Growth Performance and Histological Intestinal

http:// www.jstage.jst.go.jp / browse / jpsa
doi:10.2141/ jpsa.0130042
Copyright Ⓒ 2014, Japan Poultry Science Association.
Growth Performance and Histological Intestinal Alterations of Sanuki Cochin
Chickens Fed Diets Diluted with Untreated Whole-Grain Paddy Rice
Janjira Sittiya and Koh-en Yamauchi
Laboratory of Animal Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa-ken 761-0795, Japan
The effects of dietary untreated whole-grain paddy rice (WPR) on performance and histological intestinal alterations were investigated in Sanuki Cochin male chicks. At 2 weeks of age, chicks showing similar body weights
were randomly divided into 3 groups of 10 birds each. The control group was fed with a basal diet (starter diet: CP
21%, ME 3000 kcal/kg; grower diet: CP 18%, ME 2850 kcal/kg; finisher diet: CP 15%, ME 2800 kcal/kg) and the
other groups were fed with the basal diet diluted with WPR at 20% (starter diet: CP 17.8%, ME 2958 kcal/kg; grower
diet: CP 15.4%, ME 2838 kcal/kg; finisher diet: CP 13%, ME 2798 kcal/kg) and 40% (starter diet: CP 14.6%, ME
2916 kcal/kg; grower diet: CP 12.8%, ME 2826 kcal/kg; finisher diet: CP 11%, ME 2796 kcal/kg). They were housed
in individual cages under natural room temperature (around 5℃) with a daily lighting regimen of 16 h of light and 8 h
of dark. The growth performance, relative length of the intestines and relative weight of the visceral organs to 100 g
body weight did not differ except that the weight of the gizzard increased significantly (p＜0.05) in the WPR groups.
Most parameters of villus height, villus area, cell area and cell mitosis numbers of the WPR groups did not show a
significant decrease. In scanning electron microscopic results, the morphology of the villus apical surface in the WPR
groups did not show damage due to WPR and had similar cells to the control (protuberated cells). These results
demonstrate that WPR can be diluted by up to 40% as a feed ingredient in chicken basal diets.
Key words: growth performance, intestinal histological alterations, Sanuki Cochin, whole paddy feed rice
J. Poult. Sci., 51: 52-57, 2014
Introduction
Feed represents the largest cost of poultry production,
constituting up to 70% of the total cost. Therefore, some
poultry producers have long been focused on reducing feed
costs without negative effects on growth performance by
using various dietary management methods, such as whole
grain feedings or dietary dilutions. In many countries, feeding whole grains to poultry has become a common practice to
reduce the cost of grinding (Cumming, 1992; Svihus et al.,
2004) and to increase the use of locally grown grains (Nanto
et al., 2012). Concurrently, the cultivation and use of paddy
rice for the livestock industry has also been advocated in
Japan. Consequently, paddy rice might potentially be used
as an ingredient in poultry diets.
Chickens have the ability to process and digest whole
grains, primarily due to their gizzard function (Rose et al.,
1986; Banfield and Forbes, 2001). Numerous trials have
reported that there is no effect on weight gain when the
Received: March 12, 2013, Accepted: June 7, 2013
Released Online Advance Publication: July 25, 2013
Correspondence: Prof. K. Yamauchi, Laboratory of Animal Science, Faculty
of Agriculture, Kagawa University, Miki-cho, Kagawa-ken 761-0795,
Japan. (E-mail: [email protected])
broiler diet is diluted with up to 30% whole wheat (Covasa
and Forbes, 1994, Bennett et al., 1995). More recently,
Kiiskinen (1996) reported that during growth periods, it is
possible to dilute starter broiler diets with up to 40% whole
wheat and whole barley. Feeding whole grain wheat and
barley has primarily been practiced in the broiler industries.
However, no reports had been done on the growth performance in chickens fed basal diets diluted with untreated wholegrain paddy rice (WPR). Therefore, the objective of the
present experiment was to determine the performance and
histological alterations of intestinal villi and epithelial cells
in chickens fed basal diets diluted with WPR.
