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The objective of this study was to investigate the anticoccidial effect of apple cider vinegar added to drinking water with the anticoccidial effect of amprolium to feed broiler chicken. The study has adopted an observational approach to evaluate the anticoccidial effect of apple cider vinegar on broiler chicken. The antioxidative changes were measured adding natural apple cider vinegar to drinking water. Four hundred and fifty broiler chickens were purchased from the local market and distributed into three groups (T+vc: positive control, T-vc: negative control Tv: apple cider vinegar) with 150 chickens in each group. The three groups were further replicated into 3 blocks each containing 50 chickens. The groups were fed balanced diet, amprolium was added to the feed of positive control group, and apple cider vinegar was added to the water of Tv group. Measurements of the different variables were started from week 3, at the end of each week 3 birds were chosen randomly, blood samples were collected via the wing vein, and fecal oocysts were counted from intestinal contents of each individual bird using the McMaster technique. Broiler in the control groups T+ve and T-ve showed clinical signs of coccidiosis (blood in feces) and the number of coccidial oocytes in feces increased with time. In the vinegar group, no clinical signs of coccidiosis were observed. Concentrations of total antioxidants and catalase enzyme activity significantly increased (p≤0.05); while malondialdehyde concentration significantly decreased (p≤0.05).
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Polish Journal of Veterinary Sciences Vol. 21, No. 2 (2018), 5–12
DOI 10.24425/122605
Original article
F.M.F. Hayajneh1,, M. Jalal1, H Zakaria1, A. Abdelqader1, M. Abuajamieh1
1 College of Agriculture, Dept. of Animal Production,
The Jordan University, Amman, Jordan,
Jordan University, Amman 11942 Jordan
Correspondence to: F.M.F. Hayajneh, e-mail: firashope@gmail.com, mobile: (+) 962778486050, fixed (+) 96 26 535 50 00,
ext. 223 82, fax (+) 96 26 530 08 06
Abstract
The objective of this study was to investigate the anticoccidial effect of apple cider vinegar
added to drinking water with the anticoccidial effect of amprolium to feed broiler chicken.
The study has adopted an observational approach to evaluate the anticoccidial effect of apple
cider vinegar on broiler chicken. The antioxidative changes were measured adding natural apple
cider vinegar to drinking water. Four hundred and fifty broiler chickens were purchased from the
local market and distributed into three groups (T+vc: positive control, T-vc: negative control Tv:
apple cider vinegar) with 150 chickens in each group. The three groups were further replicated
into 3 blocks each containing 50 chickens. The groups were fed balanced diet, amprolium was
added to the feed of positive control group, and apple cider vinegar was added to the water of
Tv group. Measurements of the different variables were started from week 3, at the end of each
week 3 birds were chosen randomly, blood samples were collected via the wing vein, and fecal
oocysts were counted from intestinal contents of each individual bird using the McMaster
technique. Broiler in the control groups T+ve and T-ve showed clinical signs of coccidiosis (blood
in feces) and the number of coccidial oocytes in feces increased with time. In the vinegar group,
no clinical signs of coccidiosis were observed. Concentrations of total antioxidants and catalase
enzyme activity significantly increased (p0.05); while malondialdehyde concentration signifi-
cantly decreased (p0.05).
Keywords: apple cider vinegar, broilers, coccidial oocytes, feed, diet, disease
Anticoccidial effect of apple cider vinegar
on broiler chicken: an organic treatment
to measure anti-oxidant effect
Introduction
Coccidiosis has been attributed to the loss of about
1 to 3 billion dollars annually, on the global record
within the poultry industry (Cardenas et al. 2017: Muth-
amilselvan et al. 2016). Avian coccidiosis is character-
ized as an infectious protozoan disease, caused by gut
parasites of the genus Eimeria (Muthamilselvan et al.
2016; Williams 2005). These parasites are transmitted
into the body via oral route and multiply within muco-
sal epithelia in different parts of the gastrointestinal
tract. As a result, they may lead to the gut damage along
with other conditions like inflammation, haemorrhage,
diarrhoea, morbidity, and mortality in poultry. This dis-
ease annually causes a global loss of over 2.4 billion US
dollars in the poultry industry, accompanied with
2F.M.F. Hayajneh et al.
setbacks of poor growth performance, replacement of
chicks, and medication (Quiroz-castaneda et al. 2015).
