Influence of dietary mushroom Agaricus bisporus on intestinal morphology and microflora composition in broiler chickens

Research in Veterinary Science 89 (2010) 78–84

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Influence of dietary mushroom Agaricus bisporus on intestinal morphology and microflora composition in broiler chickens I. Giannenas a,*, D. Tontis b, E. Tsalie b, E.F. Chronis c, D. Doukas b, I. Kyriazakis a a

Laboratory of Animal Nutrition and Husbandry, Veterinary Faculty, University of Thessaly, 43100 Karditsa, Greece Laboratory of Pathology, Veterinary Faculty, University of Thessaly, 43100 Karditsa, Greece c 3rd Military Veterinary Hospital, Thessaloniki, 57 001 Thermi, Greece b

a r t i c l e

i n f o

Article history: Accepted 2 February 2010

Keywords: Mushroom Broiler chickens Agaricus bisporus Intestinal morphology Intestinal bacteria populations Lactobacilli spp.

a b s t r a c t In this study, we evaluated the intestinal morphology and bacteria populations in broiler chickens fed for six weeks diets that contained different amount of the mushroom Agaricus bisporus. Ninety day-old female chicks were randomly divided into three dietary treatments, each with three replicates kept in floor pens and fed a basal diet supplemented with the dried mushroom at levels of 0, 10 or 20 g/kg fresh feed. Feed and water were offered to birds ad libitum. The morphological examinations of the intestine were carried out on 1-cm long excised segments from duodenum, jejunum and ileum. The populations of total aerobes, total anaerobes, Lactobacilli spp., Bifidobacteria spp., Escherichia coli, Bacteroides spp. and Enterococci were enumerated in ileum and caecum by conventional microbiological techniques using selective agar media. The results of the study showed that dietary mushroom supplementation did not significantly affect intestinal morphology at either level of inclusion. Morphometrical parameters of depth of duodenum, jejunum and ileum crypt and height of villi revealed no differences amongst dietary treatments. In the ileum, Lactobacilli spp. were higher in birds supplemented at the level of 20 g/kg compared to controls; however, other measurements of bacteria loads were similar amongst the three dietary treatments. In the caecum, Lactobacilli spp. and Bifidobacteria spp. loads were higher in birds supplemented at either level of inclusion compared to control birds, although these did not differ between the two levels of supplementation. In conclusion, dietary mushroom supplementation may beneficially affect intestinal health of broiler chickens. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Over the last decade, the importance of gastrointestinal tract health in broiler chicken has been increasingly recognised due to its contribution to their overall health and performance (Mountzouris et al., 2007; Rehman et al., 2007a,b). The use of antibiotics at subtherapeutic levels has been a cornerstone of the poultry industry for the control of subclinical diseases, maintenance of gut health and growth promotion for a number of decades. As new antibiotic-resistant strains of pathogens emerge, the routine use of antibiotics in animal feed has become less common. In Europe, subtherapeutic use of antibiotics in poultry rearing has been phased out since 2006 (European Commission Regulations, 1998). In accordance to these restrictions, alternative means for preventing bacterial infections common to poultry and enhancing growth performance are required.

* Corresponding author. Tel.: +30 2441066089; fax: +30 2441066041. E-mail address: [email protected] (I. Giannenas). 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.02.003

Natural medicinal products originating from fungi or herbs have been used as feed supplements for centuries in ethnoveterinary medicine (Guo, 2003). A current estimation of the number of plants and fungi with antibacterial and immunoactive substances ranges from 200 to 300. Currently, mushrooms are considered to have both growth promoting and immuno-stimulating properties. It has been hypothesized that some of the properties of plants or fungi are carried through a prebiotic effect (Verstegen and Schaafsma, 1999; Cummings and MacFarlane, 2002) due to their polysaccharide content (Guo et al., 2003a,b). The immunoactive components of mushrooms may include polysaccharides, glycosides, alkaloids, volatile oils, and organic acids (Yang and Feng, 1998; Willis et al., 2007). Poly- and oligo-saccharides from mushrooms have been used as immune enhancers and have been shown to possess antibacterial, antiviral and antiparasitic activities in chicken (Guo et al., 2003a). Recent studies have shown that dietary extracts from Shiitake and Maitake mushroom spp. can be used in broiler chicken as alternatives to antibiotic growth promoters (Guo et al., 2004a), whilst they exert immunomodulating activity against Eimeria and Myco-

