roberfroid et al., 1998). Previous studies have revealed that dietary prebiotics may increase beneficial intestinal bacteria populations (Kim et al., 2011), alter cecal microbial activity (Rehman et al., 2008), improve gut integrity (Baurhoo et al., 2009), and promote the digestibility of proteins and fats in a maize-soybean meal based diet of broiler chickens (Rodriguez-Suarez et al., 2010). In addition, the in ovo inclusion of inulin (Tako and Glahn, 2012) and mannan oligosaccharide (Cheled-Shoval et al., 2011) has a beneficial effect on gastrointestinal functionality and development. Synbiotics are defined as a combination of pro- and prebiotics that support the host by improving the survival and implantation of newly added bacterial strains in the intestine by activating the metabolism of healthpromoting bacteria and/or selectively stimulating their growth (Gibson and Roberfroid, 1995). Synbiotics promote the growth of the probiotic organism by providing it with the substances needed to complete fermentation (Farnworth, 2001). Tako et al. (2004) concluded that the combined use of E. faecium and inulin has a

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INTRA-AMNIOTIC SYNBIOTIC ADMINISTRATION Table 1. Composition of the basal diet.1 Starter 0 to 14 d Item

Basal Diet

Synbiotic

Basal Diet

Finisher 36 to 42 d

Synbiotic

Basal Diet

Synbiotic

48.83 30.00 13.00 2.70 0.85 2.25 0.40 0.26 0.15 0.35 0.10 0.10 1.00 0.01 100.0

55.10 24.00 13.00 3.90 0.80 2.00 0.31 0.19 0.15 0.35 0.10 0.10 0.00 0.00 100.0

53.80 24.00 13.00 4.20 0.80 2.00 0.31 0.18 0.15 0.35 0.10 0.10 1.00 0.01 100.0

54.40 24.00 13.00 4.90 0.80 2.00 0.20 0.00 0.15 0.35 0.10 0.10 0.00 0.00 100.0

53.29 24.00 13.00 5.00 0.80 2.00 0.20 0.00 0.15 0.35 0.10 0.10 1.00 0.01 100.0

3,015 22.6 1.43 1.09 0.99 0.50

3,196 20.3 1.23 0.95 0.90 0.45

3,180 20.2 1.23 0.95 0.90 0.45

3,252 20.0 1.08 0.84 0.90 0.45

3,224 19.9 1.08 0.84 0.90 0.45

88.8 22.5 3,040

88.6 20.3 3,189

88.9 20.3 3,183

89.0 20.1 3,250

88.6 20.0 3,241

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As-fed basis. Provided per kilogram of complete diet: vitamin A, 12,000 IU; vitamin D3 , 2,500 IU; vitamin E, 40 IU; vitamin K3 , 5 mg; thiamin, 2.5 mg; riboflavin, 6 mg; pyridoxine, 5 mg; pantothenic acid, 15 mg; niacin, 25 mg; folic acid, 1 mg; biotin, 50 µg; vitamin B12 , 20 µg. 3 Provided per kilogram of complete diet: Cu, 5 mg; I, 1 mg; Co, 200 µg; Se, 150 µg; Fe, 60 mg; Zn, 60 mg; Mn, 80 mg. 4 Orafti IPS, Beneo Animal Nutrition, Oreye, Belgium. 5 Enterococcus faecium NCIMB 10415, Cylactin ME20, DSM Nutritional Products, Basel, Switzerland. 6 Metabolizable energy (ME) content of diets was estimated according to the equation of Carpenter and Clegg (1956). 2

similar for all replicates. After injection, the injection holes were sealed with cellophane tape, and eggs were placed in hatching trays such that each treatment was equally represented in each location of the incubator (Tako et al., 2004). Hatch Sampling. At hatch, the number of livehatched and unhatched chicks was counted to determine the hatchability of fertile eggs (%). Unhatched eggs were opened to determine the cause of death. All hatched chicks were weighed, and 12 chicks from each treatment were randomly selected to determine the internal organ and yolk weights. Tissue samples for histomorphological and immunohistochemical analysis were taken from the duodenum, jejunum, and ileum (sampling and analysis procedures are detailed in the section describing Experiment 2).

Experiment 2 Birds and Management. Hatchlings of the noninjected (NC) and synbiotic injected (S) groups were selected for Experiment 2. The NC and S groups were further divided into 2 sets, and the birds were offered a basal or synbiotic supplemented diet. One hundred ninety-six broiler hatchlings were randomly al-

located in a 2 × 2 factorial arrangement that included intra-amniotic treatment (non-injected or synbiotic injected) and dietary treatment (basal or synbiotic supplemented diet). The experiment was conducted with 7 replicate pens (90 × 80 cm; litter material consisted of 5 cm of wood shavings) containing 7 birds each. The birds were housed in a controlled environmental enclosure for 42 days. The ambient temperature was thermostatically controlled and gradually decreased from 32 to 35◦ C on the first day, and then to 22◦ C when the broilers were 3 wk old. The temperature was then maintained at 22◦ C thereafter. The relative humidity of the house during the experiment was 50 ± 5%. The house was artificially ventilated and continuous light regimens were provided. Chicks were vaccinated at hatch against Newcastle disease virus (La Sota strain) and the infectious bronchitis virus. The starter, grower, and finisher diets were based on maize-soybean meal and were offered to the birds from 0 to 14, 15 to 35, and 36 to 42 d of age, respectively (Table 1). All diets were formulated to meet or exceed NRC (1994) nutrient recommendations. Each pen was equipped with a manual plastic feeder and an automatic nipple drinker. Water and the experimental diet (in mash form) were provided ad libitum

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Ingredient, % Corn 50.03 Soybean meal 30.00 Soybean (Full fat) 13.00 Vegetable oil 2.50 Limestone 0.86 Dicalcium phosphate 2.25 Dl-Methionine (98%) 0.40 L-lysine-HCl (78%) 0.26 L-Threonine 0.15 Salt 0.35 Vitamin premix2 0.10 Mineral premix3 0.10 Inulin4 0.00 0.00 Enterococcus faecium5 Total 100.0 Chemical composition (calculated) ME, kcal/kg 3,037 CP, % 22.7 Lysine, % 1.43 Methionine + cysteine, % 1.10 Calcium, % 1.00 Available phosphorus, % 0.50 Analyzed composition DM,% 88.6 CP, % 22.7 ME, kcal/kg6 3,035