Different methods are currently being used to constrain
avian coccidiosis, which may include anticoccidial
chemicals, vaccines, and natural products. Anticoccidi-
al chemicals, coccidiocides, coccidiostats, and iono-
phores have been used as a conventional strategy to
control the avian coccidiosis in modern poultry produc-
tion (Ritzi et al. 2014; Amare et al. 2012). Moreover,
prophylactic in-feed medication for broilers and broiler
breeders are worldwide used as a treatment measure
against coccidiosis (Kitandu and Juranová 2006).
This strategy is cost-effective and successful; yet the
presence of drug resistance and public demands for
residue-free meat still encourage the development
for alternative control strategies (Amare et al. 2012).
The prophylactic use of anticoccidial chemicals in
European countries as feed additives has been regula-
ted strictly since 2006. Natural products are emerging
as an attractive way to control and manage the cocci-
diosis. Currently, there are at least four plant products,
which are available commercially in the market that can
be used as anticoccidial feed additives in chickens and/
or other animals (Muthamilselvan et al. 2016). The re-
sistance of avian coccidia to drugs has been increasing
dramatically. The limitation in the treatment and the
rising public concern about drug remains in chicken
meat have stimulated the search for new methods to
control coccidiosis (Lillehoj et al. 2007; Innes and Ver-
meulen 2006; Tan 2005). It has been evaluated that the
production of flavoured vinegar was initiated 5000
years back. The Babylonians have produced and traded
vinegars in the 6th century, along with different flavours,
which mainly include honey, malt, and fruit, medical
practitioners have mainly indicated that vinegar is help-
ful for treating various disorders, which include high
fever, stomach ache, croup, poison ivy, and oedema
(Budak et al., 2014). Therefore, the anticoccidial effect
of apple cider vinegar, added to broiler water, was mea-
sured in this study, along with the variations in the
blood antioxidant capacity induced by adding apple
cider vinegar to the broiler feed.
Materials and Methods
Study Setting
The research was conducted at the University of
Jordan. The birds were recruited from those which
were grown in an open house, on a floor system. An
observational study was conducted to investigate the
anticoccidial effects of apple cider vinegar on broiler
chicken, and measure the subsequent anti-oxidative
changes.
Birds
A total of 600 chicks of Lohmann broiler strain
were brought from local market. The birds used in this
experiment were free of coccidial infection. The tem-
perature of the skin was evaluated to investigate the
intensity of strain among chickens. The congruence
among temperature and skin patterns was perceived
through physiological observations. From 600 chickens,
a total of 450 chickens were observed with low tem-
perature and were considered to have strain. These
chickens were distributed into 3 groups (150 birds in
each group), which were further divided into 3 repli-
cates (50 birds in each). All animal handling procedures
were conducted in accordance with the guidelines, set
forth by the Jordanian Society for Animal Production.
Parasitological techniques of the infectious dose
Wet smears of the mucosa were prepared from in-
testinal and caecal scrapings for microscopic examina-
tion of Eimeria spp. oocysts. Eimeria spp. were identi-
fied according to the site of infection and oocyst
morphology including size, shape, and color after spor-
ulation according to Alnatour (2002). After collection
of the infectious dose a sample of the infectious dose
was investigated, 5 Eimeria spp. were identified inclu-
ding E. acervulina (10%), E. brunette (13 %), E. maxi-
ma, E. necatrix (12%)(12%), and E. tenella (57%).
Induced coccidiosis
The infectious coccidial oocysts (Eimeria spp.) were
isolated from intestines of naturally infected chickens
obtained from the local farms. The oocysts were sepa-
rated using sieving and sedimentation techniques
(Soulsby 1982). Oocytes were allowed to sporulate at
room temperature in 2.5% potassium dichromate solu-
tion. The sporulated oocysts were cleared and counted
per 1.0 ml of the solution using the McMaster tech-
nique (Soulsby 1982). The oocysts were administered
orally to 14 day-old birds and the dose of infectious coc-
cidian oocytes was 3x105/bird, given via oral administra-
tion (Arabkhazaeli et al. 2011). In Mc Master Method,
the faeces were mixed with sodium chloride solution,
which were then strained using sieve with an aperture
of 1mm. The resultant solute was transferred to the
McMaster slide and left for 5 minutes. Coccidiosis was
induced on 14th day orally (3x105 sporulated oocysts/
bird).
