Effect of dietary supplementation of mannan-oligosaccharides on performance, blood metabolites, ileal nutrient digestibility, and gut microflora in Escherichia coli-challenged laying hens R. Jahanian1 and M. Ashnagar Department of Animal Sciences, College of Agriculture, Isfahan University of Technology, Isfahan 84156-83111, Iran

Key words: laying hen, mannan-oligosaccharide, Escherichia coli, low-density lipoprotein, immune response 2015 Poultry Science 00:1–8 http://dx.doi.org/10.3382/ps/pev180

INTRODUCTION

testinal health (Ferket et al., 2002; Gunal et al., 2006; Buchanan et al., 2008), largely because of the manipulation of intestinal microflora and establishment of a balanced bacterial population within the gut. Among the prebiotic compounds, mannanoligosaccharides (MOS) have a good potential to affect gut microflora. In contrast to the mode of action of most antibiotics, MOS and possibly other oligosaccharides serve as alternate attachment sites for Gram-negative pathogens, preventing their attachment onto the enterocytes and subsequent enteric infection (Ofek et al., 1977; Van der Wielen et al., 2002). Adherence of the pathogenic microbes to the entrecote’s cell wall is thought to be a prerequisite for the onset

The final target of poultry production for the food chain is to obtain optimum performance and feed conversion efficiency, while maintaining optimal animal health. One the main strategies to achieve this, is maintaining gut health. Different feed additives such as antibiotics, organic acids, probiotics, and prebiotics have been investigated and it was proven that dietary supplementation with these additives could affect gastroin C 2015 Poultry Science Association Inc. Received December 10, 2014. Accepted May 25, 2015. 1 Corresponding author: [email protected]

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creases in hen-day egg production and egg mass, respectively, during the entire experimental period. Dietary supplementation with 0.1 to 0.2% MOS decreased (P < 0.01) serum triglycerides concentration compared with control birds. In addition, serum concentration of low-density lipoproteins was reduced (P < 0.001) by all supplemental MOS levels. In contrast to Newcastle antibody titer, primary antibody response against sheep red blood cell was significantly (P < 0.05) affected by supplemental MOS. Supplementation of MOS into the diet caused increases in digestibility coefficients of DM (P < 0.05) and CP (P < 0.01). In addition, there was a significant (P < 0.01) difference between dietary treatments for ileal ether extract digestibility, with the highest digestibility values assigned to the hens supplemented with 0.05% MOS. Although dietary MOS supplementation had no effect on ileal E. coli and total bacteria enumerations, it resulted in a decrease (P < 0.01) in Salmonella count and increased Lactobacillus. The present findings indicate that MOS supplementation of laying hens under bacterial challenge could improve productive performance probably through modification of intestinal bacterial populations and improving nutrient digestibility.

ABSTRACT The present study was carried out to investigate the effect of dietary supplementation of different levels of mannan-oligosaccharides (MOS) on performance, egg quality, immune responses, and gut microflora in laying hens exposed to Escherichia coli (E. coli) challenge. A total of 180 Hy-Line W-36 laying hens, 55 wk of age, were randomly distributed among 5 dietary treatments with 6 replicates of 6 hens each. Experimental diets consisted of 5 graded levels of MOS (0, 0.05, 0.1, 0.15, and 0.2% of diet). The study lasted 77 d including 7 d for adaptation and 70 d as the main experimental period subdivided into two 35d periods. The results showed increases (P < 0.05) in egg production percentage and egg mass, and a decrease (P < 0.05) in feed conversion ratio (FCR) in birds fed the diets containing 0.1 and 0.15% MOS compared with control birds during the first 35-d period. In addition, there were significant differences between dietary treatments for egg mass and FCR during the second 35-d, with the best (P < 0.05) values observed for hens fed on 0.1% MOS-supplemented diet. Feed intake and egg weight, however, were not influenced by dietary treatments throughout the experimental period. Compared to control birds, supplemental MOS resulted in 9.8% (P < 0.01) and 8.1% (P < 0.05) in-

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JAHANIAN AND ASHNAGAR

MATERIALS AND METHODS Birds, Diets, and Experimental Procedures The present study was performed in an industrial poultry farm (Isfahan, Iran) and all experimental procedures used were approved by the Isfahan University of Technology Animal Care and Use Committee. A total of 180 Hy-Line (W-36) Single Comb White laying hens, 55 wk of age, were housed in groups and randomly assigned into 5 dietary treatments with 6 cages of 6 hens each. Experimental diets consisted of a control group (not supplemented) and MOS groups at the levels of 0.05, 0.1, 0.15 and 0.2% of diet (as provided by Techno-MOS product; Biochem Co., Lohne, Germany). The basal diet (Table 1) was formulated according to the Hy-Line W-36 Management Guide (Hy-Line International, 2007); all of the experimental diets were isocaloric and isonitrogenous, and had a similar nutrient composition.

