Journal of the Saudi Society of Agricultural Sciences 20 (2021) 103–110

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Journal of the Saudi Society of Agricultural Sciences

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Effect of synbiotic from Allium sativum and Lactobacillus acidophilus on

hematological indices, antioxidative status and intestinal ecology of
broiler chicken
Prayogi Sunu a,b,⇑, Dwi Sunarti a, Luthfi Djauhari Mahfudz a, Vitus Dwi Yunianto a
a
Department of Animal Science, Faculty of Animal and Agricultural Sciences, Diponegoro University, Indonesia
b
Department of Animal Science, Faculty of Animal Science, Boyolali University, Indonesia

a r t i c l e i n f o a b s t r a c t

Article history: Synbiotic is a combination of prebiotics and probiotics that can be used as a feed additive that is safe for
Received 3 September 2020 livestock and produce healthy products for consumers because they do not cause residues. The purpose
Revised 12 October 2020 staties of this study were to examine the synbiotic potential of garlic combined with Lactobacillus aci-
Accepted 9 December 2020
dophilus for broilers. Completed randomized design (CRD) with 3 treatment and 8 replicate and each
Available online 25 December 2020
replicate was filled with 6 broiler chickens aged 7 days with a bodyweight of 218.5 g. The treatments
were synbiotic from garlic and Lactobacillus acidophilus, synbiotics preparation garlic in extract form
Keywords:
(2 ml, 4 ml), after that we put 100 ml ratio of garlic and Lactobacillus acidophilus 1 ml with the concen-
Garlic
Broiler chicken
tration of 108 Cfu. as follows: TO = basal feed; T1 = basal feed + 2 ml synbiotic; T2 = basal feed + 4 ml
Synbiotics synbiotic. The parameters observed were body weight gain, pH, digestion rate, organ immunity, blood
Blood profile profile, MDA, SOD, and intestinal microflora. The results showed that the synbiotic treatment signifi-
Microbial population cantly (P < 0.05) improved performance, nutrient digestibility, blood profile, antioxidants, and intestinal
Intestinal morphology health. The addition of synbiotics between garlic extract and Lactobacillus acidophilus with a level of 4 ml
And antioxidants improves performance, intestinal health, antioxidants and nutrient digestion in broiler chickens.
Ó 2020 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction commonly used as a growth booster and health promotion agents

for boilers (Sugiharto et al. 2018).
Antibiotics Growth Promoter (AGPs) was officially banned in Garlic (Allium sativum) is herbaceous with clumps at the bot-
2003 The USA Institute of Medicine reported an increase in harm- tom, merging into large white bulbs. Garlic contains phytochemi-
ful bacteria (superbugs) and proposed a ban on the use of antibi- cal compounds that are useful for increasing the consumption of
otics as a supplement to animal feed (Sugiharto, 2016). feed, drinking water, and protein (Sunu et al. 2019). Phytochemical
Furthermore, the European Parliament issued a regulation pro- compounds can inhibit the growth of harmful microorganisms in
hibiting the use of antibiotics as an additive to animal feed the digestive tract of chickens so that the utilization of food sub-
(Giacomo Biagi, et al., 2017). Indonesia has banned the use of AGPs stances by chickens can be optimal and growth will increase
since January 1, 2019. Therefore, it is necessary to find alternative (Sugiharto et al. 2018). Furthermore, based on several studies that
materials that can be used as a substitute for antibiotics, one of have been done, side effects and toxicity of garlic were not found
which is garlic. Among AGPs alternatives, synbiotics have been so it is safe for consumption (Einas and Ibrahim, 2017). Garlic,
besides being a phytochemical, can also play a role as a prebiotic
(Yakhkeshi et al. 2011). The use of garlic as a prebiotic is reported
⇑ Corresponding author at: Department of Animal Science, Faculty of Animal to increase host defense due to the invasion of pathogens in the
Science, Boyolali University, Indonesia. intestine (Sugiharto, 2016). The mechanism by which prebiotics
E-mail address: [email protected] (P. Sunu). of garlic can increase host defense cannot yet be explained with
Peer review under responsibility of King Saud University. certainty, garlic can probably increase the Lactic acid bacteria
(LAB) population so that it can have a positive impact on the pop-
ulation of pathogenic bacteria (Zhui et al., 2018).
Prebiotics act as substrates that can selectively stimulate the
Production and hosting by Elsevier growth of beneficial bacteria in the colon. Besides prebiotics,

https://doi.org/10.1016/j.jssas.2020.12.005
1658-077X/Ó 2020 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
P. Sunu, D. Sunarti, Luthfi Djauhari Mahfudz et al. Journal of the Saudi Society of Agricultural Sciences 20 (2021) 103–110

another alternative to AGPs is probiotics. Probiotics are live Table 1

microbes that are given as food additives and provide benefits to Basal Research Ration.

the host by improving the balance of intestinal microbial popula- Feed ingredients / Nutrients Percentage
tions (Abudabos et al. 2016). Among the probiotics that are widely Corn 51,50
used for broiler chickens today is Lactobacillus acidophilus. Rice bran 15,00
Research on broilers shows that the addition of probiotic Lacto- Soybean meal 23,00
bacillus acidophilus in the ration can increase body weight gain, Fish meal 10,00
Premix 0,50
decrease FCR, and reduce mortality (Zhui et al., 2018). To increase Total 100,00
the effectiveness of prebiotics and probiotics in improving the EM (kkal/kg) 3051,00
health and productivity of broilers, prebiotics, and probiotics are Crude Protein 22,01
often used together. The combination of the two is called synbiotic. Crude Fat 6,20
Crude Fiber 4,36
Several studies report that the use of synbiotics can increase the
Calcium 1,99
number of lactic acid bacteria in the duodenum, jejunum, and Total phosphorus 1,08
ileum, decrease the number of escherichia Colli in the ileum Arginine 1,29
(Neeraj, 2016) and increase intestinal villi in the duodenum, jeju- Methionine 0,49
num, ileum and dilate intestinal villi in the ileum (Duskaev et al. Lysin 1,21

