Livestock Science 204 (2017) 1–6

Contents lists available at ScienceDirect

Livestock Science
journal homepage: www.elsevier.com/locate/livsci

Effects of pyrimidine nucleosides on growth performance, gut morphology, MARK

digestive enzymes, serum biochemical indices and immune response in
broiler chickens
⁎
A. Daneshmanda,b, H. Kermanshahia, , M. Danesh Mesgarana, A.J. Kingb, S.A. Ibrahimc
a
Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran
b
Department of Animal Science, University of California, Davis, CA 95616, United States
c
Food and Nutritional Science Program, North Carolina A & T State University, Greensboro, NC 27411, United States

A R T I C L E I N F O A B S T R A C T

Keywords: The present study was carried out to evaluate the effects of pyrimidine nucleosides on growth, intestinal mor-
Broiler phology, digestive enzymes and humoral immunity in broilers from 0 to 21 days of age. A total number of 360
Pyrimidine nucleosides day old chicks (Cobb 500) was randomly divided into 4 treatments with 6 replications. Treatments were com-
Growth performance prised of a corn-soybean meal based control diet and diets containing 0.1% pure cytidine, 0.1% pure uridine and
Gut morphology
0.1% equal amounts of pure cytidine (0.05%) and uridine (0.05%). On days 11 and 21, two birds per cage (12
Immunity
birds per treatment) were euthanized to obtain samples of serum, intestine, bursa and spleen. The combination
Digestive enzyme
of cytidine plus uridine increased (P < 0.05) body weight and average daily gain of broilers. Supplementing
cytidine plus uridine increased (P < 0.05) villus height and width along with activities of alkaline phosphatase
and aminopeptidase; however, maltase was not affected by the experimental diets. The combination of cytidine
and uridine increased (P < 0.05) the relative weight of the bursa of Fabricius and IgA activity in the jejunum, but
there was no significant difference among treatments regarding the relative weight of the spleen. In conclusion,
the study clearly indicated that the combination of cytidine and uridine could improve health status and per-
formance of broilers.

1. Introduction
Nucleotides are ubiquitous low-molecular-weight compounds that
play crucial roles in diverse biological processes such as encoding ge-
netic information, mediating energy metabolism, and serving as coen-
zymes (Carver, 1999). De novo and salvage pathways are two biosyn-
thetic pathways which provide nucleotides required by cells. Although
these pathways can provide nucleotides for the maintenance of host
cells, such pathways are metabolically costly processes requiring energy
sources and amino acids (Zollner, 1982; Carver, 1999). On the other
hand, some cells such as the epithelial cells of the intestine and the
immune system cells have little ability to synthesize nucleotides
through biosynthetic pathways and depend on exogenous sources
(Grimble and Westwood, 2001). In addition, the nucleotide require-
ment of cells increases under special conditions such as rapid growth,
limited nutrient intake, diseases, and enhancement of the immune re-
sponses (Grimble and Westwood, 2001; Sauer et al., 2011). In a pre-
vious study, Sauer et al. (2011) showed that fast growing animals re-
quire exogenous nucleotides (combination of pure nucleotides) to
provide rapid growth rate of organs such as the intestine and immune
system which have a low nucleotides biosynthesis capacity while
playing important roles in rapid growth process during early life.
Domeneghini et al. (2004) showed that dietary nucleotides (0.05%
yeast commercial product) enhanced villus height and width and other
morphometric characteristics of various segments of the intestine re-
sulting in the development of entrocytes. Deng et al. (2005) evaluated
the effects of dietary yeast RNA (0, 5, and 10 g/kg diet) on various
immunological parameters in Leghorn layers and demonstrated that
yeast RNA could improve humoral and cellular immune response
during the experiment, but this result was unsustainable over longer
periods.
Most previous studies investigating the effects of nucleotides on
broiler performance and immunity were based on the supplementation
of yeast extract as a source of nucleotides whereas Sauer et al. (2012)
and Jung and Batal (2012) indicated that yeast extract (0.25% Torula
yeast) contains various amounts of nucleotides along with other nu-
trients like amino acids, vitamins, viable cells and cell wall components
which can confound the interpretation of the results obtained relative

⁎
Corresponding author.
E-mail addresses: kermansh@um.ac.ir, hassbird@yahoo.com (H. Kermanshahi).

http://dx.doi.org/10.1016/j.livsci.2017.08.005
Received 15 February 2017; Received in revised form 2 August 2017; Accepted 4 August 2017
1871-1413/ © 2017 Elsevier B.V. All rights reserved.
A. Daneshmand et al. Livestock Science 204 (2017) 1–6

to the role of specific nucleotides. In fact, dietary nucleotides require to Table 1