Table 1. Ingredients and chemical composition of basal diet during 55 to 66 wk of age (as-fed basis) Items Ingredients Corn, yellow Soybean meal Wheat bran Sunflower oil Limestone Oyster shell Monocalcium phosphate Mineral premix2 Vitamin premix3 Common salt Sodium bicarbonate DL-Methionine L-Lysine·HCl L-Threonine Zeolite4 Calculated Nutrient Composition AMEn (kcal/kg) CP Methionine Methionine + cysteine Lysine Threonine Calcium Nonphytate P Sodium

Percent1 56.51 24.98 3.00 2.31 5.23 5.00 1.29 0.25 0.25 0.25 0.20 0.17 0.04 0.02 0.50 2,750 16.00 0.43 0.72 0.89 0.63 4.05 0.41 0.17

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Unless stated otherwise. Mineral premix provided per kilogram of diet: Mn (from MnSO4 ·H2 O), 90 mg; Zn, 80 mg; Fe (from FeSO4 ·7H2 O), 60 mg; Cu (from CuSO4 ·5H2 O), 10 mg; I (from Ca (IO3 )2 ·H2 O), 0.8 mg; Se, 0.3 mg; cobalt, 0.15 mg. 3 Vitamin premix provided per kilogram of diet: vitamin A (from retinyl acetate), 10,000 IU; vitamin D3 , 2,500 IU; vitamin E (from dlα -tocopheryl acetate), 15 IU; vitamin B1 , 2.2 mg; vitamin B2 , 4 mg; pantothenic acid, 8 mg; vitamin B6 , 2 mg; niacin, 30 mg; vitamin B12 , 0.015 mg; folic acid, 0.5 mg; biotin, 0.15 mg; choline (from choline chloride 60%), 400 mg. 4 Different levels (0.05, 0.1, 0.15, and 0.2% of diet) of mannan-oligosaccharide product (Techno-MOS; Biochem Co., Lohne, Germany) were made at the expense of equal amount of zeolite, so that the chemical composition was similar among the experimental diets. 2

The study lasted 77 d including 7 d for adaptation (wk 55 of age) and 70 d as the main experimental period (commenced from wk 56 of age) by 35-d intervals. The birds were housed in layer wire-floored cages (total of 30 cages) at a density of 417 cm2 /bird in a windowless house, and were given artificial light (16L:8D) throughout the duration of the study. Each cage was equipped with a separated feed trough and nipple drinker, and the diets were offered ad libitum. All of the birds were challenged orally with 0.5 mL (containing 2 × 109 cfu/mL sterile saline; pH = 7.2) of an E. coli suspension (a mix of different E. coli serotypes including K1, O2, O78, and O88; Razi Vaccine and Serum Research Institute, Karaj, Iran) at the intervals of 1 to 7, 15 to 17, 29 to 31, 43 to 45, and 57 to 59 d of the main experimental period, so that the count of E. coli was always 105 cfu/g fresh digesta (measured using the spread plate method; Mahdavi et al., 2010). These serotypes are selected because it has been reported that they are pathogenic to poultry and