2018). Associated with the use of synbiotic based combinations

of garlic and Lactobacillus acidophilus, data in the literature were
not found to date. livestock every day as well as weighing the body weight of broiler
Therefore this study aimed to examine the response of the use chicken carried out on days 7, 14, 21, 28, and 35.
of prebiotics from garlic and the Lactobacillus acidophilus probi- The data collection phase included the digestion rate, small
otics on body weight gain, organ immunity, blood profile, intestinal intestine pH, and final body weight assessment. Digestion rate
microflora, and antioxidants in broiler chickens. measurement was done when chickens were 35 days old, using
the Fe2O3 indicator for 2 days alternately. One birds was taken
per unit of experiment and put in individual battery cages and
2. Materials and methods given a treatment ration that has been mixed with 0.5% indicator
Fe2O3. The indicator serves as a marker for the start of measuring
2.1. Garlic extract preparation the digestion rate. The time was recorded when colored excreta
according to the indicator first came out, Likewise when the color-
Garlic material that has been prepared as much as 3 kg of garlic less excreta was first released after the ration without the indicator
material has been prepared, washed and thereafter peeled. After was given again. The digestion rate was obtained from the time dif-
peeling the garlic is washed again and sliced with a thickness of ference when the indicated and not indicated ration was consumed
1 cm. Pieces of garlic are were put in a juicer to extract. The extract and the excreta with or without the indicator first coming out, then
obtained was then filtered. After the filtering process, the extract is the average is calculated. The measurement of the pH of the diges-
sterilized by the pasteurization method for 10 min, stored in a tion was carried out using a digital pH meter brand Senz pH (Sci-
clean container and kept in a closed place at a temperature below entific) in each part of the small intestine, namely the
20 0C. The amount of extract obtained is recorded and compared duodenum, jejunum, and ileum. The final body weight measure-
with the amount of raw material used. ment was done by weighing the chicken at the age of 35 days.
The data collection phase is carried out on the 35 days with
weighing the bodyweight of the broiler chicken carried mastery
2.2. Synbiotics preparation
for 3 h before taking blood. The blood sampling method is done
by selecting 1 sample randomly for each flock unit. Blood collec-
Garlic was extracted at different concentrations (2 ml and 4 ml),
tion is done through the brachial vein using a syringe. Blood was
then added to Lactobacillus acidophilus at a ratio of 100:1 (ml).
taken at least 1 ml and collected in EDTA tubes containing antico-
Isolates from de Man Rogosa and Sharpe (MRS) media supple-
agulants. Tubes are shaken slowly and stored at low temperatures
mented with 20% glycerol (v/v) and rejuvenated bacterial culture
(18 °C) to avoid lysis of blood clots. Blood to be tested in the lab is
on MRS media were incubated at 37 °C for 24–48 h to activate
stored in a cooler box. Hemoglobin concentrations were analyzed
the bacteria, which were then inoculated into a MRS broth media
using metric colony (Gudev et al., 2011). Measurement of hemat-
as much as 1 oose and incubated at 37 °C for 24 h [Sunu et al.,
ocrit levels is done by inserting blood into a microcapillary tube
2019].
of heparin and centrifuged at a speed of 1,300x g for 10 min
(Wakenell, 2010). Erythrocyte and leukocyte measurements were
2.3. In vivo experiment analyzed using an automatic hematology tool and for differential
measurement of leukocytes (lymphocytes, heterophils, eosino-
The material used in this study were 144 broiler chickens uni- phils, monocytes, and basophils) using blood smear preparations.
sex, each unit was filled with 6 broiler chickens aged 7 days with Preparation of blood smear preparations (blood mear) begins with
a bodyweight of 218,5 g. Birds at 1–6 days of age received the com- making blood smears on glass objects, then fixed with methanol,
mercial feed. Basal ration formulations were prepared based on the colored with Giemsa, washed with water, and allowed to dry at
needs of broiler chickens. The composition of the basal ration used room temperature. After drying is observed under a microscope
at 7–35 days is presented in Table 1. Birds were randomly placed in and the percentage of heterophils, lymphocytes, monocytes, eosi-
a 1x1 m enclosure. Vaccination was carried out using the ND B1 nophils, and basophils is calculated (Chunhong Li, et al., 2013).
strain vaccine through eye drops at the age of three days. The gum- The percentage of heterophils, lymphocytes, monocytes, eosino-
boro vaccine is given at fourteen days through eye drops and the phils, and basophils is then multiplied by the number of leukocytes
Lasota ND vaccine is given at the age of 21 days through drinking so that the number of heterophils, lymphocytes, monocytes, eosi-
water. Drinking water and rations were given to the birds was ad li- nophils, and basophils. Determination of the ratio of heterophils:
bitum. Weighing the feed given is done every day. Besides, we also lymphocytes are obtained by dividing the number of heterophils
weighed the remaining feed to determine the consumption of by the number of lymphocytes.