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plasma spp. in experimentally infected chickens (Guo et al., 2004b,c). The bacterial populations in the gut of birds can be manipulated by altering the diet composition (Smits et al., 1998). For this reason, the effects of dietary digestible carbohydrates on bacterial populations have been studied extensively (Vahjen et al., 1998; Yang et al., 2009). However, the role of fermentable carbohydrates in relation to avian gut microbial ecology is markedly unknown (Knarreborg et al., 2002). Although, dietary mannan-oligosaccharides are known to reduce Salmonella enteritidis colonization in chicken caecal microflora (Spring et al., 2000; Fernandez et al., 2002), as well as, to inhibit enteropathogenic Escherichia coli from attaching to the gut mucosa of rats at high levels of supplementation (Peuranen et al., 2004), currently, the effects of mushrooms on intestinal bacteria population and gut morphology in chicken, are largely unknown. Gut microflora can impair both growth performance and health of chickens in many ways. The ecology of the caecal microflora has received considerable attention (Mead, 1989; Apajalahti et al., 2001; Gong et al., 2002), as it is the heaviest populated site in the gastrointestinal tract. On the other hand, the microbial ecology of the small intestine has been less well studied (Barnes et al., 1972; Salanitro et al., 1978), although the jejunum and ileum are the principal sites of nutrient absorption (Noy and Sklan, 2001; Choct, 2009). The aim of this study was to introduce a natural product such as Agaricus bisporus mushroom into chicken diets and examine its consumption effects on intestinal morphology and microflora composition in the digestive tract. A. bisporus is a commonly cultivated mushroom in temperate climates and differs in its composition from oriental mushrooms previously included in chicken diets. Dietary supplementation with this mushroom was previously shown to have growth promoting and antioxidant activity on chicken (Giannenas et al., 2010). 2. Material and methods 2.1. Animals ethics The trial protocol was approved by the Institutional Committee for Animal Use and Ethics of The Veterinary Faculty of the University of Thessaly. Throughout the trial, the chickens were handled according to the principles for the care of animals in experimentation (National Research Council, 1985). 2.2. Animals and diets Ninety, one-day-old female broiler chicks were randomly allocated into one of three experimental treatments. Each treatment consisted of three replicates of 10 birds each. Each replicate was housed in separate stainless floor pens under controlled temperature and light conditions. Each pen was 100  100 cm (1 m2 per 10 birds). All birds were reared in the floor pens using wood shavings as litter at a commercial poultry farm (Kotopoula Barbagianni), Giannitsa, Greece (latitude 40.77°, longitude 22.45°). The lighting programme was set at 40–60 W/20 m2 during the first 2 weeks and 15 W/20 m2 thereafter, with 23 h light per day. The temperature was set at 36 °C during the first day, 34 °C during the first week and was gradually reduced by 3 °C per week to reach a minimum 22 °C at 28 days of age. Relative humidity was between 65% and 75%. The experiment lasted for 42 days. To meet the nutrient requirements of the broiler chickens over this period, a complete basal diet was formulated for each of the three stages of growth: starter, grower and finisher. The feeds were based on corn–soybean meal, were formulated to meet NRC recommendations

(NRC, 1994) and contained no antibacterial or anticoccidial supplements. Table 1 presents the ingredients and the composition of the basal diets that were in mash form. Proximate analysis showed no major deviation from calculated values. The birds within the control group (CON) were given the basal diet for the respective growth stage. The other two groups were given experimental diets based on the basal diets, but contained an additional of 10 g (M10) or 20 g (M20)/kg ground, dried A. bisporus mushroom. Access to feed and water was ad libitum. Feeds were prepared every second day. Chickens were euthanized by cervical dislocation at the end of the trial at the age of 42 days and six birds per replicate were randomly selected for sampling in order to take morphometrical and bacteriological measurements. 2.3. Mushroom preparation and analysis Mushrooms were obtained from a local mushroom producer (Ippotur, Lazarina, S.A., Lazarina Trikala, Greece). The intact mushrooms were dried overnight at 60 °C and ground through a 5 mm sieve before being incorporated into the feed. For chemical analysis, mushrooms were freeze dried at 76 °C and 0.023 mbar vacuum for 30 h by Telstar Cryodos (Telstar, Barcelona, Spain). Dried mushrooms were milled through a 1 mm sieve (Polymix – Kinematica PX-MFC90D, Littau, Switzerland) prior to analysis for protein, fat, fibre, and ash according to the procedures described in AOAC International (1995). Total protein content was determined by Kjeldahl, fat Table 1 Composition of basal diets on fresh basis (g/kg). Ingredients Corn grains Soybean meal Soybean oil Vegetable fat Fish meal Gluten meal Limestone Monocalcium phosphate

