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Italian Journal of Animal Science 2015; volume 14:3886

PAPER
the pH of gastro-intestinal tract (GIT) and sub-
Effect of dietary acidification sequently to reduce pathogenic bacterial Corresponding author: Dr. Seyed Mohammad
in broiler chickens: growth and provide better condition for bird Hosseini, Department of Animal Science, Faculty
of Agriculture, The University of Birjand, Birjand
gut health to optimize nutrient absorption
2. Digestive tract (Hernández et al., 2006). Additionally, OA can 6813717133, Iran.
measurements and bone penetrate the lipid membrane of the bacterial Tel. +98.9151612192.
mineralization cell and reduce intracellular pH, which leads to E-mail: Shosseini@birjand.ac.ir
the destruction of the cell wall and death
Received for publication: 4 February 2015.
Zahra Tahami, Seyed Mohammad (Ricke, 2003). In addition, organic acids also Accepted for publication: 10 April 2015.
Hosseini, Moslem Bashtani stimulate digestive secretion; improve the
solubility of the feed ingredients, digestion This work is licensed under a Creative Commons
Department of Animal Science,
and absorption of the nutrients and provide Attribution NonCommercial 3.0 License (CC BY-
University of Birjand, Iran better intestinal villus integrity (Abdel-Fattah NC 3.0).
et al., 2008; Nourmohammadi and Afzali,
2013). Recently, organic acids have been ©Copyright Z. Tahamit al., 2015
Licensee PAGEPress, Italy
extensively used in the poultry industry as feed
Abstract Italian Journal of Animal Science 2015; 14:3886
additive to enhance the growth performance doi:10.4081/ijas.2015.3886
and the immune response of birds. Inclusion
The objectives of this study were to assess of OA in the diet of broilers improved nutrient
the effects of dietary Orgacids® (organic acid; utilization, growth and feed efficiency Iran. One hundred-sixty male broiler chicks of
OA) supplementation on digestive tract meas- (Nourmohammadi et al., 2012). Hence, the the Ross strain were obtained from a commer-
urements, intestinal morphometry, pH values usage of OAs are becoming more acceptable to cial hatchery and were used in this experi-
in content of digestive tract segments and feed manufacturers and poultry producers, ment, which lasted for 35 d. A total of 4 treat-
bone mineralization of broiler chickens. The there is a growing interest in their use as ments were replicated 4 times, with 10 birds
experiment was conducted with 160 7-d-old growth promoter. per pen (0.9 × 1.2 × 0.7 m). The experimental
Ross 308 male broilers in 4 groups of 4 pens However, acidifiers have been used in broi- period was divided into 2 phases: a grower
and for both grower (7 to 21 d) and finisher ler diets for years and seem to obtain a positive phase (7 to 21 d) and a finisher phase (22 to 42
(22 to 42 d) phases. The treatments were basal response in growth performance, but there are d). Basal diets for both phases were formulat-
diet only (control) and basal diet supplement- contradictory results about the use of OA in ed to meet or exceed all nutrient recommenda-
ed with 1, 2 and 3 OA g kg–1 of feed. The results poultry and, according to Nourmohammadi et tions published in the Ross rearing guideline
showed that jejunum empty relative weight (% al. (2011b), these effects depend on the chem- (Table 1; Aviagen, 2011). For preparing the rest