be enzymically hydrolyzed to nucleosides and bases which are the only Composition of diets (as fed basis).a
forms absorbed in the intestine (Quan and Uauy, 1991). Nucleosides are
Ingredients (g/kg) Starter (0–10 d) Grower (11–21 d)
absorbed into the enterocyte by facilitated diffusion through Na-de-
pendent channels (Bronk and Hastewell, 1987). More recently, we ex- Corn 598.0 629.2
amined the effects of purine and combinations of purine and pyrimidine Soybean meal (441 g/kg CP) 338.3 299.9
Soybean Oil 22.3 32.4
nucleosides on various parameters in broilers (Daneshmand et al.,
Dicalcium phosphateb 17.8 16.6
2017a, 2017b). The results of these experiments showed that adenosine Limestonec 8.0 7.4
(Daneshmand et al., 2017a) and combination of adenosine + cytidine Salt 3.7 3.7
+ uridine (Daneshmand et al., 2017b) improved growth performance, DL-Methionine 3.3 2.8
L-Lysine 2.8 2.4
intestinal morphology and enzyme secretion and immune function in
L-Threonine 0.8 0.6
broilers. Pyrimidine nucleosides are a group of non-protein nitrogenous
Vitamin Premixd 2.5 2.5
compounds that primarily include cytidine and uridine which play Mineral Premixe 2.5 2.5
important roles in the biochemical processes such as components of
Calculated value
DNA and RNA as well as of coenzymes (D’Mello, 1982; Zollner, 1982). Metabolizable energy (MJ/kg) 12.6 12.9
Although previous studies worked on the effects of yeast extract as a Crude protein (g/kg) 210.6 190.0
source of nucleotides in poultry, the effects of pure pyrimidine nu- Available Phosphorus (g/kg) 4.5 4.2
cleosides have not been investigated, to the best of our knowledge. Calcium (g/kg) 9.0 8.4
Sodium (g/kg) 1.6 1.6
Therefore, the current study was carried out to assess the effects of Methionine (g/kg) 6.4 5.7
pyrimidine nucleosides on the growth performance, morphology and Methionine + Cysteine (g/kg) 9.8 8.9
maturity of intestinal cells, serum biochemical indices and immune Lysine (g/kg) 13.2 11.9
response in broiler chickens. Arginine (g/kg) 13.8 12.6
Threonine (g/kg) 8.6 7.8

2. Materials and methods Analyzed value

Crude protein (g/kg) 209.8 190.6
Calcium (g/kg) 8.8 8.6
2.1. Animals, treatments, housing and experimental design Total phosphorous (g/kg) 6.2 5.7

Three hundred and sixty one-day-old male chicks (Cobb 500) were
obtained from a local commercial hatchery (Foster Farms, Ellenwood,
CA, USA). All birds were raised in brooder battery cages with woven
wire floors (Petersime Inc., Gettysburg, OH, USA). The temperature was
set at 35 °C for the first 3 d and then gradually decreased by 3 °C per
week until it reached 29 °C and was maintained at that level until the
end of the experiment (d 21). Feed and water were provided ad libitum
throughout the experiment and a 23L:1D lighting program was applied.
Birds were weighed on arrival and assigned to 4 treatments of 6 re-
plications with 15 birds in each replication. Briefly, from d 0 to 21,
birds were fed diets as follows: 1) a corn-soybean meal control diet; 2)
control diet supplemented with 0.1% pure uridine; 3) control diet
supplemented with 0.1% pure cytidine; 4) control diet supplemented
with a 0.1% equal combination of pure uridine and cytidine. Pure
pyrimidine nucleosides were donated by Chemoforma AG (Augst,
Switzerland) and added to the diets at the expense of corn. All diets
were in mash form and formulated to be isonitrogenous and iso-
energetic and also meet or exceed the minimum requirements of Cobb
500 (Cobb-Vantress, 2012; Table 1). All experimental procedures were
approved by the University of California-Davis Institutional Animal
Care and Use Committee.
a
Pure nucleosides (1 g/kg) were added as a replacement for maize.
b
Dicalcium phosphate contained 210 g/kg calcium and 180 g/kg phosphorus.
c
Limestone contained 380 g/kg calcium.
d
Vitamin premix per kilogram contained vitamin A, 9000 IU; vitamin D3, 2000 IU;
vitamin E, 18 IU; vitamin K3, 2 mg; vitamin B1, 1.8 mg; vitamin B2, 6.6 mg; vitamin B3,
10 mg; vitamin B5, 30 mg; vitamin B6, 3 mg; vitamin B9, 1 mg; vitamin B12, 0.015 mg;
biotin, 0.1 mg; choline chloride, 250 mg; antioxidant, 100 mg.
e
Mineral premix per kilogram contained Mn, 100 mg; Zn, 84.7 mg; Fe, 50 mg; Cu,
10 mg; I, 1 mg; Se, 0.2 mg.
Franklin Lakes, NJ, USA) and centrifuged at 2000×g for 15 min at 4 °C
to obtain serum samples in the same day. The serum samples were
stored at − 20 °C prior to analysis. Selected birds were then euthanized
by CO2 asphyxiation, the viscera was excised, and the intestine was
removed and carefully cleaned of adherent materials. The jejunal seg-
ment was separated and gently squeezed to empty digesta content. The
bursa of Fabricius and spleen were collected to obtain relative weights
of these organs as the immunological indices.
2.4. Intestinal morphology