Grower 15 to 35 d

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throughout the experimental period. All chicks were individually weighed, and feed intake (FI) was recorded at weekly intervals. Body weight gain (BWG), FI, and feed conversion ratio (FCR) were subsequently calculated based on the performance values. Sampling Procedures. At d 7, 21, and 42, one male bird from each replicate was selected according to the average BW of each treatment group. Birds were slaughtered by exsanguination and the intestinal tract was removed immediately. Tissue samples for histomorphological and immunohistochemical analysis were taken from the jejunum and ileum. To ensure the uniformity of samples, approximately 1 cm length of the mucosal segments of the jejunum and ileum were excised at the following locations: 8 cm proximal to Meckel’s diverticulum (jejunum), and 8 cm proximal to the ileocecal junction (ileum). The tissue samples were flushed with saline solution to remove adherent intestinal contents and then fixed in 10% neutral buffered formalin solution for 24 h (Sa¸cakli et al., 2015). In addition, cecal content from each selected bird was collected in sterile tubes for bacterial enumeration of fresh samples. Approximately 2 g of cecal digesta was collected and immediately frozen for short-chain fatty acid (SCFA) and branched-chain fatty acids (BCFA) analysis on d 42. Samples were stored at –20◦ C for further analysis. Histomorphologic Measurements. Tissue samples in the formalin solution were dehydrated in graded ethanol solutions, cleared with xylol, and then embedded in paraffin. The intestinal segments were sectioned at a thickness of 5 µm with microtome. Cross sections were prepared and stained with combined Alcian BluePeriodic Acid-Schiff (AB-PAS) reagents in order to determine the jejunal and ileal morphometry and goblet cell count. Villus height was measured from the top of the villus to the crypt mouth, and crypt depth was defined as the depth of the invagination between adjacent crypt mouths. Goblet cells were identified as follows (Figure 1): acid mucin was stained by AB (blue), neutral mucin was stained by PAS (pink), and the intermediate mucin that contains acidic and neutral mucins were both stained by AB-PAS (purple) (Geier et al., 2011). All positive cells along the villus were counted regardless of the mucin type (Calik and Ergun, 2015). A total of 10 well-oriented villi and crypts were selected randomly for histological measurements. Histological sections were examined under a light microscope (Leica DM2500, Leica Microsystems GmbH, Wetzlar, Germany) and photographed with a digital microscope camera (Leica DFC450, Leica Microsystems GmbH, Wetzlar, Germany). The images were evaluated using ImageJ software (Image J, U.S. National Institutes of Health, Bethesda, MD). Proliferating Cell Nuclear Antigen (PCNA) Staining. Immunohistochemical staining was performed on the stored 5-µm thick formalin-fixed paraffin-embedded tissue sections. Tissue sections were placed on polyL-lysine microscope slides (Thermo Scientific, Braunschweig, Germany). The microscope slides were then

Figure 1. A: Intestinal villus height and crypt depth, Bar 100 µm. B: Goblet cells. Thin arrow: intermediate goblet cell; White arrow: neutral goblet cell; Thick arrow: acidic goblet cell. Bar 20 µm. Alcian blue (pH 2.5) and Periodic Acid-Schiff staining.

placed in an oven at 37◦ C overnight and deparaffinized with xylene and rehydrated through graded alcohols. Endogenous peroxidase activity was blocked by quenched with H2 O2 (3% in methanol) for 30 min. The sections were pre-treated by heating for 20 min in 0.01 M citric acid buffer (pH 6) in a microwave oven at 800 W. After cooling for 20 min at room temperature, tissue sections were washed with PBS and incubated with 10% normal goat serum for 30 min for protein blocking to prevent the non-specific binding of antibodies, followed by incubation with the primary antibody to PCNA (MAB424, mouse anti PCNA monoclonal antibody, PC10 clone; EMD Millipore, Darmstadt, Germany) at dilutions of 1:100 overnight

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at 4◦ C. After incubation with the primary antibodies, the tissue sections were washed with PBS and incubated with a biotinylated secondary antibody (goat anti-rabbit IgG, Invitrogen) for 30 min at room temperature. Negative control experiments were performed by replacing the primary antibodies with PBS. After a PBS wash, tissue sections were incubated using a streptavidin horseradish peroxidase kit (HistostainPlus IHC Kit, HRP, broad spectrum, Invitrogen, Carlsbad, CA) for 30 min at room temperature. Final PBS was followed by incubation for color development 3,3-diaminobenzidine tetrahydrochloride (DAB, Invitrogen) for 3 min at room temperature. Tissue sections were counterstained with Gill’s hematoxylin, dehydrated in graded alcohols, applied to a coverslip using Entellan (Merck, Darmstadt, Germany), and examined with a Leica DM2500 light microscope. All images were captured with a digital camera (Leica DFC450) and processed with Image J (Figure 2). Proliferating cell nuclear antigen positive nuclei of total crypt epithelial cells on ten different randomly selected intact crypts, regardless of the staining intensity, were counted as described by Bologna-Molina et al. (2011). Determination of Bacterial Population in the Cecum. One gram of fresh cecal digesta was transferred to 9 mL of sterile physiological saline solution

and homogenized. Inoculants were serially diluted up to 10−8 . Subsequently, dilutions of 10−6 , 10−7 , and 10−8 were inoculated (100 µL of each dilution) to appropriate selective agar media to determine coliform and Lactobacillus counts. MacConkey agar (Merck Millipore) and MRS agar (Merck Millipore) were used to enumerate coliform and Lactobacillus, respectively. All dilutions were inoculated to selective agars in triplicate. Bacterial colonies were counted and averaged. Data were expressed as log10 colony-forming units (cfu) per gram of cecal digesta. Short Chain Fatty Acid and BCFA Analysis. Frozen cecal contents were thawed at 4◦ C and diluted with 4-fold double-distilled water in sterile screw cap tubes. Cecal contents were homogenized and centrifuged at 4,000 × g for 15 min at 4◦ C. One mL of supernatant was then transferred to an Eppendorf tube and mixed with 0.2 mL of ice-cold 25% metaphosphoric acid solution. Subsequently, tubes were placed in an ice bath for 30 min and samples were centrifuged at 11,000 × g for 10 min at 4◦ C. Supernatants were analyzed using gas chromatography (Shimadzu GC-2010, Shimadzu Co., Kyoto, Japan) coupled with a 30 m × 0.53 mm i.d. column (Teknokroma TRB-FFAP, Barcelona, Spain) and a flame ionization detector (FID) to determine SCFA

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Figure 2. A–D: PCNA positive cells in the villus and crypt depth on d 0, 7, 21, and 42. E: Thick arrow: PCNA positive cell; Thin arrow: PCNA negative cell. Bar: 50, 100, 100, 200, 20 µm, respectively.

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CALIK ET AL. Table 2. Effects of intra-amniotic synbiotic administration on hatchability (%), hatching weight, and internal organ and yolk weights on day of hatch. Treatments1 Item Egg weight (before setting), g Hatchability, % Hatching weight, g Liver weight, g Heart weight, g Yolk weight, g Gizzard weight, g Bursa of Fabricius weight, g

Statistics

NC

PC

S

SEM

P-value

59.5 92.4 41.4 1.05 0.32 4.63 2.30 0.05

59.5 91.2 42.6 1.04 0.34 4.91 2.43 0.05

59.5 90.9 41.9 1.01 0.33 4.93 2.30 0.05

0.10 0.83 0.36 0.02 0.01 0.19 0.05 0.002

0.99 0.74 0.43 0.67 0.29 0.78 0.54 0.91

1 NC: Negative control, non-injected group; PC: Positive control, injected with 0.9% NaCl; S: Synbiotic group, injected with 0.5% inulin and 1 × 106 Enterococcus faecium NCIMB 10415.

RESULTS Experiment 1 Hatchling Performance and Organ Weights. The effects of intra-amniotic synbiotic administration on hatchability (%), hatching weight, and internal organ and yolk weights are shown in Table 2. The administration of intra-amniotic synbiotic to the embryonated eggs on E17 did not affect hatchability. In addition, there were no significant differences in internal organ and yolk weights of the hatchlings. Histomorphological and Immunohistochemical Analyses. Histomorphological and immunohistochem-

ical measurements of the duodenum, jejunum, and ileum are shown in Table 3. Villus heights and crypt depths of the duodenum, jejunum, and ileum were increased (P ≤ 0.001) in the intra-amniotic synbiotic administrated group as compared to those of the NC and PC groups. No significant differences were observed in duodenum goblet cell numbers on the day of hatch. However, a significant increase in jejunal (P = 0.018) and ileal (P = 0.015) goblet cell numbers was observed when the eggs received a synbiotic injection. Proliferating cell nuclear antigen-positive cells were higher (P ≤ 0.001) in the duodenum, jejunum, and ileum crypt regions of the intra-amniotic synbiotic administrated group versus those in the NC and PC groups.

