The Effect of Tadoples (Rana catesbeiana) Serum on Total and

Differential Leukocyte in Rats (Rattus norvegicus): That Have Been

Induced With Dimetylbenz-α-anthracene

as Animal MODEL of Skin Cancer

Nur Prabowo Dwi Cahyo

,Prima Ayu Wibawati

Hardany Primarizky

Ragil Angga Prastiya

Muhammad Thohawi Elziyad Purnama

and Ratna Damayanti

Student Faculty of Veterinary Medicine, Universitas Airlangga

Post Graduate School Universitas Airlangga

Department of Veterinary Public Health,

Department of Veterinary Clinic

Department of Veterinary Reproduction,

Department of Veterinary Anatomy,

Departement Basic Veterinary Medicine Faculty of Veterinary Medicine, Universitas Airlangga

Post Graduate School Universitas Airlangga

Keywords: Total leukocyte, differential leukocyte , Rana catesbeiana, serum, skin cancer.

Abstract: This study aimed to determine the effect of tadpoles (Rana catesbeiana) serum on total and differential

leukocyte in animal model of white rat (Rattus norvegicus) that has been induced with skin cancer by

Dimethylbenz-α-anthracene (DMBA). Male rats were induced by DMBA 20 mg/rat twice a week for 18

days to induce skin cancer. Tadpole’s serum was injected intracutaneously after cancer had been known.

Negative control (K-) was not induced with DMBA and tadpole’s serum, while positive control grup (K+)

was induced to DMBA. The treatment groups P1, P2, and P3 were induced with DMBA and injected

tadpole’s serum 100%; 75%; 25%/rat/day. This study used a Completely Randomized Design (CRD). Data

were analyzed with ANOVA and continued by Duncan multiple test. The results obtained average number ±

SD of total leukocyte K-, K+, P1, P2, and P3 were 12000.00±3814.88,3975.00±2451.36,8650.00± 5470.83,

6390.50 ± 3007.18 and 5590.00 ± 1292.18 respectively. There are significant differences regarding an

increase in number of total leukocyte on treatment, but there is not real difference between K+, P1, P2 and

P3. The results shsowed average number of limphocyte and monocyte are not significant but it is signiffant

in granulocyte. Based on the results, it can be concluded that tadpoles serum is effective to increase number

of total leukocyte and differential leukocyte (especially in granulocyte) in animal model of rats induced skin

cancer.

1 INTRODUCTION

The prevalence of cancer in the population of all

ages in Indonesia in 2003 reached 0.14 percent of

the total population or 347,792 people (Ministry of

Health, 2015). According to Indonesian Nutrition

Network (2005), in Indonesia, cancer patients

reached 6% of the population and deaths from this

malignancy ranked second after death from

infection. Based on a research conducted by

Dhaygude (2006) in Mumbai, India during the

period from January 2001 to December 2005, from

124 dogs who were autopsied or biopsy, the most

recorded skin tumor was found in 74 (59.67%)

followed by mammary gland tumor as many as 43

556

Cahyo, N., Wibawati, P., Primarizky, H., Prastiya, R., Purnama, M. and Damayanti, R.

The Effect of Tadoples (Rana catesbeiana) Serum on Total and Differential Leukocyte in Rats (Rattus norvegicus) That Have Been Induced With Dimetylbenz-Î

s-anthracene as Animal MODEL

of Skin Cancer.

DOI: 10.5220/0007546905560560

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 556-560

ISBN: 978-989-758-348-3

tails (34,67%) and subsequently transmissible

granuloma in genitals as many as 3 tails and ovarian

and testicular tumor were 2 each (1.6%).

According to Mondou and Kaltenbach (1979),

the tadpoles serum (Rana catesbeiana) contains

thyroxine hormones that can enhance cellular

immune responses. Thyroxine content in tadpole

serum is 9.4 ng / ml, while thyroxine needed as

therapy to increase immune is 0.01 - 0.1 microgram /

ml. Leukocytes have a very important role in the

body's defense, so the examination of the number of

leukocytes is to support the diagnosis of disease. The

body has a special system to remove a variety of

infectious and toxic materials, one of which is

leukocytes or white blood cells. Blood test results

can be used as a good parameter and can generally

describe the condition of the body such as the

examination of the total number of leukocytes

(Guyton and Hall, 2011).