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of infection (Gibbons and Van Houte, 1975). Adhesion leads to bacterial growth, entrapment and formation of mixed colonies, the entrapment of nutrients for growth, and the possible prevention of antibody attachment to the pathogenic organism (Costerton et al., 1978). It has been well-documented that MOS, derived from mannans on yeast cell surfaces, act as high affinity ligands, offering a competitive binding site for the bacterial attachment. Pathogens with the mannose-specific Type-1 fimbriae attach to the MOS instead of the intestinal epithelial cells and therefore move through the intestine without colonization (Ofek et al., 1977). Supplementation of poultry diets with MOS has resulted in improved performance in terms of BW gain and feed conversion (Parks et al., 2001), partly due to its nutrient sparing effect but primarily because of its influence on nutrient utilization in the gastrointestinal tract (Sonmez and Eren, 1999). Day et al. (1987) and Nursoy et al. (2004) reported no effect of dietary yeast culture on feed consumption, egg production, egg weight, and feed efficiency in laying hens. In contrast, Abou El-Ella et al. (1996) showed an increased egg production and improved feed efficiency as the result of dietary supplementation of a yeast culture in laying hens. To our knowledge, there is no study to examine the effect of supplemental MOS in laying hens under bacterial challenge. On the other hand, Escherichia coli (E. coli) infection is prevalent in some poultry farms in Iran (including laying hens) following the viral infections. Some E. coli serotypes have been shown to be pathogenic for poultry and are widespread (Menao et al., 2002). The objective of this study, therefore, was to investigate the effects of dietary supplementation of different levels of MOS on performance, internal and external egg qualities, antibody responses, and ileal microflora in Leghorn laying hens subjected to an E. coli challenge.

MANNAN-OLIGOSACCHARIDES AND E. COLI CHALLENGE IN HENS

are widespread compared with other serotypes (Menao et al., 2002). Because the birds were maintained in a common house, we avoided a negative (unchallenged) control group.

Antigen Preparation and Intestinal E. coli Enumeration

Performance Parameters Mortality was recorded as it occurred. Eggs were collected daily and egg production percentage was expressed on a hen-day basis during 56 to 61, 61 to 66, and 56 to 66 d intervals. Egg weight was recorded daily throughout the experimental period. Feed intake was recorded at the end of each 35-d period. Feed conversion ratio (FCR) was calculated each 35-d.

Egg Quality Measurements To determine egg quality indices, 5 eggs produced over the last 2 d of each 35-d period were collected. Each egg was individually weighed and broken using a quasistatic compression device to measure eggshell breaking strength. Eggshell thickness was measured at 3 different places (upper and lower ends, and middle) by using a micrometer screw gauge (Jahanian and Rasouli, 2014). The height of the albumen (average of 4 points), midway between the yolk and the edge of the thick albumen, was measured with a tripod microme-

ter; then, Haugh units (HU) were calculated using the formula: HU = 100 log (H + 7.57 − 1.7 W0.37 ), where H is the mean height (mm) of the albumen, and W is the weight (g) of the egg (Silversides, 1994). The yolk index is a measure of the standing-up quality of the yolk and was obtained by dividing the height of the yolk by its diameter. Yolk color was visually scored using a Roche yolk color fan.

Blood Biochemical Parameters At the final day of the study, blood samples were collected from 3 birds/replicate; thereafter, the serum samples were analyzed for cholesterol, low-density lipoproteins (LDL), high-density lipoproteins (HDL), and triglyceride concentrations using standard kits (Pars Azmoun, Tehran, Iran) with a biochemical analyzer (ERBA CHEM-5; Beijing Biochemical Instrument Co., Beijing, China).

Immune Responses LaSota strain vaccine (one dose/hen) was used to vaccinate the hens against Newcastle disease virus (NDV) via spraying method at d 32 of the main experimental period and serum samples were collected for antibody assay at d 6 and 12 postvaccine inoculation from 2 birds/replicate. The hemagglutination inhibition test (Marquardt et al., 1984) was performed to determine the antibody production response against NDV antigen as log2 of the reciprocal of the last dilution. In addition, 2 birds/cage were injected intraperitonealy with 0.5 cc of 5% SRBC suspension (suspended in PBS) at d 52 and 60 of the main experimental period. Thereafter, the hens (wing-banded) were bled from the wing vein at d 7 after each inoculation to measure primary and secondary antibody responses against SRBC according to the procedure described by van der Zijpp and Leenstra (1980).

Ileal Nutrient Digestibility Marker-containing diets (supplemented with 0.5% Celite as a source of acid insoluble ash) were fed for 4 d, from the 67th to 70th d of main experimental period, to investigate ileal nutrient digestibility. At the final day, 3 birds/replicate were killed by cervical dislocation and ileal contents from the Meckel’s diverticulum to the ileocecal junction were collected directly into 150-mL cups (Scott and Boldaji, 1997). Samples were held on ice, frozen (−20◦ C), freeze-dried, and stored for analysis. Feed samples and freeze-dried digesta were ground (0.5 mm screen) prior to chemical analysis. The samples were analyzed for DM (Code 934.01), CP (Code 976.06), ether extract (Code 954.02), and total ash (Code 942.05) according to the standard procedures of the AOAC (2002). Organic matter was determined through subtraction.