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P. Sunu, D. Sunarti, Luthfi Djauhari Mahfudz et al. Journal of the Saudi Society of Agricultural Sciences 20 (2021) 103–110

Observation of intestinal morphology data was carried out in F arithmetic < F table (0.05), then H0 is accepted and H1 is
the duodenum, jejunum, and ileum based on the method of rejected.
Rahman, et al., (2017). All three parts of the intestine are cut about F arithmetic  F table (0.05), then H0 is rejected and H1 is
2 cm and fixed in 10% formalin buffer, then the third piece of intes- accepted
tine is immersed in a phosphate buffer solution for 24 h. The part is
then processed in paraffin and the sample is cut thin with a size of
3. Results and discussions
4 mm and paint hematoxylin and eosin is done. Histology prepara-
tions that are ready in glass objects are observed and measured
3.1. Effect of synbiotics on growth performance
using a microscope with the help of a computer. Measurement of
villi height is done using a flat-screen computer with Microsoft
The results of the study showed that significant synbiotic
Office Picture Manager program at 10x magnification and with
administration (P < 0.05) increased body weight gain, and feed
the help of a microscope and optic camera. The histological picture
consumption (Table 2).
appears on the optical screen monitor viewer. After the intestinal
The observations in Table 2 show that the growth response of
morphology was found as expected, a photo was taken of all prepa-
broilers given synbiotics (T2) in the ration increased body weight
rations to be measured. A minimum measurement of three times
gain compared with controls (T0) and treatments (T1). This weight
per slide is made for each parameter. At first, the standard size of
gain is thought to be related to the synbiotic role itself, which is to
lm is determined in advance with the help of a computer that is
increase the survival of probiotic bacteria because a specific sub-
the value of the magnification used or desired and converted into
strate is available for fermentation so that livestock gets more per-
units of length (lm).
fect benefits from this combination of probiotics and prebiotics.
Measurement of MDA Plasma levels was carried out according
With more and more probiotic bacteria in the intestines, livestock
to the method used by Yuan, et al., (2015). Homogenate production
resistance will be better. Therefore, probiotic bacteria will produce
from blood plasma is done by taking blood in the right-wing vein
lactic acid to increase the acidity in the intestine. This high acidity
(vena vectors) using a projecting needle. The blood is then col-
level will make the condition unfavorable for pathogenic microor-
lected into a tube that has been given EDTA and shaken slowly
ganisms (Fayed, et al., 2015). These results indicate the role of pre-
and then stored in a process to avoid lysis. Homogenate added
biotics and probiotics to increase food digestibility so that weight
1 ml of TCA. Put in an Eppendorf tube and centrifuged at
gain with a high value and the highest value of feed consumption.
10,000 rpm for 10 min. The supernatant is taken and done for
Lipolytic, cellulolytic, ligninolytic, and gastric acid microbes con-
the TBA test. The determination of the level of MDA was carried
tained in probiotics are thought to have an active role in increasing
out by the TBA test. Supernatant from 400 lL blood plasma is
the digestibility of food substances. Prebiotics also provide food for
inserted into the Eppendorf tube, 400 lL sterile TBA is added,
microorganisms so they can maintain a good balance of microor-
1 ml distilled water, 200 lL HCL 1 N. The solution is homogenized
ganism ecosystems in the intestine.
and centrifuged at 500 rpm for 10 min. The solution was incubated
in handling 95 0C water for 15 min. The solution was cooled in a
refrigerator at 8 0C for 15 min, then centrifuged 10,000 rpm for 3.2. Effects of synbiotics on immune organs
10 min. Then put into the skillet to measure the absorbance at a
maximum wavelength of 512 nm. The absorbance obtained is then The results showed that the treatment is given significant syn-
plotted on the linear regression equation obtained, to obtain MDA biotic (P < 0.05) increased the weight bursa of Fabricius, thymus,
levels. spleen, and seca tonsil exchanges (Table 3).
Calculating SOD activity in a sample is done according to the The results of this study (Table 3) show that the weight of the
Peter method [Peter, 2015]. The supernatant obtained from the Fabricius stock exchanges significantly with the administration of
results of the preparation is then taken as much as 100 mL plus synbiotics at T2 and T1 compared with control treatments (T0).
100 mL Xanthine, 100 mL Xanthine oxidase, 100 mL NBT and PBS The weight of the Fabricius stock at T2 is significantly higher than
pH 7,4 as much as 1600 mL put into a 2 ml Eppendorf tube. After in other treatments. According to Farshid Kheiri et al. (2018), bursa
that vortex for 5 min with a speed of 2600 rpm. Then the samples Fabricius stock weight in 42-day broilers is 0.098%. Fekri et al.
were incubated for 30 min at 30 0C and centrifuged for 10 min at (2018) states that the weight of the Fabricius bursa decreases
3500 rpm. The blank used for this measurement is PBS pH 7.4. and enlarges with increasing absolute weight or age of livestock,
After that, the sample and blank are transferred into the cuvette and if the weight of the bursa is small it can reduce the number
and their absorbance is measured using a UV spectrophotometer of lymphocytes so that antibodies as an indicator of immunity
at a wavelength of 580 nm. become low. Toghyani et al. (2015) state that the Fabricius stock
exchange develops fully at 5–7 weeks. The factor that influences
the relative high or low weight of the Fabricius stock exchange is
2.4. Data analysis protein intake. According to Goodarzi et al. (2014), Low protein
levels can inhibit the development of Fabricius exchanges. The
A linear model that explains each observation value using a immune system needs a highly nutritious ration so that its organs
model completed randomized design (CRD) (Bereket Abraha can develop quickly and if it doesn’t develop at the age of
Gherezgihier, et al., 2017), namely: 4–7 weeks it means that there is a disturbance.
Yij = m + si + eij
Information:
Table 2
Yij = Observation value in the i-th treatment, jth replication Effects of synbiotics on body weight gain feed consumption, feed conversion and feed
m = Average value of treatment conversion.
si = Effect of i-i treatment Variable T0 (control) T1 (2 ml) T2 (4 ml)
eij = i-treatment error, j-replication
Body weight gain (g) 1104.09 ± 4,37c 1162.17 ± 3,72b 1509.78 ± 3,95 a
The data obtained were tested using analysis of variance
Feed consumption (g) 2682.32 ± 4,54c 2797.50 ± 2,85b 2908.92 ± 1,24 a

