journal of poultry science, 38: 289-301, 2001

Journal of Poultry Science, 38: 289-301, 2001

Morphological Changes of the Intestinal Villi in Chickens Fed the

Dietary Charcoal Powder Including Wood Vinegar Compounds

Mongkol Samanya and Koh-en Yamauchi*

Laboratory of Animal Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa-ken, 761-0795, Japan

To investigate the effects of dietary charcoal powder including wood vinegar compounds (4: 1, CWVC) on the intestinal villus morphology, 130-d-old male Single Comb White Leghorn chickens were randomly allotted into 0, 1, 3 and 5% dietary CWVC groups of each 8 bird. Four rations were formulated by addition of each level of CWVC (CP: 2.5%) to commercial layer finisher mash diet (CP: 14.5%, ME: 2,803 kcal/kg), and fed ad libitum for 28d. During the feeding experimental period, feedintake and body weight gain were measured. After the end of feeding experiments, 4birds were randomly selected per each group, and intestinal villus height, epithelial cell area and cell mitosis in each intestinal segment were compared using a light microscope. Besides, the morphological change of villus tip surface was observed using a scanning electron microscope.

Although the feed intake did not differ among each group, the body weight gain tended to be higher in 1 and 3% dietary CWVC groups than that of 0% group, resulting in the feed conversion ratio being insignificantly improved in these groups.

Values of the intestinal villus height, epithelial cell area and cell mitosis were higher in 1% dietary CWVC group but lower in 5% dietary CWVC group than those of another

groups in each intestinal segment.The comparatively smooth surface of the duodenal villus apex in the 0% dietary

CWVC duodenum changed to the rough surface with a clear cell outline between each epithelial cell due to the conspicuous cell protuberances after feeding 1% dietary CWVC. However, such conspicuous cell protuberances disappeared and cells having no microvilli and deep cells at the sites of recently exfoliated cells were observed after feeding 3% dietary CWVC, and the latter was much increased after feeding 5% dietary CWVC. Fundamentally, the villus apex surface in the jejunum and ileum revealed an almost similar morphological alteration to that in the duodenum except that cells having no microvilli and deeper cells due to recently exfoliated cells were not seen even in 5% dietary CWVC and that the cell protuberances of 1% dietary CWVC became faint with moving caudally.

The present morphological changes of intestinal villi in chickens fed the dietary

CWVC diets demonstrate that the villus function could be activated also in the ileum at 1% level, and that such an activated villus function in all small intestinal segment parts

might improve the feed conversion ratio.

Key words: charcoal with wood vinegar compounds, growth performance, villi, light microscopy, electron microscopy

Received: March 23, 2001 Accepted: May 30, 2001 * Corresponding author; Tel, Fax: 087-891-3053, E-mail: yamauchi@ag. kagawa-u. ac. jp

290 J. Poult. Sci., 38 (4)

Introduction

Chemical medicines such as disinfectant and insecticide, vaccine, and antibiotic

have been frequently used for safeguard against chicken disease in poultry industry.

However, every effort should be made also to produce high quality animal products

without using these medicines and to reduce environmental contamination by efficient

utilization of natural substances. Charcoal is a solid fuel made by dry distillation of

wood, and powder of which is traditionally scattered on the floor in chicken house to

reduce the smell of feces by adsorbing ammonia. A mixed powder of wood vinegar

compounds and amorphous charcoal carbon (CWVC) has been used as an oral antidote

to produce high animal productions. The dietary addition of CWVC to diets induced

a significant increase in hen-day egg production and feed conversion ratio (Sakaida et

al., 1987a) and in broiler hatchability (Sakaida et al., 1987b). Also in our prelimi-

nary feeding experiment, feed intake did not show a difference among 0, 1, 3 and 5%

dietary CWVC diets, but the body weight gain of birds fed 1% dietary CWVC diet was

increased, improving the feed conversion ratio (unpublished).

Although the improved growth performance of chicken fed the dietary CWVC

have been reported, the reason why CWVC induces such a powerful effective stimula-

tion on growth performances has not been determined, but it may be related to their

intestinal functions. The intestinal histological alterations are known to be induced by

the fed diets (Langhout et al., 1999; Yasar and Forbes, 1999), and be intimately

related to intestinal functions (Shamoto et al., 1999; Shamoto and Yamauchi, 2000;

Yamauchi et al., 1996; Yamauchi and Tarachai, 2000). Therefore, it was thus of

great interest to investigate the histological alterations of the intestinal villi in chickens

fed the non-nutritive sorptive CWVC.