BW) increased with increasing inclusion of OA ical structure and compound of the acid, pKa of experimental diets, the basal diet was sup-
(linear effect, P<0.05). For histological meas- values, animal species and the site of action of plemented with OA (Orgacids; Sunzen
ures, intestinal villi were larger and longer in acids. Combinations of some acidifiers seem Biotech, Selangor, Malaysia) at the levels of 0,
birds fed dietary OA (linear; P<0.01), crypts to be more effective, which comes to no sur- 1, 2 and 3 g kg–1. Orgacids® is a combination of
were deeper (linear; P<0.05) and the ratios of prise because there are several modes of six acids that are blend into a fine granular
villus length to crypt depth were higher (lin- action which contribute to antibacterial effects carrier (formic acid, lactic acid, malic acid, tar-
ear; P<0.05) for the duodenum and jejunum and nutrient utilization. There is sufficient taric acid, citric acid and orthophosphoric
segments, but there was no effect on the ileal data in the scientific literature on the growth acid).
morphometric indices. The mucosal epithelial promoting effects of OA, while few literatures To keep the diets isocaloric, OA was added to
layers were thinner (linear; P<0.05) in the are available on the effects of OA on digestive the basal diets in place of corn starch. All
fore parts of small intestine. A linear (P<0.05) organs development and morphometric experimental diets were free of antibiotics and
effect was observed for the pH values in con- indices. were provided in mash form. All chicks were
tents of proventriculus, gizzard and duodenum Therefore, the aim of the present study was fed a typical commercial broiler starter ration
contents of broilers. Tibia ash, phosphorus (P) to investigate the effect of dietary OA for the first 6 days prior to the start the exper-
content and tibia-breaking strength in broilers (Orgacids®) on the characteristics of the gas- iment. Experimental diets were offered to the
given diets with OA were greater (linear; trointestinal tract (GIT), intestinal morpholo- birds from d 7 of age (BW=121 g ± 1; P>0.05).
P<0.05) than those of the control group. gy, pH values of GIT contents, and bone miner- Birds had free access to feed and water.
Intestinal adaptation revealed by jejunum alization of broiler chickens.
weight histological modifications in birds fed Samples collection and measure-
diets supplemented with OA can be viewed as ments
an attempt to compensate for the low function- On d 42, two broilers were randomly select-
ality of the gastric area. Materials and methods ed from each pen (8 birds/treatment), individ-
ually weighed and then killed by cervical dislo-
Experimental birds and diets cation, defeathered, and eviscerated. Different
The treatment protocols and procedures in segments of the small intestine including the
Introduction this study were conducted according to local duodenum (from gizzard to the end of pancre-
ethical guidelines, and were approved by the atic loop), jejunum (part between pancreatic
Organic acids (OA), which have positive Animal Ethics Committee of the Research loop and Meckel’s diverticulum), ileum (part
effects on broiler performance indices, reduce Council of the Birjand University, Birjand, between Meckel’s diverticulum and ileocaecal

