THE EFFECT OF NONI FRUIT Morinda citrifolia L.

EXTRACT IMMERSION
ON THE HEMATOLOGY OF TILAPIA Oreochromis niloticus

M. Tando Ridho Pratama1, Woro Hastuti Satyantini2*, Sudarno2

1Aquaculture, Faculty of Fisheries and Marine Universitas Airlangga, Surabaya 60115

2Department of Fish Health Management and Aquaculture, Faculty of Fisheries and Marine,
Universitas Airlangga, Surabaya 60115
*Correspondence : [email protected]

Abstract
Immersion method using noni fruit Morinda citrifolia L. extract is an attempt to
improve the fish immune response to disease attack on tilapia O. niloticus cultivation. The
influence of different noni fruit extract concentration on hematology parameters has not been
widely known, thus conducting this research for determining the hematological study of nile
tilapia after immersed by noni fruit extract. This research used complete randomized design
(CRD) method with six treatments and four replications, namely A (0 g/L), B (3.6 g/L), C (4.2
g/L ), D (4.8 g/L), E (5.4 g/L) and F (6 g/L). Main parameters observed were total erythrocyte,
total leukocyte, hemoglobin level, and blood glucose level. The result indicated that noni fruit
extract immersion was significantly different (P<0.05) on blood profiles, such as total
erythrocytes, total leukocytes, hemoglobin level and blood glucose level of tilapia. 4.8 g/L noni
fruit extract concentration showed the best concentration to improve immunity and survival rate
of tilapia more than 50%.

Keywords : Blood profiles, immersion method, noni fruit extract, tilapia

Introduction
Tilapia fish farming O. niloticus is inseparable from the high mortality risk caused by
disease, namely viruses, bacteria, fungi, or other pathogen (Aisiah, 2012). The existence of the
disease in tilapia fish farming requires more handling with immunostimulant. Noni fruit Morinda
citrifolia L. proved an effective use as an immunostimulant for disease preventive effort by
affecting the immune response against pathogenic diseases (Chan-Blanco et al., 2006).
Some contents of the noni fruit extract which is useful to affect the fish immune system
of the fish are flavonoid, saponin, and scopoletin. Flavonoid increases erythrocyte and
hemoglobin production, lowering the blood glucose level (Wahjuningrum et al., 2008). Saponin
serves as α-glucosidase enzyme inhibitor which was able to lower the blood glucose levels and
stimulate pancreatic β cells to produce more insulin (Nayak et al., 2009). Scopoletin increases
the macrophage activity for phagocytosis, serotonin binding, and blood vessel widening
(Solomon, 1998). Noni fruit extract was often used as an immunostimulant for fish, but not much
was known about the toxicity level in tilapia which correlated with fish blood profiles. Toxicity
test noni fruit extract on tilapia was observed based on the lethal concentration that causes 50%
mortality in fish (LC50) (Al-Attar, 2005).
Noni fruit extract can be given by immersion method. Haryani et al. (2012) stated that
immersion method on fish seed eased the medication process on the giant scale. This condition
would increase the modulation function of noni fruit extract and immune system activity by
stimulating and improving the immune system function, causing a change that affects the
physiology of blood profiles (Zumrotul, 2013). Effective noni fruit extract concentration is
administered by blood examination method.
Blood profile examination is beneficial to help examine the immune system, disease
diagnosis, and observe the fish health status (Salasia et al., 2001). The influence of noni fruit
extract in different concentration for immersion method against the blood profile of tilapia had
not been much reported, hence it was necessary to conduct a research on total erythrocytes and
leukocytes, as well as hemoglobin and blood glucose level of tilapia after immersed by noni fruit
extract.

Materials dan Method

Place and Period
This research was conducted on February – July 2018 in Anatomy and Microbiology
laboratory, Faculty of Fisheries and Marine, Universitas Airlangga Surabaya.
Tools and Materials
Tools used in this research were knives, net, fabric, glass bottle, Beaker glass, cover glass,
microscope, DO (dissolved oxygen) meter, pH meter, syringe, glucometer, haemocytometer,
handcounter, microtube, Hb-meter tube, HCl, stirrer, Sahli pippette, micropippette, Thoma
pippett for erythrocyte dan leukocyte. Tools used for tilapia rearing were aquarium sized
40x30x30cm3, aerator, aeration hose, and aeration stone. Materials used this research were 240
tilapia sized 7-9 cm retrieved from UPT PBAT Umbulan, Pasuruan, East Java. Riped noni fruits
were obtained from Surabaya. Other materials were sterile freshwater, ammonia test kit, pellet,
Hayem solution, Turk solution, EDTA, aquadest, and lable paper.
Treatment
This research used complete randomized design (CRD) experimental method using six
treatments and four replications (Kusriningrum 2010). Treatments given were A (0 g/L), B (3.6
g/L), C (4.2 g/L ), D (4.8 g/L), E (5.4 g/L) and F (6 g/L).

