Arch. Pol. Fish.

(2017) 25: 103-115

DOI 10.1515/aopf-2017-0010
RESEARCH ARTICLE

Terminalia catappa L. extract improves survival, hematological

profile and resistance to Aeromonas hydrophila in Betta sp.

Rudy Agung Nugroho, Hetty Manurung, Firman M. Nur, Widha Prahastika

Received – 16 September 2016/Accepted – 09 June 2017. Published online: 30 June 2017; ©Inland Fisheries Institute in Olsztyn, Poland
Citation: Nugroho R.A., Manurung H., Nur F.M., Prahastika W. 2017 – Terminalia catappa L. extract improves survival, hematological pro-
file and resistance to Aeromonas hydrophila in Betta sp. – Arch. Pol. Fish. 25: 103-115.

Abstract. To determine the effects of Terminalia catappa extract Introduction

(TCE) immersion on the survival and hematological profile of
Betta sp, a group of fish was immersed in 500 ppm TCE and Aeromonas species have been identified as causative
compared with the control group. After three days of immersion,
agents and serious pathogens to various aquatic ani-
survival, leukocyte (WBC), erythrocyte (RBC), hemoglobin (Hb),
hematocrit (Hct), platelet (PLT), and differential leukocyte counts mals (Carriero et al. 2016, Maliwat et al. 2016, Ni et
were compared between the two groups. Surviving fish from each al. 2016, Yang et al. 2016) resulting in mass mortali-
group were then divided into three subgroups: subgroup without ties to many cultured ornamental fish (Majtán et al.
injection; subgroup injected with normal saline; subgroup injected 2012, Abdi et al. 2014, Harikrishnan et al. 2015).
with Aeromonas hydrophila. Survival, WBC, RBC, Hb, Hct, PLT, Among various Aeromonas species, Aeromonas
the percentage and number of lymphocyte, monocyte, and
hydrophila is known to cause disease outbreaks in
granulocytes post injection were evaluated for 48 h. The results
showed that the survival of immersed fish was significantly higher Labeo rohita (Hamilton) (Das et al. 2015), goldfish,
than that in the control. No significant differences in the Carassius auratus (L.) (Anusha et al. 2014, Sahoo et
hematological profile were noted between the control and the al. 2016), and red hybrid tilapia (Oreochromis spp.)
immersed fish. The WBC of control subgroup (A. hydrophila (Kirubakaran et al. 2016, Lee et al. 2016, Musthafa
injection) was significantly increased after 24 h. The fish et al. 2016). Intramuscular injection of A. hydrophila
immersed and injected with A. hydrophila had the highest PLT.
can produce a virulent reaction and reduce immune
The number of lymphocytes of all subgroups of fish was stable
while the percentage of monocytes and granulocytes of the responses in Pangasianodon hypophthalmus
subgroups of immersed fish were increased. This finding (Sauvage) (Tamamdusturi and Yuhana 2016). To
suggested that 500 ppm of TCE is beneficial for improving overcome high mortalities of cultured animals,
survival, blood profile, and resistance to A. hydrophila. aquaculturists and researchers use antibiotics to pre-
vent virulent reactions to A. hydrophila infection
Keywords: Terminalia catappa, survival, blood profiles,
(Guz and Kozinska 2004, Sinclair et al. 2016).
Betta sp., Aeromonas hydrophilla
However, increasing global demand for the pres-
ervation of eco-friendly environments, the applica-
R.A. Nugroho [+], H. Manurung, F.M. Nur, W. Prahastika
tion of antibiotics, which are notorious for increasing
Animal Physiology, Development and Molecular Laboratory,
Department of Biology, Faculty of Mathematic and Natural Science, antibiotic-resistant pathogens and inducing environ-
Mulawarman University, Jl. Barong Tongkok No 4, Gn Kelua, mental deterioration, is being questioned
Samarinda, East Kalimantan 75123, Indonesia (Samanidou and Evaggelopoulou 2007, He et al.
e-mail: [email protected]
2016, Uchida et al. 2016). Alternatively, various

© Copyright by Stanis³aw Sakowicz Inland Fisheries Institute in Olsztyn.