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Organic acid in broiler diets

junction) and cecum were removed and their supports 60 mm apart and a load applied at 50 tive lengths (cm/100 g of BW) and relative
weights and lengths were recorded. The mm/min to the midpoint of the long axis of the weights (g/100 g of BW) of different parts of
weight and length of each organ was bone (Knowles and Broom, 1990). The parts of the small intestine are presented in Table 2.
expressed relative to the total body weight. the tensiometer that were in contact with the Organic acid supplementation did not have a
A sample of approximately three cm was cut bone were covered in soft rubber tubing to significant effect on the relative lengths and
from middle part of each segment of the duo- avoid point stresses. The breaking strength weights of different parts of the small intes-
denum, jejunum and ileum from the same was recorded as the peak load before the bone tine, except for relative jejunum weight (RJW).
birds used for intestine weights. Intestinal breakage. Inclusion of OA linearly increased RJW
samples were flushed with ice-cold saline and One gram of the crop contents and 1 g of (P<0.05) compared with the control group. In
placed in 10% neutral buffered formalin for 48 digesta from the proventriculus, gizzard, duo- general, digestive development indices were
h. After fixation, a single 1-cm sample was cut denum, jejunum, ileum, and rectum segments slightly higher in birds fed with the addition of
from each section, dehydrated with increasing of each bird were immediately collected and OA in their diet, as compared to the control.
concentrations (70, 80, 90, and 100%) of placed into clean Falcon tubes. The samples The effects of dietary OA supplementation
ethanol, and cleared with xylene. In the histo- were mixed with deionized water (1:10 wt/vol), on small intestine morphometry of broiler
morphology study, images were captured using and the pH of the solution was measured using chickens are summarized in Table 3. Inclusion
a light microscope and a system that analyzes a digital pH meter (Model 827, Metrohm, of 3 g OA/kg of diet linearly increased villus
computerized images (Yu et al., 1998). Villus Herisau, Switzerland) at room temperature. length (VL; P<0.01), crypt depth (CD; P<0.05)
length, crypt depth, villus width and the thick- and VL: CD ratio (P <0.01), but epithelial
ness of epithelium were measured at Statistical analysis thickness (ET) was linearly reduced (P<0.05)
100×magnification using computer software Data were analyzed using GLM procedure at the duodenum and jejunum. The results
(Sigma Scan, Jandel Scientific, San Rafael, (SAS, 2006) in a completely randomized showed that ileal morphometric indices were
CA, USA). The ratio of villus length to crypt design. All of the treatment means were com- not affected by dietary groups.
depth was calculated. The slides holding the pared using Tukey-Kramer’s test. For the dif- The effects of dietary acidification on the pH
fixed tissue sections were deparaffinized, ferent statistical tests, significance was values in content of gastro intestinal tract
rehydrated, incubated with 5 g/L of periodic declared at P<0.05. (GIT) of broiler chickens are shown in Table 4.
acid solution for 15 min, washed, and finally The data show that feeding the OA treatment
incubated with Schiff’s reagent (1 g of basic compared with the control diet linearly
fuchsin, 200 mL of distilled water, 20 mL of 1 decreased the pH values in proventriculus, giz-
mol/L HCl, 6 g of sodium pyrosulfite) for 30 Results zard and duodenum contents of broilers
min. The sections were then washed in dis- (P<0.05) but did not affect pH values in con-
tilled water, dehydrated and mounted. Goblet The effects of dietary treatment on the rela- tents of the crop, jejunum, ileum and rectum.
cell counts were collected from 5-mm sections
stained with periodic acid-Schiff reagent
(Armed Forces Institute of Pathology, 1992).
Table 1. Feed ingredients and nutrient composition of basal diets for experimental broiler
Briefly, tissues were deparaffinized and chicks. Based on Aviagen (2011).
hydrated, oxidized in periodic acid (5 g/L) for 5
min, rinsed in distilled water, and placed in Finisher, d 22 to 42 Grower, d 7 to 21
Coleman’s Schiff reagent (Sigma Chemical
Ingredient, g/kg
Co.) for 30 min. After a 15-min rinse in luke-
Corn 527 502
warm tap water, tissues were counterstained
Soybean meal (43% CP) 289 346
in hematoxylin, rinsed, dehydrated, and
Wheat 80 50
mounted. Positively stained periodic acid-
Fish meal 30 30
Schiff cells were enumerated on 30 villi per
Soybean oil 40 35
sample, and the means were considered for
Dicalcium phosphate 14 16
statistical analysis.
Oyster shell 10 11
The right tibia of the two killed birds was
Salt 2 2
removed and stored at -18 °C to assess the
Methionine 2 2
total ash and mineral content. Tibia ash was
Lys 1 1
determined by removing the adhering tissue,
Premix° 5 5
drying the bone at 110 °C for 12 h and extrac-
Calculated nutrient composition
tion of fat with ether. The dry fat-free bones
Metabolizable energy, MJ/kg 12.98 12.56
were ashed in a muffle furnace at 550°C for 3
Crude protein, % 20.0 22.0
h. Ash weight was calculated as a percentage
Calcium, % 0.9 1.0
of dry fat-free bone weight. The tibia contents Available phosphorus, % 0.45 0.50
of Ca, P and Mg were measured using dry- Methionine, % 0.35 0.37
ashed bone samples following the same proce- Lysine, % 1.1 1.25
dure of mineral estimation in feed and faeces Methionine + Cystine, % 0.85 0.95
(AOAC, 2007). Bone strength was determined
°Provided (per kilogram of diet): vitamin A, 11,000 U; cholecalciferol, 1800 U; vitamin E, 11 mg; vitamin K, 5.7 mg; vitamin B2, 2 mg;
by breaking the right tibia on an Instron 4301 vitamin B6, 2 mg; vitamin B12, 0.024 mg; folic acid, 0.5 mg; niacin, 28 mg; pantothenic acid, 12 mg; choline chloride, 250 mg; manganese,
tensiometer, using a three-point bend with 100 mg; selenium, 0.22 mg; copper, 5 mg; iodine, 0.5 mg; cobalt, 0.5 mg; zinc, 62.

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Tahami et al.