Procedures
Noni fruit extract
Noni fruit extract produced was the aqueous extract. Riped noni fruits were chosen as
containing higher bioactive compounds possessed than the unriped ones (Chan-Bkanco et al.
2006). Noni fruits were cleaned and cut into pieces, then soluted 100 g of noni furit into 1 L
aquadest. Noni fruits were blended and filtered using fabric. Aqueous extract of noni furit was
stored in 4°C for 1-2 days, based on Berkovich et al. (2013).

Preparation
Aquariums were disinfected using 150 mg/L chlorine, then washed until clean. Water
media used for tilapia rearing was firstly precipitated on the water stock tank and filled on the
aquarium along with aeration setting. Sample fish were selected based on the length and weight,
health status, and body morphology.

Blood sampling
Every aquarium was given the same treatment and condition, but different noni fruit
extract concentration given. Each aquarium was filled with 10 tilapia or 1 fish/L stocking density
(Rahayu et al., 2013). Tilapia was immersed in the noni fruit extract under different
concentration for 96 hours and hematologically observed at 24 and 96 hours after immersion by
taking three samples from each treatment (Khabakhsh et al., 2014). Mardin (2011) explained that
tilapia blood was retrieved from caudalis vein by 1 ml syringe injection with EDTA 10% and put
inside 1 ml microtube for conducting the blood profile observation. Tilapia were not fed to
maintain the water quality. Survival rate observation and water quality measurement were
performed at the beginning and end of the research.

Total erythrocyte count

Erythrocytes were counted based on Blaxhall and Daisley (1973) method. Blood sample
from microtube was absorbed using 0.5 ml erythrocytes capillary pipette and diluted with Hayem
solution in 101 ml scale. Both ends were closed parallelly and shaken forming number eight for
3-5 minutes until all solution is mixed. Blood was disposed in two drops, while another drop was
put in haemocytometer count covered with glass. Erythrocyte was observed under the
microscope with 400x maginification and calculated at five small box with the following
formula:
1
Ʃ Erythrocyte = N x small box volume x P
Note :
N : Counted cell
P : Dilution number

Total leukocyte count

Leukocyte count was based on Blaxhall and Daisley (1973) method. Blood sample from
microtube was absorbed using 0.5 ml leukocyte capillary pipette and diluted with Turk solution
in 11 ml scale. Both ends were closed parallelly and shaken forming number eight for 3-5
minutes until all solution is mixed. Blood was disposed in two drops, while another drop was put
in haemocytometer count covered with glass. Leukocyte was observed under the microscope
with 400x maginification and calculated at five small box with the following formula:
1
Ʃ Leukosit = N x big box volume x P

Keterangan :
N : Counted cell
P : Dilution number

Hemoglobin
Wedemeyer and Yasutake (1977) stated that the procedure for hemoglobin calculation
was referred to Sahli method. Blood sample was absorbed onto Sahli pippette until reaching 20
mm3 or 0.2 ml. Blood in the pipette was moved to the Hb-meter tube filled with 0.1 N HCl until
scale 10 (red sign). Blood was stirred with a condenser for approximately 5 minutes. Blood was
added aquadest into the tube until the blood color matched with the existing standard solution in
Hb-meter. Hemoglobin level was expressed in g%.

Blood glucose
Blood sample was taken intravenously using 1 ml. Blood sample was collected in the
One Touch Horizon blood glucose monitoring system (BGMS) to read the blood glucose level of
the sampled fish. Blood glucose level was expressed in mg/dl.
Survival rate
Fish survival rate is comparison between the amount of fish survived at the beginning and
the end of the research. Tilapia survival rate was calculated using the following formula:
Nt
SR = N0 x 100%

Note :
SR : Survival rate
Nt : Number of fish survived at the beginning of the research
N0 : Number of fish survived at the end of the research

Data analysis
The data obtained were processed using Analysis of Variance (ANOVA) to observe
whether the noni fruit extract affected the blood profiles of tilapia. Data processing was
continued using Duncan's multiple range test, when there were significant difference found (p <
0.05) among treatments (Kusriningrum, 2010). The data were analyzed using SPSS 17.0
software application.