2.2. Growth parameters
On days 10 and 21, BW and feed consumption of each cage were
recorded to calculate the average daily gain (ADG), average daily feed
intake (ADFI) and feed conversion ratio (FCR). Mortality for each cage
was recorded daily in order to modify the abovementioned parameters
accordingly. The average daily mortality was less than 1% (2 from 360
chicks).
2.3. Feed analysis and sample collection
The basal diet was analyzed for CP, calcium, and total phosphorus
according to AOAC (2003) procedures.
Two birds from each cage (12 birds per treatment) were randomly
selected at 11 and 21 days of age. Blood samples (5 ml) were collected
from a cardiac puncture of selected birds (without applying fasting)
using Vacutainer tubes and a One-Use Holder (Becton Dickinson,
About 2 cm of the same position of mid-jejunal samples were stored
in a 10% formaldehyde phosphate buffer for 48 h. The sample was then
embedded in paraffin, fixed on microtome (Leica HI1210, Leica
Microsystems Ltd., Wetzlar, Germany), sliced to a thickness of 3 µm,
mounted on a slide and dehydrated on a hotplate (Leica ASP300S, Leica
Microsystems Ltd., Wetzlar, Germany). Then, the prepared sample was
dyed with hematoxylin and eosin (Leica Autostain BRXL, Leica
Microsystems Ltd., Wetzlar, Germany), and examined under a micro-
scope (Olympus BX41, Olympus Corporation, Tokyo, Japan). A total of
9 slides were prepared from each jejunal segment per bird, and 10 in-
dividual well-oriented villi were measured per prepared slide (90 villi
per bird). The average of slide measurements per sample was expressed
as a mean for each bird. Villus width (VW) was measured at the base of
each villus; villus height (VH) was measured from the top of the villus
to the villus-crypt junction, crypt depth (CD) was measured from the
base of the adjacent villus to the sub-mucosa, and the ratio of VH to CD
was calculated.

2
A. Daneshmand et al. Livestock Science 204 (2017) 1–6

2.5. Digestive enzymes
Holmes and Lobley (1989) demonstrated that the activity of brush
border enzymes indicates the maturity of epithelial cells. In addition,
Martínez del Rio (1990) showed that using tissue homogenates rather
than mucosal samples can help to avoid underestimation of enzyme
activity. Thus, about 2 cm of mid-jejunum was cut, emptied through
washing with normal saline (9 g/L Sodium Chloride), frozen in liquid
nitrogen and stored at − 80 °C. The enzyme activity in the jejunum
samples was determined based on the method described by
Daneshmand et al. (2017a, 2017b). In brief, jejunal samples were
thawed at 4 °C and homogenized in 10 volumes of cold normal saline.
Then, the homogenates were centrifuged at 15,000×g for 15 min at
4 °C and the supernatant was collected for enzyme assays. Before de-
termination, the protein content of supernatants was measured by the
BCA assay (Thermo Fisher Scientific, Rockford, IL). Aminopeptidase-N
(EC 3.4.11.2) activity was measured at 450 nm with L-alanine-4-ni-
troanilide in Na2HPO4, (40 mM pH 7.0). Alkaline phosphatase activity
(EC 3.1.3.1) was determined by colorimetric assay with a commercial
assay kit (MyBioSource, San Diego, CA, USA) using an ELISA reader
(Perkin-Elmer, Shelton, CT, USA). Maltose (250 mM) was dissolved in
sodium maleate buffer (62.5 mM, pH 6.0) and used as a substrate for
maltase (EC 3.2.1.20) activity which was spectrophotometrically de-
termined at 450 nm. Enzyme activity was expressed as µmol/mg pro-
tein/min.
2.6. Serum metabolites
The concentrations of triglycerides (TG) (Fisher Diagnostics,
Middletown, VA, USA), cholesterol (TC) (AbCam, Cambridge, MA,
USA), high-density lipoproteins (HDL) (AbCam, Cambridge, MA, USA)
and uric acid (UA) (Fisher Diagnostics, Middletown, VA, USA) in serum
samples were analyzed using commercial assay kits according to the
manufacturer's instructions. The intra CV% was evaluated for each
plate and obtained CVs were less than 10%.
2.7. Immune response
The mucus samples of jejunum was collected based on the method
described by Tsirtsikos et al. (2012). Briefly, about 5 cm of mid-jejunal
sample were longitudinally opened and carefully cleaned of the luminal
content. Then, the tissue was rinsed 3 times with cold phosphate-buf-
fered saline (PBS) and a glass slide was gently pulled on luminal surface
to obtain mucosal scrapings. Next, mucus sample was mixed with 5 mM
EDTA and subsequently homogenized. Finally, mucus sample was
centrifuged at 15,000×g for 15 min at 4 °C and the supernatant con-
taining mucus was collected. The concentrations of immunoglobulin G
(IgG) and immunoglobulin A (IgA) in the mucus were determined ac-
cording to the procedures provided in the ELISA kit (Bethyl Labora-
tories Inc., Montgomery, TX, USA) with goat anti-chicken IgG and IgA,
respectively. The absorbance wavelength for both procedures was
450 nm. The concentrations of IgG and IgA were presented as micro-
grams per milliliter of mucosa. The intra CV% was measured for IgA
and IgG, separately and the data showed that CVs were less than 10%.
2.8. Statistical analysis
All data were analyzed in a completely randomized design by
ANOVA using the General Linear Model (GLM) procedure of SAS
Institute (2004). Tukey's test was used to compare differences among
means of treatments and P values less than 0.05 were considered to be
significant.
3. Results
3.1. Growth performance
Table 2 presents the effects of pyrimidine nucleosides on perfor-
mance measurements including BW, average daily gain (ADG), average
daily feed intake (ADFI) and FCR. Experimental diets did not affect
performance parameters during the starter period (1–10 days). Sup-
plementing diets with a combination of cytidine and uridine increased
(P < 0.05) BW and ADG and improved (P < 0.05) FCR as compared to
the control group at 11–21 days of age. The effect of dietary pyrimidine
nucleosides on growth parameters at whole period of experiment (d
0–21) showed that birds receiving cytidine plus uridine in their diets
had higher (P < 0.05) BW and ADG compared to control birds, while
other treatments did not affect performance parameters at the same
time.
3.2. Intestinal morphology and digestive enzymes
Table 3 summarizes the effects of pyrimidine nucleosides on various
histological measurements of the jejunum. Villus width of birds re-
ceiving combination of cytidine and uridine increased (P < 0.05) as
compared to those of birds in the control group at 11 days of age. The
incorporation of cytidine plus uridine to the broilers’ diet increased
(P < 0.05) VH, VW and VH/CD compared to control diets at day 21,
while other treatments had no significant effect on morphological
parameters.
In order to investigate the results of pyrimidine nucleosides on the
maturation of villi, the activity of digestive enzymes secreted from villi
were measured (Table 4). Dietary pyrimidine nucleosides did not affect
the activity of intestinal enzymes at sampling day 11, whereas birds
receiving diets supplemented with combination of cytidine plus uridine
exhibited a higher (P < 0.05) alkaline phosphatase and aminopeptidase
activity in jejunal samples compared to control birds at 21 days of age.
No significant differences were observed for maltase activity at sam-
pling day 21.

