The effect of fermented feed on intestinal morphology and ...€¦ · conducted to evaluate the effect of feed wetting and fermented feed on the intestinal flora and immunity response

The effect of fermented feed on intestinal

morphology and histology of broiler

S.A.H. Naji(1)

J.K.M. Al-Gharawi(2)

I.F.B. Al-Zamili(2)

(1) Department of animal resource- college of agriculture- Al-Qadisia University- Iraq.

(2) Department of animal resource- college of agriculture- Al-Mthanna University- Iraq.

Corresponding author: [email protected]

Abstract:

During recent years solid state fermented feed (SSFF) has been introduced with

great success in poultry nutrition, where it has shown to have some beneficial

properties in particular considering animal health. The present experiment was

conducted to evaluate the effect of feed wetting and fermented feed on the

intestinal flora and immunity response parameters of broiler chicks. The SSFF

were prepared in two stages; at the first stage the commercial broiler feed were

moistened with tap water at the rate 1:1 (1 liter water for each one kg. feed). At the

second stage the wetting feed was placed in a plastic tray and inoculated with Iraqi

probiotic (IP) at the rate 11 grams of IP for each one one kg. of feed. Then the

plastic tray was closed ad incubate for 33 h. at 3±72 ºC for complete fermentation.

Significant value (P<1015) in the percentage of the weight, length of the fine

intestine including Duodenum, Jejunum, Ileum, Ceca. Significant value increase

(P<1015) villi height, crypt depth, Percentage of villi height to crypt depth in

duodenum, jejunum and ileum.

In conclusion, it can be started that feed fermentation generally improves

bacterial ecology of the gastrointestinal tract and immunity response in broiler

chicks, therefore be a new handle on future strategy to control chicken disease.

In conclusion, the results of the current experiment indicated that feed

fermentation could improve the intestinal structure of the broilers, enhance villus

height and increased villus height to crypt depth ratio in the duodenum, jejunum,

and ileum.

Key words: fermented feed, broiler, intestinal morphology, histology.

Introduction:

Fermentation is the chemical transformation of organic substances into simpler

compounds by the active enzymes, complex organic catalysts, which produced by

microorganisms such as bacteria, yeasts, or molds. Enzymes act by hydrolysis, a

process of breaking down or predigesting complex organic molecules to form

smaller (more easily digestible) compounds and nutrient (Shurtleff and Aoyagi,

211±). The word “fermentation” is derived from the Latin meaning “to boil”, from

the budding and foaming of early fermenting beverage seemed closely akin to

boiling. Although most microbial fermentations are an accomplished in Liquid

phase, several advantages occur for solid state fermentations (SSF): (1) Low

medium cost, (2) Low water output, (3) Low capital investment, (4) More practical

when carried out in the fields (Adams et. al., 2112).

Fermentation has been practiced for quite a long time as a means to improve the

quality of food. Fermentation process has been applied to improve the nutritive

value of soybean (Mathivanan et. al., 2116) copra meal (Hatta and Sunda, 2112)

and tofu waste (Rasud, 2112).

The fermentation process can create conditions for the growth of

microorganisms that break down fiber and anti-nutrients. Fermented feed

influences the bacterial ecology of the gastrointestinal tract and reduced the level

of Enterobacteriaceae in different parts of the gastrointestinal tract in pigs (Winsen

et. al., 2111) and broiler chicks (Heres et. al., 2113). Lactobacilli and yeast in the

kefir which supplemented in drinking water were significantly increased the

population of Lactobailli spp. and total aerobic bacteria and decreasing the

population of Enterobactciaceae and coliform in the geese intestine (Yaman et. al.,

2116).

Primarily fermented feed causes a reduction of pathogenic bacteria, including

Salmonella and Campylobacter in the digestive tract, most particularly in the crop

and gizzard. Because the crop often ruptures during slaughter, the decrease level of

pathogens in this area in particular makes contamination of meatless likely

(Donkor et. al., 2116)

Antibiotic have been used as feed additives to improve growth performance and

control disease in animals.