Experiment 2 Posthatch Performance. Birds were in good health throughout the entire experimental period. The effects of intra-amniotic synbiotic administration and subsequent posthatch dietary synbiotic supplementation on the growth performance of chicks are shown in Table 4. Intra-amniotic synbiotic inclusion had a positive effect on FCR at d 0 to 42 (P = 0.041) and d 22 to 42 (P = 0.036). There was no significant interaction effect on the growth performance of the birds in terms of BWG, FCR, and FI, between intra-amniotic and dietary synbiotic treatments during different experimental periods, or the entire course of the experiment. Morphological Measurements of the Jejunum and Ileum. Posthatch morphological measurements of the jejunum and ileum are shown in Table 5. Intra-amniotic synbiotic administration significantly increased ileum villus height (P = 0.001) and villus height (VH):crypt depth (CD) ratio (P = 0.001) on d 7. In addition, the VH:CD ratios of the jejunum (P = 0.022) and ileum (P ≤ 0.001) were increased by dietary synbiotic supplementation on d 7. At d 21, intra-amniotic synbiotic administration increased jejunum and ileum villus height (P = 0.024 and 0.030, respectively) and crypt depth (P < 0.001 and P = 0.001, respectively). However, they were not influenced by dietary synbiotic supplementation. Both intra-amniotic synbiotic administration and posthatch dietary synbiotic

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and BCFA concentrations of cecal digesta (Zhang et al., 2003; Rebole et al., 2010). The column temperature was programmed to increase gradually from 120◦ C to 160◦ C during the analysis. In addition, injector port and FID temperatures were fixed at 230◦ C and 250◦ C, respectively. Injection volume was set to 1 µL, and analyses were performed in duplicate (Calik and Ergun, 2015). Statistical Analysis. In Experiment 1, a randomized complete block design was employed during incubation with each of the tray levels of the setter and each of the hatching basket levels in the hatcher, and with all treatments being equally represented in each block. Treatments were viewed as a fixed effect, and blocks as a random effect in the one-way ANOVA. An arcsine square root transformation was used for hatchability data to obtain normally distributed data. In Experiment 2, chicks from the NC and S groups were randomly assigned to 4 experimental groups with 7 replicate floor pens (90 × 80 cm), each containing 7 birds. Data were subjected to a 2-way ANOVA using the GLM procedure of SPSS 14.01 (SPSS Inc., Chicago, IL, USA). The models included the intra-amniotic administration (NC and S) and the posthatch dietary supplementation (basal and synbiotic) as the main factors, and the 2-way interaction. Post hoc testing was only carried out for significant interactions and was performed using simple effect analysis. Mortality rates were compared using a chi-square test. A probability value of less than 0.05 was considered significant, unless otherwise noted.

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INTRA-AMNIOTIC SYNBIOTIC ADMINISTRATION Table 3. Effects of intra-amniotic synbiotic administration on intestinal histomorphology and goblet cell and PCNA-positive cell numbers at day of hatch.1 Treatments2 Item

NC

PC

S

SEM

P-value

172b 47.2b 3.64b 32.2

186b 43.8b 4.27a 32.3

257a 64.9a 3.95a,b 32.1

7.86 1.89 0.07 0.59

< 0.001 < 0.001 < 0.001 0.98

191b 48.7b 3.96 31.5b

190b 45.3b 4.21 33.0a,b

228a 57.0a 4.03 35.3a

3.84 0.99 0.06 0.57

< 0.001 < 0.001 0.24 0.018

138b 40.8b 3.39 23.0b

139b 40.5b 3.43 23.5b

161a 48.3a 3.34 26.4a

2.28 0.74 0.05 0.53

< 0.001 < 0.001 0.71 0.015

0.12 0.07 0.10

< 0.001 < 0.001 0.001

8.14b 7.28b 10.0b

8.02b 7.43b 10.3b

9.01a 7.85a 10.9a

Means with different superscripts in the same row are significantly different (P < 0.05). Data represent mean values of 12 replicates per treatment. 2 NC: Negative control, non-injected group; PC: Positive control, injected with 0.9% NaCl; S: Synbiotic group, injected with 0.5% inulin and 1 × 106 Enterococcus faecium NCIMB 10415. 3 Villus height to crypt depth ratio. 4 Proliferating cell nuclear antigen. a,b 1

supplementation significantly influenced villus height and crypt depth of the jejunum and ileum on d 42. In addition, intra-amniotic applications increased the VH:CD of the jejunum (P = 0.020) and ileum (P < 0.001) on d 42. No significant interaction effect on villus height and VH:CD was observed between intraamniotic and dietary synbiotic treatment during the experimental period. Goblet Cells. Posthatch goblet cell counts of the jejunum and ileum are shown in Table 6. Intra-amniotic synbiotic administration had no effect on goblet cell numbers of the jejunum and ileum during different experimental periods. Dietary synbiotic supplementation significantly increased goblet cell numbers in both the jejunum (on d 7; P = 0.007) and ileum (on d 21; P = 0.004). No significant interaction effect on goblet cell number was observed between intra-amniotic and dietary synbiotic treatment during the experimental period. PCNA-Positive Cells. Posthatch PCNA-positive cells in the crypt depths are shown in Table 6. A significant interaction effect was observed in PCNA-positive cells in both the jejunum and ileum on d 7 (P = 0.017 and 0.031, respectively), d 21 (P < 0.001), and d 42 (P < 0.001). Cecal Microflora. The microflora composition of the cecal samples is shown in Table 7. Cecal coliform bacterial populations were not affected by intraamniotic or dietary synbiotic treatments on d 7. The number of Lactobacillus bacteria was consistently higher for both intra-amniotic and dietary synbiotic treatments on d 7 (P = 0.001), d 21 (P < 0.001), and

d 42 (P < 0.001). Furthermore, there was a significant interaction between intra-amniotic and dietary synbiotic treatments, in terms of Lactobacillus number, on d 21. The coliform count was significantly decreased by dietary synbiotic supplementation on d 21. A positive significant interaction (P = 0.001) was observed in the coliform count between intra-amniotic and dietary synbiotic treatments on d 42. Cecal Concentrations of SCFA and BCFA. Cecal concentrations of SCFA and BCFA (µmol/g of digesta) are shown in Table 8. Dietary synbiotic supplementation significantly increased cecal butyrate concentration at the end of the experiment. No significant interaction was seen in acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, BCFA, and total SCFA concentrations on d 42.

DISCUSSION The intestinal lumen, mucosal surface, and cecum of broiler chickens are densely populated with microorganisms. The management of these gut microorganisms is of vital importance to the nutrition and health of modern antibiotic-free broiler production systems (Lan et al., 2013). In addition, the early colonization of hatchling intestines with healthy microorganisms has a significant effect on future broiler performance and intestinal health (Higgins et al., 2008; Flint and Garner, 2009). After the ban of growth-promoting antibiotics, the use of probiotics, prebiotics, or synbiotics in broiler diets has become common. In this context, the present study aimed to determine the effects of intra-amniotic

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Duodenum Villus height (µm) Crypt depth (µm) VH:CD3 Goblet cell number Jejunum Villus height (µm) Crypt depth (µm) VH:CD ratio Goblet cell number Ileum Villus height (µm) Crypt depth (µm) VH:CD Goblet cell number PCNA-positive cell4 Duodenum Jejunum Ileum