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The E. coli serotypes K1, O2, O77, and O88 were purchased from Razi Vaccine and Serum Research Institute (Karaj, Iran). To ensure the viability of the mixed culture, bacteria were grown in tryptic soy broth (Merck, Darmestadt, Germany) for 6 h (until late log phase) at 37◦ C with shaking at 180 rpm. Cells were harvested by centrifugation at 5,000 × g for 15 min at 4◦ C (Sigma 6K15, Laboratory Centrifuges, Germany). The pellet was washed 3 times with PBS (pH = 7.2) and resuspended in a quantity of PBS to achieve a concentration of 2 × 109 cfu/mL. For intestinal E. coli enumeration, 3 birds/treatment were euthanized at d 24 and 52 of the main experimental period and intestinal contents from the Meckel’s diverticulum to the ileocecal junction were carefully collected into the sterile cups. The cups were kept in an ice-covered container, then immediately transferred to the laboratory. A volume containing 1 g digesta was serially (1:10) diluted to 10−6 using 0.85% NaCl solution, and 0.1 mL each dilution was plated in duplicate onto eosin methylene blue agar plates (Merck, Darmstadt, Germany). The inoculated plates were incubated at 37◦ C for 24 h and E. coli colonies were confirmed using biochemical tests of IMViC (indole, methyl red, Voges–Proskauer, and citrate tests). The plates were counted by spread plate method to determine the total number of bacterial cfu/gram fresh digesta.

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JAHANIAN AND ASHNAGAR Table 2. Effect of supplemental mannan-oligosaccharides (MOS) on performance parameters of laying hens during 56 to 66 wk age1 MOS Level (% of Diet) 0

1

0.1

62.2 73.5b,c 45.7c,d 106 2.31a,b

63.9 80.6a,b 51.5a,b 106 2.06c

63.5 71.8b,c 45.6b 108 2.37a,b 62.9 72.6b,c 45.7b,c 107 2.34a,b

0.15

0.2

SEM

Treatments

Control vs. MOS

63.0 82.8a 52.2a 105 2.00c

63.1 77.3a,b,c 48.8b,c 104 2.14b,c

0.89 2.60 1.46 1.67 0.09

0.482 0.025 0.030 0.832 0.036

0.999 0.029 0.037 0.322 0.042

63.6 76.3a 48.6a 109 2.25b

63.0 72.9a,b 45.9b 109 2.36b

62.5 71.6b,c 44.7b 108 2.41a,b

0.89 1.64 1.01 2.64 0.10

0.955 0.047 0.033 0.821 0.048

0.940 0.013 0.035 0.559 0.613

63.8 78.5a 50.0a 108 2.15c

63.0 77.9a 49.1a,b 107 2.17b,c

62.8 74.4a,b 46.8b,c 106 2.27a,b,c

0.86 1.99 1.25 1.85 0.09

0.828 0.003 0.011 0.786 0.039

0.982 0.005 0.022 0.370 0.385

Dietary MOS supplementation was made using a commercial MOS product (Techno-MOS; Biochem Co., Lohne, Germany). Means with no common superscripts within each row are significantly (P < 0.05) different.

a–d

Ileal Bacterial Count In addition to ileal digestibility, the subsamples of ileal contents of euthanized birds were collected into the specific sampling cups to count populations of total bacteria, E. coli, Salmonella spp., and Lactobacillus spp. according to the methods described by Baurhoo et al. (2007). Briefly, the ileal contents were serially diluted in 0.85% sterile saline solution and used to enumerate bacterial populations. All microbiological analyses were performed in duplicate, and the average values were used for statistical analyses. Lactobacilli were anaerobically assayed using the Lactobacillus (deMan, Rogosa, and Sharpe) agar and incubated at 37◦ C for 48 h. Escherichia coli was assayed using Rapid E. coli 2 agar. Ileal population of Salmonella was enumerated using the Salmonella–Shigella agar (Baurhoo et al., 2007).