In this study, effects of dietary CWVC on feed intake, body weight gain, and feed

conversion ratio were examined in chickens. Then, villus height, cell area and cell

mitosis number in each intestinal segment of these birds were observed using light

microscope. Besides, fine structural alterations of the villus apex surface were com-

pared using scanning electron microscope.

Materials and Methods

Birds and experimental design

Male Single Comb White Leghorn chickens (callus gallus domesticus) (Julia

strain) were placed into individual cages in an environmentally controlled room with a

13-h photoperiod (06.00 to 19.00h) at mean environmental temperature of 22.1•Ž (first

week, around 17.0•Ž; final week, around 21.0•Ž). Table 1 shows the primary ingredi-

ents and chemical composition of commercial finisher mash diet (Nippon Formula Feed

Manufacturing Company, Ltd., Kanagawa, Japan) used in this experiment as a basal

diet. At 130-d-old, 32 birds were randomly divided into 4 groups of each 8 bird as

follows: dietary addition of CWVC (Nekkarich(R) Table 2, Miyazaki Midori Seiyaku

Co., Ltd, Miyazaki, Japan) to basal diets at 0, 1, 3 and 5%. Commercial Nekkarich(R)

Samanya and Yamauchi: Villus Change of Chickens Fed Charcoal with Wood Vinegar Compounds 291

Table 1. Composition of fed diet (air dry basis)

1Including 6% premix; Premix provided the following per kg

of diet: vitamin A, 2.6IU; vitamin D3, 7IU; vitamin E, 2.5mg;

vitamin K3, 3mg; vitamin B,, 2mg; vitamin B2, 1.5mg;

vitamin B6, 2mg; vitamin B12, 0.003mg; biotin, 2mg; folic

acid, 2mg; pantothenate, 1.5mg; niacin, 2mg; choline, 2mg;

iodine, 3mg; manganese, 2 g; ferrous, 4mg; zinc, 2mg;

copper, 2mg.

Table 2. Composition of charcoal powder including wood

vinegar compounds (4:1)

was produced in the company by as follows; wood vinegar solution obtained after

cooling smoke during making charcoal from broad-leaf tree by dry distillation at 300•`

450•Ž was kept for 2•`3 years; then the skimmed solution was distilled to remove the

harmful substances such as tar; this wood vinegar compounds were absorbed into

292 J. Poult. Sci., 38 (4)

amorphous charcoal carbon (1:4). Birds were given ad libitum access to water and

each experimental diet for 28d. Feed intake and body weight gain were measured

every day and weekly, respectively.

Tissue sampling

Four birds randomly selected per each group were killed by decapitation under

light anesthesia with diethyl ether at the end of the experiment. All experimental

treatments were performed according to the humane care guidelines provided by the

Faculty of Agriculture of Kagawa University. Whole small intestine was removed

immediately and put into a mixture of 3% glutaraldehyde and 4% paraformaldehyde

fixative solution in 0.1M cacodylate buffer (pH7.4). The same fixative solution was

also injected into the intestinal lumen. The intestinal part from the ventriculus to

pancreatic and bile ducts was recognized as the duodenum; jejunum from the ducts to Meckel's diverticulum and; ileum from the diverticulum to ileo-cecal-colonic junction.

Middle part of each segment was taken as tissue samples.

Light microscopic examination

A 1-cm length of each intestinal segment for light microscopic observation was

fixed with Bouin's solution, prepared for paraplast embedding, cut at 5-um cross-

section, and stained with hematoxylin-eosin.

All villus heights having the lamina propria were measured from villus tip to the

base excluding the crypt in one transverse section. An average of these values was

expressed as a mean villus height per one section. A total of 8 sections were counted

from one bird, and an average of 8-villus height per each 8 section was also expressed

as a mean villus height for each bird. Finally, these 4 mean villus heights from 4 birds

were expressed as a mean villus height for one group.