Statistics

102 133 1.30

354 506 1.44

793 1,148 1.45

2,118 3,392 1.60

2,912 4,540 1.56 4.1

348 530 1.52

797 1,187 1.49

2,071 3,446 1.66

2,868 4,633 1.62 2.0

S

100 138 1.38

NC

2,862 4,567 1.60 4.1

2,094 3,454 1.65

769 1,113 1.45

344 508 1.48

97.0 129 1.33

NC

Synbiotic

2,878 4,545 1.58 2.0

2,136 3,447 1.61

742 1,098 1.48

327 482 1.49

92.6 126 1.37

S

2,865 4,600 1.61

2,082 3,450 1.66

783 1,150 1.47

346 519 1.50

98.5 134 1.36

NC

2,895 4,543 1.57

2,127 3,420 1.61

768 1,123 1.47

341 494 1.46

97.5 130 1.34

S

Intra-amniotic administration3

2,890 4,587 1.59

2,095 3,419 1.63

795 1,168 1.47

351 518 1.48

101 136 1.34

Basal

Diet3

2,870 4,556 1.59

2,115 3,451 1.63

756 1,106 1.47

335 495 1.48

94.7 128 1.35

Synbiotic

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IAA 0.72 0.43 0.70 0.53 0.009 0.31 0.45 0.35 0.88 0.17 0.62 0.036 0.51 0.47 0.041

RMSE 7.64 12.3 0.12 25.8 23.6 0.11 51.7 74.1 0.08 84.1 160 0.05 116 207 0.04

0.66 0.70 0.99

0.54 0.61 0.95

0.060 0.037 0.94

0.12 0.017 0.94

0.082 0.087 0.86

D

P-value

Statistics4

0.75 0.65 0.23

0.94 0.70 0.54

0.56 0.66 0.22

0.25 0.92 0.27

0.27 0.83 0.23

IAA × D

1 Experiment 1. NC: Negative control, non-injected group; S: Synbiotic group, injected with 0.5% inulin and 1 × 106 Enterococcus faecium NCIMB 10415; Experiment 2. Basal: Corn–soybean meal basal diet (free of synbiotic); Synbiotic: basal diet containing 1% inulin and 2 × 109 Enterococcus faecium cfu/kg feed. 2 Data represent mean values of 7 replicates per treatment. 3 Data represent mean values of 14 replicates per treatment. 4 IAA: Intra-amniotic administration; D: Diet 5 BW: Body weight; FCR: Feed conversion ratio. 6 Mortality rates were compared using a chi-square test (P = 0.88).

0 to 7 d BW gain (g) Feed intake (g) FCR 0 to 14 d BW gain (g) Feed intake (g) FCR 0 to 21 d BW gain (g) Feed intake (g) FCR 22 to 42 d BW gain (g) Feed intake (g) FCR 0 to 42 d BW gain (g) Feed intake (g) FCR Mortality6 (%)

Item5

Basal

Treatments2

Table 4. Effects of intra-amniotic synbiotic administration and posthatch dietary synbiotic supplementation on body weight gain, feed intake, and feed conversion ratio of broiler chickens.1

8 CALIK ET AL.

540 96.8 5.59 367 85.6 4.30 837 117 7.16 570 111 5.14

1,050 144 7.30

799 118c 6.77

332 85.8 3.86

790 109 7.27

513 100 5.20

921 134 6.87

721 119c 6.06

S

527 96.3 5.47

NC

769 127b 6.04

989 147 6.73

564 106 5.24

776 109 7.15

343 79.6 4.32

485 94.2 5.14

NC

584 110 5.32

867 121 7.14

372 79.2 4.69

514 102 5.04

S

897 138a 6.50

1,174 158 7.24

Synbiotic

745 123 6.05

955 141 6.80

539 103 5.22

783 109 7.21

337 82.8 4.09

506 95.3 5.31

NC

848 128 6.64

1,112 151 7.27

577 110 5.23

852 119 7.15

370 82.4 4.50

527 99.1 5.32

S

Intra-amniotic administration3

760 119 6.42

986 139 7.08

542 106 5.17

814 113 7.22

349 85.7 4.08

534 96.6 5.53

Basal

Diet3

833 133 6.27

1,081 152 6.99

574 108 5.28

821 115 7.15

358 79.4 4.51

500 97.8 5.09

Synbiotic

1

a-c

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0.061 0.24 0.37 0.001 < 0.001 0.62 < 0.001 < 0.001 0.26

0.001 0.85 0.001 0.024 < 0.001 0.79 0.030 0.001 0.97 < 0.001 < 0.001 0.020 < 0.001 0.018 < 0.001

22.8 4.75 0.27 73.9 3.94 0.57 42.6 4.99 0.33 65.5 6.29 0.51 43.1 4.96 0.34

0.79 0.17 0.75

0.35 0.002 < 0.001

0.088 0.60 0.022

0.28 0.11 0.95

49.6 5.97 0.45

D

IAA

RMSE

P-value

Statistics4

0.15 0.007 0.33

0.27 0.97 0.82

0.27 0.064 0.56

0.44 0.14 0.80

0.71 0.96 0.76

0.66 0.16 0.55

IAA × D

Means with different superscripts in the same row are significantly different (P < 0.05). Experiment 1. NC: Negative control, non-injected group; S: Synbiotic group, injected with 0.5% inulin and 1 × 106 Enterococcus faecium NCIMB 10415; Experiment 2. Basal: Corn–soybean meal basal diet (free of synbiotic); Synbiotic: basal diet containing 1% inulin and 2 × 109 Enterococcus faecium cfu/kg feed. 2 Data represent mean values of 7 replicates per treatment. 3 Data represent mean values of 14 replicates per treatment. 4 IAA: Intra-amniotic administration; D: Diet 5 Villus height to crypt depth ratio.

d7 Jejunum Villus height (µm) Crypt depth (µm) VH:CD5 Ileum Villus height (µm) Crypt depth (µm) VH:CD d 21 Jejunum Villus height (µm) Crypt depth (µm) VH:CD Ileum Villus height (µm) Crypt depth (µm) VH:CD d 42 Jejunum Villus height (µm) Crypt depth (µm) VH:CD Ileum Villus height (µm) Crypt depth (µm) VH:CD

Item

Basal

Treatments2

Table 5. Effects of intra-amniotic synbiotic administration and posthatch dietary synbiotic supplementation on histomorphological parameters of the jejunum and ileum on d 7, d 21, and d 42.1

INTRA-AMNIOTIC SYNBIOTIC ADMINISTRATION

9

94.5 157 204

84.7 125 152 19.9b 51.9b 92.3b 18.8b 50.2b 86.1b

85.0 118 155

19.6b 52.9b 91.4b

18.0c 49.7b 85.4b

S

93.2 160 209

NC

18.7b 49.8b 86.1b

19.9b 52.1b 91.7b

84.9 142 153

99.0 153 208

NC

94.2 147 162

107 175 211

S

20.3a 56.2a 90.0a

22.0a 60.0a 101a

Synbiotic

18.3 49.8 85.8

19.8 52.5 91.6

85.0 130 154

96.1 157 209

NC

19.6 53.2 88.0

20.9 56.0 96.6

89.4 136 157

101 166 208

S

Intra-amniotic administration3

18.4 50.0 85.8

19.8 52.4 91.9

84.9 122 154

93.9 159 207

Basal

Diet3

19.5 53.0 88.1

20.9 56.0 96.3

89.6 145 158

103 164 210

Synbiotic

1

a-c

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0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001

0.010 0.40 0.57

0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001

6.54 19.0 12.7

0.81 1.51 1.09 0.44 1.57 1.03

0.081 0.004 0.44

0.007 0.39 0.73

0.15 0.17 0.90

8.45 16.7 23.2

D

IAA

RMSE

P-value

Statistics4

0.031 < 0.001 < 0.001

0.017 < 0.001 < 0.001

0.077 0.91 0.23

0.28 0.064 0.71

IAA × D

Means with different superscripts in the same row are significantly different (P < 0.05). Experiment 1. NC: Negative control, non-injected group; S: Synbiotic group, injected with 0.5% inulin and 1 × 106 Enterococcus faecium NCIMB 10415; Experiment 2. Basal: Corn–soybean meal basal diet (free of synbiotic); Synbiotic: basal diet containing 1% inulin and 2 × 109 Enterococcus faecium cfu/kg feed. 2 Data represent mean values of 7 replicates per treatment. 3 Data represent mean values of 14 replicates per treatment. 4 IAA: Intra-amniotic administration; D: Diet 5 Proliferating cell nuclear antigen.