Statistical Analysis All data were subjected to ANOVA using general linear model procedures of SAS statistical software (SAS Institute, 1999) and treatment means were separated by the least significant difference test at P < 0.05 statistical level. Also, single-degree-of-freedom contrast comparisons were made among the experimental groups to compare control hens with those supplemented with MOS.

RESULTS AND DISCUSSION Performance Data on production performance are shown in Table 2. As noted, the records of egg production are

definitely less than those of Hy-Line W-36 standards. Before the study commenced, the average egg production percentage was 86.13% and records well-indicate that E. coli challenge has significantly suppressed laying performance. Dietary treatments did not affect feed intake and egg weight during the first and second 35-d periods (Table 2). On the other hand, egg production percentage and egg mass were increased (P < 0.05) as the result of dietary supplementation of 0.1 or 0.15% MOS compared with control birds during the first 35-d period. In addition, dietary supplementation with 0.1 and 0.15% MOS improved (P < 0.05) FCR values throughout the experimental period. Although supplemental MOS had no marked effect on egg weight and feed intake, it increased egg production (P < 0.01) and egg mass (P < 0.05) during the entire experimental period. Contrast comparisons showed that compared with control hens, supplemental MOS resulted in 9.8 and 8.1% increases in hen-day egg production and egg mass, respectively, throughout the experimental period. In contrast to our results, Gao et al. (2008) reported that dietary supplementation of a yeast culture product at the level of 0.25% decreased feed intake compared with control birds. Other researchers, however, reported that supplemental yeast culture had no effect on feed intake of laying hens (Day et al., 1987; Nursoy et al., 2004; and Yal¸cın et al., 2008). Also, Hassanein and Soliman (2010) reported that the average egg weight was not influenced by adding yeast culture into the laying diets. However, dietary supplementation of yeast culture resulted in a significant increase in egg weight in the study by Yal¸cın et al. (2008). It has been reported that feed conversion efficiency was improved by dietary supplementation of yeast culture in laying hens (Abou El-Ella et al., 1996),

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Values at 56 to 61 Wk of Age Egg weight (g) 63.5 Egg production (%) 69.5c Egg mass (g/d/hen) 44.1d Feed intake (g/d/hen) 104 FCR (g feed/g egg) 2.35a Values at 61 to 66 Wk of Age Egg weight (g) 62.7 Egg production (%) 67.3c Egg mass (g/d/hen) 42.2c Feed intake (g/d/hen) 108 FCR (g feed/g egg) 2.55a Values at 56 to 66 Wk of Age Egg weight (g) 63.1 Egg production (%) 68.4c Egg mass (g/d/hen) 43.1c Feed intake (g/d/hen) 106 FCR (g feed/g egg) 2.45a

0.05

Probability

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MANNAN-OLIGOSACCHARIDES AND E. COLI CHALLENGE IN HENS Table 3. Effect of supplemental mannan-oligosaccharides (MOS) on egg quality measurements at 61 and 66 wk age1 MOS Level (% of Diet)

Values at 61 Wk of Age Shell strength (kg/cm2 ) Eggshell thickness (mm) HU Yolk index Yolk color Values at 66 Wk of Age Shell strength (kg/cm2 ) Eggshell thickness (mm) HU Yolk index Yolk color