Materials and Methods
Animals and Diets
A total of 40 day-old Sanuki Cochin male chicks were
obtained from a farm in Kagawa prefecture. To determine
when the chicks were capable of eating and digesting WPR
(Momiroman), 10 day-old chicks were fed only WPR. The
remaining 30 birds were fed a basal diet. Although the 10
birds ate a little WPR around 4-days old, remains of rice
husks were not found in the feces. Feed intake volume increased gradually and at levels similar to that of the basal diet
group by around 10 days old. Therefore, at 2 weeks of age,
Sittiya and Yamauchi: Intestine and Whole Paddy Feed Rice
Table 1.
53
Composition of the basal diets diluting with untreated whole-grain paddy rice (WPR)
Grower (29 to 70 d)
Starter (1 to 28 d)
Item
Ingredients (%)
Maize
Milo
Soybean meal
Rapeseed meal
Gluten meal
Fish meal
Rice bran
Animal fat
Calcium carbonate
Dicalcium phosphate
Salt
Vitamin/mineral premix1
Total
Basal diet
WPR
Calculated composition
Crude protein (%)
Metabolizable energy (kcal/kg)
Crude fat (%)
Crude fiber (%)
Crude ash (%)
Calcium (%)
Phosphorus, available (%)
0%
WPR
20%
WPR
40%
WPR
0%
WPR
20%
WPR
Finisher (71 to 77 d)
40%
WPR
0%
WPR
20%
WPR
40%
WPR
59 . 0
2.0
27 . 0
─
2 .0
7.0
─
1.1
1.0
0.3
0.2
0.4
100 . 0
100 . 0
0.0
─
─
─
─
─
─
─
─
─
─
─
─
─
80 . 0
20 . 0
─
─
─
─
─
─
─
─
─
─
─
─
─
60 .0
40 . 0
59 . 0
2.0
27 .0
─
2.0
7.0
─
1.1
1.0
0.3
0.2
0.4
100 . 0
100 . 0
0.0
─
─
─
─
─
─
─
─
─
─
─
─
─
80 .0
20 . 0
─
─
─
─
─
─
─
─
─
─
─
─
─
60 . 0
40 . 0
59 . 0
2.0
27 . 0
─
2 .0
7.0
─
1.1
1.0
0.3
0.2
0.4
100 . 0
100 . 0
0.0
─
─
─
─
─
─
─
─
─
─
─
─
─
80 . 0
20 . 0
─
─
─
─
─
─
─
─
─
─
─
─
─
60 . 0
40 . 0
21 . 0
3000
3.0
6.0
8.0
0.7
0.5
17 .8
2958
2.8
6.9
7.4
0.6
0.4
14 . 6
2916
2.5
7.8
6.8
0.5
0.4
18 . 0
2850
3.0
6.0
9.0
0.7
0.5
15 . 4
2838
2.8
6.9
8.2
0.6
0.4
12 . 8
2826
2.5
7.8
7.4
0.5
0.4
15 . 0
2800
2.5
8.0
9.0
0.5
0.4
13 . 0
2798
2.4
8.5
8.2
0.5
0.4
11 . 0
2796
2.2
9.0
7.4
0.4
0.3
1
Vitamin and mineral premix including (per kg of diet): retinyl acetate, 2106 μg; cholecalciferol, 35 μg; DL-α-tocopherol acetate, 12.5 mg;
menadione, 1.5 mg; thiamine, 2.6 mg; riboflavin, 2.7 mg; pyridoxine, 6 mg; cobalamine, 9 μg; biotin, 0.2 mg; folic acid, 0.5 mg; pantothenic
acid, 15 mg; niacin, 22 mg; choline, 1000 mg; iodine, 1.05 mg; manganese, 50 mg; iron, 160 mg; zinc, 70 mg; copper, 8 mg.
the 30 birds were randomly divided into 3 groups of 10 birds
each. They were housed in individual cages under natural
conditions with a daily lighting regimen of 16 h of light and 8
h of dark (with a mean temperature of around 5℃).
Commercial starter (1-28 days), grower (29-70 days) and
finisher (71-77 days) diets were used as the basal diet in this
experiment. The control group was fed with basal diet and
the other groups were fed with basal diet diluted with WPR at
20 and 40% (Table 1). Feed and water were provided ad
libitum throughout the experimental period of 11 weeks.
Feed intake and body weight gain were measured weekly.