Goblet cell number Jejunum d7 d 21 d 42 Ileum d7 d 21 d 42 PCNA-positive cell5 Jejunum d7 d 21 d 42 Ileum d7 d 21 d 42

Item

Basal

Treatments2

Table 6. Effects of intra-amniotic synbiotic administration and posthatch dietary synbiotic supplementation on goblet cell number and proliferating cell nuclear antigen-positive cells of the jejunum and ileum on d 7, d 21, and d 42.1

10 CALIK ET AL.

7.10 7.43

7.92 9.04b

7.60b 9.18

8.04 8.74c

7.98a 9.07

S

7.20 7.11

NC

7.42b 9.35

7.75 9.14a

7.09 7.42

NC

Synbiotic

7.53b 9.42

7.75 9.21a

7.19 7.84

S

7.70 9.21

7.90 8.94

7.15 7.27

NC

7.57 9.30

7.84 9.13

7.14 7.64

S

Intra-amniotic administration3

7.79 9.13

7.98 8.89

7.15 7.30

Basal

Diet3

7.48 9.39

7.75 9.18

7.14 7.63

Synbiotic

IAA 0.90 0.001 0.19 < 0.001 0.016 < 0.001

RMSE 0.11 0.26 0.12 0.06 0.13 0.05

0.060 0.58 0.18 < 0.001 < 0.001 0.54

< 0.001 < 0.001 < 0.001 < 0.001

IAA × D

0.80 0.001

D

P-value

Statistics4

35.0 3.91 7.44 1.01 1.09 1.04 3.15 49.5

Acetate Propionate Butyrate Isobutyrate Valerate Isovalerate BCFA5 Total SCFA6

42.1 4.82 10.4 0.86 1.16 0.87 2.88 60.2

S 40.6 4.11 12.3 0.90 1.13 0.90 2.94 59.9

NC

Synbiotic

37.1 3.57 12.1 0.81 1.07 0.81 2.69 55.5

S 37.8 4.01 9.86 0.96 1.11 0.97 3.04 54.7

NC

39.6 4.19 11.3 0.83 1.11 0.84 2.78 57.9

S

Intra-amniotic administration3

38.5 4.36 8.94 0.94 1.12 0.95 3.01 54.8

Basal

Diet3

38.9 3.84 12.2 0.86 1.10 0.86 2.81 57.7

Synbiotic

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IAA 0.65 0.77 0.24 0.049 0.98 0.058 0.16 0.55

RMSE 9.95 1.49 2.92 0.15 0.17 0.16 0.45 13.2

0.93 0.38 0.011 0.19 0.73 0.15 0.27 0.59

D

P-value

Statistics4

0.19 0.23 0.18 0.62 0.35 0.54 0.97 0.16

IAA × D

1 Experiment 1. NC: Negative control, non-injected group; S: Synbiotic group, injected with 0.5% inulin and 1 × 106 Enterococcus faecium NCIMB 10415; Experiment 2. Basal: Corn–soybean meal basal diet (free of synbiotic); Synbiotic: basal diet containing 1% inulin and 2 × 109 Enterococcus faecium cfu/kg feed. 2 Data represent mean values of 7 replicates per treatment. 3 Data represent mean values of 14 replicates per treatment. 4 IAA: Intra-amniotic administration; D: Diet 5 BCFA (Branched Chain Fatty Acids) = isobutyrate + valerate + isovalerate. 6 Total SCFA (Short Chain Fatty Acids) = acetate + propionate + butyrate + isobutyrate + valerate + isovalerate.

NC

Item

Basal

Treatments2

Table 8. Effects of intra-amniotic synbiotic administration and posthatch dietary synbiotic supplementation on cecal SCFA and BCFA composition (µmol/g of digesta) on d 42.1

1

a–c

Means with different superscripts in the same row are significantly different (P < 0.05). Experiment 1. NC: Negative control, non-injected group; S: Synbiotic group, injected with 0.5% inulin and 1 × 106 Enterococcus faecium NCIMB 10415; Experiment 2. Basal: Corn–soybean meal basal diet (free of synbiotic); Synbiotic: basal diet containing 1% inulin and 2 × 109 Enterococcus faecium cfu/kg feed. 2 Data represent mean values of 7 replicates per treatment. 3 Data represent mean values of 14 replicates per treatment. 4 IAA: Intra-amniotic administration; D: Diet.

d7 Coliform Lactobacillus d 21 Coliform Lactobacillus d 42 Coliform Lactobacillus

Item

Basal

Treatments2

Table 7. Effects of intra-amniotic synbiotic administration and posthatch dietary synbiotic supplementation on cecal bacterial counts (log10 cfu/g of cecal content) on d 7, d 21, and d 42.1

INTRA-AMNIOTIC SYNBIOTIC ADMINISTRATION

11

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CALIK ET AL.

tory results of the effects of dietary probiotics, prebiotics, or synbiotics on the zootechnical performance of broilers. Awad et al. (2008) observed improvement in broiler performance when E. faecium and a chicoryderived prebiotic that is rich in inulin was added to the basal diet. In contrast, Rodriguez et al. (2012) showed that the addition of E. faecium and inulin to a wheatand barley-based diet did not influence the BWG and FI of the broilers. Another study showed that a multibacterial species probiotic product resulted in beneficial modulation of cecal microflora, as evidenced by significant increases in the concentrations of beneficial bacteria (Mountzouris et al., 2007). However, Mountzouris et al. (2007) concluded that the dietary addition of probiotics did not improve broiler BW on d 42. Similarly, the addition of inulin at 5 to 20 g/kg to a maize-soybean meal based diet improved the apparent ileal digestibility of protein and fat, but did not enhance broiler performance (Alzueta et al., 2010). We did not observe any significant interaction between intra-amniotic administration and dietary synbiotic supplementation in broiler performance under the experimental conditions, even if the applications positively influenced intestinal integrity and cecal microflora. The lack of consistency between the performance and improved intestinal health might be explained by a study by Timmerman et al. (2006), who showed that the administration of probiotics via drinking water improved the FCR of birds (1.93 vs. 1.87) in field trials. However, unlike the field trials, FCR (1.66 vs. 1.67) was not influenced in the controlled trial. The authors suggested that the effect of probiotics becomes smaller when the productivity rates of broilers are higher. In addition, Patterson and Burkholder (2003) suggested that environmental stress influenced the efficacy of such feed additives, which tend to be more effective when animals are producing well below their genetic potential. Another possible explanation for the unaffected performance may be related to the supplementation levels of probiotic and prebiotic because the inclusion level of such products in diets can influence efficiency (Patterson and Burkholder, 2003). In modern broiler production, improvements in intestinal architecture and the mucus layer can have a beneficial influence on the performance and health of the broilers. The intestinal mucus layer that is synthesized and secreted by goblet cells protects the brush border area and acts as the first line of defense against attack by enteric pathogens by decreasing their adherence to the intestinal mucosa. The layer also provides protection against bacterial and environmental toxins and other dietary components that may damage the mucosa. In addition, the mucus aids in digestion and absorption (Solis De Los Santos et al., 2007; Baurhoo et al., 2009; Cheled-Shoval et al., 2014). As hypothesized in this study, intra-amniotic synbiotic administration and posthatch dietary synbiotic supplementation had a pronounced effect on intestinal integrity and goblet cell count in the jejunum and ileum. Furthermore,