Probability

0

0.05

0.1

0.15

0.2

SEM

Treatments

Control vs. MOS

2.93 0.41 84.5 0.44a,b 8.22

2.91 0.42 83.7 0.44a,b 8.19

2.84 0.43 83.9 0.46a 8.23

2.92 0.42 82.6 0.42b 8.12

2.95 0.44 84.0 0.45a,b 7.83

0.13 0.01 1.04 0.01 0.15

0.794 0.433 0.621 0.043 0.492

0.744 0.483 0.485 0.862 0.859

2.71 0.41 80.6 0.44 8.11

2.94 0.40 85.7 0.45 8.26

2.79 0.40 87.2 0.45 8.30

2.73 0.40 86.4 0.45 8.54

2.84 0.41 84.8 0.43 8.20

0.10 0.01 1.71 0.01 0.09

0.176 0.646 0.107 0.071 0.202

0.240 0.467 0.038 0.248 0.390

laying quails (Yıldız et al. 2004), and broiler chicks (Onifade and Babatunde, 1996; Parks et al., 2001). It has been hypothesized that the improvement in feed efficiency may partially be attributed to the establishment of an intestinal bacterial population that favored improved nutrient retention (Ferket et al., 2002; Gunal et al., 2006). It has been reported that MOS have at least 3 possible modes of action by which performance is improved: 1) adsorption of pathogenic bacteria containing Type-1 fimbriae with mannose-sensitive lectins, sometimes referred as the “receptor analog” mechanism, or stated another way, different bacterial strains can agglutinate MOS (Spring et al., 2000); 2) improving intestinal function and gut health via increase in villus uniformity and integrity (Spring et al., 2000; Baurhoo et al., 2007); and 3) modulating the gut immune system by acting as a nonpathogenic microbial antigen giving an adjuvantlike effect (Ferket et al., 2002). The growth-promoting effect of MOS is attributed to their ability to limit the growth of potentially pathogenic bacteria in the digestive tract of animals (Bozkurt et al., 2008). Thus, the digestive tract remains healthy, functions more efficiently, and more nutrients are available for absorption.

Egg Quality Indices Most indices of egg internal and external quality were not influenced by adding MOS into the experimental diets (Table 3). Single-degree-of-freedom contrasts showed that only HU was influenced (P < 0.05) by supplemental MOS compared with that of control eggs during the second 35-d period. Consistent with our findings, Nursoy et al. (2004) did not find any effect on albumen or egg yolk qualities of laying hens fed a yeast-supplemented diet. Also, it was reported that egg breaking strength (Day et al., 1987; Nursoy et al., 2004), shell thickness (Abou El-Ella et al., 1996; Nursoy

et al., 2004), HU, and yolk index (Nursoy et al., 2004) were not affected by dietary supplementation of yeast culture. In contrast to the present results, Shashidhara and Devegowda (2003) reported that supplemental MOS improved egg shell quality in old breeder females. In addition, egg shell percentage and eggshell thickness were improved due to the feeding various levels of yeast culture to laying hens in the study of Hassanein and Soliman (2010).

Serum Lipid Metabolites Blood glucose and lipid profiles of animals reflect their physiological and nutritional status according to their internal and external environments (Meng et al., 2010). Therefore, we measured serum lipid metabolites to determine whether MOS can affect blood biochemical parameters in laying hens. Contrast comparisons showed that serum concentrations of triglycerides (P < 0.01) and LDL (P < 0.001) were decreased as the result of dietary inclusion of MOS compared with those of control hens, with the lowest LDL values observed for birds fed the diet containing 0.2% MOS (Table 4). Decrease in serum triglycerides concentration may be, in part, attributed to the transportation of blood triglycerides into the ovary (as phospholipids from liver) to support yolk development and egg production (Stevens, 1996). The fact that why this lipid transportation to ovary did not increase egg weight, is probably due to increasing egg production percentage and egg mass. In agreement with the present observation, Kannan et al. (2005) reported that feeding prebiotics reduced serum triglycerides concentration and abdominal fat content in broiler chicks. In addition, serum HDL level was affected (P < 0.05) by dietary treatments, and supplemental MOS levels of 0.1 to 0.2% increased serum HDL concentration

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1 Dietary MOS supplementation was made using a commercial MOS product (Techno-MOS; Biochem Co., Lohne, Germany). The Haugh unit (HU) was calculated using the formula: HU = 100 log (H + 7.57 − 1.7 W0.37 ), where H is the mean height (mm) of the albumen, and W is the weight (g) of egg. Yolk index was measured by dividing the height of the yolk by its diameter. Yolk color was visually scored using a Roche yolk color fan. a,b Means with no common superscripts within each row are significantly (P < 0.05) different.