© 2017 Author(s). This is an open access article licensed under the Creative Commons Attribution-NonCommercial-NoDerivs License
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
104 Rudy Agung Nugroho et al.

plant extracts, such as oat (Avena sativa), oyster an antioxidant enzyme that is responsible for pre-
mushroom (Pleurotus ostreatus), nettle (Urtica venting cellular damage and improving immune
dioica), sea grass (Cymodocea serrulata), turmeric competence (Middleton and Kandaswami 1992,
(Curcuma longa), spirulina (Spirulina platensis), and Middleton 1998, Saroja et al. 2012).
beet root (Beta vulgaris) have been tested and used as The immune status of fish is related to their he-
alternatives to antibiotics (Baba et al. 2016, Bilen et matological parameters, which are important in fish
al. 2016, Devi et al. 2016). There has been a surge in culture because of their value in monitoring the
the use of plant extracts to enhance the survival and health status of fish (Chandel et al. 2009). Hemato-
immune competence of cultured aquatic animals logical parameters such as red blood cell (RBC), he-
(Reverter et al. 2014, Adel et al. 2016, Kakaei and moglobin count (Hb), hematocrit (Hct), platelet
Shahbazi 2016). (PLT), and total and differential leukocyte counts
One potential plant that can be used as an (TLC and DLC) in fish are effective tools that can be
antimicrobial to enhance survival and used to evaluate physiological and pathological
immunocompetence is the Indian almond tree (T. changes in fish (Inama et al. 1993). Leukocyte counts
catappa). Terminalia species are widely distributed are used regularly as indicators of fish health status
in both tropical and sub-tropical regions, including because leukocytes are key components of innate im-
in Asian (Hyttel et al. 2010). In Indonesia, T. catappa mune defense and are involved in the regulation of
L. is an attractive, long-lived tree well suited to orna- immunological function in fish (Zhou et al. 2010,
mental and amenity plantings. The water extract of T. Ekman et al. 2013). Moreover, immunity related
catappa leaf is well known as a folk medicine for physiological responses measured by alterations in
antipyretic, hemostatic, hepatitis, and liver-related TLC and DLC can be used as indicators of immune
diseases in the Philippines, Malaysia, and Indonesia competence and the health status of several fish spe-
(Meena and Raja 2006, Vuèuroviæ and Razmovski cies (Alvarez et al. 1988, Modrá et al. 1998, Do Huu
2012). et al. 2016).
According to Rakholiya and Chanda (2012), T. The ornamental fish Betta sp. is a member of the
catappa extract (TCE) shows the highest and stron- Labyrinth fish family (Belonitiidae) and is known as
gest synergistic effect against the bacterial strains a colorful species, especially in males (Ekman et al.
tested. Meanwhile, a study in fish revealed that TCE 2013). As an important cultured ornamental species
can be used as an alternative antibacterial remedy in Indonesia, Betta sp. is recognized as a substantial
against tilapia, Oreochromis niloticus (L.) parasites ornamental fish commodity (Alfian 2010). In-
and bacterial pathogens (Öztop et al. 2002, Goh et al. creasing market demand for this fish has led to a sig-
2010). According to Pandey (2013), TCE is also a po- nificant boost in research to improve the survival and
tential plant biomedicine for improving the health performance of it.
non-specific defense mechanism in fish and elevat- However, information regarding the effects of
ing the specific immune response. The use of TCE in TCE immersion on the survival and immune compe-
O. niloticus (Chitmanat et al., 2003) and post-larval tence of ornamental fish when challenged with A.
black tiger shrimp, Penaeus monodon, (Ikhwanuddin hydrophila is unknown. Thus, the current study was
et al. 2014) also improved their survival and immu- designed to evaluate the effects of TCE immersion on
nity. Further, TCE also increased the hematological survival, RBC, Hb, Hct, PLT, TLC, and DLC, includ-
profiles of African catfish, Clarias gariepinus ing the number and percentage of lymphocytes,
(Burchell) (Julius et al. 2015). Additionally, TCE monocytes, and granulocytes in Betta sp. when chal-
with phytochemical active ingredients such as lenged with A. hydrophila.
flavonoid is useful for boosting immune function and
 Terminalia catappa L. extract improves survival, hematological profile and resistance to... 105