Gastrointestinal Organ Measurements
At the end of the feeding experiment, 5 birds from each
group were collected, weighed individually and killed by
decapitation. The visceral organs were removed. The lengths
of the duodenum, jejunum, ileum and ceca were measured
and then the contents of these segments, including the gizzard and proventriculus, were removed. Subsequently, the
proventriculus, gizzard, duodenum, jejunum, ileum and ceca
were weighed without their contents and recorded relative to
100 g body weight.
Microscopic Examinations
Another 4 birds per group were used for histological
intestinal observations. Immediately following decapitation,
the birds’ whole small intestines were removed and placed in
a mixture of 3% glutaraldehyde and 4% paraformaldehyde
fixative solution in 0.1 M cacodylate buffer (pH 7.4). The
same fixative solution was also injected into the intestinal
lumen. The middle parts of the duodenum, jejunum and
ileum were prepared for light and scanning electron microscopy.
Light Microscopy
Each intestinal segment was transversally cut (length, 3
cm), washed with 0.1 M phosphate-buffered saline (pH 7.4)
and fixed in Bouin’s fixative solution. The samples were
dehydrated with varying concentrations of alcohol and then
embedded in paraffin wax. Transverse sections were cut at 4
μm thickness (8 sections per sample) and then stained with
hematoxylin-eosin. Subsequently, the following results were
measured using an image analyzer (Nikon Cosmozone 1S;
Nikon Co., Tokyo, Japan).
Measurement of Villus Height
Two villi having a lamina propria were randomly selected
per transverse section and measured from villus tip to base,
excluding the crypt. The average villus height from the 4
birds (16 villi from 8 different sections in each bird) was
expressed as a mean villus height for one group.
Measurement of Villus Area
The villus area was calculated from the villus height, basal
width and apical width. A total of 16 calculations of the
Journal of Poultry Science, 51 (1)
54
villus area were measured from different sections in each
bird. The average villus area from 4 birds was expressed as a
mean villus area for one group.
Measurement of Epithelial Cell Area
The epithelial cell layer was measured at the middle of the
villi and the number of cell nuclei within this layer was
counted. Then the area of the epithelial layer was divided by
the number of cell nuclei. A total of 8 sections were counted
per bird.
Measurement of Cell Mitosis Number
Mitotic cells with homogeneous, intensely haematoxylinstained basophilic nuclei were counted. Cell mitosis within
the crypt was counted from 4 different sections in each bird;
from these 4 values was calculated a mean cell mitosis
number for each bird. Then, these 4 mean cell mitosis numbers from the 4 birds were expressed as a mean cell mitosis
number for each group.
Scanning Electron Microscopy
Each intestinal segment (length: 2 cm) was slit longitudinally and the intestinal contents were removed with 0.1 M
phosphate-buffered saline (pH 7.4). The tissue samples were
fixed with a mixture of 3% glutaraldehyde and 4% paraformaldehyde fixative solution in 0.1 M cacodylate buffer (pH
7.4) at room temperature for 2 h. Next, the tissue samples
were cut into 5×5-mm2 squares, washed with 0.1 M cacodylate buffer, and post-fixed for 2 h in 1% osmium tetroxide.
Then these specimens were washed with deionized distilled
water, dehydrated with varying concentrations of alcohol,
and freeze-dried (Hitachi freeze dryer, Hitachi Ltd., Tokyo,
Japan). After being coated with platinum (E-1030 ion sputter, Hitachi Ltd., Tokyo, Japan), all villi were observed with a
scanning electron microscope (Hitachi S-4300SE/N, Hitachi
Ltd., Tokyo, Japan).
All of the experiment and collection protocols in the present study were conducted in accordance with the guidelines
and rules for animal experiments, Kagawa University, Japan.
Statistical Analysis
The data from the experimental groups were statistically
analyzed using one-way analysis of variance (ANOVA) in
the SPSS statistical software package (version 10.0 for Windows, SPSS, Inc., Chicago, IL). Significant differences among
the treatments were determined with Duncan’s multiple
range test. Statistical significance was accepted at P＜0.05.
Results
Growth Performance
Feed intake, body weight and feed efficiency (Table 2)
were not significantly different between the control group
and the experimental groups.
Visceral Organ Measurements
No significant differences were found in the relative intestinal length (Table 3). The relative weights of the gizzard in
Growth performance of chickens fed basal diet diluted with whole paddy
feed rice (WPR) at 20 and 40% during 2 to 13 weeks of age (n＝4)
Table 2.