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synbiotic administration and subsequent posthatch dietary synbiotic supplementation on the intestinal integrity and cecal microflora of broiler chickens. In the present study, intra-amniotic synbiotic administration did not affect hatchability or hatching weight of the birds. These findings are consistent with those of previous studies that reported that the inclusion of 5 × 109 cfu/per egg E. faecium (de Oliveira et al., 2014), 4% inulin (Tako and Glahn, 2012), and 0.1% mannan oligosaccharide (MOS) (Cheled-Shoval et al., 2011) did not reduce hatchability and hatching weight of chickens, as compared to those hatched from noninjected eggs. However, to our knowledge, the combined intra-amniotic application of E. faecium and inulin has not been reported. Our results indicate that the intra-amniotic administration of inulin and E. faecium could help establish healthy microflora in the intestines without any negative effect on hatchability and hatchling performance. As indicated in previous studies (de Oliveira et al., 2014; Madej et al., 2015; Pruszynska-Oszmalek et al., 2015), intra-amniotic inoculation of probiotics, prebiotics, or synbiotics can have beneficial effects on broiler hatchings under experimental conditions. However, more research is required to set the commercial standards and observe their widespread effects in practice. Morphological changes in the small intestine, such as increased villus height and VH:CD ratio, can improve the performance of birds by enhancing the absorptive surface area, which is important when alternative growth stimulators are applied (Calik and Ergun, 2015). The results of Experiment 1 showed that intra-amniotic synbiotic administration improved the morphological development of the intestine on day old hatches, as indicated by an increase in villus height and goblet cell and PCNA positive cell numbers. Similarly, Cheled-Shoval et al. (2011) observed a significant increase in villus height, crypt depth, and goblet cell numbers in hatchlings administered MOS as compared to the NaCl-injected group. However, the present study is the first report of the effect of intra-amniotic synbiotic administration on intestinal morphological parameters and PCNA positive cell numbers. The positive effects of inulin and E. faecium may overcome the physiological limitations of the digestive tract due to the delayed access to feed during the posthatch period. In the present study, intra-amniotic synbiotic administration improved FCR on d 0 to 42 and d 22 to 42, a trend that may be attributed to morphological changes in the intestines due to beneficial bacterial colonization at a young age. Previous research has shown that the development of the digestive tract at an early stage may affect the future performance of chicks (Tako et al., 2004). Contrary to our expectation, the current study showed that intra-amniotic synbiotic administration followed by posthatch dietary synbiotic supplementation of E. faecium and inulin had no significant interaction effect on the growth performance of broilers. However, several studies have produced contradic-

INTRA-AMNIOTIC SYNBIOTIC ADMINISTRATION

early life stage and help reduce pathogens. To the best of our knowledge, the results for cecal Lactobacillus and coliform bacteria in this study provide new insights regarding the intra-amniotic effects of E. faecium and inulin in broilers. Further research using molecular techniques could reveal more details regarding cecal microflora. In poultry, the cecum harbors a wide variety of microbiota and provides the most stable environment for bacterial growth in the gastrointestinal tract (Mead, 1989; Meimandipour et al., 2010). Bacterial fermentation in the cecum leads to the formation of SCFAs that are necessary for intestinal functionality and integrity (Meimandipour et al., 2010). In addition, these fermentation by-products contribute to broiler energy metabolism and lower the pH of the intestinal environment, which may limit the growth of bacterial pathogens (van Der Wielen et al., 2000). Meimandipour et al. (2010) suggested that lactate, produced by Lactobacillus species in the cecal digesta, promotes the growth of butyrate-producing bacteria, and consequently increases the cecal butyrate concentration. Among the SCFAs, butyric acid both stands out as the preferred energy source for enterocytes and takes part in cellular differentiation and proliferation within the intestinal mucosa (Rinttil¨ a and Apajalahti, 2013). Our results showed that cecal butyric acid concentration increased on d 42 in accordance with the increase in cecal Lactobacillus count in chicks. Therefore, the observed improvement in intestinal architecture might be related to the trophic effects of butyric acid on enterocytes. In conclusion, the present study demonstrated that intra-amniotic E. faecium and inulin inoculation and their subsequent supplementation in the diet improved broiler intestinal morphology by selectively stimulating intestinal beneficial microflora and increasing cecal butyrate concentrations. In addition, intra-amniotic synbiotic administration influenced future broiler performance by improving overall FCR. This method establishes a healthy microflora in the intestines, without any negative effect on hatchability and hatchling performance. In addition, this study suggests that both pre- and posthatch use of a synbiotic is more effective than supplementing only in the posthatch period. However, this in ovo feeding application remains in the experimental stage, and its potential impact on commercial standards unknown. In ovo application standards in hatcheries must be determined before there is a practical use for this method in commercial facilities. Thus, these applications should be optimized with larger numbers to estimate their impacts more precisely.

ACKNOWLEDGMENTS This study was funded by the Ankara University Coordination of Scientific Research Projects with project number 13B3338008. The authors thank Beypili¸c A.S ¸ ., DSM Nutritional Products and Artısan Gıda A.S ¸ . for providing materials. We also recognize Ahmet

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previous studies found that intestinal integrity improved in birds fed diets containing several synbiotics (Awad et al., 2010; Sen et al., 2012). These enhancements can be attributed to the synbiotic administration supporting beneficial bacterial growth, which influences cellular turnover (Awad et al., 2009; Mookiah et al., 2014) and mucin dynamics (Cheled-Shoval et al., 2014). The absorptive and protective functions of the intestine are dependent on a fully functional and healthy epithelial lining (Umar, 2010). Intestinal epithelial cells have a short lifespan and need to be replaced rapidly and continuously via the replication of undifferentiated cells. Proliferating cell nuclear antigen, also known as cyclin or DNA-polymerase delta auxiliary protein, is an endogenous nuclear protein that is used to identify replicating cells in tissues (Foley et al., 1993; Uni et al., 1998; Gulbahar et al., 2005). In most mammals, enterocyte proliferation is restricted to the crypt region; however, in chickens, enterocyte proliferation is localized predominantly in the crypt region, but also occurs to a lesser extent in the villi (Uni et al., 1998). Similar to Uni et al. (1998), our results showed that PCNA positive cells are not confined to the crypt depth, but are also found along the villi (Figure 2). Increased villus height is directly related to higher epithelial turnover (Fan et al., 1997) and activated cell mitosis (Samanya and Yamauchi, 2002). The improvements in intestinal integrity in the present study are presumably related to the positive effects of pre- and posthatch synbiotic administration on intestinal epithelial cell turnover in the crypt region, which supports the growth of beneficial bacteria. Intestinal microflora and their metabolic activity have significant effects on broiler health and performance (Calik and Ergun, 2015). Bacterial colonization patterns are unstable in young animals, which makes them more susceptible to bacterial infections (Gaggia et al., 2010), and is why early beneficial bacterial colonization is so important. Previous studies have shown that dietary E. faecium (Samli et al., 2007) and inulin (Rebole et al., 2010) alter the intestinal bacterial population as evidenced by a significant increase in lactic acid bacteria count. Moreover, Mookiah et al. (2014) revealed that cecal Lactobacillus and Bifidobacterium counts increased and E. coli numbers significantly decreased with dietary synbiotic supplementation. However, published data on the response of intestinal bacteria to intra-amniotic and posthatch dietary E. faecium and inulin supplementation are limited. Interestingly, our results indicated that intra-amniotic synbiotic injection followed by dietary supplementation with a synbiotic significantly increased Lactobacillus colonization and decreased coliform population in the broiler cecum. This beneficial effect may be attributed to early bacterial colonization facilitated by intra-amniotic synbiotic administration. In this manner, our findings showed that pre- and posthatch use of a synbiotic might contribute to the colonization of beneficial bacteria at an

13

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CALIK ET AL.