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JAHANIAN AND ASHNAGAR Table 4. Effect of supplemental mannan-oligosaccharides (MOS) on serum biochemical parameters (mg/dL) and antibody responses (log2 ) against different antigens in laying hens1 MOS Level (% of Diet) 0 Serum Metabolites Cholesterol Triglycerides HDL LDL Antibody Titers NDV, 6 dpi NDV, 12 dpi SRBC, primary SRBC, secondary

153 1487a 32.0b 88.5a 5.42 6.26 4.61b 5.22

0.05 181 1315a,b 37.6a,b 58.4b 5.93 6.47 5.82a 5.84

0.1 119 1244b,c 45.2a 44.3b,c 6.36 7.69 5.30a,b 6.29

Probability

0.15 104 1130c 42.4a 34.8c 5.24 7.35 5.46a,b 5.75

0.2

SEM

Treatments

Control vs. MOS

110 1211b,c 44.5a 35.8c

36.0 73.7 2.91 6.88

0.287 0.009 0.011 0.001

0.996 0.005 0.002 0.001

0.48 0.88 0.41 0.52

0.330 0.188 0.049 0.774

0.418 0.064 0.036 0.291

5.85 8.20 5.31a,b 5.92

compared with control hens. Although serum concentrations of HDL and LDL were influenced by supplemental MOS, serum cholesterol level was not significantly affected. Of course, the serum cholesterol levels were numerically lower in hens fed the diets containing 0.15 and 0.2% MOS. In agreement with the present finding, Basmacioglu et al. (2005) reported that supplementation of esterified glucomannan to aflatoxinchallenged broiler chicks did not markedly affect plasma cholesterol concentration. On the other hand, Kannan et al. (2005) and Sohail et al. (2010) reported that serum total cholesterol level was significantly lower in broiler chicks fed on MOS-supplemented diets when compared to control birds. The decrease in serum cholesterol level has been attributed to its assimilation by Lactobacillus (Fernades et al., 1987; Zarate et al., 2002), as prebiotics (such as MOS) have been reported to enhance Lactobacillus count (Gilliland et al., 1985) or Lactobacillus’ ability to synthesize bile salt hydrolase, which deconjugates bile salts (making them less absorbable), and since cholesterol is the precursor of bile salts, more cholesterol is taken out of blood circulation.

Immunological Responses The effects of dietary MOS supplementation on antibody responses against NDV and SRBC are shown in Table 4. As presented, dietary treatments had no considerable impact on antibody responses to NDV at d 6 and 12 postvaccination. Of course, contrast comparisons showed that supplemental MOS tended (P = 0.064) to increase NDV antibody titer at d 12 post vaccine inoculation. In contrast to NDV, primary antibody response against SRBC was increased (P < 0.05) by dietary supplementation of 0.05% MOS. During the secondary response, however, supplemental MOS did not affect SRBC antibody titer. Consistent to the present findings, Sadeghi et al. (2013) reported that dietary supple-

mentation of a MOS and β -glucan-based prebiotic had no marked effect on antibody response against NDV in Salmonella enteritidis-challenged broiler chicks. In contrast, G´ omez–Verduzco et al. (2009) reported that dietary supplementation of 0.05% yeast cell wall increased local mucosal immunoglobulin-A secretion and humoral and cell-mediated immune responses, and reduced parasite excretion in feces of Eimeria-challenged broiler chicks. Oliveira et al. (2009) showed improved antibody responses against NDV and infectious bursal disease virus as the result of dietary MOS supplementation. It seems that supplemental MOS could effectively suppress enteric pathogens, whereby promote immune system and its responses, and improve the integrity of intestinal mucosa, as proposed by Spring et al. (2000).

Ileal Nutrient Digestibility As noted in Table 5, inclusion of MOS into the diet caused 5.1 and 7.6% increases in digestibility coefficients of DM (P < 0.05) and CP (P < 0.01), respectively, when compared to control hens. In addition, there was a significant (P < 0.01) difference between the experimental groups for ileal digestibility of ether extract, with the highest digestibility values observed for the hens fed the diet containing 0.05% MOS. Dietary MOS supplementation resulted in a nearly significant (P = 0.0853) increase in ash digestibility with the highest value observed for hens fed 0.10% MOSdiets. Consistent with this, Meng et al. (2010) reported that supplementation of chito-oligosaccharide to laying hens’ diet improved DM and nitrogen digestibility. In addition, Sonmez and Eren (1999) stated that improved weight gain and feed efficiency as a result of supplemental prebiotic products is partly due to improved nutrient utilization across the gastrointestinal tract. The improvements in ileal digestibility by supplemental MOS can be attributed to the improvements of morphological indices of intestinal epithelium, as indicated by Baurhoo et al. (2007), who reported that

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1 Dietary MOS supplementation was made using a commercial MOS product (Techno-MOS; Biochem Co., Lohne, Germany). HDL = High density lipoproteins; LDL = Low-density lipoproteins; NDV = Newcastle disease virus; dpi = Days postvaccine inoculation; and SRBC = Sheep red blood cell. SRBC titers refer to antibody titers during primary and secondary responses after boosted SRBC inoculation. a–c Means with no common superscripts within each row are significantly (P < 0.05) different.