Tibia ash and bone mineralization of broiler et al. (2010) reported significantly higher jejunum weight (Tortuero and Fernandez,
chickens are presented in Table 5. The results length and weight of small intestines in broiler 1995; Abdel-Fattah et al., 2008). Moreover,
indicated that no linear or quadratic response chickens receiving diets supplemented with 20 increases in jejunum weight may indicate that
was found in Ca and Mg content of tibia, or 30 g/kg OA supplementation, which are con- this segment of small intestine utilized the
whereas tibia ash content of chicks increased siderably higher levels compared to levels used nutrients for its growth with higher priority.
both linearly and quadratically (P<0.05) with in present research. On the other hand, the The increase in jejunum weight can also be
the increasing dietary concentrations of OA, increased feed intake leads to reach more related to the increased digestion of nutrients
and the response was maximized at 3 g kg–1 nutrients in the small intestine, which selec- (Nourmohammadi et al., 2012).
supplemental OA. Compared with control tively increases protein synthesis in the A lengthening of the villus length increases
group, P content of tibia was decreased (lin- jejunum (main site for nutrient absorption) the surface area for better nutrients absorp-
ear; P<0.05) by the addition of OA in broiler and promotes the appropriate growth of small tion. The large crypt depth indicates fast tissue
diet. Further analysis showed that the tibia- intestine, and subsequently results in increase turnover and a high demand for new tissue
breaking strength also showed a quadratic
increase (P<0.05) with the increase in the lev-
els of dietary OA (Figure 1).

Discussion
There are a limited number of studies that
have evaluated the influence of OA on relative
organ weights. The relative weight and length
of different parts of broiler gut, except relative
jejunum weight, in the current study were not
affected by dietary treatments. Furthermore,
the effect was more outstanding with addition
of OA at 3 g kg-1. These results were in general
agreement with those reported by Denli et al.
(2003) and Nourmohammadi et al. (2012) as
confirmed that supplementing the diet with
organic acids affects the digestive organs
weight and length. The reasons for these find-
ings are not clear, but an explanation for this
result and also the higher weight of jejunum
may be provided by taking into account that
only little dietary OA reaches the hind parts of
the GIT, and therefore their effect on acidity is Figure 1. Tibia-breaking strength (breaking force divided by bone weight expressed as
limited to upper parts of the digestive tract, Newton per mm2) by dietary inclusion of organic acid (OA; Orgacids®). Values are
particularly crop and gizzard (Thompson and mean±SE (n=4). The value is significantly lower for acidified diets compared with control
Hinton, 1997). In the literature, however, Adil (P<0.05).

Table 2. Effect of organic acid supplementation on digestive tract measurements in broiler chickens.
Item Control Dietary acidification, g kg–1 SEM Orthogonal polynomials
1 2 3 Linear Quadratic
Relative weight, g/100 g BW
RDW 0.83 0.83 0.80 0.76 0.05 ns ns
RJW 1.74b 2.12a 2.26a 2.32a 0.20 0.05 ns
RIW 1.46 1.48 1.75 1.89 0.25 ns ns
RCW 0.44 0.54 0.51 0.52 0.05 ns ns
Relative length, cm/100 g BW
RDL 1.48 1.56 1.51 1.56 0.09 ns ns
RJL 3.67 3.14 3.23 3.71 0.03 ns ns
RIL 3.53 3.42 3.17 3.42 0.09 ns ns
RCL 0.74 0.75 0.75 0.85 0.06 ns ns
BW, body weight; RDW, relative duodenum weight; RJW, relative jejunum weight; RIW, relative ileum weight; RCW, relative cecum weight; RDL, relative duodenum length; RJL, relative jejunum length; RIL,
relative ileum length; RCL, relative cecum length; Each value represents the mean of 8 observations (four replicates × 2 birds/replicate). a,bMean values within a row with no common superscript differ
significantly from each other (P<0.05); ns, not significant.

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Organic acid in broiler diets

Table 3. Effect of organic acid supplementation on intestinal morphometry in broiler chickens.