Starter diet (1– 14 days)

Grower diet (15–28 days)

Finisher diet (29–42 days)

525.1 340.0 38.0 0.0 35.0 25.0 10.1 14.1

542.5 340.0 35.0 12.5 25.0 12.5 9.8 12.7

571.6 320.0 35.0 25.0 15.0 0.0 11.4 14.2

L-Lysine

3.5

2.3

0.5

DL-Methionine

2.5

2.2

2.2

L-Threonine

0.5

0.4

0.0

Sodium chloride Sodium bicarbonate Vitamin premixa Trace-mineral premixb Natuphos phytase

1.9 3.2 0.5 0.5

2.0 2.0 0.5 0.5

2.1 1.9 0.5 0.5

0.1

0.1

0.1

891.3 230.0

892.2 221.2

890.3 205.1

54.4 32.1 54.3

71.1 35.1 55.2

87.7 34.7 55.3

9.3 7.0 14.0 11.2 3060.0

9.1 7.0 13.0 10.1 3180.0

9.0 6.7 11.5 9.5 3200.0

Chemical analysisc Dry matter Crude protein (N  6.25) Crude fat Crude fibre Ash Calculated analysis Ca P (total) Lysine Methionine + cystine Metabolizable energy (kcal/kg)

a Supplying per kg feed: vitamin A 12,000 IU, vitamin D3 5000 IU, vitamin E 30 mg, menadione 3 mg, thiamine 1 mg, riboflavin 8 mg, pyridoxine 3 mg, vitamin B12 0.02 mg, niacin 20 mg, pantothenic acid 20 mg, folic acid 2 mg, biotin 0.2 mg, vitamin C 10 mg, and choline chloride 480 mg. b Supplying per kg feed: zinc 100 mg, manganese 120 mg, iron 40 mg, copper 20 mg, cobalt 0.2 mg, iodine 1 mg, and selenium 0.3 mg. c According to AOAC International (1995) methods.

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content was extracted from the samples with petroleum ether in a Soxhlet apparatus, crude fibre content was analysed in Dosi Fibre (Selecta) apparatus and ash by incinerating dried samples at 600 °C for about 6 h in a furnace (Selecta, Barcelona, Spain) (AOAC International, 1995). Starch and glucose- and fructose-monosaccharide contents were determined by Megazyme kits (Megazyme Int., Ireland Ltd.). Water soluble polysaccharides in mushroom extracts were determined according to Guo et al. (2004a). The freeze–dried mushroom material was ground and extracted with hot water, and protein was partially removed with an equal volume of 10% tri-chloroacetic acid. Finally, three volumes of 96% ethanol were used to extract the polysaccharide precipitate. The yield of the polysaccharide fraction was collected and total sugars were determined according to the method of Dubois et al. (1956). Total phenolic (TP) content in mushrooms was measured using Folin–Ciocalteau reagent (Merck, Germany). Ethanol extracts were used to analyse their phenolic compounds following a modification of the method of Fu et al. (2002). Five gram of the freeze–dried mushroom powder was added to 60 ml of 80% ethanol and heated to 60 °C for one hour using a water bath. The sample was filtered after one hour and the procedure was repeated two additional times. After a total of three hours, the extract was filtered, combined with the previous extracts and diluted with 80% ethanol to a final volume of 200 ml, which was then vortexed for 30 s. One milliliter of the ethanol extract was added to 4 ml of the Folin–Ciocalteau reagent, which was diluted with distilled water (1:10). After 3 min, 5 ml of a 7.5% aqueous sodium carbonate solution was added and after further 30 min, absorbance was measured by an UV–Vis spectrophotometer at a wavelength of 765 nm. Gallic acid was used as the standard in order to create a calibration curve by plotting absorbance against concentration. TP content was standardized against gallic acid and the data was expressed as mg of gallic acid equivalents (GAE) per gram of dry weight (mg GAE/g dw). The linearity range for this assay was determined as 0.025–0.3 mg/ml GAE (R2 = 0.9964), yielding an average absorbance range of 0.15– 2.7 AU. Each sample was extracted in triplicate. The spectrophotometer used was a Hitachi U-1900 model, tissue homogenizer was Ultraturrax IKAÒ T18 basic and the centrifuge was a Centurion model by Scientific Ltd. Company. The enzyme kits for starch and sugar monosaccharide determination, containing alpha amylase, beta glucosidase, glucose and starch standards were purchased from Megazyme Int. (Ireland Ltd.). The freeze-dryer apparatus was by Telstar Cryodos (Telstar, Barcelona, Spain).