Materials and methods Animals and experimental setup

Three hundred and sixty two-month old fish (average

Plant material initial weight 0.62 g) were purchased from a local
breeding farm in Samarinda, East Kalimantan (Indo-
Dried, cut brown T. catappa leaves were collected on nesia) and acclimated at the Animal Physiology, De-
the campus of Mulawarman University, Barong velopment, and Molecular Laboratory, Mulawarman
Tongkok, East Kalimantan, Indonesia. To eliminate ex- University, East Kalimantan for three days. The fish
were then randomly distributed into two groups (C
traneous matter, the collected T. catappa leaves were
and P) with triplicate groups of twenty fish per repli-
washed with deionized water and immediately dried in
cate group. Each fish was then placed in an individual
an oven at 40°C for 12 h. T. catappa powder was ob-
glass tank (0.5 L capacity, 0.4 L of freshwater in each
tained using a mill. The powder was extracted with eth- tank). For three days, 500 ppm TCE was added to the
anol 95% for 3 days (100 g per 100 mL). After filtration, fish in group P (Immersion), while there was no im-
the extract was evaporated using a rotary evaporator mersion in group C (Control). Temperature, pH, and
and stored at 4°C until it was used as a crude extract. dissolved oxygen (DO) were measured daily with
To detect the presence of possible phyto- a thermometer and a HM-7 pH meter (TOA-DKK
chemicals in the crude extract of T. catappa leaves, Corporation, Japan). Nitrate, nitrite, and ammonium
some preliminary phytochemical tests for alkaloid, were measured and recorded weekly using chemical
saponin, steroid, triterpenoid, quinon, phenolic, tan- test kits (Aquarium Pharmaceuticals™, Inc., USA).
nin, and flavonoid were performed as follows: (1) Al- The fish in each glass tank was fed with frozen blood
kaloid-Dragendorff Test – 2 mL of the filtrate was worm at a ratio of 1% of body weight every day. The
added to 1 mL of Dragendorff reagent along the side optimum immersion (500 ppm) and feeding rations
of the test tube, an orange or orange-reddish-brown were determined during previous experiments
precipitate indicated a positive test, (2) test for (Nugroho et al. 2016). Uneaten food and feces were si-
saponin – 1 mL of extract + 5 mL distilled water was phoned out before renewing the water. The water vol-
shaken vigorously, the appearance of stable froth for ume was renewed every day at a concentration of 500
15 minutes indicated the presence of saponin, (3) ppm (only for group P) and maintained at 0.4 L of wa-
test for steroids and (4) triterpenoid (Lieber- ter in every glass tank.
mann-Burchard): – 2 mL extract + 1 mL chloroform
+ a few drops of acetic anhydride + concentrated Challenge test
sulphuric acid added along the side of the test tube,
the appearance of a blue or green color indicated the At the end of the trial, both groups of fish (C and P)
presence of steroids, while the appearance of a red or were further divided into three triplicate subgroups
brown color indicated the presence of triterpenoid, each (20 fish per replicate). Two subgroups, one from
(5) test for Quinon – 1 mL of extract + 1 mL of con- each group, were injected with 20 µL of A.
centrated sulfuric acid, a red color indicated the pres- hydrophila stock suspension (106 CFU mL-1) that
ence of quinon, (6) test for phenols and tannins – was obtained from the Department of Biology, Fac-
crude extract was mixed with 2 mL of 2% solution of ulty of Mathematics and Natural Sciences,
FeCl3, a blue-green or black color indicated the pres- Mulawarman University, Indonesia; two subgroups
ence of phenols and tannins, (7) test for flavonoids – from each group were injected with 20 µL normal sa-
2 mL extract + concentrated hydrochloric acid + line solution and; the third and final two subgroups
magnesium ribbon, the appearance of pink-red color from each group were not injected (control sub-
indicated the presence of flavonoids. group). All injections were performed
106 Rudy Agung Nugroho et al.

intraperitoneally. All fish were then monitored for Results

survival, RBC, Hb, Hct, PLT, TLC, and DLC, includ-
ing the number and percentage of lymphocytes, The screening test for bioactive compounds in TCE
monocytes, and granulocytes at 0, 24, and 48 h must be done to determine the benefit of
post-injection time. phytochemical bioactive agents in fish. The current
study showed the presence of saponin, triterpenoid,
quinon, phenolic, tannins, and flavonoid in the TCE
Sampling and analytical procedure (Table 1).