Table 2
Effects of pyrimidine nucleosides on growth performance of broiler chickens from 0 to 21 days of age.

Treatment BWa (g) ADG (g/b/d) ADFI (g/b/d) FCR (g/g)

10 21 0–10 11–21 0–21 0–10 11–21 0–21 0–10 11–21 0–21

b b b a
Control 293 784 25.3 50.5 37.3 26.7 71.3 50.8 1.06 1.42 1.36
Cytidineb 284 805ab 24.4 53.7ab 38.3ab 27.3 72.8 52.3 1.12 1.35ab 1.36
Uridine 292 832ab 25.2 55.5ab 39.6ab 26.6 70.9 50.4 1.05 1.27ab 1.26
Cytidine + Uridine 297 851a 25. 7 56.9a 40.5a 26.1 69.7 49.2 1.02 1.22b 1.21
SEMc 2.4 9.6 0.27 0.89 0.46 0.45 1.21 1.21 0.019 0.027 0.029
P-value 0.428 0.046 0.428 0.042 0.039 0.852 0.852 0.852 0.351 0.039 0.185

a-b
Values within a column with different letters differ significantly (P < 0.05).
a
BW: body weight; ADG: average daily gain; ADFI: average daily feed intake; FCR: feed conversion ratio; g/b/d/: grams/bird/day.
b
Cytidine (0.1%), uridine (0.1%) and cytidine (0.05%) + uridine (0.05%) were added to diets in expense of corn.
c
SEM: standard error of means (results are given as means of 6 pens of 15 birds in each pen).

3
A. Daneshmand et al. Livestock Science 204 (2017) 1–6

Table 3
Effects of pyrimidine nucleosides on gut morphology (µm) of broiler chickens at 11 and 21 days of age.

Treatment Day 11 Day 21

VHa VW CD VH/CD VH VW CD VH/CD

Control 868.7 201.2b 248.2 3.57 1148.4b 137.9c 213.8 5.40b

Cytidineb 902.0 211.9b 231.4 4.05 1177.1b 154.5bc 229.9 5.27b
Uridine 899.8 237.1ab 203.7 4.43 1256.8ab 158. 8bc 179.2 6.59ab
Cytidine + Uridine 909.9 269.1a 203.3 4.54 1343.8a 202.6a 209.4 7.070a
SEMc 8.45 8.82 9.14 0.187 28.81 8.33 9.19 0.290
P-value 0.352 0.011 0.232 0.263 0.041 0.013 0.279 0.038

a-c
Values within a column with different letters differ significantly (P < 0.05).
a
VH: villus height; VW: villus width; CD: crypt depth; VH/CD: the ratio of villus height to crypt depth.
b
Cytidine (0.1%), Uridine (0.1%) and Cytidine (0.05%) + Uridine (0.05%) were added to diets in expense of corn.
c
SEM: standard error of means (results are given as means (n = 12) for each treatment).