Items
Control
20%WPR
40%WPR
Feed intake (g)
Initial weight (g)
Final weight (g)
Body weight gain (g)
Feed efficiency
8217 . 77±155 . 01
94 . 44±3 . 66
2029 . 44±43 . 05
1935 . 00±41 . 65
0 . 23±0 . 005
8192 . 77±302 . 81
94 . 11±3 . 59
2016 . 11±56 . 64
1922 . 00±55 . 15
0 . 23±0 . 01
7996 . 87±264 . 61
98 . 50±3 . 73
1986 . 25±63 . 54
1887 . 75±63 . 63
0 . 23±0 . 008
There are no significant differences between each groups (p＞0.05).
Length of intestine and weight of visceral organs in chickens
fed basal diet diluted with whole paddy feed rice (WPR) at 20 and 40%
during 2 to 13 weeks of age (n＝5)
Table 3.
Items
Length (cm/100 g BW)
Duodenum
Jejunum
Ileum
Ceca
Weight (g/100 g BW)
Duodenum
Jejunum
Ileum
Ceca
Proventriculus
Gizzard
a, b
Control
20%WPR
40%WPR
1 . 55±0 . 10
3 . 19±0 . 14
3 . 41±0 . 19
1 . 68±0 . 08
1 . 53±0 . 11
3 . 17±0 . 38
3 . 17±0 . 26
1 . 66±0 . 11
1 . 44±0 . 09
3 . 06±0 . 28
2 . 97±0 . 21
1 . 44±0 . 05
0 . 60±0 . 02
0 . 99±0 . 05
0 . 82±0 . 04
0 . 40±0 . 02
0 . 38±0 . 02
2 . 09±0 . 10b
0 . 57±0 . 01
1 . 07±0 . 04
0 . 76±0 . 05
0 . 40±0 . 06
0 . 46±0 . 07
2 . 87±0 . 24a
0 . 52±0 . 08
0 . 88±0 . 84
0 . 72±0 . 04
0 . 31±0 . 05
0 . 41±0 . 04
3 . 02±0 . 28a
Means within a row with different superscripts are significantly different (p＜0.05).
Sittiya and Yamauchi: Intestine and Whole Paddy Feed Rice
55
Fig. 2. Epithelial cells on the duodenal villus apical surface in the chickens fed basal diet diluted with WPR at
0%, 20% and 40%. Arrows, protuberated cells. Scale bar,
50 μm.
Scanning Electron Microscopic Observation
As on the duodenal villus apical surface of the control
group (Fig. 2A), the epithelial cells of the 20% (Fig. 2B) and
40% (Fig. 2C) WPR groups showed a rough surface with
protuberated cells; no damage was found on the villus apical
surface. Also, on the jejunum and ileum, protuberated cells
were observed but damage was not found.
Discussion
Fig. 1. Villus height, villus area, cell area and cell mitosis number of duodenal, jejunal and ileal parts in the
chickens fed basal diet diluted with WPR at 0, 20 and
40% (n＝4). a, b Means with different superscripts are significantly different from each other (p＜0.05).
the 20% and 40% WPR groups were significantly higher than
that of the control (P＜0.05), while no significant differences
in weight of the other visceral organs was shown.
Light Microscopic Observations
The villus height and cell mitosis numbers of all the intestinal segments did not differ among all groups (Fig. 1).
Compared with the control, the ileal villus area was narrower
(P＜0.05) in the 40% WPR group. The duodenal cell area
decreased significantly (P＜0.05) in both the 20 and 40%
WPR groups.
Numerous studies have reported on the effects on bird
growth performance of substituting whole grains in chicken
diets, but relatively little work has been carried out to study
the effects of diluting chicken diets with whole grains
compared to the effects of undiluted diets. Moreover, these
studies have mostly been performed using whole wheat or
whole barley (Covasa and Forbes, 1994; Bennett et al.,
1995; Kiiskinen, 1996; Yasar, 2003). To our knowledge,
this is the first report indicating the effects of diets diluted
with WPR at different levels on the growth performance and
intestinal histology in chickens (Sanuki Cochin).