(ANOVA) level of 5%. If there is a real effect of the treatment (H1 Feed conversion (g) 1.47 ± 0,07 a 1.38 ± 0,06b 1.24 ± 0,04c
is accepted) then it is continued by Duncan’s Double Region Test. abc
Superscripts in the line shown significant (P < 0.05).
The statistical analysis testing criteria used are:
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P. Sunu, D. Sunarti, Luthfi Djauhari Mahfudz et al. Journal of the Saudi Society of Agricultural Sciences 20 (2021) 103–110

Table 3 The results of this study (Table 4) showed that the number of
Effects of synbiotics on the relative weight of organs. erythrocytes was significantly given to synbiotic compared with
Immune organs T0 (control) T1(2 ml) T2 (4 ml) control treatment (T0). The erythrocytic value of broilers in this
Bursa of Fabricius (g) 1,74 ± 0,29c
1,93 ± 0,23b
2,43 ± 0,49 a study is within the normal hematological range. Scanes and
Thymus (g) 1,71 ± 0,14b 2.06 ± 0,29 a 1.76 ± 0,13b Christensen (2014) reported that five-week-old broilers had
Spleen (g) 1.74 ± 0,18 a 1,59 ± 0,14b 1.75 ± 0,25 a erythrocyte values of 3,2  106/lL. According to Ning, et al.
Seca tonsil (g) 0.106 ± 0,003b 0.116 ± 0,001b 0.123 ± 0,003 a
(2014) The normal value of broiler red blood cells is around
ab
Superscripts in the line shown significant (P < 0.05). 2.0–3.2  106 / mm3, whereas according to Tamzil et al. (2014)
the average red blood cell under normal conditions in 26-day-old
chickens is 3,2  106 per mm3. According to Rehman, et al.,
The results of this study (Table 3) show that thymus weights (2017) the value of erythrocytes in broilers is 2.5–3.2  106 /
were significantly synbiotic compared with control treatments mm. If the red blood cells are above the average or excess, can
(T0). The thymus relative weights at T0, and T2 are in the low range experience erythrocytosis. Erythrocytosis is caused by
while T1 is in the normal range. Sugiharto et al. (2018) state that dehydration, if the red blood cells are below average, it can be
the normal range of thymus relative weights is 0.16–0.38%. anemic. Anemia can also be caused by injury, erythrocyte damage,
Ahmadi et al. (2018) state that the thymus that experiences rapid and air pollution (Ali Asghar Saki, et al., 2016).
atrophy is a reaction to stress so that have small thymus means a The results of this study (Table 4) show that leukocyte counts
disturbance is indicated. Although the weight of the thymus that is were significantly synbiotic compared with the control treatment
produced tends to be small, the livestock is still in a healthy (T0). The results of this study indicate the range of broiler
condition. chicken leukocytes ranges from 7,43  103 / ml (T0),
The results of this study (Table 3) show that spleen weights 9,11  103 / ml (T1), and 11,60  103 / ml (T2), where the average
were significantly given synbiotic compared with control treat- number is in the condition healthy. Normal leukocyte counts in
ments (T0). The spleen percentage in the T0, T1, and T2 treatments broiler chickens are in the range of 12–30  103 / ml (Ullah,
was normal, and by the opinion of Ola et al. (2017) that the per- et al. 2018) According to Ferdous, et al. (2016) reported that nor-
centage of broiler spleen ranged from 0.18 to 0.23%. Besides, the mally the number of leukocytes in the blood of broilers is in the
percentage of spleen obtained is not much different from the range between 6 and 40  103 / ml. The average number of
results submitted by Hatab et al. (2016) that the spleen weight leukocytes in the blood of the broiler shows a healthy condition
in broiler chickens ranges from 0.12 to 0.14%. The comfortable con- so that the chicken does not make efforts to fight pathogenic
dition of the cage makes the performance of the spleen not too bacteria or viruses that enter the body. Bacteria infected cattle
heavy so that the relative weight of the spleen tends to be normal. will cause the health of these chickens to decrease with a
The results of this study (Table 3) show that tonsillar Seca marked increase in white blood cells.