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MANNAN-OLIGOSACCHARIDES AND E. COLI CHALLENGE IN HENS Table 5. Effect of supplemental mannan-oligosaccharides (MOS) on ileal nutrient digestibility (%) and bacterial counts (log10 cfu/g fresh digesta) in laying hens1 MOS Level (% of Diet) 0 Ileal Digestibility DM Organic matter CP Ether extract Total ash Ileal Microflora Escherichia coli Salmonella spp. Lactobacillus spp. Total bacteria

64.6b 70.4 59.9c 63.5b,c 59.0 5.23a,b 6.12a 3.57c 6.87a

Probability

0.05

0.1

0.15

0.2

SEM

Treatments

Control vs. MOS

66.9b 71.6 62.1b,c 67.6a 59.4

67.4b 75.3 65.7a 66.1a,b 65.3

70.7a 75.3 63.5a,b 66.3a,b 64.1

68.2a,b 70.1 64.4a 61.6c 63.9

1.43 2.13 1.08 1.37 2.65

0.021 0.086 0.002 0.008 0.072

0.025 0.169 0.003 0.589 0.085

0.24 0.75 0.30 0.53

0.036 0.008 0.028 0.031

0.375 0.001 0.004 0.406

5.39a 4.35b 4.05b,c 6.93a

4.32c 3.76b,c 4.93a 5.85b

4.82b 3.21c 4.26b 6.61a,b

5.46a 3.49b,c 4.69a,b 6.89a

1 Dietary MOS supplementation was made using a commercial MOS product (Techno-MOS; Biochem Co., Lohne, Germany). a–c Means with no common superscripts within each row are significantly (P < 0.05) different.

Intestinal Bacterial Colonization There were marked differences (P < 0.05) between dietary treatments for all microbial subpopulations enumerated in this study, with the highest changes observed for Salmonella count. The single-degree-offreedom contrasts, however, showed that dietary MOS supplementation had no effect on E. coli and total bacteria enumerations when compared to control group. The finding of interest was that supplemental MOS at the levels of 0.1 to 0.2% resulted in a marked increase in Lactobacillus count compared with control diet. The colonization of bacteria on mucosal tissues is recognized as an important and prerequisite step in the infections. To colonize the mucosal surface, bacteria must first bind to the epithelial cells. One of the main mechanisms for binding to the epithelial cells is through attachment of Type-1 fimbriae (Ofek et al., 1977). In this regard, it has been indicated that MOS and maybe other oligosaccharides serve as the attachment sites for Gram-negative pathogens, whereby preventing attachment of bacteria onto the enterocytes, subsequently avoiding enteric infection (Gibbons and Van Houte, 1975). Consistent with the present findings, Baurhoo et al. (2007) reported that dietary MOS supplementation caused increases in populations of Lactobacillus and Bifidobacteria in the cecal content of broiler chicks. Moreover, these researchers showed that cecal E. coli enumeration was reduced by supplemental MOS (Baurhoo et al., 2007). On the other hand, Spring et al. (2000) observed that MOS did not significantly change the populations of cecal Lactobacillus and coliforms, although coliforms were numerically lower in MOS groups. Of course, the prevalence of Salmonella in ceca was reduced by dietary MOS supplementation (Spring et al. 2000).

CONCLUSIONS The present findings indicated that supplemental MOS at the levels of 0.1 and 0.15% improved productive performance and feed conversion efficiency in E. colichallenged laying hens. In addition, supplemental MOS (especially 0.15 and 0.2% of diet) revealed a potent impact on blood lipid metabolites, whereby decreased serum concentrations of LDL and triglycerides, and increased serum HDL level. In addition, dietary MOS reduced ileal Salmonella and increased Lactobacilli in hens subjected to E. coli challenge. One could achieve the beneficial impacts of MOS by supplementation of 0.1 to 0.15% MOS into the diet.

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