for 10 villi per section. Crypt depth (CD) (the vertical distance from the villous–crypt junction to the lower limit of the crypt) was estimated for 10 corresponding crypts per section. 2.5. Enumeration of bacteria populations in ileum and caecum Intestinal samples were collected and fresh digesta samples from ileum and caecum were taken for bacterial analyses within an hour from collection. Digesta samples were serially diluted in 0.85% sterile saline solution for enumeration of total aerobes, total anaerobes, Lactobacilli spp., Bifidobacteria spp., E. coli, Bacteroides spp. and Enterococci by conventional microbiological techniques using selective agar media. All microbiological analyses were performed in duplicate and the average values were used for statistical analysis. In particular, Lactobacilli spp. were anaerobically assayed using MRS agar (Fluka 80961). In cases of doubt confirmation of Lactobacilli spp. was performed by using API 50 CH kit (BiomerieuxÒ SA, Marcy-l’Etoile/France). Bifidobacteria spp. were anaerobically assayed using Reinforced Clostridial Agar (RCA) (Fluka 27546) according to Rybka and Kailasapathy (1996) and RCA medium supplemented with Prussian blue dye (Fluka 27546 03899) according to Moriya et al. (2006). The media is selective for Bifidobacteria which form white colonies, while Lactobacilli spp. and Streptococci form colonies with a blue halo and white centre. For the confirmation, colonies were identified as members of the genus Bifidobacterium by the following criteria: they were gram positive, pleomorphic rods with characteristic bifurcated Bifidobacterium cell morphology; they were unable to grow under aerobic conditions; they were catalase negative; and they showed fructose-6-phosphate phosphoketolase (EC 4.1.2.22) activity as described by Scardovi (1986). Aerobes were enumerated using Plate Count MUG Agar (Fluka 80961). Anaerobes were enumerated by using Reinforced Clostridial Medium (Fluka 27546) and (Tryptone Sulphite Agar without cyclocerine). Bacteroides spp. were enumerated using Bacteroides Bile Esculin Agar (the composition was concordant with the Difco Manual) (Bioprepare S.A., Athens). E. coli were enumerated through the use of Plate Count MUG Agar (Fluka 80961) and TBX Agar (Fluka 92435). Enterococci were enumerated using Slanetz and Bartley Agar (Fluka 45183). Results were expressed as base-10 logarithm colony-forming units per gram of ileal or caecal digesta.

2.4. Histology and morphometric analysis of the small intestine

2.6. Statistical procedures

During necropsy of the selected birds, the gastrointestinal tract was removed and the small intestine was divided into three parts: duodenum (from the gizzard outlet to the end of the pancreatic loop), jejunum (from the pancreatic loop to Meckel’s diverticulum) and ileum (from Meckel’s diverticulum to the ileo-caeco-colic junction). Segments one centimeter long were taken from the central of each part and fixed in 10% buffered formalin for morphometrical assays under light microscopy. Formalin-fixed intestinal tissues were processed, embedded in paraffin wax, sectioned at 3 lm and stained by the haematoxylin and eosin methods. Histological sections were examined with a Nikon phase contrast microscope coupled with a Microcomp integrated digital imaging analysis system (Nikon Eclipse 80i, Nikon Co., Tokyo, Japan). Images were viewed (4) to measure morphometric parameters of intestinal architecture. For that purpose, three favourably orientated sections cut perpendicularly from villus enterocytes to the muscularis mucosa were selected from each animal and measurements were carried as follows. Villous height (VH) was estimated by measuring the vertical distance from the villous tip to villous–crypt junction level