Apple cider vinegar
Apple Cider Vinegar (ACV) was used by organic
broiler producers. It was made by crushing apples and
3
Anticoccidial effect of apple cider vinegar on broiler ...
squeezing out the liquid. Bacteria and yeast were added
to the liquid to start the alcoholic fermentation process.
During alcoholic fermentation process, the sugars were
turned into alcohol. In a second fermentation process,
the alcohol was converted into vinegar by acetic acid
forming bacteria (acetobacter). Acetic acid and malic
acid gave vinegar its sour taste. Unpasteurized or or-
ganic apple cider vinegar has a cobweb-like appearance
and can make the vinegar look slightly congealed (5%
concentrated). Other constituents of vinegar included
polyphenolic compounds, vitamins, minerals, mineral
salt, amino acids, and organic acids (Omar et al. 2015).
The apple cider vinegar was chemically analysed and its
pH, density, and content of organic acids were evaluat-
ed before administrating it to the chickens. The pH of
apple cider vinegar was evaluated to be 2.5, density was
1.02 grams/ml; whereas the content of organic acids
were within normal limits.
Experimental Design and Treatments
Three treatments (T-vec, T+vec, Tv) with three repli-
cates were allocated to the birds. The apple cider was
added to the drinking water of the chickens in the vine-
gar group Tv as follows:
1-2 weeks: 10 ml/litter of water,
3-4 weeks: 20 ml/litter of water,
5-7 weeks: 30 ml/litter of water.
Amprolium was added to the feed of T+vec group (at
concentration of 1 ppm in anticoccidial drug) (El-Ban-
na et al. 2005). Neither coccidiosis treatment nor am-
prolium was added to the feed for T-vec group. Table 1
presents the composition of feed (g/kg) of the basal di-
ets.
The treatments (T-vec, T+vec, Tv) were arranged in a
Randomized Complete Block Design (RCBD) with
three replicates. A total of 9 blocks were made; howev-
er, the number of blocks represented the number of
replications. The treatments were assigned within the
Table 1. The composition of feed (g/kg) of the basal diets.
Starter
(0-21 d)
Grower
(22-35 d)
Finisher
(36-49 d)
Ingredients ------------------------------------
Corn 58.5 36.3 67.05
Soybean meal (48% CP) 35.65 31 26
Palm oil 1.84 1.79 3
Limestone (ground) 1 0.96 1.68
Dicalcium phosphate 0.2 0.2 1.02
NaCl 0.11 0.12 0.42
DL-methionine (98%) 0.10 0.1 0.2
L-Lysine-HCL (98.5%) 0.10 0.1 0.13
Coccidiostat 0.10 0.1 --
Vitamin premix 0.1 0.1 0.1
Mineral premix 0.1 0.1 0.1
Choline chloride 0.1 0.1 0.1
Antioxidant 0.1 0.1 0.1
Antifungal 0.1 0.1 0.1
Calculated nutrient composition
ME, kcal/kg feed 3 3.075 3.15
Protein 22 20 18
TSSA (g/kg) 0.009 0.0086 0.0081
Methionine (g/kg) 0.0054 0.0051 0.005
Lysine (g/kg) 0.0131 0.012 0.0107
Therionine (g/kg) 0.0084 0.0076 0.0068
Tryptophan (g/kg) 0.0029 0.0027 0.0023
Ca (g/kg) 0.0103 0.0098 0.0095
P, nonphytate (g/kg) 0.0045 0.0042 0.004
Na (g/kg) 0.0018 0.0018 0.0018
Tv group: no coccidiostats drug was added (apple cider vinegar was added); T-vec group: no coccidiostats drug was added.
4F.M.F. Hayajneh et al.
blocks randomly. A single treatment has been applied
per block. The chickens have been grouped into blocks
according to the suspected variations that isolate them.
The conditions are homogenous within each block, but
large differences may exist between different blocks.
The faeces of chickens were evaluated to assess the
number of coccidial oocytes that appear in their faeces.