The survival rate of the fish in each group was re-

corded daily during the pre-challenge test and every Table 1
Phytochemical screening test of T. catappa leaf extract.
24 h post injection for 48 hours. At day three after im- (+) Present; (-) Absent
mersion and every 24 h interval during the challenge
Phytochemicals Results
test, blood samples were taken by caudal
venipuncture after anesthetizing the fish with MS-222 Alkaloid -
(200 mg L-1). Total RBC (106 per mm3) and WBC (103 Saponin +
per mm3) were determined manually with the im- Steroid -
proved Neubauer counting chamber. Hemoglobin
Triterpenoid +
(Hb) was determined according to the
Quinon +
cyanmethemoglobin procedure (Blaxhall and Rao
Phenolic +
1973) and expressed in g dL-1. The number and per-
centage of lymphocytes, monocytes, granulocytes, Tanin +
Htc, and PLT were determined using an Mindray Flavonoid +
BC2800 Auto Hematology Analyser (Mindray®
Shenzhen, China). During the three days of the immersion treat-
ment, the temperature, pH, DO, nitrate, nitrite, and
ammonia levels were recorded as follow: tempera-
Statistical analysis
ture – 27.9°C ± 0.3, pH – 7.37±0.2, and dissolved
oxygen level – 8.2 ± 0.3 mg L-1. The nitrate, nitrite,
Results are expressed as means ± standard error
and ammonia levels in the water of the control and
(SE) and the data were analyzed using SPSS version
500 ppm TCE treatment groups were below the lim-
21 (SPSS, Inc., USA). The data on survival, the per-
its of detection.
centage of lymphocytes, monocytes, and granulo-
The survival and hematological profiles of the
cytes were transformed to arcsine. The WBC, RBC,
control group and the group of fish immersed in 500
Hb, the number of lymphocytes, monocytes, and
ppm TCE are presented in Table 2. After three days
granulocytes on day three after immersion were sub- of immersion, the survival of fish in the TCE immer-
jected to the T-test to evaluate the significance of dif- sion was significantly higher (T-test, P < 0.05) than in
ferences among groups. Meanwhile, survival, WBC, the control group, whereas blood parameters such as
RBC, Hb, Htc, PLT, the percentage and number of WBC, RBC, Hb, Htc, PLT, and the number of lym-
lymphocytes, monocytes, and granulocytes during phocytes, monocytes, and granulocytes were not af-
the challenge test were subjected to two way fected by 500 ppm TCE immersion.
ANOVA, followed by Tukey’s post hoc test to evalu- After being challenged with A. hydrophila, the
ate significant differences among the groups of treat- survival rates of fish without immersion were signifi-
ments. All tests were significant at P < 0.05. cantly lower (P < 0.05) than those of the other fish
 Terminalia catappa L. extract improves survival, hematological profile and resistance to... 107

Table 2
Mean ± SE (standard error) survival and blood profile of Betta sp. after three-day immersion in 500 ppm T. catappa leaf
extract

Groups

Parameters Control 500 ppm

Survival (%) 88.88a ± 0.5 98.88b ± 2.42

WBC (103 µL-1) 8.32 ± 0.21 8.38 ± 0.12
6 -1
RBC (10 µL ) 1.64 ± 0.14 1.72 ± 0.04
Hb (g dL-1) 6.22 ± 0.46 6.11 ± 0.38
Htc (%) 18.66 ± 1.40 16.66 ± 0.47
3 -1
Lymphocytes (10 µL ) 7.00 ± 1.01 5.00 ± 0.23
3 -1
Monocytes (10 µL ) 1.22 ± 0.22 1.66 ± 0.23
Granulocytes (103 µL-1) 1.77 ± 0.22 1.88 ± 0.20
3 -1
PLT (10 µL ) 34.88 ± 1.04 35.77 ± 1.81

WBC – White blood cell; RBC – Red blood cell; Hb – Hemoglobin; Htc – Hematocrit; PLT – Platelet. Different letter indexes (a, b)
indicate significantly different means for different treatments at P < 0.05.

(Fig. 1). The WBC counts of the control fish increased in the number of PLT at 48 h post challenge. The
significantly after the challenge with A. hydrophila. RBC, Hb, and Hct of all subgroups of fish were not
The highest WBC count (12.36 ± 0.19 × 103 µL-1) significantly different during the challenge test.
was noted in the control fish subgroup at 48 h post During the challenge test, the 500 ppm
challenge (Table 3). Meanwhile, the PLT of TCE-immersed fish had the lowest percentage of
TCE-immersed fish that were injected with the A. lymphocytes. However, the highest percentage of
hydrophila pathogen was significantly the highest (P monocytes and granulocytes were found in the sub-
< 0.05) at 24 h post challenge, followed by a decline groups of infected fish that had been immersed in

100

80
Survival (%)

60
C1
C2
40 C3
P1
20 P2
P3

0
0 24 48
Hour
Figure 1. Survival of Betta sp. challenged with A. hydrophila. Note: C1 – control with no injection; C2 – control with 20 µL normal saline in-
jection; C3 – control with 20 µL A. hydrophila injection; P1 – 500 ppm T. catappa leaf extract (TCE) immersion with no challenge; P2 – 500
ppm immersion with 20 µL normal saline injection; P3 – 500 ppm immersion with 20 µL A. hydrophila injection. *Significant difference at P
< 0.05.
108 Rudy Agung Nugroho et al.