All data concerning intestinal villous height, crypt depth and bacterial count numbers were analysed by ANOVA appropriate for randomized complete block designs in the General Linear Model of the SPSS statistical package (SPSS 12.00, SPSS Ltd., Woking, Surrey, UK). The replicates (10 birds) were considered to be the experimental unit for the analyses, with each replicate being used as a block. The homogeneity of the variances, including variances of bacterial species ratios, was tested by Levene’s test. As bacterial numbers were not normally distributed, they were log transformed to create a normal distribution prior to analysis. Means are presented on transformed basis (Wellock et al., 2008). The Tukey’s test was carried out to assess any significant differences at a probability level of P < 0.05 amongst the experimental treatments. 3. Results 3.1. Mushroom polysaccharides, phenolics and constituents The fresh mushrooms contained 89.7% moisture, and protein 29.7%, ether extract 2.1%, ash 8.4%, nitrogen-free extract 42.4%

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and starch 6.7% as percentages of the dry matter. Phenolic content was found to be 8.85 mg of Gallic acid equivalents per gram expressed as dry weight. Yield of polysaccharide extract expressed on the basis of the dry matter of the intact mushroom was found to be 155 g/kg. The dry matter content of the polysaccharide extract was determined by freeze drying and found to be 962 g/kg. Total sugar content of the extract was determined using the phenol–sulphuric method and found to be 643 g/kg of the polysaccharide extract. The physicochemical properties of the mushroom composition, with emphasis on their phenolic content and polysaccharide fractions could be the basis for these results. The chemical composition of A. bisporus mushroom was in accordance to the values reported in literature (Vetter, 1993; Manzi et al., 2001; Guo et al., 2004a). 3.2. Morphometric analysis of the gut Animals of all groups had a normal intestinal structure; the highest villi were in duodenum followed by lower villi in jejunum

Fig. 1. Photomicrograph of cross section of ileum full thickness from M20 animal (a: indicating villous height and b: indicating crypt depth as measured) H–E, 4 (bar = 100 lm).

Table 2 Effect of dietary mushroom supplementation on small intestine morphology of female broiler chicken at 42 days of agea. b

CON

a

M10

M20

Pooled SEMc

P value

Duodenum Villous height (lm) Crypt depth (lm) Villous height to crypt depth ratio

1746.7 179.7 9.72

1851.2 183.1 10.11

1830.6 172.4 10.61

59.9 8.7 0.5

0.161 0.971 0.736

Jejunum Villous height (lm) Crypt depth (lm) Villous height to crypt depth ratio

1345.5 143.6 9.36

1415.6 152.6 9.27

1458.9 156.9 9.29

36.8 4.4 0.4

0.167 0.780 0.661

Ileum Villous height (lm) Crypt depth (lm) Villous height to crypt depth ratio

824.9 116.8 7.06

781.4 107.9 7.24

792.7 114.5 6.92

30.3 9.4 0.2

0.760 0.507 0.308

Results are given as means of groups (n = 3 = subgroups). CON, M10, M20 represent groups of broiler chicken fed basal diet supplemented with ground dried Agaricus bisporus mushroom at the level of 0, 10 or 20 g/kg of feed. c SEM = standard error of the mean. b

and the lowest being in ileum. The intestinal villi were slim and finger-shaped and the intestinal mucosa revealed no histopathological changes in animals from all groups (CON, M10, and M20) (Fig. 1). Dietary supplementation with mushrooms did not affect the duodenum, jejunum or ileum villous height or crypt depth (Table 2) in comparison to the untreated control group.