Management of birds
Broiler chicken were purchased from local market
and distributed into 3 groups with 3 replicates each
containing 50 chicks. All the groups were fed balanced
diet, amprolium was added to the feed of the positive
control group T+ve, and apple cider vinegar was added
to the water of Tv group, nothing was added to the feed
of the negative control group T-ve. The infectious dose
of coccidian oocytes was 3x105/bird given via oral ad-
ministration (Arabkhazaeli et al. 2011).
Collecting blood samples
Blood samples were collected at the end of each
week, via wing veins, using sterile gauge 19 needles and
syringes. About 3.5 ml of blood used to be collected in
tubes, containing ethylene diamineate acetic acid
(EDTA).
Measurements
Three birds from each replicate were randomly
chosen to measure coccidial count in faeces. Blood
samples from the three birds were taken to measure:
1. Total antioxidant capacity (Allarad et al. 1998):
The Cell Biolabs’ Creative proteomics TAC Assay Kit
was used to measure the total antioxidant capacity of
sample. The kit detects total antioxidant capacity
through colorimetric method. This is suitable to be
used with serum, cell lysates, plasma, urine, food ex-
tracts, and tissue homogenates. It also works with wide
range of antioxidants.
2. The catalase enzyme activity was determined by
Catalase Activity Assay Kit from Creative proteomics.
It is a simple yet sensitive method to measure catalase
activity of variety of biological samples. The catalase
present in the sample reacts with hydrogen peroxide.
However, the catalase activity present in the sample is
inversely proportional to the obtained signal.
3. The malondialdehyde (an indicator of lipid per-
oxidation) was measured using Lipid Peroxidation
(MDA) Assay Kit from Creative proteomics, USA. The
kit is considered as a sensitive tool for detecting
malondialdehyde. Malondialdehyde in the sample re-
acts with thiobarbituric acid (TBA) to produce MDA-
TBA adduct, which can be easily quantified colorimet-
rically or fluorometrically.
Three birds per replicate were slaughtered to make
investigative slides and examine the histopathological
changes in the affected part of the chicken intestines.
Defecation was also observed for individual chickens.
A whole fecal bolus was collected and dissected after
observing defecation. The samples were analyzed soon
after the collection.
Oocysts Output
The fecal samples were collected and stored at 4°C
to determine the Oocyst per gram (OPG) count and
perform Fecal oocyst reduction test (FORT). This
method is carried on through the McMaster counting
chamber technique that uses saturated NaCl as the flo-
tation medium.
Histopathological examination
Classical lesions were taken for the histopathologi-
cal preparation. The cecum part was histopathologicaly
analysis. Haematoxylin and eosin (H&E) staining was
used to demonstrate the developmental stages in the
cecum. Tissues sampled were fixed in 10% neutral buff-
ered formalin, sectioned at 5-6 μm thicknesses and
stained with haematoxylin eosin stain. (Kadhim et al.
2014).
Statistical analysis
The collected data were properly coded and en-
tered into an excel spreadsheet, which was later entered
into SPSS version 17; SPSS Inc. Chicago. The data were
analysed by repeated measure of analysis (RMA). Re-
peated measure of ANOVA has been selected as the
parameter for testing. The treatments and noted vari-
ables were apparently dependent on each other. RMA
is an extension of dependent t-test as it is considered
ideal for the assessment. The mean values were further
assessed to identify any false occurrence among the
data. The differences among group means were consid-
ered significant at p<0.05.
Results
In the present study, doses of natural apple cider
vinegar administered in drinking water have shown an-
ticoccidial effects against Eimeria species. The effects
have been noted in terms of decreasing number of coc-
cidial oocytes in faeces, increasing antioxidative status,
and lowering concentration of malondialdehyde in the
blood of broil. Coccidial oocyte count in broiler chicken
is presented in Table 2. Investigations of broilers’ blood
indicated that no clinical signs of coccidiosis appeared
in chicken infected with coccidial oocytes.
5
Anticoccidial effect of apple cider vinegar on broiler ...
Figure 1a presents total antioxidant capacity (TAC),
catalase enzyme activity (CAT) and malondialdehyde
(MAD) in blood of the apple cider vinegar group.