80
70
fish culture causing heavy economic losses because
60 C1 of high mortalities. Plant-based extracts have been
Lymphocyte (%)

C2
50
C3
proven to enhancesurvival and immunocompetence
40
P1 in cultured fish. A number of plant extracts that have
30 P2
* active ingredients and various biological activities
20 P3

10 have been reported as suitable for use as supple-

0
0 24 48
ments in aquaculture (Citarasu 2010, Madhuri et al.
2013, Chakraborty et al. 2014, Sivasankar et al.
40
35 2015, Syahidah et al. 2015). The current study indi-
*
30 C1 cates that the application of 500 ppm TCE, which
C2
Monocyte (%)

25
C3
contains active phytochemical compounds such as
20
15
P1 saponin, triterpenoid, quinon, phenolic, tannins, and
P2
10 P3 flavonoid, can significantly improve the survival of
5 Betta sp. even when they are challenged with A.
0
0 24 48
hydrophila. Similarly, improved survival of A.
50
hydrophila-infected goldfish (C. auratus) fed herbal
* plant extract (Ixora coccinea) has also been reported
40 C1
Granulocyte (%)

C2
30 (Anusha et al. 2014).
C3
20 P1 Plant-based bioactive compounds are mostly bio-
P2
10 P3 degradable in nature, ecofriendly, safe for humans,
0 biocompatible, and usually less expensive than syn-
0 24 48
thetic compounds (Immanuel et al. 2009). Several
Hour
phytochemical compounds such as saponin,
Figure 2. Lymphocyte, monocyte, and granulocyte count percent- triterpenoid, quinon, phenolic, tannins, and flavonoid
ages in the blood of Betta sp. Note: C1 – control with no injection; are found in various parts of some plants, including
C2 – control with 20 µL normal saline injection; C3 – control with
the leaves of T. catappa (Citarasu 2010, Chakraborty
20 µL A. hydrophila injection; P1 – 500 ppm T. catappa leaf extract
(TCE) immersion with no challenge; P2 – 500 ppm immersion with et al. 2014). The phytochemicals which are contained
20 µL normal saline injection; P3 – 500 ppm immersion with 20 µL in the plant may boost the innate immune system and
A. hydrophila injections. *Significant difference at P < 0.05. could be used widely in fish culture (Chakraborty and
Hancz 2011). Some previous studies have been con-
500 ppm TCE (Fig. 2). The number of lymphocytes of ducted to determine the use of phytochemicals from
all subgroups of fish was stable during the challenge various parts of plant extracts to increase survival
test, whereas the number of monocytes and granulo- rates of marine ornamental fish (Balachandran and
cytes of all the subgroups of fish were significantly in- Tissera 2013), Nile tilapia (O. niloticus)
creased 24 h post challenge. The highest number of (Abdel-Tawwab et al. 2010, Akinwande et al. 2011)
monocytes and granulocytes were noted in the and common carp, Cyprinus carpio L. (Soltanian and
TCE-immersed fish challenged with A. hydrophila Fereidouni 2016) against A. hydrophila.
48 h post injection (Fig. 3).

Survival

Discussion The current study found that immersing fish for three
days in TCE and the subgroups of TCE-immersed
Over the past few years, infectious diseases caused fish with or without injections exhibited significantly
by A. hydrophila have become a major problem in higher (P < 0.05) survival rates than did the control
 Terminalia catappa L. extract improves survival, hematological profile and resistance to... 109

12 1
b
10 1
a 1
1 b
1 1 ab 1
Numbers (10 µL )

1
-1

8
a a 1 ab ab
1 1 1
3

1 1 1 1
b a 1 1 a a
6 a ab a a
a ab

0
0 24 48
C1 C2 C3 P1 P2 P3

5 2 2
ab b
4.5
2 2
4 b
b
3.5
Numbers (10 µL )
-1

3 2
3

1 ab
2.5 1 b 2
1 2 2 2
a a 2
2 1 b a a 2 a 2
1 1 a
a a a
1.5 a a
1
0.5
0
0 24 48
C1 C2 C3 P1 P2 P3
2
6 c 2
2 bc 2
bc c
5
2
2 b 2
Numbers (10 µL )
-1