However, the continued use of antibiotics has resulted in common problems

such as the development of drug resistant bacteria, imbalance of normal microflora

and drug residues in animal products (Chen et. al., 2112). Since 2116, antibiotics

have been banned for use as feed additives in the European Union. Probiotics have

therefore become important as replacement feed additives (Steiner, 2116). A

probiotics is a live microbial feed supplement that beneficially affects the host

animal by improving its intestinal microbial balance.

After feeding of probiotics, improvements in growth performance, feed

efficiency, immunity parameters and disease resistance have been reported (Al-

Gharawi, 2112).

The major probiotic strains include Lactobacillus, Saccharomyces,

Streptococcus and Aspergillus (Tannock, 2111). Presently Bacillus, Lactobacillus

and Saccharomyces are the major strains applied in broilers (Zhang et. al., 2115;

Chen et. al., 2112).

Since there have been few investigations of the fermentation of feed with

probiotics and used wet feeding in broiler chicks. The objectives of this study were

to clarify the effect of wet and fermented on intestinal microflora populations and

immunity in broiler chicks.

Materials and Methods

Preparation of fermented feed:

A commercial broiler starter and finisher diet (Table 1) were purchased from

local markets. The chicks were fed on a starter diet during the first three weeks,

and then transferred to finisher diet were used for the reminder of the experimental

period which was lasted for 6 weeks.

Table1. Composition of basal diet.

Basal Diet

Items 23 to 35 d 1 to 22 d

53011 4402 Corn

15 1301 Wheat

2± 33 Soybean meal (454)

1 1 Mineral and vitamin premix

3 2 Oil

106 103 Limestone

103 103 Dicalcium phosphate

111 % 111 % Total

Calculated analysis

120±1 21022 Crude protein (%)

3111 2221 Metabolism energy (kilo

calorie per kg. Diet)

1035 1023 Calcium (%)

1045 1043 Phosphorus (%)

1051 1055 Methionine (%)

1025 1035 Lysine (%)

1021 1035 Methionine + Cysteine (%)

102 101 Folic acid * produced by Ghadeer Babylon, calculated analysis according to NRC (1224).

The fermented feed (SSFF) was prepared in two stages; at the first stage the feed

were moisted with water (water: feed, 1:1). At the second stage the wetting feed

was placed in a plastic tray and inoculated with probiotic (IP) at the rate 11 grams

of IP for each one kilogram of feed. Then the plastic trays were closed and

incubate for 43 h. at 3±72 ºC for complete fermentation.

The IP was purchased from laboratory of poultry technology at agriculture

faculty, university of Baghdad. According to the manufacture information label,

each one gram of IP contains at least 112

cfu of Lactobacillus acidophilus, Bacillus

subtilis, Bifidobacterium and at least 113 cfu of Saccharomyces cervisia.

Fermented feed was characterized by high lactic acid concentration (up to 261

mmol/ kg feed) and moderate amounts of acetic acid (21-31 mmol/ kg feed), high

number of lactic acid bacteria (Log 2-11 cfu/ g. feed) and PH of approximately

405-501 as described by Cutlure et. al. (2115).

Broiler husbandry and experimental design:

The experiment was carried out at poultry research farm-faculty of agriculture-

university of Al-Mothanna, Iraq, during the period from 2nd

– November- 2113 to

±th

- December- 2113 and aimed to study the appropriate proportion of dry feed

replacement with fermented feed. A total of 361 one day old Ross313 broiler

chicks were randomly assigned (CRD) chicks in the six experimental groups were

fed as follow:

T1: Control group fed on dry feed.

T2: Fed on wetting feed (1:1, feed: water).

T3: 254 fermented feed + ±54 dry feed.

T4: 514 fermented feed + 514 dry feed.

T5: ±54 fermented feed + 254 dry feed.

T6: 1114 fermented feed throughout the experimental period.

Each treatment group was replicated three times with 21 chicks per replicate. The

chicks were reared in battery cages (105 × 101 m) with four tears. The chicks were

raised in a temperature and humidity controlled room with a 24-h. constant light

schedule and ad. libitum access to water and feed throughout the experiment.