Figures 1b and 1c present results in negative and posi-
tive control groups respectively. Histopathological
changes in intestine of 5 and 6 weeks broiler control
group are presented in Fig 2. Sections A, C, E, and G
show submucosal parasitic stages; section B shows par-
asitic stages in the glandular epithelium (arrows); sec-
tion D shows thickening in the submucosal tissue and
destruction of intestinal cells; F section presents in-
flammatory cells influxes into the submucosa; and sec-
Fig. 1. (A) Total antioxidant capacity (TAC), Catalase enzyme activity (CAT) and Malondialdehyde (MAD) in blood of apple cider
vinegar group; (B) Negative control group; (C) Positive control group
6F.M.F. Hayajneh et al.
tion H shows sloughing in the epithelial cells (Fig 2-I).
At the same time, sections A, C, and D show normal
intestinal cells; section C shows normal submucosal tis-
sue; and B section shows no parasitic stages in the glan-
dular epithelium. Crypt hyperplasia and increased leu-
kocyte infiltration, which is normally found in the
intestine of broiler infected with coccidiosis, were not
observed in the group treated with apple cider vinegar
from day 1 of the experiment (Fig 2-II). Some of the
chickens did not produce enough wet fecal material for
applying the OPG counting technique. The chickens
were capable of ingesting oocyts from an infected cage,
despite of range in oocyts output when the chickens were
spray inoculated.
An increase in the number of coccidial oocytes was
observed in faeces of the control group. On the other
hand, a significant decrease (p0.05) in the number of
coccidial oocytes in faeces of broiler treated with apple
cider vinegar has been found. Moreover, Table 2 shows
a significant increase (p0.05) in the catalase enzyme
activity in blood of broilers treated with apple cider
vinegar; whereas the catalase enzyme activity displays a
decrease in the control group (Fig 2). Tables 3 and 4
show coccidial oocyte count in broiler chickens in the
control group and within the group that was adminis-
tered with apple cider vinegar. Figure 2 also shows a
significant increase (p0.05) in the total antioxidant
capacity in blood of broilers treated with apple cider
Fig. 2. (A) Histopathological changes in the intestine of 5 and 6 weeks old broiler control group, A, C, G X200, B, D, F and H X100;
(B) Histopathological changes in the intestine of 5 and 6 weeks old broilers treated with apple cider vinegar, X100.
Table 2. Coccidial oocyte count in broiler chicken (Tv:vinegar group).
Vinegar group Tv (EPG)
Week Week Std. Error P valuea95% Confidence Interval for Differencea
Lower Bound Upper Bound
1
2 60.09 P0.05 -13879.98 -12186.69
3 116.67 P0.05 3272.94 6560.39
4 44.10 P0.05 4895.40 6137.94
5 104.08 P0.05 4383.57 7316.44
2
3 150.00 P0.05 15836.64 20063.36
4 50.00 P0.05 17845.55 19254.45
5 158.99 P0.05 16643.32 21123.35
34 100.00 P>0.05 -808.91 2008.91
5 166.67 P>0.05 -1414.84 3281.51
4 5 145.30 P>0.05 -1713.76 2380.42
Based on estimated marginal means,
* The mean difference is significant at the 0.05 level, a Adjustment for multiple comparisons: Bonferroni.
Dependent variable: coccidial oocyte count egg per gram (EPG).
7
Anticoccidial effect of apple cider vinegar on broiler ...
vinegar; whereas the total antioxidant capacity in blood
of broiler control group shows a decrease (Fig 1).
Figure 2 shows a significant decrease (p0.05) in the
malondialdehyde concentration in blood of the broiler
control group whereas the malondialdehyde concentra-
tion in blood of the apple cider vinegar group depicts a
decrease.
Discussion
The present findings are supported by the reported
by Abbas et al. (2011) and Nidullah et al. (2010). These
researchers have stated that the lower dose of hydro-
chloric acid (HCl) has the potential to be used as an
alternative to the chemotherapeutic drugs for Eimeria
tenella control. Apple cider vinegar also contains other
group of acidsincluding citric, formic, lactic, malic, and
succinic acids (Budak et al. 2014). The main histo-
pathological lesions were observed in the control group,
which were similar to those observed by McDougald
and Reid (1997) Associated with the infection induced
by E. tenella were very severe. The general effects
included changes in the morphology of the villi.
The pathological changes were mainly due to the
second generation schizonts (Soulsby 1982). Parasites
in various stages of development were located throug-
Table 3. Coccidial oocyte count in broiler chicken (T+vepositive control group).