3.3. Serum profile and immunological parameters
The effect of dietary pyrimidines on serum profile is shown in
Table 5. Cytidine plus uridine increased (P < 0.05) concentration of
HDL compared to the control group, while the concentration of other
lipid particles and uric acid were not affected by dietary treatments at
11 days of age. Diets containing cytidine with uridine reduced
(P < 0.05) concentrations of TC and increased (P < 0.05) the con-
centration of HDL and UA in serum samples in comparison to those of
the control diets at 21 days of age.
Table 6 summarizes the results for the relative weights of immune
organs. Dietary treatments did not affect the relative weight of the
spleen during the entire experimental period. The combination of cy-
tidine and uridine increased (P < 0.05) the relative weight of the Bursa
of Fabricius compared to the control group at sampling days 11 and 21.
In order to examine the effects of nucleosides on broilers’ immunity,
the concentration of immunoglobulin was examined in the jejunal
mucus (Table 7). Dietary treatments did not affect the concentration of
IgG in the jejunal mucus at days 11 and 21. Birds fed diets containing a
combination of cytidine with uridine showed an increased (P < 0.05)
concentration of IgA in the jejunal mucus in comparison to control birds
at sampling days 11 and 21.
4. Discussion
The overall performance results showed that the combination of
uridine and cytidine increased (P < 0.05) BW and ADG at whole ex-
perimental period and improved FCR at grower interval (d 11–21).
Although there is no other data reporting the effects of pyrimidine
nucleosides on chicken growth parameters, some inconsistencies were
reported on the effects of nucleotides. Jung and Batal (2012) added
nucleotides originated from yeast to broilers diet and demonstrated that
nucleotides did not influence growth performance. Other investigators
(Esteve-Garcia et al., 2007; Superchi et al., 2012) demonstrated that
adding nucleotides could beneficially affect productive performance
such as body weight, weight gain and feed intake. Different environ-
mental and management reasons such as animal model, age, strain,
type of challenge and stressors and even source of nucleotides might
explain these inconsistencies observed by other researchers. To clarify
the latter factor, previous studies evaluated the effects of yeast extract
as a source of nucleotides in animals, whereas Sauer et al. (2012) and
Jung and Batal (2012) revealed that yeast extract comprises the mixture
of nucleotides along with amino acids, minerals, vitamins and viable
cells and cell wall components which can interfere with the specific
effect of nucleotides.
An investigation of the literature reveals no well-documented
reason(s) for enhanced growth in birds receiving the combination of
uridine and cytidine compared to the control group; however, two
hypotheses are posited to explain the results. One hypothesis is related
to the cumulative effects of exogenous uridine and cytidine on the in-
hibition of de novo pathway (D’Mello, 1982; Zollner, 1982). Because the
main components of the de novo pathway are various amino acids such
as glutamine, aspartate and glycine (Zollner, 1982), the blockage of the
pathway in epithelial cells by exogenous uridine plus cytidine might
induce an amino acid sparing effect. The importance of amino acid
sparing effects of the combination of uridine and cytidine becomes
more prominent due to the fact that about 1% of the glutamine flux is
absorbed by the de novo pathway (Grimble, 1996).
The second hypothesis points to the beneficial effects of the com-
bination of uridine and cytidine on gut morphology. The results showed
that the uridine plus cytidine treatment increased VH and VW in the
jejunum of birds at the end of the experiment which is in agreement
with previous investigations (Moore et al., 2011; Jung and Batal, 2012).
Sauer et al. (2011) demonstrated that the addition of nucleotides to
pigs’ diet increased VH and CD. The growth rate in broilers depends
mainly on the amount of various nutrients absorbed by the intestine
(Sauer et al., 2011). The villi in the intestine play an important role in
the absorption of nutrients from the intestinal lumen (Sato et al., 1999),

Table 4
Effects of pyrimidine nucleosides on the digestive enzymes (µmol/mg protein/min) in the jejunum samples of broiler chickens at 11 and 21 days of age.

Treatment Day 11 Day 21

Alkaline phosphatase Aminopeptidase Maltase Alkaline phosphatase Aminopeptidase Maltase

b b
Control 238.0 3.2 89.3 183.9 2.9 87.5
Cytidinea 244.4 3.3 90.9 196.9ab 3.1ab 90.1
Uridine 250.5 3.5 98.7 191.2ab 3.4ab 93.0
Cytidine + Uridine 299.9 3.5 99.9 214.7a 3.7a 95.1
SEMb 17.02 0.08 2.91 4.85 0.12 1.66
P-value 0.622 0.653 0.524 0.039 0.042 0.439

a-b
Values within a column with different letters differ significantly (P < 0.05).
a
Cytidine (0.1%), Uridine (0.1%) and Cytidine (0.05%) + Uridine (0.05%) were added to diets in expense of corn.
b
SEM: standard error of means (results are given as means (n = 12) for each treatment).

4
A. Daneshmand et al. Livestock Science 204 (2017) 1–6

Table 5
Effects of pyrimidine nucleosides on serum metabolites (mg/dl) of broiler chickens at 11 and 21 days of age.