Result
Total erythrocyte count
Total erythrocyte was measured to observe the change of tilapia total erythrocyte after
immersed with noni fruit extract. Average total erythrocyte of tilapia is shown on Table 1.

Table 1. Total erythrocyte of tilapia after noni fruit extract immersion on different concentration
Total erythrocyte (x105 sel/mm³)
Treatment
24 hours 96 hours
A 3.11 ± 0.01a 5.58 ± 0,01d
B 6.48 ± 0.02c 3.99 ± 0,03c
b
C 5.53 ± 0.00 3.22 ± 0,03b
D 7.99 ± 0.01d 7.61 ± 0,00e
f
E 13.15 ± 0.03 10.1 ± 0,01f
F 11.22 ± 0.03e 2.83 ± 0,01a
Note : Different superscript letter on the same column shows signiifcant difference (p<0.05), A (0 g/L), B (3.6 g/L),
C (4.2 g/L ), D (4.8 g/L), E (5.4 g/L), F (6 g/L)

Noni fruit concentration extract treatments provide significant difference (p < 0.05)
against total erythrocyte of tilapia. The average level of erythrocyte during research was around
2.83-13.15 x 105 cells/mm3. The total erythrocyte at 24 and 96 hours experienced a decrease
level B, C, D, E and F. Total erythrocytes on treatment D and E was higher compared to the total
erythrocytes on A (control). The best concentration for noni fruit extract immersion was 4.8 g/L
(D), which showed stabilized total erythrocyte.

Total leukocyte count

Total leukocyte was measured to observe the change of tilapia total leukocyte after
immersed with noni fruit extract. Average total leukocyte level of tilapia is shown on Table 2.
Table 2. Total leukocyte of tilapia after noni fruit extract immersion on different concentration
Total leukocyte (x105 sel/mm³)
Treatment
24 hours 96 hours
A 2.96 ± 0.03a 2.83 ± 0.04a
B 4.18 ± 0.01b 5.10 ± 0.01b
c
C 5.75 ± 0.02 5.90 ± 0.04c
D 6.13 ± 0.01c 7.42 ± 0.01d
d
E 8.06 ± 0.01 10.65 ± 0.02f
F 11.40 ± 0.01e 9.07 ± 0.04e
Note : Different superscript letter on the same column shows signiifcant difference (p<0.05), A (0 g/L), B (3.6 g/L),
C (4.2 g/L ), D (4.8 g/L), E (5.4 g/L), F (6 g/L)

Noni fruit extract provides significant difference (p < 0.05) against total leukocytes of
tilapia. Total leukocyte of tilapia was ranged 2.83 - 11.40 x 104 cells/mm3. Total leukocyte of
tilapia at 24 and 96 hours showed decreased level on A and F, while increased level was
observed on B, C, D and E. Total leukocytes on B, C, D, E and F were compared to A (control).
The best concentration for noni fruit extract immersion was 4.8 g/L (D), which showed stabilized
rise on the total leukocyte after immersion.

Hemoglobin
Hemoglobin level was measured to observe the change of tilapia hemoglobin level after
immersed with noni fruit extract. Average hemoglobin level of tilapia is shown on Table 3.

Table 3. Hemoglobin level of tilapia after noni fruit extract immersion on different concentration
Hemoglobin level (g%)
Treatment
24 hours 96 hours
a
A 5.5 ± 0.39 6.2 ± 0.92b
a
B 4.7 ± 0.89 4.6 ± 0.57a
C 5.2 ± 0.47a 4.9 ± 0.42a
a
D 5.1 ± 0.64 5.0 ± 0.31a
E 5.2 ± 0.63 a 4.5 ± 0.83a
F 5.0 ± 0.60a 4.3 ± 0.59a
Note : Different superscript letter on the same column shows signiifcant difference (p<0.05), A (0 g/L), B (3.6 g/L),
C (4.2 g/L ), D (4.8 g/L), E (5.4 g/L), F (6 g/L)

The analysis result of hemoglobin level showed that the noni fruit extract gave significant
difference (p < 0.05) against tilapia hemoglobin level until the end of the research. Hemoglobin
level during the research was ranged 4.3 – 6.2 g%. Hemoglobin level of tilapia at 24 and 96
hours decreased on B, C, D, E and F. Hemoglobin level on D was closed to the hemoglobin level
observed on A (control). The best concentration for noni fruit extract immersion was 4.8 g/L (D),
which showed stabilized hemoglobin level.