Item Control Dietary acidification, g kg–1 SEM Orthogonal polynomials
1 2 3 Linear Quadratic
Duodenum
Villus length, µm 1286b 1304b 1315b 1375a 12 0.01 ns
Villus width, µm 181 182 181 182 4 ns ns
Crypt depth, µm 177b 179b 178b 183a 3 0.05 ns
Villus length:crypt depth 7.26b 7.28b 7.39b 7.52a 0.4 0.05 ns
Epithelial thickness, µm 56a 55a 55a 51b 0.2 0.05 ns
Goblet cell number° 10 11 11 12 0.7 ns ns
Jejunum
Villus length, µm 1165b 1172b 1176b 1210a 11 0.01 ns
Villus width, µm 108 112 110 109 5 ns ns
Crypt depth, µm 159b 160b 161b 165a 4 0.05 ns
Villus length:crypt depth 7.32b 7.32b 7.30b 7.36a 0.4 0.05 ns
Epithelial thickness, µm 35a 34a 35a 31b 0.1 0.05 ns
Goblet cell number° 10 11 10 12 0.7 ns ns
Ileum
Villus length, µm 856 849 854 857 12 ns ns
Villus width, µm 69 68 67 66 4 ns ns
Crypt depth, µm 152 150 151 152 3 ns ns
Villus length:crypt depth 5.63 5.66 5.65 5.64 0.5 ns ns
Epithelial thickness, µm 34 34 33 32 0.2 ns ns
Goblet cell number° 8 7 8 6 0.6 ns ns
Each value represents the mean of 8 observations (four replicates × 2 birds/replicate). °Numbers in area of epithelial cells. a,bMean values within a row with no common superscript differ significantly
from each other (P<0.05); ns, not significant.

Table 4. Effect of organic acid supplementation on pH values in content of digestive tract segments in broiler chickens.
Item Control Dietary acidification, g kg–1 SEM Orthogonal polynomials
1 2 3 Linear Quadratic
Crop 5.00 5.02 4.82 4.90 0.06 ns ns
Proventriculus 4.15a 3.82b 3.95ab 3.95ab 0.07 0.05 ns
Gizzard 3.70a 3.57b 3.62ab 3.37b 0.17 0.05 ns
Duodenum 6.22a 6.07b 6.00b 6.02b 0.04 0.01 ns
Jejunum 6.00 6.02 5.95 6.02 0.06 ns ns
Ileum 6.10 6.00 6.05 6.02 0.04 ns ns
Rectum 6.17 6.17 6.17 6.22 0.06 ns ns
Each value represents the mean of 8 observations (four replicates × 2 birds/replicate). a,bMean values within a row with no common superscript differ significantly from each other (P<0.05); ns, not
significant.

Table 5. Effect of organic acid supplementation on bone minerliztion in broiler chickens.


Item Control Dietary acidification, g kg–1 SEM Orthogonal polynomials
1 2 3 Linear Quadratic
b a a a
Tibia ash, % 39.75 43.32 44.25 42.00 1.81 0.05 0.05
Ca, % 33.25 33.75 34.50 35.75 0.90 ns ns
P, % 12.52b 14.93a 15.93a 16.81a 1.97 0.05 ns
Mg, % 0.74 0.75 0.76 0.76 0.01 ns ns
Each value represents the mean of 8 observations (four replicates × 2 birds/replicate). °Mineral content in bone is calculated on the basis of the tibia ash. a,bMean values within a row with no common
superscript differ significantly from each other (P<0.05); ns, not significant.

[Ital J Anim Sci vol.14:2015] [page 413]


Tahami et al.

(Yason et al., 1987). Changes in intestinal uted to their effects on decreasing the GIT pH acids. Int. J. Poult. Sci. 7: 215-222.
morphometry, such as longer villi and deeper (Ao et al., 2009). Therefore, the pH values of Adil, S., Bandary, T., Bhat, G.A., Mir, M.S.,
crypts and thinner epithelial thickness, can different segments of the GIT were measured. Rehman, M., 2010. Effect of dietary supple-
contribute to the enhanced of nutrient absorp- As expected, OA decreased the pH of proven- mentation of organic acids on perform-
tion, increased secretion in the gut, increased triculus, gizzard and duodenum contents, in ance, intestinal histomorphology, and
disease resistance, and growth performance agreement with the report by serum biochemistry of broiler chicken.
improvement (Hu et al., 2012). In the present Nourmohammadi et al. (2011a). However, Vet. Med. Int. 10:4061-4067.
study, supplementation with OA increased the dietary treatments did not affect the pH of the Ao, T., Cantor, A.H., Pescatore, A.J., Ford, M.J.,
villus length, the crypt depth and the villus crop, jejunum, ileum, and rectum, in agree- Pierce, J.L., Dawson, K.A., 2009. Effect of
height to crypt depth ratio at the duodenum ment with results reported by Smulikowska et enzyme supplementation and acidification
and jejunum segments, but had no effect on al. (2010). It has been known that little dietary of diets on nutrient digestibility and
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in enterocytes development and villi structure experiment regarding the bone mineralization Chemists, Gaithersburg, MD, USA.
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