Day 11 Day 21

Treatment TGa TC HDL Uric acid TG TC HDL Uric acid

Control 116.4 157.9 80.3b 6.0 161.3 137.9 80.7b 6.6b

Cytidineb 108.9 145.5 88.5ab 6.4 121.9 134.8 88.0ab 7.1ab
Uridine 90.8 133.3 100.5ab 6.9 116.3 122.2 89.3ab 8.1ab
Cytidine + Uridine 94.5 138.0 111.3a 7.0 106.9 130.1 99.2a 9.4a
SEMc 4.46 5.67 4.75 0.36 11.53 3.90 2.73 0.46
P-value 0.124 0.498 0.046 0.778 0.076 0.575 0.020 0.033

a-b
Values within a column with different letters differ significantly (P < 0.05).
a
TG: triglyceride; TC: total cholesterol; HDL: high density lipoprotein.
b
Cytidine (0.1%), Uridine (0.1%) and Cytidine (0.05%) + Uridine (0.05%) were added to diets in expense of corn.
c
SEM: standard error of means (results are given as means (n = 12) for each treatment).

jejunal alkaline phosphatase. Results of other studies (Sauer et al.,

Table 6
Effects of pyrimidine nucleosides on the relative weight (% organ weight to body weight) 2012) have shown that supplementing nucleotides into weaning pigs
of immune organs of broiler chickens at 11 and 21 days of age. did not influence intestinal enzymes, which is inconsistent with the
findings of the present study and might be due to the type of additives
Treatment Day 11 Day 21 (pure nucleosides vs. yeast derivatives) and animal model (chicks vs.
Spleen Bursa Spleen Bursa
pigs). Sanchez-Pozo et al. (1994) demonstrated that the maturity of
epithelial cells in the intestine leads to higher secretion and activity of
Control 0.091 0.122b 0.059 0.157c brush border enzymes containing alkaline phosphatase, aminopepti-
Cytidinea 0.101 0.128ab 0.061 0.169bc dase and maltase. Because the inclusion of uridine and cytidine en-
Uridine 0.102 0.137ab 0.063 0.172bc
Cytidine + Uridine 0.113 0.148a 0.064 0.195a
hanced VH and VW, it can be postulated that uridine plus cytidine
SEMb 0.0060 0.0041 0.0012 0.0055 might also support the growth, development and maturity of epithelial
P-value 0.696 0.034 0.562 0.026 cells and pertinent brush borders as shown by the higher secretion and
activity of brush border enzymes in broilers fed uridine plus cytidine.
a-c
Values within a column with different letters differ significantly (P < 0.05). Dietary treatments had no effect on TC and TG, while combination
a
Cytidine (0.1%), Uridine (0.1%) and Cytidine (0.05%) + Uridine (0.05%) were
of uridine with cytidine increased HDL and UA in the serum samples.
added to diets in expense of corn.
b
SEM: standard error of means (results are given as means (n = 12) for each treat- Yousefi et al. (2012) reported that TC and TG were not influenced by
ment). dietary nucleotides, which is in agreement with the current findings. In
addition, the higher concentration of HDL in birds supplemented with
uridine plus cytidine may be explained through the results of study
Table 7
conducted by Sanchez-Pozo et al. (1994) who suggested that dietary
Effects of pyrimidine nucleosides on the concentration of immunoglobulin (µg/ml) in the
jejunal mucus of broiler chickens at 11 and 21 days of age. nucleotides are taken up by the intestinal cells in order to synthesize
DNA and RNA in the epithelial cells. The higher concentration of nu-
Day 11 Day 21 cleotides may result in an increased apo synthesis and the production of
a
phospholipids through increasing the availability of diphosphate deri-
Treatment Ig A Ig G Ig A Ig G
vatives which subsequently improves apolipoprotein A-I (apoA-I),
Control 76.3 c
371.8 61.3 b
322.0 which is the main component of HDL (Sanchez-Pozo et al., 1994).
Cytidineb 94.3bc 343.0 73.2ab 323.6 It is well-documented that surplus amounts of pyrimidine nucleo-
Uridine 89.3bc 373.8 76.2ab 328.5 side can convert to UA and be transferred through the bloodstream to
Cytidine + Uridine 116.0a 390.4 81.8a 338.6
SEMc 5.15 21.45 3.27 13.40
the liver for further metabolism and provide more nucleosides for he-
P-value 0.005 0.918 0.045 0.980 patocytes (Grimble and Westwood, 2001). Thus, it can be hypothesized
that the concentration of nucleosides absorbed by the enterocytes were
a-c
Values within a column with different letters differ significantly (P ≤ 0.05). more than the amount needed, the extra nucleosides were converted
a
Ig A: immunoglobulin A; Ig G: immunoglobulin G. into a form that could be transported (i.e. UA) and were released into
b
Cytidine (0.1%), Uridine (0.1%) and Cytidine (0.05%) + Uridine (0.05%) were
the bloodstream to be carried to the liver for further metabolism.
added to diets in expense of corn.
c
SEM: standard error of means (results are given as means (n = 12) for each treat- The effects of dietary treatments on the immune system showed that
ment). the inclusion of uridine plus cytidine increased the relative weight of
Bursa and improved IgA concentration in the jejunum. Sauer et al.
so the number and structure of villi are the fundamental factors in (2012) reported that supplementing pig diet with combination of pure
growth performance. The development of villi, and subsequently epi- nucleotides increased the concentration of Ig A which is in consistent
thelial cells, depends on the sufficiency of genomic materials such as with the present findings. The immune system and subset organs like
nucleosides in nucleotide pools to produce DNA and RNA for more the Bursa of Fabricius protect the body against a wide range of chal-
growth (Sato et al., 1999). Therefore, it is the combination of uridine lenges and pathogens which drastically increase the cell turnover rate
and cytidine that might support the high demand for genetic materials, of immune system. Zhou et al. (2015) demonstrated that different cells
improve villi and epithelial cells development, enhance nutrient ab- such as macrophages, epithelial cells, lymphocytes, and plasma cells
sorption and consequently improve the growth performance in birds as exist in the Bursa and need sufficient amounts of DNA and RNA for
explained above. maintenance and growth. In addition, Hess and Greenberg (2012) re-
The enzyme activity increased in the intestine of birds supple- ported that an exogenous source of nucleosides can be absorbed from
mented with combination of uridine plus cytidine compared to the birds the intestinal lumen and migrate into immune organs such as the Bursa.
received control diet. Similar results were reported by Lee et al. (2007) This migration is critical for the growth and maturation of immune
who showed that the inclusion of nucleotides in the diet increased system cells (Hess and Greenberg, 2012). Thus, the high relative weight