Sampling procedure and analytic methods:

At the end of the feeding trial, six birds per treatment were selected at random and

slaughtered for sampling. The intestinal parts weight and length and relative parts

weight and length were calculated according to live body weight.

The intestinal parts were separated carefully; duodenum loop, jejunum the middle

sections of the intestine between duodenum and mackles diverticulum, and ileum

which located between mackles diverticulum to the ileocecal junction. The two

cecum were also separated from the site of ileocecal junction and their relative

weight and length were calculated. Samples from intestinal parts were taken for

histological study. Samples were fixed in 114 buffered formalin and embedded in

paraffin. Three micron sections were microtome cut and stained with haematoxylin

and eosin. Slides were measured with light microscopy to measure crypt depth,

villi height and villi height/ crypt depth. Measurements of villus height and crypt

depth were taken only from sections were the plane of section ran vertically from

the top of villus to the base of an adjacent crypt.

Values presented are means from ± samples of villi measured from the tip to the

crypt mouth and ± associated crypts measured from the crypt mouth to the base

(Xu et. al. 2113).

Statistical analysis:

Data generated from the present experiment was subjected to statistical analysis

using the GLM procedure of SAS (2111) statistical software package. When

significant differences were noted, mean were compared using Duncan’s multiple

range test (1255).

Results and discussion:

Morphological measurements of the duodenum, jejunum, ileum, and

cecum were shown in table 2 and 3. Birds fed on fermented feed had

higher (P≤1015) relative weight and relative length of duodenum,

jejunum, ileum, and cecum when compared with birds fed on the control

dry feed. As the percentages of the fermented feed in the diet increased

these parameters were significantly (P≤1015) increased.

Table (2) the effect of fermented feed of the Iraqi probiotic on the

relative weight of the small intestine and cecum (%) of broiler.

The relative weight Treatments

Cecum Ileum jejunum Duodenum The small

intestine

1045 1 114 10±1 1 113 1046 1 121 105± 1 11± 30±3 1 131 T1

1051 1 115 1034 1 122 1052 1 112 1062 10115 c 4012 1 126 T2

105± 1 116 2012 1 121 103± 1 121 1034 10113 b 4031 10122 b T3

1061 1 115 2013 1 113 1032 1 123 1033 1 11± 4021 10121 b T4

1062 1 11± 2016 1 122 1021 1 111 1032 10116 b 4026 1 115 T5

10±2 1 112 2043 1 111 201± 1 112 1014 1011± a 50±2 1 113 T6

*

*

*

*

*

Significant

level

T1: Control group fed on dry feed. T2: fed on wetting feed (1:1, feed: water). T3: 254 fermented feed + ±54 dry feed.

T4: 514 fermented feed + 514 dry feed. T5: ±54 fermented feed + 254 dry feed. T6: 1114 fermented feed throughout

the experimental period. a,b Means within columns with no common superscript differ significantly (P < 1015).

Dietary treatment had a significant (P≤1015) effect on villus height, crypt depth,

and villus height to crypt depth ratio in the duodenum (table 4.), jejunum (table 5.)

and ileum (table 6.). Birds fed the fermented feed had higher (P≤1. 15) Villus

heigh and crypt depth in the duodenum, jejunum, and ileum than birds fed entire

control dry feed. These birds also had a higher (P≤1015) villus high to crypt depth

ratio in all these three parts as well.

Table (3) effect fermented feed by the Iraqi probiotic in the relative

length of the small intestine and cecum (cm / 111 g body weight) of

broiler.

The relative length Treatments

Cecum Ileum Jejunum Duodenum The small

intestine

1011 1 11 4035 1 42 3026 1 41 1066 1 12 202± 1 14 T1

1014 1 11 4041 1 4± 4012 1 43 10±1 1 21 11013 1 13 T2

1014 1 13 40±3 1 51 4013 1 4± 1036 1 12 11032

1 16

T3

1015 1 11 4031 1 43 4022 1 3± 1032 1 1± 11021

1 21

T4

1021 1 1± 4033 1 43 4024 1 51 1021 1 21 11023

1 16

T5

1034 1 14 4026 1 32 4042 1 22 201± 1 13 11033 1 21 T6

*

*

*

*

*

Significant

level




Table (4) Effect of fermentation feed by the Iraqi probiotic on villus

height, crypt depth (μm) and the ratio of villus height to crypt depth in

the duodenal of broiler.