Blood glucose
Blood glucose level was measured to observe the change of tilapia blood glucose level
after immersed with different concentration of noni fruit extract. Average blood glucose level of
tilapia is shown on Table 4.
Table 4. Tilapia blood glucose level after noni fruit extract immersion on different concentration
Blood glucose level (mg/dl)
Treatment
24 hours 96 hours
a
A 57.5 ± 12.82 66.3 ± 5.25b
a
B 58.5 ± 6.14 31.8 ± 4.57a
C 62.3 ± 7.93a 28.5 ± 4.65a
a
D 75.3 ± 0.06 27.8 ± 1.71a
E 109.3 ± 0.10b 29.3 ± 10.87a
b
F 103.8 ± 0.07 29.8 ± 7.58a
Note : Different superscript letter on the same column shows signiifcant difference (p<0.05), A (0 g/L), B (3.6 g/L),
C (4.2 g/L ), D (4.8 g/L), E (5.4 g/L), F (6 g/L)

The analysis result of hemoglobin level showed that the noni fruit extract gave significant
difference (p < 0.05) against tilapia blood glucose level. Tilapia blood glucose level was ranged
2.78 – 109.3 mg/dl. Tilapia blood glucose level at 24 and 96 hours decreased on B, C, D, E, and
F. Blood glucose level at D was lower than A (control). The best concentration for noni fruit
extract immersion was 4.8 g/L (D), which showed stabilized blood glucose level.

Water quality
Water quality has an important role on the aquaculture production, as it will affect the life
of cultured commodities. Water quality parameters measured during this research were
temperature, dissolved oxygen (DO), pH, and ammonia content. The average level of water
quality parameters are shown on Table 5.

Table 5. Water quality of tilapia O. niloticus rearing

Treatment
Water quality
A B C D E F
Temperature (oC) 29 29 29 29 29 28.5-28.9
DO (mg/L) 6.2-6.3 5.5-5.9 5.8-5.9 5.4-5.7 5.2 5-5.1
pH 7-7.1 6.9-7.1 6.9-7.2 6.8-7.2 6.8-7.2 6.6-7.2
Ammonia (mg/L) 0 0.5 0.5 0.5 1.0-2.0 2
Note : A (0 g/L), B (3.6 g/L), C (4.2 g/L ), D (4.8 g/L), E (5.4 g/L), F (6 g/L)

Water quality measurement result during the research showed temperature was 28.5-
29.4°C, DO 5.07-6.37 mg/L, pH 6.8-7.2, and ammonia content was 0-2 mg/L.

Survival rate
Survival rate observation was done to determine the influence of noni fruit extract
immersion against the survival rate of tilapia after immersion. Average result of survival rate is
shown on Table 6.
Tabel 6. Survival rate of tilapia O. niloticus
Blood glucose level (mg/dl)
Treatment
24 hours 96 hours
a
A 100 ± 0 100 ± 0a
a
B 100 ± 0 100 ± 0a
C 100 ± 0a 90 ± 0b
b
D 97.5 ± 5 85 ± 5.8c
E 87.5 ± 5c 47.5 ± 5d
d
F 50 ± 8.2 12.5 ± 5e
Note : Different superscript letter on the same column shows signiifcant difference (p<0.05), A (0 g/L), B (3.6 g/L),
C (4.2 g/L ), D (4.8 g/L), E (5.4 g/L), F (6 g/L)

Statistical analysis result showed that survival rate percentage after 24 hours on A had no
significant different (P>0.05) against B and C (100%), while having significant difference
(p<0.05) against D (97.5%), E (87.5%), and F (50%). Survival rate of tilapia on A showed
significant difference (p<0.05) against C (90%), D (85%), E (47.5%), and F (12.5%), while
having no significant difference (p>0.05) against B (100%).