Control group T+ve (EPG)
95% Confidence Interval for Differencea
Std. Error P value.aLower Bound Upper Bound
3041.38 P0.05 -78350.16 7350.16
1922.09 P0.05 -66413.80 -12252.86
1589.90 P0.05 -70233.49 -25433.18
927.96 P0.05 -75740.75 -49592.58
1691.48 P>0.05 -27664.70 19998.04
3929.94 P>0.05 -67702.47 43035.81
2166.67 P>0.05 -57692.94 3359.60
3329.16 P>0.05 -55404.75 38404.75
1452.97 P0.05 -43804.24 -2862.42
1922.09 P>0.05 -41913.80 12247.14
Based on estimated marginal means,
* The mean difference is significant at the 0.05 level. a Adjustment for multiple comparisons: Bonferroni.
Dependent variable: coccidial oocyte count egg per gram (EPG).
Table 4. Coccidial oocyte count in broiler chicken (T-ve:negative control group).
T-ve control group (EPG)
95% Confidence Interval for Differencea
Std. Error P value.aLower Bound Upper Bound
3041 P0.05 -78351 7350
1922 P0.05 -66414 -12253
1590 P0.05 -70234 -25434
928 P0.05 -75741 -49593
1691 P>0.05 -27665 19998
3930 P>0.05 -67703 43035
2167 P>0.05 -57693 3359
3329 P>0.05 -55405 38404
1453 P0.05 -43805 -2863
1922 P>0.05 -41914 12247
Based on estimated marginal means,
* The mean difference is significant at the 0.05 level. a Adjustment for multiple comparisons: Bonferroni.
Dependent variable: coccidial oocyte count egg per gram (EPG).
8F.M.F. Hayajneh et al.
hout the mucosa. Epithelial cell necrosis was more
severe, when there were massive accumulations of schi-
zonts with merozoites, marked proliferation of epitheli-
al cells of crypts, and foci of intense mononuclear infil-
trates in the submucosal membrane. Moreover,
multifocal and discrete interstitial edema at the submu-
cosal membranes was associated with various intrale-
sional forms of the parasite within epithelial cells.
(Adamu et al. 2013; Patra et al. 2009; Shirley et al. 2005).
The main histopathological lesions were observed
in the Tv group and they were mild (Fig 2). It has been
characterized principally by the inflammatory cell in-
fluxes that the submucosa along with the thickening of
mucosal and submucosal layer showed slight conges-
tion of blood vessel necrosis and thickened epithelial
mucosa (Kadhim 2014). It is advised to use apple cider
vinegar, which is commercially available for the preven-
tion and treatment of coccidiosis (Quiroz-castañeda
and Dantán-gonzález 2015). Nidaullah (2010) utilized
aqueous extracts from different medicinal plants, which
have been reported to cause a reduction in oocysts
count (p<0.05) with increased concentration of herbal
plants in the respective recipe. Similar approach has
been applied by Tipu et al. (2002) who fed Neem fruit
to broilers and observed reduction in coccidial oocysts
count per gram of feces.
Anticoccidial drug development has increased due
to urgent need to control this disease. Several strategies
have been used to treat or prevent the occurrence of
coccidiosis. Moreover, new alternatives to these drugs
were emerging, where some of these alternatives were
being obtained from the plants. An advantage of using
natural extracts like apple cider vinegar, is the approach
of lowering the risk of developing resistance towards
drugs. Also, the residues of such natural products in
meat are friendly to human consumers and may have
no adverse effects on their health. This research has
proven that medicinal herbal alternatives like natural
apple cider vinegar, can be used to prevent and treat
infectious diseases in broiler chicken like coccidiosis.
Limitations
The study results cannot be considered accurate be-
cause a small population of chickens has been recruit-
ed. Only three replicate of chickens were given the
three diets. The most important measurement of the
experiment was that of the coccidial counts and only
three birds were used for this. The number of coccidial
oocytes considered in the study is not sufficient to con-
clude the anticoccidial effect. The vinegar was adminis-
tered to the chickens through water and coccidial oo-
cytes were administered orally, but the water and feed
intakes of the chickens were not monitored.
Acknowledgment
The authors would like to thank the Jordan Univer-
sity for the financial support.
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