The ratio of villus

height to crypt depth

Crypt depth

(μm)

Villus height

(μm)

Treatments

±065 1 15 15035 1 13 11±042 1 15 T1

±035 1 15 15052 1 21 122033 2 16 T2

3016 1 14 1±052 1 12 141016 1 23 T3

3025 1 13 1±0±2 1 13 146021 2 33 T4

3035 1 1± 1±02± 1 21 151015 1 1± T5

3034 1 15 13032 1 13 166036 1 42 T6

*

*

*

Significant

level






the jejunum of broiler.

The ratio of villus


Crypt depth

(μm)

Villus height

(μm)

Treatments

±016 1 12 14032 1 15 111012 1 1± T1

±013 1 1± 14044 1 12 11306± 1 12 T2

±046 1 16 15013 1 13 113022 1 61 T3

±055 1 15 15025 1 21 115. 1± 1 22 T4

±062 1 16 1503± 1 21 11±021 1 16 T5

3015 1 13 16041 1 12 132. 13 1 36 T6

*

*

* Significant

level






the ileum of broiler.

The ratio of villus


Crypt depth

(μm)

Villus height

(μm)

Treatments

40±± 1 15 3062 1 ±2 41014 1 53 T1

4031 1 14 30±1 1 63 410±± 1 61 T2

5012 1 16 2031 1 ±2 51013 1 54 T3

5012 1 1± 203± 1 ±1 51025 1 65 T4

5025 1 16 11011 1 31 53012 1 42 T5

5053 1 15 11014 1 64 61055 1 56 T6

*

*

* Significant

level




In the current study, the morphology and histology parameters of the digestive

tracts parts were improved in broiler chicks feed on fermented feed than broilers

fed on control diet. These results are in agreement with that of Chiang et. al.

(2111), and Xu et. al. (2112).

The villus height to crypt depth is a very useful measure to estimate the

absorption capacity of the small intestine. Maximum digestion and absorption is

believed to occur as villus height to crypt depth ratio increased (Montagne et. al.,

2114; Chiang et. al., 2111). Changes in the intestinal morphology such as reduced

villus height and deeper crypt may also indicate the presence of toxins (Xu et. al.,

2113) in the present study, increased villus height and increased villus height to

crypt depth ratio were observed in broilers fed on fermented feed compared with

unfermented feed. The increased villi height and villus heigh to crypt depth ratio

might be associated with the increased number of beneficial bacteria like

Lactobcilli, Bifidobacterium, Bacillus subtilis, and Sacchromyces cervisia (Xu et.

al., 2113; Naji et. al., 2114). Fermented feed was characterized by a high number

of lactic acid bacteria (Log 2-11 cfu/ g feed) and pH of approximately 405-501 as

described by culter et. al. (2115). The increased villus height and villus height to

crypt depth ratio produced an intestinal structure more oriented to digestion, with

improved absorptive and hydrolysis potential, as well as requiring fewer nutrients

towards intestinal maintenance. Thus, the intestinal structure of duodenum,

jejunum, and ileum is more favorable for the bird and may help to explain the

improvement in weight gain and feed conversion (Hu et. al., 2113; him et. al. ,

2111). The better results obtained of SSF feed might be attributable to the higher

production of secondary microbial metabolites during solid fermentation. These

metabolites include organic acid (Lactic acid) produced by Lactobacilli, enzymes

(amylase and protease) and antimicrobial substances like iturin and surfactin which

produced by Bacillus subtilis bacteria during solid fermentation.

In conclusion, the results of the current experiment indicated that feed

fermentation could improve the intestinal structure of the broilers, enhance villus

height and increased villus height to crypt depth ratio in the duodenum, jejunum,

and ileum.

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The effect of fermented feed on intestinal morphology and ...€¦ · conducted to evaluate the effect of feed wetting and fermented feed on the intestinal flora and immunity response