5
A. Daneshmand et al.
of the Bursa might be due to the transfer of exogenous uridine and
combined cytidine to provide sufficient nucleosides for the synthesis of
more cells in this organ.
The activity of IgA in the gut is considered as an important factor in
intestinal mucosal immunity, because IgA stretches a supportive layer on
the mucosal layer of the intestine and protects this layer from various
pathogenic invaders (Sauer et al., 2012). Lymphocytes are important
members of the immune system that require nucleosides for their
maintenance and proliferation (Jyonouchi, 1994). Dietary nucleosides
stimulate the production of cytokines from lymphocytes such as inter-
feron-γ, TNF-α and IL-2 (Yamauchi et al., 1996) that differentiate in-
testinal B cells into plasma cells which are responsible for the production
and secretion of IgA on the mucosal membrane of the gut that the se-
cretory IgA enables further protection against pathogens (Sauer et al.,
2012; Zhou et al., 2015). These findings are in agreement with the results
of the present study in which combination of uridine with cytidine in-
creased the relative weight of the bursa and secreted IgA.
In conclusion, the present results have shown that a combination of
pyrimidine nucleosides could improve growth performance, intestinal
morphology and enzyme activity in broilers. The lipid profile, IgA ac-
tivity and relative weight of Bursa were enhanced in chicks receiving
uridine plus cytidine in their diets. Therefore, supplementing broilers’
diet with uridine plus cytidine enhanced economically important at-
tributes such as performance, intestinal health and immunity in broi-
lers. Because the present study was carried out under strictly controlled
sanitary conditions, further work is warranted in order to examine the
proficiency of pyrimidine nucleosides under more challenging and au-
thentic poultry farm conditions.
Conflict of interest
Authors declared that there is no conflicts of interest.
Acknowledgments
The authors would like to thank Ferdowsi University of Mashhad,
Iran, for its financial support of this project. The authors appreciate
Sadia Nasim and Ketwee Saksrithai (graduate students in Dr. King's
laboratory) for their assistance, James Graham (research assistance at
the central laboratory in the Department of Nutrition, UC Davis) for his
technical assistance, researchers in the Department of Animal Science
for discussions and advice and Dr. Klaus Hoffmann (Chemoforma Co.)
for providing pure nucleosides.
References
AOAC, 2003. Official Methods of Analysis, 17th ed. Assoc. Off. Anal. Chem.,
Arlington, VA.
Bronk, J.R., Hastewell, J.G., 1987. The transport of pyrimidines into tissue rings cut from
rat small intestine. J. Physiol. 382, 475–488.
Carver, J.D., 1999. Dietary nucleotides: effects on the immune and gastrointestinal sys-
tems. Acta Peadiatr. Suppl. 430, 83–88.
Cobb-Vantress, 2012. Cobb 500 Broiler Management Guide. Blueprint for Success. Cobb-
Vantress, Siloam Springs,, AR, USA.
D’Mello, J.P.F., 1982. Utilization of dietary purines and pyrimidines by non-ruminant
animals. Proc. Nutr. Soc. 41, 301–308.
Livestock Science 204 (2017) 1–6
Daneshmand, A., Kermanshahi, H., Danesh Mesgaran, M., King, A.J., Klasing, K.C., 2017a.
Combination of purine and pyrimidine nucleosides influences growth performance,
gut morphology, digestive enzymes, serum biochemical indexes and immune func-
tion in broiler chickens. Anim. Feed Sci. Technol. 228, 186–193.
Daneshmand, A., Kermanshahi, H., King, A.J., Ibrahim, S.A., 2017b. Effect of pure purine
nucleosides on growth performance, gut morphology, digestive enzymes, serum li-
pids and immune response in broiler chickens. Br. Poult. Sci. http://dx.doi.org/10.
1080/00071668.2017.1335859.
Deng, K., Wong, C.W., Nolan, J.V., 2005. Carry-over effects of dietary yeast RNA as a
source of nucleotides on lymphoid organs and immune responses in Leghorn-type
chickens. Br. Poult. Sci. 46, 673–678.
Domeneghini, C., Di Giancamillo, A., Savoini, G., 2004. Structural patterns of swine ileal