4 2 bc bc
b
3

2 2 2
3 1 1 1 a
1 1 1 a a
b bc c
a bc a
2 2
a
1

0
0 24 48
Time (h)
C1 C2 C3 P1 P2 P3
Figure 3. Lymphocyte, monocyte, and granulocyte count numbers in the blood of Betta sp. challenged with A. hydrophila. Different letter
indexes (a, b, c) over bars indicate significantly different means for different treatments at P < 0.05. Different numerical indexes (1, 2, 3)
over bars indicate significantly different means at different times at P < 0.05. Note: C1 – control with no injection; C2 – control with 20 µL
normal saline injection; C3 – control with 20 µL A. hydrophila injection; P1 – 500 ppm T. catappa leaf extract (TCE) immersion with no
challenge; P2 – 500 ppm immersion with 20 µL normal saline injection; P3 – 500 ppm immersion with 20 µL A. hydrophila injections.
110 Rudy Agung Nugroho et al.

Table 3
Mean ± SE blood profile of Betta sp. challenged with A. hydrophila

Groups

Control 500 ppm

Parameters Hour C1 C2 C3 P1 P2 P3

WBC
(103 µL-1) 0 8.00±0.50a1 8.43±0.31b1 8.53±0.43c1 8.13±0.32a1 8.13±0.27a1 8.03±0.34a1
24 8.50±0.18a2 10.43±0.42b2 11.70±0.41c2 8.43±0.22a2 8.56±0.22a2 8.36±0.22a2
48 8.33±0.19a2 9.13±0.44b2 12.36±0.19c2 8.43±0.13a2 8.90±0.25a2 8.86±0.22a2
RBC
(106 µL-1) 0 1.43±0.13a1 1.83±0.50a 1.66±0.27a 1.70±0.18a 1.76±0.22a 1.70±0.25a
a a a a a
24 1.76±0.26 1.80±0.18 1.66±0.19 1.76±0.22 1.70±0.18 1.73±0.13a
48 1.66±0.13a 1.73±0.13a 1.73±0.22a 1.80±0.18a 1.63±0.22a 1.66±0.13a
Hb
(g dL-1) 0 6.00±1.41a 6.66±1.49a 6.00±1.41a 6.33±1.45a 5.33±1.33a 6.66±1.49a
24 6.33±1.45a 5.33±1.33a 6.33±1.45a 6.66±1.49a 5.66±1.37a 6.33±1.45a
a a a a a
48 6.66±1.49 6.66±1.49 6.00±1.41 7.00±1.52 5.66±1.37 6.33±1.45a
Hct (%) 0 16.33±0.91a 20.66±1.53a 19.00±1.57a 15.66±0.43a 17.66±0.71a 16.66±0.71a
24 18.33±0.83a 17.00±0.57a 17.00±0.75a 18.33±0.43a 17.00±0.57a 17.33±0.71a
48 16.66±0.62a 18.00±0.57a 17.33±0.71a 16.66±0.43a 18.33±0.43a 16.66±0.43a
PLT
(103 µL-1) 0 36.66±1.20a1 34.00±0.93a1 34.66±1.03a1 33.00±0.57a1 40.00±1.20b1 34.33±1.58b1
24 41.66±1.78a2 32.00±1.47a2 40.00±1.07a2 33.33±0.43a2 46.00±1.81b2 49.33±1.28b2
48 31.66±0.71a1 32.00±0.75a1 33.33±0.71a2 31.66±0.98a1 33.00±0.57b1 34.00±0.57b1

Different letter indexes (a, b, c) indicate significantly different means for different treatments at P < 0.05. Different numerical
indexes (1, 2) indicate significantly different means at different times at P < 0.05. Note: WBC – White blood cell; RBC – Red blood
cell; Hb – Hemoglobin; Htc – Hematocrit; PLT – Platelet. C1 – control with no injection; C2 – control with 20 µL normal saline
injection; C3 – control with 20 µL A. hydrophila injection; P1 – 500 ppm T. catappa leaf extract (TCE) immersion with no
challenge; P2 – 500 ppm TCE immersion with 20 µL normal saline injection; P3 – 500 ppm TCE immersion with 20 µL A.
hydrophila injection.