3.3. Results enumeration of intestinal microflora composition The composition of ileal microflora of broiler chickens at the end of the experiment is shown in Table 3. The Lactobacilli spp. population was significantly higher in the broilers supplemented with mushrooms at the level of 20 g/kg (M20: 6.24 log10/g) compared to the broilers fed the control diet (CON: 5.55 log10/g of wet ileal digesta) or the M10 group. However, total aerobes, total anaerobes, Clostridia spp., Bifidobacteria spp., E. coli, Bacteroides spp. and Enterococci loads were similar amongst dietary treatments. The ratio of E. coli populations to Lactobacilli spp. was significantly lower in birds supplemented with mushrooms at the level of 20 g/kg compared to control (M20: 0.63 ± 0.04 vs. CON: 0.73 ± 0.04, P = 0.029). M10 group had Lactobacilli spp. counts that did not differ either from the M20 or the control group (M10: 5.68 log10/g). On average, total aerobes, total anaerobes, Clostridia spp., Bifidobacteria spp., E. coli, Bacteroides spp. and Enterococci numbers were unaffected by the dietary source. In caecum, the microflora composition at the end of the experiment is shown in Table 3. The Lactobacilli spp. populations were significantly higher in the mushroom-supplemented broilers at both levels 10 and 20 g/kg compared to the broilers fed the control diet up to 1.3 log10/g of wet caecal digesta (M20: 8.05; M10: 7.82 and CON: 6.45 log10/g). The Bifidobacteria populations profile was similar to that of Lactobacilli spp. (M20: 7.77; M10: 7.58 and CON: 6.28 log10/g). M10 group had Lactobacilli spp. and Bifidobacteria spp. counts that did not differ from M20 group. Total aerobes, total anaerobes, Clostridia spp., E. coli, Bacteroides spp. and Enterococci loads were similar amongst dietary treatments. The ratio of E. coli populations to Lactobacilli spp. was lower in mushroom-sup-

Table 3 Effect of dietary mushroom supplementation on ileum and caecum bacteria populations of female broiler chicken at 42 days of ageA.

a,b

CONB

M10

M20

Pooled SEMC

P value

Ileum Total aerobes Total anaerobes Bacteroides spp. E. coli Enterococci Bifidobacteria spp. Lactobacilli spp.

6.27 6.19 6.39 4.04 6.05 5.92 5.55b

6.71 6.24 6.47 4.16 6.06 6.14 5.68b

6.12 6.08 6.29 3.93 6.00 6.26 6.24a

0.122 0.072 0.068 0.063 0.041 0.088 0.123

0.213 0.925 0.844 0.175 0.916 0.313 0.005

Caecum Total aerobes Total anaerobes Bacteroides spp. E. coli Enterococci Bifidobacteria spp. Lactobacilli spp.

8.27 6.69 6.69 7.23 6.94 6.28b 6.45b

8.54 7.14 7.46 7.66 7.05 7.58a 7.82a

8.21 7.47 7.39 7.04 7.00 7.77a 8.05a

0.107 0.147 0.132 0.110 0.053 0.245 0.250

0.507 0.129 0.055 0.066 0.079 0.005 0.005

Values in the same row with a different superscript differ significantly at P 6 0.05. A Results are given as means of groups (n = 3 = subgroups). B CON, M10, and M20 represent groups of broiler chicken fed basal diet supplemented with ground dried Agaricus bisporus mushroom at the level of 0, 10 or 20 g/ kg of feed. C SEM = standard error of the mean.

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plemented birds compared to control (M20: 0.87 ± 0.08 and M10: 0.98 ± 0.06 vs. CON: 1.12 ± 0.05, P = 0.036).