A single cell area on a 5-um transverse section was measured at the middle part of the villi. At first, the epithelial cell layer was randomly measured, then the number of

cell nuclei within this measured epithelial cell layer were counted. Finally, the epithe-

lial cell layer area was divided by the number of cell nuclei to obtain an epithelial cell

area. Two cell areas were calculated per one transverse section, and an average of

these two values was expressed as a mean cell area per one section. A total of 8

sections were counted from one bird, and an average of 8 cell areas per each 8 section

was also expressed as a mean cell area for each bird. Finally, these 4 mean cell areas

from 4 birds were expressed as a mean cell area for one group.

As cell mitosis numbers in the proliferative zone (crypt), different stages of

karyokinesis from mitotic cell division were counted as described previously (Tarachai

and Yamauchi, 2000): cytoplasmic division having the definite formation of two

separate daughter cells, mitotic cells having nuclear division showing chromatids

separated in daughter chromatids, mitotic cells having nucleus pulled apart and moved

toward the poles of cells, and mitotic cells having the homogenous, intensely stained

basophilic nuclei. All cell mitoses of the crypt seen in one transverse section was

measured. A total of cell mitosis numbers were counted from 5 different sections for

each bird, and these 5 values were used to calculate a mean cell mitosis for one bird.

Finally, these 4 mean cell mitoses from 4 birds were expressed as a mean cell mitosis in


one group. These values were measured also in each intestinal segment using an image

analyzer (Nikon Cosmozone iS, Nikon Co., Tokyo, Japan).

Statistical analysisThe average of villus height, cell area and cell mitosis number of each bird from

each treatment group were analyzed across all treatment groups by one-way analysis

with a Duncan's multiple range test using Stat View program (Abacus Concepts, Inc.,

HULINKS, Inc., Tokyo, Japan). Differences at P<0.05 were considered significant.

Scanning electron microscopic examination

A 2-cm tissue sample of each intestinal segment lying next to the light microscopic

examination was transversely cut, slit longitudinally and washed with 0.01M phosphate

buffered saline (pH7.4) to remove intestinal contents. Tissue samples were pinned flat

to prevent curling within a mixture of 3% glutaraldehyde and 4% paraformaldehyde in

0.1M cacodylate buffer (pH7.4) at room temperature for 1h. Then, samples were cut

into 4-x 7-mm squares and fixed for 1h more. After rinsing with 0.1M cacodylate

buffer (pH7.4) for 3-4 times, the pieces were postfixed with 1% osmium tetroxide in

ice-cold buffer for 2h. Specimens were washed by distilled deionised water and

dehydrated in graded ethanol solution. After keeping in the isoamyl acetate, the

specimens were dried in a critical point drying machine (Hitachi HCP-1, Hitachi Ltd.,

Tokyo, 100-8220 Japan) using liquid carbon dioxide as the medium. The dried

specimens were placed on aluminum stubs, mounted with electrically conducting

cement (silver paste), coated with platinum (RMC-Eiko RE vacuum coater, Eiko Engineering Co., Ltd., Tokyo, Japan) at 100 millitorr under 7 milliamperes for 15min

and observed using a scanning electron microscope (Hitachi S-800, Hitachi Ltd.,

Tokyo, Japan) at 8kV.

Results

Feed intake, body weight gain, and feed conversion ratio

Table 3 shows feed intake, body weight gain and feed conversion ratio of chickens

fed 0, 1, 3 and 5% dietary CWVC diets. Although the feed intake of each week

decreased with increasing feeding period in all groups, it did not show a significant

difference among each group after feeding the dietary CWVC. Body weight gain

tended to increase at 1% and 3% dietary CWVC but to decrease at 5% level, resulting

in the feed conversion ratio being insignificantly improved at 1 and 3% dietary CWVC.

Villus height, cell area, and cell mitosis

Figure 1 shows alterations of villus height, cell area and cell mitosis number in each

intestinal segment of chickens fed 0, 1, 3, and 5% dietary CWVC diets. Villus height

did not show a significant difference among each group, although that of the 1% dietary

CWVC group tended to be slightly higher than another groups in all intestinal segments.