weights were significantly given synbiotic compared with control The results of this study (Table 4) showed that the number of
treatments (T0). The spleen percentage in the T0, T1, and T2 treat- hemoglobin was significantly given to synbiotic compared with
ments is normal. Rezaian and Hamedi (2007) states that Sekal ton- control treatment (T0). The results of this study indicate that the
sil will respond to foreign agents that enter the body of lymphoid range of broiler chicken hemoglobin ranges from 7.91 g / dL (T0),
tissue so that cattle become healthy. According to Akter, et al. 8,42 g / dL (T1), and 9,45 g / dL (T2), where the average amount
(2006), once tonsillar structures similar to the intestine are intesti- is in a healthy condition. Normal levels of chicken hemoglobin
nal branches and contain lymphocytes. The structure of these according to Jola et al. (2018) range from 7.3 to 10.9 g / 100 ml,
organs is kept, lymphatic nodules (lymphoid follicles), external whereas according to Ajay, et al. (2015) is 7.0–13.0 g/dl. Hemoglo-
extrafollicular, epithelial, and villi musculus muscular. bin is important for survival because it carries and delivers oxygen
to body tissues (Agboola, et al. 2014). Two forms of hemoglobin
that perform this function are oxyhemoglobin and deoxyhe-
3.3. Effects of synbiotics on blood profile
moglobin. The ability of blood to carry oxygen is produced by
hemoglobin levels in the blood and chemical characteristics of
Data on the effect of synbiotic on blood profile in broilers are
hemoglobin (Olaiya, et al. 2014). Synthesis and destruction of
presented in Table 4. The results showed that significant synbiotic
hemoglobin are balanced by physiological conditions and one of
(P < 0.05) on the levels of erythrocytes, leukocytes, hemoglobin
the disorders will lead to hematological abnormalities.
(Hb), Packed cell value (PSV), Total plasma protein (TPP), Hetero-
The results of this study (Table 4) showed that the number of
phyll segments, Eosinophils, Basophils and Lymphocytes, and
hematocrits was significantly synbiotic compared with the control
Monocytes.
treatment (T0). The hematocrit value of treatment (T0) is 22,87%,
(T1) 28,62%, and (T2) 25,25%. has a hematocrit value that is higher
Table 4 than T1 and T0. According to Sugiharto, et al. (2018), the normal
Effects of synbiotics on blood profile in broiler chickens and antioxidant. value of chicken hematocrit is between 22 and 35% with an aver-
Variable T0 (Control) T1 (2 ml) T2 (4 ml)
age of 30%. This increase can not be said to occur erythrocytosis
because hematocrit values are still in the normal range. According
Eritrosit (jt/ml) 2,34b 3,05 a 2,79 a
to Ajayi and Imouokhome (2015), erythrocytosis is characterized
Leukosit (../ml) 7,43b 9,11 a 11,60 a
Hb (g/dL) 7,91b 8,42 ab 9,45 a by an increase in hematocrit, hemoglobin, and the number of ery-
PCV (%) 22,87b 28,62 a 25,25b throcytes above the normal range. Erythrocytosis can be absolute
TPP (g/dl) 1,70b 2,55 a 2,97 a or relative. Erythrocytosis is relatively normal with erythrocytes.
Heterofil segmen 18,39b 20,13 ab 27,24 a
The situation is caused by contraction of the spleen or dehydration.
Eosinofil 0,75b 1,10b 4,10 a
Limfosit 52,03b 78,44 a 81,40 a
Spleen contractions are stimulated by epinephrine release that
Monosit 2,31b 5,63 a 4,62 a occurs when fear, pain, or exercise. Absolute erythrocytosis is char-
Ratio Heterofil/Limfosit 0,35 a 0,25b 0,33b acterized by high hematocrit values due to an increase in the num-
MDA (nmol/ml) 0,77 a 0,48b 0,40b ber of erythrocytes due to increased erythropoietin production.
SOD (U/mL) 44,79b 59,88 a 66,49 a
According to Ogunwole et al. (2013), an increase in hematocrit
ab
Superscripts in the line shown significant (P < 0.05). value has limited benefits because it can increase the viscosity
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P. Sunu, D. Sunarti, Luthfi Djauhari Mahfudz et al. Journal of the Saudi Society of Agricultural Sciences 20 (2021) 103–110