4. Discussion The present study was designed to evaluate the effects of sustained consumption of a natural product such as A. bisporus mushroom, with the view of its potential inclusion in chicken diets as an alternative to infeed antimicrobials. Previous studies with Lentinus edodes and Tremella fuciformis mushrooms had suggested a putative beneficial effect on broiler chicken performance and in particular an immune enhancing effects on Eimeria challenged chickens (Guo, 2003; Guo et al., 2004b). These studies used oriental mushrooms that are not common in many parts of the world and/or are not grown on commercial scale. In our experiment, we used A. bisporus mushroom which is the most common cultivated mushroom species worldwide, including temperate climates (Chang and Buswell, 1996). In addition, the amount of mushrooms added to the basal diet corresponded to a relatively low dietary inclusion level 10 or 20 g/kg and was consumed as a part of the usual feeding regime of broiler chicken. The question addressed in this paper was whether the intake of mushrooms and their constituents could beneficially affect the host, by selectively stimulating the growth or the activity or both of a number of bacterial species in the small intestine and caecum, thus improving host gut health through maintaining gut’s epithelial integrity. In a previous study conducted to investigate the effects of dietary supplementation of A. bisporus mushroom on the growth performance of broilers, we found that supplementation had growth promoting and carcass antioxidant effects on broilers of the same age range (Giannenas et al., 2010). Mushroom-supplemented broilers at the level of 20 g/kg had significantly higher body weights (2205 g) than broilers fed the unsupplemented diet (2011 g, P = 0.048). Lipid antioxidative stability, as well as glutathionebased enzyme activity, especially at the supplementation level of 20 g mushroom per kg, was also significantly improved (P < 0.050). Given these effects on growth and oxidative status we aimed to further investigate the effects of dietary supplemented mushrooms on intestinal microflora and integrity. In the literature, very limited information exists on the effects of mushroom and their extracts on poultry gut health. In the present study, we found that dietary supplementation with Agaricus mushroom at the level of 20 g mushroom per kg of feed increased Lactobacilli spp. loads in the ileum, both Lactobacilli spp. and Bifidobacteria spp. loads in the caecum and affected the ratios of Lactobacilli spp. to E. coli compared to control diet. The microflora composition present in birds fed mushroom-supplemented diets are similar to those usually common in healthy younger birds of two-week-old in duodenum, jejunum and ileum content (Ochi et al., 1964; Smith, 1965; Barnes et al., 1980; Rehman et al., 2006). Increased numbers of Lactobacilli spp. and Bifidobacteria spp. as well as reduced numbers of coliforms suggest a more favourable intestinal microflora that may improve gastrointestinal function, feed digestibility and animal performance (Rehman et al., 2007a,b, 2008). It is possible that mushroom fermentable polysaccharides favoured Lactobacilli spp. and Bifidobacteria spp. populations. However, there was no significant difference between the two groups supplemented with 10 or 20 g/kg mushrooms and the control group for any other examined bacteria species. Other researchers have reported that Lactobacilli spp. and Bifidobacteria spp. increased in poultry intestinal content when birds were fed specific prebiotics or oligosaccharides. Patterson et al. (1997) found that dietary administration of thermally produced saccharides increased the number of Bifidobacteria spp. and Lactobacilli spp. in

the chicken caecum. Orban et al. (1997) found a significant increase in the number of Bifidobacteria spp. in the caecum of broilers fed a diet supplemented with sucrose thermal oligosaccharides in a 4-week trial. Rada et al. (2001) found a highly significant 1-log increase in Bifidobacteria spp. when an inulin-supplemented diet was fed to 1-week-old laying hens. In the current experiment, mushroom also reduced ileal luminal E. coli counts, although this difference was not statistically significant. Jamroz et al. (2003) has reported a significant reduction of coliforms in the ileal digesta when mannan-oligosaccharides were fed to broiler chickens, while Yang et al. (2007a,b) found no difference on coliform numbers with mannan-oligosaccharide supplementation. Differences in basal diets when birds are fed diets containing different ingredients (Apajalahti et al., 2004) and/or rearing environments when birds are raised in cages instead of deep litter (Willis et al., 2002) could explain inconsistent results on intestinal microbiota. Willis et al. (2007) found that chickens given Shiitake mushroom extract presented improved performance, promoted Bifidobacteria spp. growth and possible health-enhanced attributes. Gut microflora has significant effects on host nutrition, health, and, growth performance (Barrow, 1992) by interacting with utilization and the development of gut system of the host. This interaction is very complex and, depending on the composition and activity of the gut microflora, it can have either positive or negative effects on the health and growth of birds. For example, when pathogens attach to the mucosa, gut integrity and function are severely affected (Droleskey et al., 1994) and immune system is challenged (Neish, 2002). Chickens grown in a pathogen-free environment grow 15% faster than those grown under conventional conditions where they are exposed to bacteria and viruses (Klasing et al., 1987). Furthermore, it is generally accepted that gut microflora is a nutritional ‘‘burden” in fast-growing broiler chickens (Dibner and Richards, 2005; Lan et al., 2005) since an active microflora component may have an increased energy requirement for maintenance and a reduced efficiency of nutrient utilization (Yang et al., 2009). Feed additives such as prebiotics or probiotics can modulate the gut microflora and performance of broiler chickens. In a recent study, Yang et al. (2008) demonstrated considerable changes in the ileal populations of Lactobacilli spp. and coliforms in broilers fed on diets containing mannan-oligasaccharides or zinc-bacitracin. In addition, bird performance was enhanced and energy utilization improved. Dawson (2001) suggested that an ideal flora should promote the absorption of nutrients during the digestive process, whilst also ensuring that the host is capable of mounting an effective immune response in the event of pathogenic challenge. However, defining what an ‘ideal microflora’ means in terms of their interactions with each other as well as with the host to yield favourable health and nutritional outcomes remains a challenge. This is because even with the advent of molecular techniques making it possible to identify up to 640 different species of bacteria in poultry gut, 90% of these species are previously unidentified organisms and their role and functions are totally uncharacterised (Apajalahti et al., 2004). In our study, mucosal architecture in terms of villus height and crypt depth was not influenced by mushroom consumption, despite differences in microbial populations. The structure of the intestinal mucosa can reveal some information on gut health. Stressors that are present in the digesta can lead relatively quickly to changes in the intestinal mucosa, due to the close proximity of the mucosal surface and the intestinal content. Changes in intestinal morphology, such as shorter villi and deeper crypts have been associated with the presence of toxins (Yason et al., 1987) or higher tissue turnover (Miles et al., 2006). In the present study, no increase in duodenal, jejunal or ileal villus height and (crypt depth ratio at the jejunum and ileum) in mushroom – fed chickens were