Erg¨ un (Turkish Poultry Meat Producers and Breeders Association-Secretary General), Duygu Yılmaz (DSM Nutritional Products), and Esra K¨ ose (Artısan Gıda A.S ¸ ) for their support. This manuscript was edited by English Paper Editors of Editage.

REFERENCES

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Alzueta, C., M. L. Rodriguez, L. T. Ortiz, A. Rebole, and J. Trevino. 2010. Effects of inulin on growth performance, nutrient digestibility and metabolisable energy in broiler chickens. Br. Poult. Sci. 51:393–398. Awad, W., K. Ghareeb, and J. Bohm. 2008. Intestinal structure and function of broiler chickens on diets supplemented with a synbiotic containing Enterococcus faecium and oligosaccharides. Int. J. Mol. Sci. 9:2205–2216. Awad, W. A., K. Ghareeb, S. Abdel-Raheem, and J. Bohm. 2009. Effects of dietary inclusion of probiotic and synbiotic on growth performance, organ weights, and intestinal histomorphology of broiler chickens. Poult. Sci. 88:49–56. Awad, W. A., K. Ghareeb, and J. Bohm. 2010. Effect of addition of a probiotic micro-organism to broiler diet on intestinal mucosal architecture and electrophysiological parameters. J. Anim. Physiol. Anim. Nutr. (Berl). 94:486–494. Baurhoo, B., P. R. Ferket, and X. Zhao. 2009. Effects of diets containing different concentrations of mannanoligosaccharide or antibiotics on growth performance, intestinal development, cecal and litter microbial populations, and carcass parameters of broilers. Poult. Sci. 88:2262–2272. Bohorquez, D. V. 2007. Dietary and housing effects on growth performance, gut health and Salmonella colonization of Salmonellachallenged broilers. MS. NC State University, Raleigh, NC. Bologna-Molina, R., P. Dami´ an-Matsumura, and N. MolinaFrechero. 2011. An easy cell counting method for immunohistochemistry that does not use an image analysis program. Histopathology. 59:801–803. Calik, A., and A. Ergun. 2015. Effect of lactulose supplementation on growth performance, intestinal histomorphology, cecal microbial population, and short-chain fatty acid composition of broiler chickens. Poult. Sci. 94:2173–2182. Cao, G. T., X. F. Zeng, A. G. Chen, L. Zhou, L. Zhang, Y. P. Xiao, and C. M. Yang. 2013. Effects of a probiotic, Enterococcus faecium, on growth performance, intestinal morphology, immune response, and cecal microflora in broiler chickens challenged with Escherichia coli K88. Poult. Sci. 92:2949–2955. Carpenter, K. J., and K. M. Clegg. 1956. The metabolizable energy of poultry feeding stuffs in relation to their chemical composition. J. Sci. Food Agr. 7:45–51. Cheled-Shoval, S. L., E. Amit-Romach, M. Barbakov, and Z. Uni. 2011. The effect of in ovo administration of mannan oligosaccharide on small intestine development during the pre- and posthatch periods in chickens. Poult. Sci. 90:2301–2310. Cheled-Shoval, S. L., N. S. Gamage, E. Amit-Romach, R. Forder, J. Marshal, A. Van Kessel, and Z. Uni. 2014. Differences in intestinal mucin dynamics between germ-free and conventionally reared chickens after mannan-oligosaccharide supplementation. Poult. Sci. 93:636–644. de Oliveira, J. E., E. van der Hoeven-Hangoor, I. B. van de Linde, R. C. Montijn, and J. M. van der Vossen. 2014. In ovo inoculation of chicken embryos with probiotic bacteria and its effect on posthatch Salmonella susceptibility. Poult. Sci. 93:818–829. Fan, Y. K., J. Croom, V. L. Christensen, B. L. Black, A. R. Bird, L. R. Daniel, B. W. McBride, and E. J. Eisen. 1997. Jejunal glucose uptake and oxygen consumption in turkey poults selected for rapid growth. Poult. Sci. 76:1738–1745. Farnworth, E. R. 2001. Probiotics and prebiotics. Pages 407–422 in Handbook of Nutraceuticals and Functional Foods. R. E. C. Wildman, ed. CRC Press, Boca Raton, FL. Flint, J. F., and M. R. Garner. 2009. Feeding beneficial bacteria: A natural solution for increasing efficiency and decreasing pathogens in animal agriculture. J. Appl. Poult. Res. 18:367–378.

Foley, J., T. Ton, R. Maronpot, B. Butterworth, and T. L. Goldsworthy. 1993. Comparison of proliferating cell nuclear antigen to tritiated thymidine as a marker of proliferating hepatocytes in rats. Environ. Health Perspect. 101 Suppl. 5:199–205. Gaggia, F., P. Mattarelli, and B. Biavati. 2010. Probiotics and prebiotics in animal feeding for safe food production. Int. J. Food Microbiol. 141 Suppl. 1:S15–S28. Geier, M. S., V. A. Torok, P. Guo, G. E. Allison, M. Boulianne, V. Janardhana, A. G. Bean, and R. J. Hughes. 2011. The effects of lactoferrin on the intestinal environment of broiler chickens. Br. Poult. Sci. 52:564–572. Gibson, G. R., and M. B. Roberfroid. 1995. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J. Nutr. 125:1401–1412. Gong, J., H. Yu, T. Liu, J. J. Gill, J. R. Chambers, R. Wheatcroft, and P. M. Sabour. 2008. Effects of zinc bacitracin, bird age and access to range on bacterial microbiota in the ileum and caeca of broiler chickens. J. Appl. Microbiol. 104:1372–1382. Gulbahar, M. Y., H. Yuksel, T. Guvenc, and H. Okut. 2005. Assessment of proliferative activity by AgNOR and PCNA in prostatic tissues of ram lambs implanted with zeranol. Reprod. Domest. Anim. 40:468–474. Higgins, S. E., J. P. Higgins, A. D. Wolfenden, S. N. Henderson, A. Torres-Rodriguez, G. Tellez, and B. Hargis. 2008. Evaluation of a Lactobacillus-based probiotic culture for the reduction of Salmonella enteritidis in neonatal broiler chicks. Poult. Sci. 87:27–31. Kim, G. B., Y. M. Seo, C. H. Kim, and I. K. Paik. 2011. Effect of dietary prebiotic supplementation on the performance, intestinal microflora, and immune response of broilers. Poult. Sci. 90:75–82. Lan, Y., M. W. A. Verstegen, S. Tamminga, and B. A. Williams. 2013. The role of the commensal gut microbial community in broiler chickens. World’s Poult. Sci. J. 61:95–104. Madej, J. P., T. Stefaniak, and M. Bednarczyk. 2015. Effect of in ovo-delivered prebiotics and synbiotics on lymphoid-organs’ morphology in chickens. Poult. Sci. 94:1209–1219. Maiorano, G., A. Sobolewska, D. Cianciullo, K. Walasik, G. Elminowska-Wenda, A. Slawinska, S. Tavaniello, J. Zylinska, J. Bardowski, and M. Bednarczyk. 2012. Influence of in ovo prebiotic and synbiotic administration on meat quality of broiler chickens. Poult. Sci. 91:2963–2969. Mead, G. C. 1989. Microbes of the avian cecum: types present and substrates utilized. J. Exp. Zool. Suppl. 3:48–54. Meimandipour, A., M. Shuhaimi, A. F. Soleimani, K. Azhar, M. Hair-Bejo, B. M. Kabeir, A. Javanmard, O. Muhammad Anas, and A. M. Yazid. 2010. Selected microbial groups and short-chain fatty acids profile in a simulated chicken cecum supplemented with two strains of Lactobacillus. Poult. Sci. 89:470–476. Mookiah, S., C. C. Sieo, K. Ramasamy, N. Abdullah, and Y. W. Ho. 2014. Effects of dietary prebiotics, probiotic and synbiotics on performance, caecal bacterial populations and caecal fermentation concentrations of broiler chickens. J. Sci. Food Agric. 94:341– 348. Mountzouris, K. C., P. Tsirtsikos, E. Kalamara, S. Nitsch, G. Schatzmayr, and K. Fegeros. 2007. Evaluation of the efficacy of a probiotic containing Lactobacillus, Bifidobacterium, Enterococcus, and Pediococcus strains in promoting broiler performance and modulating cecal microflora composition and metabolic activities. Poult. Sci. 86:309–317. NRC. 1994. Nutrient Requirements of Poultry. 9th ed. National Academies Press, Washington, DC. Patterson, J. A., and K. M. Burkholder. 2003. Application of prebiotics and probiotics in poultry production. Poult. Sci. 82:627–631. Pruszynska-Oszmalek, E., P. A. Kolodziejski, K. Stadnicka, M. Sassek, D. Chalupka, B. Kuston, L. Nogowski, P. Mackowiak, G. Maiorano, J. Jankowski, and M. Bednarczyk. 2015. In ovo injection of prebiotics and synbiotics affects the digestive potency of the pancreas in growing chickens. Poult. Sci. 94:1909–1916. Rebole, A., L. T. Ortiz, M. L. Rodriguez, C. Alzueta, J. Trevino, and S. Velasco. 2010. Effects of inulin and enzyme complex, individually or in combination, on growth performance, intestinal microflora, cecal fermentation characteristics, and jejunal histomorphology in broiler chickens fed a wheat- and barley-based diet. Poult. Sci. 89:276–286.