group. Similar results reported on previous work in- (Balachandran and Tissera 2013), and tilapia (O.
dicate that Nile tilapia immersed in extracts of Aspar- niloticus) (Akinwande et al. 2011). Plants, especially
agus racemosus (Mukherjee et al. 2015) and T. catappa, are rich in a wide variety of secondary
Neobenedenia sp. with Allium sativum (Militz et al. metabolite phytochemicals such as tannins and
2014) had significantly better survival than did con- saponin (Citarasu 2010, Nugroho et al. 2016). Cul-
trols without immersion. The high survival percent- tured post-larval black tiger shrimp (P. monodon)
age of fish in different treatments could indicate that treated with the tannins in TCE had significantly
the phytochemical content in the plant extracts con- higher survival rates (Ikhwanuddin et al. 2014). Ac-
tributed to improved fish health. cording to Van-Sumere et al. (1975) and Ashraf and
Phytochemical extracts from various parts of Bengston (2007), tannin can absorb harmful chemi-
plants were shown to enhance survival rates in com- cals and provide a soothing, suitable environment
mon carp (C. carpio), marine ornamental fish that is fairly benign for fish. Besides tannin, the
 Terminalia catappa L. extract improves survival, hematological profile and resistance to... 111

biological effects of saponin, including survival and reported that flavonoids from plant metabolites may
immune system stimulation in fish, have also been contribute to antibacterial activity. Flavonoids con-
widely studied and reviewed. Saponins are found in tained in TCE were able to inhibit bacterial growth.
plants containing either a steroid or triterpenoid Thus, it is clear that the WBC counts of the subgroup
aglycone to which one or more sugar chains are at- of fish immersed in TCE did not change. Additionally,
tached (Oda et al. 2003). Furthermore, the applica- flavonoid has a positive effect in reducing RBC
tion of 1 and 2 mg L-1 saponin in white shrimp, hemolysis which can be caused by bacterial infection,
Litopenaeus vannamei, increased their survival rate by protecting the biological membranes of the RBC
(Su and Chen 2008). On the other hand, it was ob- (Kitagawa et al. 1992, Asgary et al. 2005). Some in-
served that pure saponin at high levels caused stress vestigators also report that the antioxidants present in
symptoms and mortality following exposure to 200 plant extracts might trigger erythropoiesis. This seems
mg L-1 of pure saponin (Nagesh et al. 1999). to be in agreement with the present study as TCE has
flavonoid that, as an antioxidant, might maintain the
heme iron in its ferrous state (Akah et al. 2007, Uboh
Blood Profile
et al. 2010, Shatoor 2011).
Determining how phytochemicals are related to he- Besides total WBC, differential WBC such as lym-
matological indices is common in the culture of phocytes, monocytes, and granulocytes produce anti-
aquatic animals, including fish. Hematological pa- bodies to acknowledge and respond to the antigen and
rameters are used as indicators of the response of fish to act as mediators of cellular and humoral immune
health status, especially in the presence of disease responses (Abbas et al. 2010, Soltanian and
and under stressful conditions (Osman et al. 2010, Fereidouni 2016). The current results found that fish
Karimi et al. 2013, Suely et al. 2016). Hematological immersed in 500 ppm TCE had the lowest percentage
indices such as WBC, RBC, Hb, the number and per- of lymphocytes but the highest percentage of
centage of lymphocytes, monocytes, and granulo- monocytes and granulocytes. The number of lympho-
cytes play important roles in assessing the cytes of all the subgroups of fish was stable during the
physiological condition of fish. challenge test, whereas the number of monocytes and
Based on the current results, it was found that the granulocytes of all the subgroups of fish were signifi-
WBC count of control subgroups of fish challenged cantly increased 24 h post challenge. The highest
with A. hydrophila had significantly increased 24 h number of monocytes and granulocytes were noted in
post injection, whereas the number of WBC of all the the TCE-immersed fish challenged with A. hydrophila
subgroups of immersed-fish were not significantly dif- 48 h post injection. The significant increase in
ferent. Meanwhile, the RBC, Hb, and Hct of all the monocytes and granulocytes in this study is in agree-
subgroups of fish during the challenge test were not ment with Gabriel et al. (2015) who reports that the
affected. These results are in agreement with previous percentage of monocytes and granulocytes in tilapia
findings that indicate there was an increasing number (GIFT) infected with Streptococcus iniae increased af-
of WBC in Nile tilapia after infection (Martins et al. ter the challenge test. Additionally, Aly et al. (2015)
2009). Pourmoghim et al. (2015) also found similar report that Nile tilapia vaccinated with A. hydrophila
results in that dietary Origanum vulgare extract incor- had no significant different lymphocyte values com-
porated into test diets had no significant effect on pared to unvaccinated Nile tilapia.
RBC, WBC, Hct, hemoglobin, or Hb. The increase in monocyte and granulocytes in
The underlying mechanism(s) whereby the WBC TCE-immersed fish can be attributed to the improve-
of TCE-immersed fish did was not altered is not prop- ment of non-specific immune responses. However,
erly understood. However, Rattanachaikunsopon and the number of lymphocytes that was stable during
Phumkhachorn (2010) and Reverter et al. (2014) the challenge test may indicate that there was no
112 Rudy Agung Nugroho et al.