There has been no research or data on the total

type of white rat (Rattus norvegicus) leukocytes

induced in DMBA and given serum tadpoles (Rana

catesbeiana). Based on this, a research needs to be

done to find out the effect of tadpoles serum (Rana

catesbeiana) on total white rat leukocytes (Rattus

norvegicus) which suffer from cancer induced

DMBA.

2 MATERIALS AND METHODS

This study used 20 male white rats (Rattus

norvegicus) which weighed around 150-250 grams.

The experimental animals were then divided into 2

control groups (negative and positive) and 3

treatment groups.

The tools used in this research are weight

weighing rats, rat cage, drink container, food

container, litter or cage from wood powder, 1 ml

disposable syringe with tuberculin needle, 100ml

beaker glass, 100 ml measuring cup, analytical

scales, filter paper, and camera. The ingredients for

the cancer-trigger used in this study are DMBA

(7,12-Dimethylbenz-α-anthracene) dissolved in corn

oil. DMBA injection (7,12-Dimethylbenz - α-

anthracene) was done using 1 ml disposable syringe

with tuberculin needle and sterile cotton with 70%

alcohol.

Tadpoles blood-collecting (Rana

catesbeiana) was done using syringe 1 ml disposable

with tuberculin needle. Blood was collected using an

EDTA tube without anticoagulation. The tube was

covered with aluminum foil and centrifuged. Serum

dilution was done using PZ or NaCL physiological

0.9% then injected in white rat (Rattus norvegicus)

using 1 ml disposable syringe with tuberculin

needle.

Dilution of DMBA (7,12-Dimethylbenz-α-

anthracene) was performed before inducing cancer.

Dilution was done using corn oil. Corn oil served as

a solvent of DMBA (7,12-Dimethylbenz-α-

anthrancene). Dosage for DMBA induction (7,12-

Dimethylbenz-α-anthrace) as a trigger for raising

cancer cells was 20 mg / kg BW (Cabecas et al,

2014). DMBA induction (7,12-Dimethylbenz-α-

anthracene) to induce cancer cells was injected

subcutaneously using a 26G size needle. DMBA

powder (7,12-Dimethylbenz-α-anthrance) was

dissolved in advance with corn oil in order to

facilitate the induction process. Comparison of

DMBA powder (7,12-Dimethylbenz-α-anthrance)

with corn oil is 1 ml of corn oil containing 20 mg

DMBA (7.12-Dimethylbenz-α-anthrance). Induction

was done for 14 days with duration of twice a week.

Subcutaneous induction was performed

subcutaneously on the nape of white rats (Rattus

norvegicus). Cancer observations were performed

after the first injection of DMBA (7,12-

Dimethylbenz-α-anthrance) by palpation of the

injection or nape and skin portions of other white

mice (Rattus norvegicus). Cancer observation was

also carried out by the measurement of diameter and

number of nodules that arised. The expected nodule

is a cancer nodule, not an abscess nodule. Palpation

and measurements were made daily.

The negative control group was not induced with

DMBA, whereas the positive control group was

induced with 20 mg/kg BW DMBA. All treatment

groups were induced by 20 mg/kg BW DMBA. The

treatment stage after 14 days was induced with

DMBA and after the appearance of skin nodules, the

white rats treated group were injected with 1.06 ml

tadpoles serum (Mondou dan Kaltenbach, 1979), in

P1(100%), P2 (75%) and P3 (25%). Injecting

tadpoles serum was done once a day for seven days.

Blood sampling was performed through the heart

(Cardiac puncture) using a 2 ml disposable syringe

in rats on the 44

day. Blood was then

accommodated in an EDTA tube as to not affect the

size and shape of the erythrocytes or the shape of the

leukocytes (Bijanti et al., 2010). Blood examination

was done using Hematology Analyzer HORIBA

ABX MICROS 60 instrument and then connected

with computer. Blood was homogenized first using

Roller-Mixer for 1-2 minutes before checking using

Hematology Analyzer.

The Effect of Tadoples (Rana catesbeiana) Serum on Total and Differential Leukocyte in Rats (Rattus norvegicus) That Have Been Induced

With Dimetylbenz-Î

s-anthracene as Animal MODEL of Skin Cancer

557

3 RESULT AND DISCUSSION

Table 1 : Mean of Total Number of Leukocytes in White

Rat due to Tadpole Serum (Rana catesbeiana) influence.