INTRA-AMNIOTIC SYNBIOTIC ADMINISTRATION

are affected by in ovo feeding of carbohydrates. Poult. Sci. 85:669– 673. Solis De Los Santos, F., A. M. Donoghue, M. B. Farnel, G. R. Huff, W. E. Huff, and D. J. Donoghue. 2007. Gastrointestinal maturation is accelerated in turkey poults supplemented with a mannan-oligosaccharide yeast extract (Alphamune). Poult. Sci. 86:921–930. Tako, E., P. R. Ferket, and Z. Uni. 2004. Effects of in ovo feeding of carbohydrates and beta-hydroxy-beta-methylbutyrate on the development of chicken intestine. Poult. Sci. 83:2023–2028. Tako, E., and R. P. Glahn. 2012. Intra-amniotic administration and dietary inulin affect the iron status and intestinal functionality of iron-deficient broiler chickens. Poult. Sci. 91:1361–1370. Timmerman, H. M., A. Veldman, E. van den Elsen, F. M. Rombouts, and A. C. Beynen. 2006. Mortality and growth performance of broilers given drinking water supplemented with chicken-specific probiotics. Poult. Sci. 85:1383–1388. Umar, S. 2010. Intestinal stem cells. Curr. Gastroenterol. Rep. 12:340–348. Uni, Z., P. R. Ferket, E. Tako, and O. Kedar. 2005. In ovo feeding improves energy status of late-term chicken embryos. Poult. Sci. 84:764–770. Uni, Z., R. Platin, and D. Sklan. 1998. Cell proliferation in chicken intestinal epithelium occurs both in the crypt and along the villus. J. Comp. Physiol. B. 168:241–247. van Der Wielen, P. W., S. Biesterveld, S. Notermans, H. Hofstra, B. A. Urlings, and F. van Knapen. 2000. Role of volatile fatty acids in development of the cecal microflora in broiler chickens during growth. Appl. Environ. Microbiol. 66:2536–2540. Yair, R., and Z. Uni. 2011. Content and uptake of minerals in the yolk of broiler embryos during incubation and effect of nutrient enrichment. Poult. Sci. 90:1523–1531. Zhai, W., D. E. Rowe, and E. D. Peebles. 2011. Effects of commercial in ovo injection of carbohydrates on broiler embryogenesis. Poult. Sci. 90:1295–1301. Zhang, W. F., D. F. Li, W. Q. Lu, and G. F. Yi. 2003. Effects of isomalto-oligosaccharides on broiler performance and intestinal microflora. Poult. Sci. 82:657–663.

Downloaded from http://ps.oxfordjournals.org/ at Serials Division on July 24, 2016

Rehman, H., P. Hellweg, D. Taras, and J. Zentek. 2008. Effects of dietary inulin on the intestinal short chain fatty acids and microbial ecology in broiler chickens as revealed by denaturing gradient gel electrophoresis. Poult. Sci. 87:783–789. Rinttil¨ a, T., and J. Apajalahti. 2013. Intestinal microbiota and metabolites—Implications for broiler chicken health and performance. J. Appl. Poult. Res. 22:647–658. Roberfroid, M. B., J. A. Van Loo, and G. R. Gibson. 1998. The bifidogenic nature of chicory inulin and its hydrolysis products. J. Nutr. 128:11–19. Rodriguez, M. L., A. Rebole, S. Velasco, L. T. Ortiz, J. Trevino, and C. Alzueta. 2012. Wheat- and barley-based diets with or without additives influence broiler chicken performance, nutrient digestibility and intestinal microflora. J. Sci. Food Agric. 92:184– 190. Rodriguez-Suarez, E., E. Gubb, I. F. Alzueta, J. M. Falcon-Perez, A. Amorim, F. Elortza, and R. Matthiesen. 2010. Virtual expert mass spectrometrist: iTRAQ tool for database-dependent search, quantitation and result storage. Proteomics. 10:1545–1556. ¨ S Sa¸caklı, P., A. Calik, A. G. Bayraktaro˘ glu, A. Erg¨ un, O. ¸ ahan, and ¨ S. Ozaydin. 2015. Effect of clinoptilolite and/or phytase on broiler growth performance, carcass characteristics, intestinal histomorphology and tibia calcium and phosphorus levels. Kafkas Univ. Vet. Fak. Derg. 21:729–737. Samanya, M., and K. E. Yamauchi. 2002. Histological alterations of intestinal villi in chickens fed dried Bacillus subtilis var. natto. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 133:95–104. Samli, H. E., N. Senkoylu, F. Koc, M. Kanter, and A. Agma. 2007. Effects of Enterococcus faecium and dried whey on broiler performance, gut histomorphology and intestinal microbiota. Arch. Anim. Nutr. 61:42–49. Sen, S., S. L. Ingale, Y. W. Kim, J. S. Kim, K. H. Kim, J. D. Lohakare, E. K. Kim, H. S. Kim, M. H. Ryu, I. K. Kwon, and B. J. Chae. 2012. Effect of supplementation of Bacillus subtilis LS 1–2 to broiler diets on growth performance, nutrient retention, caecal microbiology and small intestinal morphology. Res. Vet. Sci. 93:264–268. Smirnov, A., E. Tako, P. R. Ferket, and Z. Uni. 2006. Mucin gene expression and mucin content in the chicken intestinal goblet cells

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