(thickness) of blood which will slow down blood flow to the capil- can cause a decrease in the health of broilers characterized by an
laries and increase the work of the heart. increase in the number of white blood cells (Saputro et al. 2013).
The results of this study (Table 4) show that the actual TPP Bacterial infections will require an inflammatory response so the
amount was given by synbiotic compared to the control treatment bone marrow will be stimulated to produce heterophils. Hetero-
(T0). The results of this study indicate the range of TPP numbers of phyll is approved to be able to respond to pathogens more quickly
broiler chickens range from 1,70 g/dl (T0), 2,55 g/dl (T1) and during initial inflammation. The occurrence of the disease will
2,97 g/dl (T2), where the average number is in a healthy condition. decrease the response will increase can also be increased (Farnell
Normal levels of chicken TPP according to Al-Daraji and Salih et al. 2006)
(2012) range from 2.25 to 3.00 g/dl, according to Abudabos et al.
[38] 2.57–2.96 g/dl, while according to Sugiharto et al (2016) it is 3.4. Synbiotic effects of antioxidant levels
2.87–4.18 g/dl. According to El-Katcha et al. (2014) protein levels
are affected by age, hormonal, nutrition, stress, and fluid loss. Pro- The mean antioxidant data is presented in Table 4. The results
tein is also important for regulating body water balance. Therefore, of the study showed that significant synbiotics (P < 0.05) increased
the protein serves to help spread body fluids evenly between the the content of superoxide dismutase (SOD) to decrease the content
blood and body tissues. of malonaldehyde (MDA).
The results of this study (Table 4) show that the number of het- The results of this study (Table 4) showed that the number of
erophile segments significantly synbiotic administration compared MDA was significantly given by the treatment of synbiotics (T0).
with the control treatment (T0). The average percentage of broiler Malondialdehyde (MDA) is an indicator of lipid peroxidation in
heterophile in this study for all treatments was 18,39% (T0), 20,13% the body that is often used and is associated with oxidative stress
(T1), and 27,42% (T2). the percentage of normal heterophile (Binder, et al. 2016). High MDA concentration indicates the oxida-
according to Rahman et al, (2017), which is 25.0 ± 2.3%. while for tion process in the cell membrane. Research using synbiotics can
other treatments the same compared to the recommendation. reduce MDA because of the role of antioxidants which inhibit lipid
The results of this study indicate the occurrence of heterophilia peroxidation. According to Uránek et al. (2013), MDA in the body is
(number of heterophile) and chickens are in a comfortable and formed as a result of oxidative stress, which is an imbalance
not stressed condition. Mohamed et al. (2012) ported that stress between the formation of reactive oxygen species (ROS) with the
occurs when heterophile percentages are above 31.95%. A high per- presence of antioxidants, where free radicals are higher than
centage of heterophils is because in the blood there is an increase antioxidants. According to Zhao et al. (2014), a decrease in plasma
in the production of heterophils. Heterophyll is the main line of MDA levels indicates inhibition by antioxidants; and high antioxi-
defense against infection (Pandian et al. 2012). dant status is usually followed by a decrease in plasma MDA levels.
The results of this study (Table 4) show that the actual eosino- The results of this study (Table 4) show that the number of SOD
phil amount was given by synbiotic compared to the control treat- was significantly given by the synbiotic treatment (T0). Decreased
ment (T0). The average percentage of broiler eosinophil in this SOD levels occur due to an increase in ROS levels due to E. tenella
study for all treatments was 0,75% (T0), 1,10% (T1), and 4,10% infection (Ayala, et al. 2014). Oxidative stress can also be caused by
(T2). Purwandari et al. (2014) state that in the normal range eosi- the presence of ischemia (Bhattacharyya, et al. 2012). Ischemia can
nophil counts are 2–8% of white blood cell counts and can survive be caused by a high density of cages so that oxygen levels in the
3–5 days. The high percentage of eosinophils in the blood cannot cage are reduced. Lack of oxygen levels causes chickens to not
be assumed that the chicken is in sick condition. The high produc- get enough oxygen intake. This results in reduced oxygen content
tion of eosinophils can also show the functioning of the body’s in the blood. Besides other factors that can cause oxidative stress
defense system in the face of disease agents are environmental pollution, climate, and feed nutrition (Rahal,
The results of this study (Table 4) show that lymphocyte counts et al. 2014).
were significantly synbiotic administration with control treatment
(T0). The average percentage of broiler lymphocyte in this study for 3.5. Effect of synbiotics on microbial population
all treatments was 52,03% (T0), 78,44% (T1), 81,40% (T2). The
increase in the number of lymphocytes is thought to be caused Data on the effect of synbiotics on E. coli bacteria contamination
by the role of bioactive compounds contained in garlic. Ahmed and coliform bacteria contamination are presented in Table 5. The
et al (2011) and Asgarpanah (2012) stated that some types of garlic results of the study showed that significant synbiotics (P < 0.05) on
are known to contain flavonoid compounds, such as routine, quer- pH, E. coli bacteria count, and the number of coliform bacteria.
cetin, luteolin, synarosida, saliprena, eugenol, and naringenin. Fla- The results of this study (Table 5) showed that the E. coli bacte-
vonoids can inhibit the development of parasites by acting as an ria contamination was significantly given by T2 synbiotics with T1
enzyme inhibitor. Chickens have lymphocyte counts between and control treatments (T0). According to Albazaz and Buyukunal
5.45 and 17.3 ( 103/ mm3) or 5.91–18.4 ( 103 / mm3) (2014), the number of bacteria in the duodenum and small intes-
(Mazagova, et al. 2015). tine is relatively low at < 108 CFU/g. in the cecum where microbial
The results of this study (Table 4) showed that the number of fermentation occurs the number of bacteria is 1011 CFU/g. Micro-
monocytes was significantly given to synbiotic by control treat- bial composition in the digestive tract is not always stable and dif-
ment (T0). The average percentage of broiler monocytes in this fers between species, individuals, and parts of the digestive tract.
study for all treatments was 2,31% (T0), 4,62% (T2), 5,63% (T1). Changes in the intestinal microbial composition are very large in
the percentage of monocytes from this study is the same compared poultry aged 2–3 weeks and will be stable at the age of 5–6 weeks.
to that stated by Scanes and Christensen (2014) of 5.6 ± 1.0%. The According to Sari et al. (2013), factors that influence microbial
results of this study are in line with research by Mazagova et al. growth in the digestive tract of chickens are age, immune response,
(2015) with a monocyte range of 1.6 to 5.6%. Clinically a low mono- feed, and antibiotic administration.
cyte count (monocytopenia) is not important and difficult to docu- The results of this study (Table 5) showed that coliform bacteria
ment because of varied poultry data (Mohamed, et al. 2012) contamination was significantly given by T2 synbiotics with T1 and
The results of this study (Table 4) show the number of H/L ratios control treatments (T0). Total coliform bacteria in the control (T0)
which are synbiotic administration with control settings (T0). This were 7,85 log CFU/g, (T1) 3,36 log CFU/g and (T2) 0,62 log CFU/g.
increasing H/L ratio can increase stress and show an increase in Khosravifar et al. (2014) stated that the number of coliform
infections detected by the immune system. Bacterial infections bacteria in the intestine broilers of the finisher period was 6.04
107
P. Sunu, D. Sunarti, Luthfi Djauhari Mahfudz et al. Journal of the Saudi Society of Agricultural Sciences 20 (2021) 103–110