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found compared to control birds. The crypt can be regarded as the villus factory and a large crypt indicates rapid tissue turnover and a high demand for new tissue. Demand for energy and protein for gut maintenance is high compared with other organs. Cook and Bird (1973) reported a shorter villus and a deeper crypt when the counts of pathogenic bacteria increase in the gastrointestinal tract, which result in fewer absorptive and more secretory cells (Schneeman, 1982). In a review, Guo et al. (2004a) have reported that polysaccharides like those from the mushrooms L. edodes and T. fuciformis, can affect both health and growth performance in broiler chickens. Different mushrooms with various bioactive properties have been studied by the scientific community, as alternatives to pharmaceutics (Lindequist et al., 2005). Mushrooms are rich sources of neutraceuticals (Elmastas et al., 2007; Ribeiro et al., 2007) that appear to exert antioxidant (Lo and Cheung, 2005; Barros et al., 2007a,c, 2008), antitumor (Wasser and Weis, 1999), and antimicrobial activities (Smonia et al., 1995; Barros et al., 2007b). In recent in vitro studies, it was found that Agaricus spp. possess antioxidant (Barros et al. 2007a) and antimicrobial activity (Barros et al., 2007b), which are influenced by mushroom conservation treatment or cooking (Barros et al., 2007d) and stage of maturity (Barros et al., 2007e). The differences in sugar composition, molecular weights, chemical structure and other properties of the polysaccharides may have some impact on their bio-activities, medicinal properties, and other effects, which, however, have not been yet fully understood. In our study we used A. bisporus mushroom. The chemical composition of A. bisporus mushroom was in accordance to the values reported in literature (Vetter, 1993; Manzi et al., 2001). Total sugar content of the polysaccharide extracts was similar to the values of Guo et al. (2004a) for other mushroom species and total phenolic content was also in accordance to values reported in literature (Manzi et al., 2001). Oriental mushrooms contain higher levels of polysaccharides and fibre (Guo et al., 2004a), while cultivated edible mushrooms contain higher levels of protein, vitamins and minerals (Vetter, 2003). It should be noted, however, that the relationship between the polysaccharide structure and its bioactive function is not well understood (Guo et al., 2004a). In conclusion, the results presented in this study showed that dietary mushroom supplementation significantly increased the numbers of Lactobacilli spp. in ileum and Lactobacilli spp. and Bifidobacteria spp. in the caecum of chickens. Dietary mushroom supplementation did not affect intestinal morphology and the mechanisms at the intestinal level responsible for the observed effects in the present of mushrooms are largely unknown. Changes in the species present in the ileal or caecal bacterial community after fermentation of mushroom constituents may underlie an effective mechanism. Whether A. bisporus can be used as potential feed additive with prebiotic activity to poultry diets is still unknown. Therefore, future in vitro and in vivo experiments are needed to further investigate the effects of mushroom components, to isolate the active substances and test them as growth and health promoters in great scale experiments with chickens, in normal or pathogen-challenged birds and elucidate the mechanisms as potential health enhancers. Acknowledgements The authors gratefully acknowledge Research Committee of University of Thessaly for partially funding of this work, IppoturLazarina S.A. for donation of Agaricus bisporus mushrooms and Messieurs Nikolaos and Anastasios Tsalkantis for donation of broiler chickens and experimental feeds. The authors also thank Stavros Lalas and Olga Gortzi for the usage of Telstar Cryodos freeze drier equipment.

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