mucosa following L-glutamine and nucleotide administration during the weaning
period. A histochemical and histometrical study. Histol. Histopathol. 19, 49–58.
Esteve-Garcia, E., Martinez, D., Borda, E., Chetrit, C., 2007. Efficacy of nucleotide pre-
paration in broiler chickens. In: 16th Euro. Sym. Poult. Nut. p. 511–514.
Grimble, G.K., 1996. Why are dietary nucleotides essential nutrients? Br. J. Nutr. 76,
475–478.
Grimble, G.K., Westwood, O.M., 2001. Nucleotides as immunomodulators in clinical
nutrition. Curr. Opin. Clin. Nutr. Metab. Care 4, 57–64.
Hess, J.R., Greenberg, N.A., 2012. The role of nucleotides in the immune and gastro-
intestinal systems: potential clinical applications. Nutr. Clin. Pract. 27, 281–294.
Holmes, R., Lobley, R.W., 1989. Intestinal brush border revisited. Gut 30, 1667–1678.
Jung, B., Batal, A.B., 2012. Effect of dietary nucleotide supplementation on performance
and development of the gastrointestinal tract of broilers. Br. Poult. Sci. 53, 98–105.
Jyonouchi, H., 1994. Nucleotide actions on humoral immune responses. Nutr. J. 124 (1
Suppl.), 138S–143S.
Lee, D.N., Liu, S.R., Chen, Y.T., Wang, R.C., Lin, S.Y., Weng, C.F., 2007. Effects of diets
supplemented with organic acids and nucleotides on growth, immune responses and
digestive tract development in weaned pigs. J. Anim. Physiol. Anim. Nutr. 91,
508–518.
Martínez del Rio, C., 1990. Dietary, phylogenetic, and ecological correlates of intestinal
sucrase and maltase activity in birds. Physiol. Zool. 63, 987–1011.
Moore, K.L., Mullan, B.P., Pluske, J.R., Kim, J.C., D'Souza, D.N., 2011. The use of nu-
cleotides, vitamins and functional amino acids to enhance the structure of the small
intestine and circulating measures of immune function in the post-weaned piglet.
Anim. Feed Sci. Technol. 165, 184–190.
Quan, R., Uauy, R., 1991. Nucleotides and gastrointestinal development. Sem. Pediar.
Gastr. Nutr. 2, 3–6.
Sanchez-Pozo, J., Morillas, L., Molt, R., Robles, D., Gil, A., 1994. Dietary nucleotides
influence lipoprotein metabolism in newborn infants A. Pediar. Res. 35, 112–116.
SAS Institute, 2004. SAS User Guide. Version 9.1. SAS Institute Inc., Cary, NC.
Sato, N., Nakano, T., Kawakami, H., Idota, T., 1999. In vitro and in vivo effects of exo-
genous nucleotides on the proliferation and maturation of intestinal epithelial cells. J.
Nutr. Sci. Vitaminol. 45, 107–118.
Sauer, N., Mosenthin, R., Bauer, E., 2011. The role of dietary nucleotides in single-
stomached animals. Nutr. Res. Rev. 24, 46–59.
Sauer, N., Eklund, M., Bauer, E., Gänzle, M.G., Field, C.J., Zijlstra, R.T., Mosenthin, R.,
2012. The effects of pure nucleotides on performance, humoral immunity, gut
structure and numbers of intestinal bacteria of newly weaned pigs. J. Anim. Sci. 90,
3126–3134.
Superchi, P., Saleri, R., Borghetti, P., De Angelis, E., Ferrari, L., Cavalli, V., Amicucci, P.,
Ossiprandi, M.C., Sabbioni, A., 2012. Effects of dietary nucleotide supplementation
on growth performance and hormonal and immune responses of piglets. Animal 6,
902–908.
Tsirtsikos, P., Fegeros, K., Balaskas, C., Kominakis, A., Mountzouris, K.C., 2012. Dietary
probiotic inclusion level modulates intestinal mucin composition and mucosal mor-
phology in broilers. Poult. Sci. 91, 1860–1868.
Yamauchi, K., Adjei, A.A., Ameho, C.K., Chan, Y.C., Kulkarni, A.D., Sato, S., Okamoto, K.,
Yamamoto, S., 1996. A nucleoside-nucleotide mixture and its components increase
lymphoproliferative and delayed hypersensitivity responses in mice. J. Nutr. 126,
1571–1577.
Yousefi, M., Abtahi, B., Abedian Kenari, A., 2012. Hematological, serum biochemical
parameters, and physiological responses to acute stress of Beluga sturgeon (Huso
huso, Linnaeus 1785) juveniles fed dietary nucleotide. Comp. Clin. Pathol. 21,
1043–1048.
Zhou, B., Liu, L., Liu, J., Yuan, F., Tian, E., Wang, H., 2015. Effect of diclazuril on the
bursa of fabricius morphology and SIgA expression in chickens infected with Eimeria
tenella. Korean J. Parasitol. 53, 675–682.
Zollner, N., 1982. Purine and pyrimidine metabolism. Proc. Nutr. Soc. 41, 329–342.