Table 5 intestinal villi height, Crypt Depth (mm), and the ratio of villi
Effects of synbiotics on microbial population and intestinal morphology. height: crypt depth. in duodenum, jejunum, and ileum of broiler
Parameter Treatment chickens.
T0 T1 T2 The results of this study (Table 5) showed that the height of
duodenal intestinal villi was significantly synbiotic with control
Microbial Population
Lactic Acid Bacteria
treatment (T0). Duodenal villi height at T2 is 1552,55 lm higher
Ileum 3,17 ± 0,16b 4,70 ± 0,31 ab 6,47 ± 0,40 a than T1 and T0 namely 1310,37 lm and 1160,54 lm. Decreased
Cecum 3,34 ± 0,23b 4,91 ± 0,32b 6,86 ± 0,45 a
pathogenic bacterial colonies greatly affect the level of small
E.coli intestinal villi, and vice versa, if the pathogenic bacteria increase
a
Ileum 5,53 ± 0,047 3,45 ± 0,150b 1,42 ± 0,027c
a growth in height of small intestinal villi. According to Dehghani
Cecum 5,86 ± 0,098 3,65 ± 0,084b 1,47 ± 0,031c
Coliform and Jahanian (2012) factors such as pathogenic bacteria, and stress
Ileum 7,85 ± 0,15 a
3,36 ± 0,09b 0,62 ± 0,06c hurts intestinal microflora or intestinal epithelium, which causes
Cecum 8,17 ± 0,16 4,01 ± 0,08 1,02 ± 0,05 cell permeability as the body’s natural defenses undergo changes
Intestinal Morphology
making it easier for harmful compounds and pathogenic bacteria
Doudenum
Villi Height (mm) 1160,54b 1310,37 ab
1552,55 a to penetrate small intestinal cells, which will disrupt metabolism,
Crypt Depth (mm) 31,06c 36,10b 48,70 a polluting and absorbing nutrients. This condition can cause chronic
Villi Height: Crypt Depth 37,36 a 36,29 a 32,34b inflammation of the intestinal mucosa, which ultimately causes
Jejenum villi height, disturbance, and absorption.
Villi Height (mm) 800,60b 840,60b 998,82 a
The results of this study (Table 5) showed that the height of
Crypt Depth (mm) 34,88c 41,40b 52,84 a
Villi Height: Crypt Depth 22,95 a 20,30 a 18,90b intestinal villi in the ileal portion was significantly synbiotic with
Ileum control treatment (T0). The height of the ileal villi at T2 is
811,28b 994,05b 1473,21 mm higher than T1 and T0 which is 994,05 mm and
a
Villi Height (mm) 1473,21
Crypt Depth (mm) 34,83b 39,34b 51,45 a 811,28 mm. This shows that the higher the addition of synbiotics
Villi Height: Crypt Depth 23,29c 25,26b 28,63 a
pH
in the ration the shorter the small intestinal villi of the ileum. This
Doudenum 6,70 ± 0,034 a 6,37 ± 0,021b 6,42 ± 0,024b is caused by inflammation and inflammation cells in the small
Jejunum 6,76 ± 0,043a 6.38 ± 0,021b 6,43 ± 0,020b intestine, causing the villi to be damaged and shortened. This is
Ileum 6,70 ± 0,028 a 6,33 ± 0,024b 6,40 ± 0,021b in line with the opinion of Incharoen et al. (2010) who said that
abc
Superscripts in the line shown significant (P < 0.05). inflammation of the digestive tract causes the intestinal villi to
shorten and enlarge cecum accompanied by infiltration of inflam-
log CFU/g. Giving synbiotics in T2 can reduce coliform bacteria. Fol- matory cells. Shortening and enlarging villi will reduce villous den-
lowing the opinion of Varmaghany et al. (2015) states that Allium sity (Houshmand, et al., 2012). Although the intestinal villi height
sativum can be used as a natural antibiotic because it contains in the ileum produced tends to be small, but the livestock is still in
active compounds that can suppress non-pathogenic microbes. a healthy condition.
The results of this study (Table 5) showed that the growth of The results of this study (Table 5) showed that the high intes-
lactic acid bacteria was significantly given by T2 synbiotics with tine villi of the jejunum were significantly synbiotic with control
T1 and control treatments (T0). Total lactic acid bacteria in the con- treatment (T0). The height of duodenal villi at T2 is 988,82 lm
trol (T0) were 3,17 log CFU/g, (T1) 4.70 log CFU/g, and (T2) 6.47 log higher than T1 and T0 namely 840,60 lm and 800,60 lm. Jejunum
CFU/g. This living microorganism is beneficial for its host so that it where digestion occurs but with a small absorption frequency. In
can maintain balance in the intestine, survive in the digestive tract the jejunum, there is a process of absorption of food substances
and stick to intestinal cells and detoxify its toxins or metabolites that have not been completed in the duodenum until only material
thereby increasing the absorption of nutrients more fully. The that cannot be digested (Xu, et al., 2004). The jejunum is similar to
results of this study are in line with Yakhkeshi’s, et al. (2011) the duodenum but the villi are smaller and fewer, whereas in the
research An increasing number of Lactic acid bacteria (LAB) will jejunum there are not very visible submucosal glands but they
produce lactic acid production and short-chain fatty acids (SCFA). have many goblet cells on the surface of their villi. Jejunum has a
The metabolite compounds produced by LAB such as organic acids, characteristic histological pattern such as the entire small intestine
hydrogen peroxide, and carbon dioxide (CO2). Organic acids (lactic mucosa, submucosa, muscular, and serosa. The mucosa is covered
acid) Lactic acid bacteria also produce antimicrobial (bacteriocin) by a simple columnar epithelium leading to the lumen (lamina
which are antagonistic to the growth of pathogenic bacteria and epithelial). It contains enterocytes and goblet cells, an epithelial
repair beneficial bacteria in the small intestine. layer followed by a layer of connective tissue (lamina propria)
The results of this study (Table 5) show that synbiotics signifi- and a muscular layer (lamina muscular mucosa). The submucosa
cantly reduce the pH of the small intestine of the duodenum, jeju- consists of loose connective tissue with blood vessels (Mile, et al.,
num, and ileum. The results of this study are in line with the 2006).
research of Adil and Magray (2012) who reported that synbiotics The results of the study in (Table 5) show that the depth of the
provide an advantage to the host because it provides a specific sub- crypt in the duodenum, jejunum, and ileum is significantly synbi-
strate for the fermentation process that supports the growth of otic with control treatment (T0). The height ratio of villi: the depth
probiotics so as to improve the health life of beneficial bacteria of the crypt in the duodenum, jejunum, and ileum also showed sig-
in the intestine. The fermentation process of lactic acid bacteria nificantly the administration of synbiotics by control treatment
produces a high concentration of lactic acid so that it will affect (T0). Peng et al. (2016) revealed that the efficient use of feed by
the decrease in pH and reduce the growth of harmful bacteria poultry depends on the digestive tract, especially villi and crypt,
(Nkukwana, et al. 2015). which play an important role in nutrient digestion. Laudadio
et al. (2012) also state that the higher the villi, the wider the
absorption area of nutrients will be greater and affect the increase
3.6. Effects of synbiotics on intestinal morphology in growth and metabolic rate. Besides, digestive capacity and nutri-
ent absorption can also be determined from the ratio of villi/ crypt.
The mean intestinal morphology data are presented in Table 5. The higher the ratio of villi/ crypt, the higher the level of digestion
The results showed that synbiotic was significant (P < 0.05) to and absorption of nutrients
108
P. Sunu, D. Sunarti, Luthfi Djauhari Mahfudz et al. Journal of the Saudi Society of Agricultural Sciences 20 (2021) 103–110