enhancement in specific immune induction. Abdel-Tawwab M., Ahmad M.H., Seden M.E.A., Sakr S.F.M.
2010 – Use of green tea, Camellia sinensis L., in practical
Monocytes play a pivotal role in the immune defense
diet for growth and protection of nile tilapia, Oreochromis
system of fish. They can transform into macrophages niloticus (L.), against Aeromonas hydrophila infection – J.
and exhibit phagocytosis activity against pathogens World Aquacult. Soc. 41: 203-213.
at first recognition and subsequent infections. Abdi K., Azadikhah D., Fard A.N. 2014 – Occurrence
Granulocytes, such as neutrophils, are the primary reported the Aeromonas sobria infection in Goldfish
(Carassius auratus) – Iranian journal congress, At Tehe-
cells responsible for first infections and the
ran.
phagocytosis of bacterial pathogens during challenge Adel M., Pourgholam R., Zorriehzahra J., Ghiasi M. 2016 –
tests (Sivagurunathan et al. 2011). Hemato-Immunological and biochemical parameters,
skin antibacterial activity, and survival in rainbow trout
(Oncorhynchus mykiss) following the diet supplemented
with Mentha piperita against Yersinia ruckeri – Fish
Conclusions Shellfish Immunol. 55: 267-273.
Akah PA., Okpi O., Okoli C.O. 2007 – Evaluation of the
In summary, this study indicates that 500 ppm TCE anti-inflammatory, analgesic and antimicrobial activities
of bark of Afzelia africana – Nig. J. Nat. Prod. Med. 11:
immersion can potentially be used to combat A.
48-52.
hydrophila as it increases the survival and hemato- Akinwande A., Dada A.A., Moody F.O. 2011 – Effect of
logical profile, such as the number and percentage of dietary administration of the phytochemical
monocytes and granulocytes, of Betta sp. The study "genistein"(3,5,7,3,4 pentahydroxyflavone) on masculine
has also shown that the application of TCE might also tilapia, Oreochromis niloticus – Elixir Aqua. 33:
2231-2233.
be of practical use in disease management strategies
Alfian. 2010 – Indonesian ornamental fish: Redifining the
in fish, especially the ornamental fish Betta sp. Fur- global status – In: Export News Indonesia (Eds) H.I.
ther research needs to be conducted to examine the Kresnarini, H.I. Widayanti, Z. Bachtar, Jakarta, Indone-
lysozyme activity, respiratory burst, and the antioxi- sia: 10 p.
dant profiles of fish immersed in 500 ppm TCE. Alvarez F., Razquin B., Villena A., Lopez Fierro P., Zapata A.
1988 – Alterations in the peripheral lymphoid organs and
Acknowledgement. The authors are grateful to the Re- differential leukocyte counts in Saprolegnia-infected
brown trout, Salmo trutta fario – Vet. Immunol.
search, Technology, and General Higher Education Minis-
Immunop. 18: 181-193.
try, Government of Indonesia for financial support No.
Aly S.M., Albutti A.S., Rahmani A.H., Atti N.M. A. 2015 – The
079/UN17.41/LT/2016 SP DIPA-042.06.1.401516/ response of New-season Nile tilapia to Aeromonas
2016. The authors also extend their gratitude to the follow- hydrophila vaccine – Int. J. Clin. Exp. Med. 8:
ing: Mulawarman University, especially the Department of 4508-4514.
Biology, Faculty of Mathematics and Natural Sciences for Anusha P., Thangaviji V., Velmurugan S., Michaelbabu M.,
their support. Citarasu T. 2014 – Protection of ornamental gold fish
Author contributions. R.A.N. and H.M. conceived and Carassius auratus against Aeromonas hydrophila by
treating Ixora coccinea active principles – Fish Shellfish
designed the experiments, and performed the experi-
Immunol. 36: 485-493.
ments, R.A.N., H.M., F.M.N., and W.P. collected the data,
Asgary S., Naderi G.H., Askari N.M.S. 2005 – Protective effect
R.A.N., H.M., and W.P. wrote the paper, R.A.N., and H.M. of flavonoids against red blood cell hemolysis by free rad-
read and approved the final manuscript. icals – Exp. Clin. Cardiol. 10: 88-90.
Ashraf M., Bengtson D.A. 2007 – Effect of tannic acid on feed
intake, survival and growth of striped bass (Morone
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