Discussion
Total erythrocytes in this research was still in the normal range, as the normal fish had
total erythrocyte level ranged 150,000 – 3,000,000 cells/mm3 (Salasia et al., 2001). The
calculation result of erythrocytes at the end of the research decreased significantly on treatment
B, C, D, E and F (Table 1). This was suspected as the bioactive content in the form of saponin
affected decreased level in the total erythrocytes. Muharrama et al. (2015) explained that
hemolytical activity of saponin was able to lyse sterol content in erythrocytes, causing decreased
total erythrocytes level, while flavonoid maintaining the erythrocyte level. Haryani et al. (2012)
stated that flavonoid affected the productivity of erythrocytes by inducing the erythropoietin
hormone synthesis. Erythropoietin is required to stimulate eritropoiesis in kidney (Secombes,
1988).
Decreased level in total erythrocytes on all treatments given allegedly was influenced by
the decreased performance of kidney and liver. Jayaraman et al. (2008) reported that the
concentration of noni fruit extract on high concentration would become toxicant, thus disturbing
kidney and liver function as erythrocytes-producing organs. Interrupted kidney performance
would cause decreased productivity of erythrocytes impaired with liver disfunction which altered
the hemoglobin transfer, lowering the erythrocyte number in fish blood (Shalan et al., 2016).
The calculation result of total leukocytes in tilapia was equal with the total erythrocytes
that experienced declining level, yet only happened on A and F (Table 2). Low total leukocytes
at F was occurred allegedly because of high noni fruit concentration extract which caused
immunosuppression or declining immune response characterized by decreased total leukocytes.
This decline, however, was still in the normal range, compared to the optimum total leukocytes
of normal fish that ranged 32,000-146,000 cells/mm³ (Lagler et al., 1977).
Increased the total leukocytes on four treatments (B, C, D, and E) was allegedly
influenced by the bioactive compunds of noni fruit, namely flavonoids, saponins, scopoletin, and
others. Scopoletin was able to increase the phagocytic activity and macrophage capacity (Aldi et
al., 2016). Purwatiningsih et al. (2014) reported that flavonoid increased the phagocytosis and
production of leukocytes.
 The calculation result of hemoglobin level in tilapia was in line with the total
erythrocytes, showing decreased levels on all treatments, namely treatment B, C, D, E and F
(Table 3). Low hemoglobin levels was caused by some physiological factors, environmental
condition, and disease infections (Wells et al., 2005). Muharrama et al. (2015) mentioned that
saponin on noni fruit had an hemolytic activity that may result in the loss of hemoglobin in
erythrocytes. Saponin could also lower the hemoglobin level in the blood by hampering fish to
breathe (Kinasih et al., 2013). The hemoglobin level is an indicator of fish adaptability against
the physiological changes caused by foreign substances that induce stess or declined
environmental conditions (Mardin, 2011). Hemoglobin level in this research was still in the
normal range, based on Salasia et al. (2001), who stated that the normal hemoglobin level was
5.05-8.33 g%.
The calculation result of blood glucose level in tilapia was declined on all treatments,
namely treatment B, C, D, E and F (Table 4). Decrease blood glucose level was allegedly due to
the existence of saponin, which functioned as α-glucosidase inhibitor by delaying the blood
glucose absorption towards blood vessels, thus reducing the blood glucose level (Wahjuningrum
et al., 2008). Saponin stimulates pancreatic β cells to produce more insulin to help maintain the
homeostatic condition at a time when fish experiencing stress (Nayak et al., 2009). Blood
glucose level of this research was still in the normal range, based on the normal blood glucose
level 40-90 mg/dl reported by Rahardjo et al. (2011).
High noni fruit concentration extract given was capable of lowering the survival rate of
tilapia. This was demonstrated by the survival rate percentage occured on C, D, E, and F,
compared to A and B (Table 6). Decreased survival rate percentage of tilapia influenced the
change of blood profiles observed (total erythrocytes, total leukocytes, hemoglobin level, and
blood glucose level). Al-Attar (2005) explained that blood profile parameters indicated the
survival rate level after medication.
The level of survival rate on D gave higher value compared to other treatments, showing
capability to maintain stabilized blood profile parameters in the normal range. This made D (4.8
g/L noni extract given) was suitable as the best immersion concentration, as producing more than
50% survival rate.
DO content during this research was still in the normal range, namely 5.07 – 6.37 mg/l.
DO standard for maintaining tilapia rearing should be more than 3 mg/L (Djarijah, 1995). pH
level during this research was also still within the tolerated limit for tilapia ranging 6.6 – 7.2, as
the optimum pH level for maintaining tilapia rearing should be 6.5 – 8.5 (Djarijah, 1995).
Temperature in each treatment was still in the normal range for tilapia, which was 28.5 –
29°C, based on the standard for rearing tilapia ranging 25 – 32°C (Djarijah, 1995). The ammonia
content during this research began to increase at F (2 mg/L), which exceeded the optimum
ammonia content for tilapia rearing that should be ≤ 1 mg/L, based on Hidayah (2015). This
happened allegedly because of tilapia experiences stress, removing a lot of urine and feces.

Conclusion
Noni fruit extract immersion on had significant influence against the blood profiles of
tilapia O. niloticus, such as total erythrocytes, total leukocytes, hemoglobin, and blood glucose
level. The best Noni fruit concentration extract for immersion method was found at 4.8 g/L, as
the blood profiles showed stabilized range followed with increased leukocyte level.
Acknowledgments
Authors would like to thank parents, sibling, mengkudu squad, and colleagues for giving
a lot of supports in writing this article.

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