Cell area of the 1% dietary CWVC group had a tendency to be higher in the duodenum and was significantly higher (P<0.05) in the jejunum than that of 0% group, but those

of 3 and 5% dietary CWVC groups tended to be clearly lower than 0% group with

increasing CWVC levels, resulting in significant decrease (P<0.05) in 5% group in the

duodenum and ileum. Cell mitosis showed almost similar phenomenon with the case

294 J. Poult. Sci., 38 (4)

Table 3. Effects of 0, 1, 3, and 5% dietary charcoal powder including wood

vinegar compounds (4:1) (CWVC) diets on feed intake, body weight

gain and feed conversion ratio in chickens (means•}SE, n=8)

a, b Means with different superscripts within same row are significantly different

from each other (p<0.05).

of cell area except that that of 1% dietary CWVC group was significantly higher (P<

0.05) than 0% group in the duodenum and those of 1 and 3% dietary CWVC groups

tended to be higher than 0% group in the jejunum.

Alterations of the villus surface

The surface morphology of the villus apex in the control duodenum (Fig.2A)

revealed only a faint cell outline between each epithelial cell due to the cell protuber-

ance, showing a comparatively smooth surface. After feeding 1% dietary CWVC

(Fig.2B), a conspicuous protuberance of each epithelial cell was observed, becoming clearer cell outline and resulting in a rough surface. However, in the case of 3%

dietary CWVC (Fig.2C), such conspicuous cell protuberances disappeared and

became faint of cell outline. Cells having no microvilli and deeper cells at the sites of recently exfoliated cells were seen in the exfloliative zone of villus tip (arrows in Fig.

2C). Furthermore, after feeding 5% dietary CWVC diet a lot of cells having no

microvilli and deeper cells due to recently exfoliated cells were found (arrows in Fig.

2D).

Fundamentally, the villus apex surface in the jejunum (Fig.3) and ileum (Fig.4)

revealed an almost similar morphological alteration to that in the duodenum except that

cells having no microvilli and deeper cells due to recently exfoliated cells were not seen

even in 5% dietary CWVC and that the cell protuberances of 1% dietary CWVC


Fig.1 Alterations of villus height (upper), cell area (middle) and cell

mitosis number (lower) in each intestinal segment of chickens fed

0, 1, 3, and 5% dietary charcoal powder including wood vinegar

compounds (4:1) (CWVC)(mean•}SE, n=4). These intestinal

morphologies tend to be activated in the 1% dietary CWVC

group.

a, b Means with different superscripts are significantly different

from each other (P<0.05).

296 J. Poult. Sci., 38 (4)

Fig.2. Villus tip surface of the duodenum in chickens fed 0 (A), 1 (B), 3

(C) and 5% (D) dietary charcoal powder including wood vinegar

compounds (4:1) (CWVC). A faint cell protuberance of the 0%

dietary CWVC group (arrows in A) is activated after feeding 1%

dietary CWVC (arrows in B), but cells having no microvilli and

deeper cells at the sites of recently exfoliated cells are found in

the 3 and 5% dietary CWVC groups (arrows in C and D). Scale

bar is common to all pictures=26um(•~195).

became faint with moving caudally.

Discussion

Gradually decreased feed intake with the feeding period was observed even in the

0% dietary CWVC group in this study. Mean environmental temperature was about

17.0•Ž at the first week during the feeding period (early in May), and 22.0•Ž at the

second week. As feed intake and body weight gain were significantly decreased in

broilers kept under hot cycling temperature (26.0•`34.0•Ž) than moderate temperature


Fig.3. Villus tip surface of the jejunum in chickens fed 0 (A), 1 (B), 3 (C)

and 5% (D) dietary charcoal powder including wood vinegar

compounds (4:1) (CWVC). A lot of cell protuberances are seen

in the 1% dietary CWVC group. Scale bar is common to all

pictures=26um(•~195).

(24.0•Ž) (Al-Batshan and Hussein, 1999), the present decreased feed intake and body

weight gain may be affected by increased environmental temperature. As the present

groups were studied in the same environmental conditions except the different dietary

CWVC levels, the statistical differences in growth performance and intestinal

morphological changes among feeding experimental groups might be induced by the

dietary CWVC levels.

The dietary CWVC diets induced a significant increase in hen-day egg production

and feed conversion ratio (Sakaida et al., 1987 a) and in broiler hatchability (Sakaida et

al., 1987 b). Also in our preliminary feeding experiment (unpublished) and the

present study, a trend of improvement in feed conversion ratio was observed in 1%

dietary CWVC group. This suggests that the ingested feed might be effectively

298 J.Poult. Sci., 38 (4)

Fig.4. Villus tip surface of the ileum in chickens fed 0 (A), 1 (B), 3 (C)

and 5% (D) dietary charcoal powder including wood vinegar

compounds (4:1) (CWVC). A lot of cell protuberances are seen

even in the ileum in the 1% dietary CWVC group. Scale bar is

common to all pictures=26um(•~195).

absorbed from the intestinal epithelial cells, being in harmony with the morphological

changes such as increased villus height, cell area and cell mitosis numbers in these birds.