4. Conclusion cortex extract and probiotic substances on the immunity and productivity of
broiler chickens. Veterinary World 11 (10), 1416–1422.
El-Katcha, M.I., Soltan, M.A., El-Kaney, H.F., Karwarie., and El-Sayed, R. 2014. Growth
The results showed that the synbiotic between garlic and Lacto- performance, blood parameters, immune response and carcass traits of broiler
bacillus acidophilus level 4 ml in the ration could increase weight chicks fed on graded levels of wheat instead of corn without or with enzyme
supplementation. Alexandria Journal of Veterinary Sciences 40: 95-111.
gain, organ immunity, blood profile, and antioxidants in broilers.
Einas A. and Ibrahim. 2017. In vitro Antimicrobial Activity of Allium sativum
(Garlic) against Wound Infection Pathogens. African Journal of Medical
Declaration of Competing Interest Sciences, 2(8): 17-25
Farnell, M.B., Donoghue, A.M., Solis, de los., Santos, F., Blore, P.J., Hargis, B.M., Tellez,
G., and Donoghue, D.J. 2006. Upregulation of oxidative burst and degranulation
The authors declare that they have no known competing finan- in chicken heterophils stimulated with probiotic bacteria. Poult Sci. 85 (11):
cial interests or personal relationships that could have appeared 1900-1906.
to influence the work reported in this paper. Farshid, K., Mostafa, F., Nasir, L., 2018. Evaluation of thyme and ajwain as antibiotic
growth promoter substitutions on growth performance, carcass characteristics
and serum biochemistry in Japanese quails (Coturnix japonica). Animal
Acknowledgments Nutrition. 4 (2018), 79–83.
Fayed, R.H., Razek, A.H.A., Ouf, M., 2015. Effect of dietary garlic supplementation on
performance, carcass traits, and meat quality in broiler chickens effect of
Thanks to the Research Ministry, Technology, and Higher Edu- dietary garlic supplementation on performance, carcass traits, and meat quality
cation of the Republic of Indonesia who helped carry out this in broiler chickens. J. Parameters. 100, 1000–1004.
research through doctoral dissertation research grants and the Fekri, Y.F., Ghalamkari, G.H., Toghiani, M., Modaresi, M., Landy, N., 2018. Efficiency
of Tribulus terrestris L. as an antibiotic growth promoter substitute on
Domestic Postgraduate Education Scholarship Program (BPP-DN performance and immuneresponses in broiler chicks. Asian Pac. J. Trop Dis. 4
DIKTI), Contract no. 258-41/UNJ.P4.3/PP/2019. (2). S1014e8.
Ferdous, F., Saski, C., Bridges, W., Burns, M., Dunn, H., Elliott, K., Scott, T.R., 2016.
Transcriptome profile of the chicken thrombocyte: New implications as an
advanced immune effector cell. PLoS ONE 11. e0163890.
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