Banfield and Forbes (2001) reported that the weight gain
did not decrease after feeding diets diluted with 40% whole
wheat. Furthermore, the chickens fed whole grains did not
show any decrease in their growth rates (Rose and Michie,
1982; Covasa and Forbes, 1994; Bennett et al., 1995). All
these reports suggest that some dilution with whole grains in
chicken diets might not interfere with growth performance in
poultry.
The gross anatomical observations of the gastrointestinal
organs did not show differences among groups, except that
the relative gizzard weight significantly increased with increasing amounts of WPR. The increased gizzard weight
56
Journal of Poultry Science, 51 (1)
was also observed in chickens fed whole grain (Svihus and
Hetland, 2001; Plavnik et al., 2002; Svihus et al., 2002;
Santos et al., 2006). This increased gizzard weight is due to
the increase in grinding necessary to process the particle size
of whole grains (Roche, 1981; Gabriel et al., 2003). Therefore, the present developed gizzard might also be induced by
the need to process WPR to a smaller particle size.
Intestinal morphology was markedly affected by the fed
diets (Langhout et al., 1999; Yasar and Forbes, 1999). The
chickens resected duodenum showed an almost similar body
weight, nitrogen retention, and ether extract digestibility, an
improved dry matter digestibility and a much greater absorption of protein and ether extract by the remnant jejunum
and ileum compared with intact control chickens, suggesting
an enhanced absorptive function of the remnant intestine
(Yamauchi et al., 2010). In these birds, with an increase in
the intestinal resection area, significantly increased significantly light microscopic parameters, increased frequency of
anastomosing of each villus, and increased numbers of
protuberated epithelial cells appeared. In fasted chickens,
villus morphology was governed neither by intraluminal
physical stimulation nor by parenteral alimentation, but by
enteral nutrient absorption (Tarachai and Yamauchi, 2000).
Also, in normal chickens showing an significantly increased
body weight gain with increased feeding levels of dietary
fermented plants, significantly increased values of light
microscopic parameters and many protuberated epithelial
cells were observed (Lokaemanee et al., 2012). On the other
hand, in chickens fed a low-protein diet (CP 9.4%) a significantly decreased villus height was observed compared
with those fed a control diet (CP 18.1%) (Incharoen et al.,
2010). Likewise, chickens refed a semi-purified proteinfree pellet diet (CP 0.1%) have demonstrated that intestinal
villi were shorter and narrower than those of chickens refed a
semi-purified well-balanced diet (CP 17%) (Maneewan and
Yamauchi, 2004). These findings demonstrate that the
intestinal histology was closely related to intestinal function.
In this study, body weight gain and most light microscopic
parameters tend to decrease with increasing of WPR. This
phenomenon is thought to correlate to the decrease in CP in
the diets with increasing WPR levels. However, body
weight gain and most light microscopic parameters did not
show a significant decrease; furthermore, feed efficiency was
identical in all groups (0.23). In addition, the surface of the
villus tip was not damaged after feeding WPR rich in fiber,
although dietary fiber was shown to disrupt the villus apical
surface in chickens (Green, 1988), pigs (Moore et al., 1988)
and rabbits (Chiou et al., 1994). On the contrary, the present
epithelial cells on the villus apical surface of the WPR groups
were protuberated into the intestinal lumen in all intestinal
segments, as in the control group. Such protuberated cells
suggest that the function of the epithelial cells was similar to
the control group. The present lack of damage in the villus
tip, as well as the protuberated cells, might have been induced by the gizzard’s ability to sufficient digest the fibers in
WPR, due to the weight of the gizzard being significantly
heavier in the WPR groups. In addition, it seems that the
hard rice husks of WPR functioned as the grit, resulting in
smaller particles being ingested as part of the diet. Such
smaller particles might be easily absorbed, resulting in protuberated cells. These results suggest that WPR can be
supplemented with commercial formula feed up to 40%.
In conclusion, no significant differences were observed in
growth performance or in most light microscopic parameters
of all intestinal segments; as well, protuberated epithelial
cells with no damage on the villus apical surface were observed, suggesting that chickens can be fed a basal diet
diluted with WPR up to a level of 40% (whole-paddy form)
without negative effects on growth performance or intestinal
histology.
Acknowledgments
This study was supported by a grant from the research for
production of valuable livestock by feeding self-sufficient
forage crops.
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