The aim of the present study was to observe the effects of dietary CWVC on the villus

morphological changes in these birds, because a close relationship between morpholog-

ical change and intestinal functional feature is well known. The intestinal morphology

was markedly affected by the fed diets (Langhout et al., 1999; Yasar and Forbes,

1999). Histologically, continuously renewed cells from stem cells located near the

bases of proliferative crypt zone are known to embark on a 2•`4-day migration along

the villus surface and arrive the villus tip where they exfoliate (Leblond and Walker,

1956; Leblond, 1981). The increased villus height, cell area and cell mitosis numbers

indicated an activated villus function (Shamoto et al., 1999; Shamoto and Yamauchi,


2000; Yamauchi et al., 1996; Yamauchi and Tarachai, 2000). Especially, the present

significant increase of cell mitosis numbers in 1% dietary CWVC group might empha-

size the activated absorptive function of villi in these birds. This activated cell mitosis

correlates well with the results of scanning electron microscopic observations that the

clearer cell outline due to conspicuous cell protuberances was appeared on the villus tip.

Such a fine structural feature has been also demonstrated to show an activated function

of the villi (Shamoto and Yamauchi, 2000; Shamoto et al., 1999; Tarachai and

Yamauchi, 2000). Profitability of activated charcoal was reported in reducing the

effects of toxin in diets by adsorbing it and there by preventing its absorption from the

intestine (Anjaneyulu et al., 1993). On the other hand, it was demonstrated that the

promoting effect of wood vinegar compounds was induced by not only crude wood vinegar but by its components such as acetic acid, propanoic acid, butanoic acid,

dimethylphenol and methoxyphenol (Yoshimura and Hayakawa, 1993; Yoshimura et

al., 1995). The villus morphology was not governed either intraluminal physical

stimulation or parenteral alimentation but enteral nutrient absorption (Tarachai and

Yamauchi, 2000). A consideration of these literatures and the present observations

leads to the general conclusion that the activated function of the villi might be

multiplicatively induced by adsorbing toxic and fungal contamination in diets and there

by activating intestinal absorptive function.

The activated morphological changes were observed even in the ileum at 1%

dietary CWVC, indicating the activated function of the ileal villi. In intact chickens,

very little nitrogen absorption was observed beyond the jejunum in chickens (Imondi

and Bird, 1965). The ileum appears to be inactive in absorptive function in intact

normal condition (Yamauchi et al., 1995) and rich in intestinal microbial population

than in the duodenum and jejunum (Gorden and Pesti, 1971; King and Toskes, 1979;

Smith, 1965). In the ileum, a low population or few species of bacteria may produce

the acceleration of the cell migration without the enhancement of cell mitotic activity

(Ishikawa et al., 1986). The acetic acid included in wood vinegar of CWVC

(Yoshimura and Hayakawa, 1993; Yoshimura et al., 1995) was reported to control the balance of intestinal microflora and pathogen (Pinheiro et al., 1968; Sorrells and

Speck, 1970). It is not clear whether the activated morphological changes of the ileal

villi at the 1% dietary CWVC was direct or indirect effect from the intestinal microflora

but these morphological changes suggest that the 1% dietary CWVC might elevate the

absorptive function of all intestinal epithelial cells beyond the jejunum. This demon-

strates morphologically that the epithelial cells in the ileum also participate in absorptive

function in case of need. The increase of hen-day egg production and feed conversion

ratio (Sakaida et al., 1987 a), and hatchability in broilers fed CWVC (Sakaida et al.,

1987 b) are thought to be induced by the activated functions of villi in all small

intestinal segment parts due to the dietary CWVC.

In conclusion, the present morphological changes of intestinal villi in chickens fed

the dietary CWVC diets demonstrate that the villus function could be activated even in

the ileum at 1% level, and that such an activated villus function in all small intestinal

segment parts might improve the feed conversion ratio.

300 J. Poult. Sci., 38 (4)

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