Treatment

Mean ± SD

K-

12000,00

± 3814,88

K+

3975,00

± 2451,36

8650,00

± 5470,83

6390,50

± 3007,18

5590,00

± 1292,18

Description : Different Superscript in the same

Coloumn shows significant differences (p<0.05)

Figure 1: Diagram of White Rat Leukocyte Due to Giving

Tadpole Serum (Rana catesbeiana).

The K+ group (DMBA 20 mg / kg BB + solvent

corn oil) showed the lowest number of 3,975,00

2,451,36 compared with the negative group and all

treatment groups, this proves that DMBA can

suppress the activity of bone marrow and splenosit

so that it can reduce the number of leukocytes

especially neutrophils, monocytes and lymphocytes

(Akrom et al., 2013)

In P1, it showed the highest increase of total

leukocyte number which is 8650,00

± 5470,83,

then followed by P2 that is 6,390,50

± 3,007,18,

and P3 that is 5,590,00

± 1,292,18. The increase in

total leukocytes in the treatment group 1 to treatment

3 showed that the addition of young tailed frog

serum increased the number of cancer-induced white

rat leukocytes (Rattus norvegicus) using DMBA

(Dimethylbenz-α-anthracene). An increase in the

number of leukocytes is estimated as a result from

an increase in the number of natural killer cells.

Tadpoles serum acts as an immunomodulator that

stimulates the immune system, such as improving

macrophage activity, increasing antibodies and

activating natural killer cells. The increase is also

associated with the entry of foreign bodies in the

body and the response of leukocytes as a mean of

body defense (Gigena et al., 2017).

The treatment group showed that higher doses of

tadpoles serum in DMBA-induced rats would

increase the number of leukocytes in the blood. This

result is consistent with the Akrom and Ermawati

(2009) study stated that by administering black

cumin as immunopreventive at higher doses can

increase the number of leukocytes in DMBA-

induced Sprague Dawley rats. The higher the dose of

the tadpoles serum, the higher the number of

leukocytes. It can be said that higher serum doses

can inhibit the development of cancer so that the

number of leukocytes increases. This illustrates the

correlation between the serum content of tadpoles,

thyroxine hormones, and increased immune system

in animals. With the higher doses, the thyroxine

hormone content in tadpole serum is higher, so the

ability to increase the immune system is getting

bigger.

Table 2 : Average and Standard Deviation of White Rat

Leucocyte Types Due to Tadpole Serum (Rana

catesbeiana) Influence.

Treat

ment

Lymphocyt

es (/ mm

)

(X ± SD)

Monocyt

e (/ mm

)

(X ± SD)

Granulocyt

es (/ mm

)

(X ± SD)

K-

9900,00

3402,94

600,00

182,57

1500,00

496,65

K+

3275,00

2087,06

250,00

191,48

450,00

208,16

4575,00

4601,72

1050,00

± 858,29

1225,00

917,87

5058,50

2213,10

793,00

465,25

677,50

384,74

4549,00

1298,10

684,00

138,41

495,50

189,24

Description: Different superscripts in the same

column show significant differences (p <0.05)

According to Kusumawati (2004), the normal

monocyte value of rats is 0.00 - 0.10 (x 10

/ mm

It can be seen from the average number of

monocytes that positive control group has the lowest

average monocyte count of all treatments. This is

because the positive control of rats were given

cancer by DMBA-induced without any serum

ICPS 2018 - 2nd International Conference Postgraduate School

558

treatment, so that DMBA suppresses bone marrow

activity resulting in decreased monocyte count

(Akrom et al., 2013).

Granulocytes are white blood cells characterized

by granules in the cytoplasm. Granulocytes such as

neutrophils, eosinophils, and basophils are very few

in normal circumstances, but when there is an

antigen the amount will increase (Fitria and Sarto,

2014). In this study, the average number of

granulocytes of the lowest rat in the K+ group was

450,00

± 208.16 compared with the K- group and

the all-treatment group. This study shows that

DMBA induction in rats can decrease the average

number of animal granulocytes. In the K- group with

the K+, P1, P2, and P3 groups, there was a marked

difference (p <0.05). The P1, P2, and P3 groups

showed an increase in the number of granulocytes

compared to the K- groups, although the statistics

showed that the increase was not significantly

different (p> 0.05).

4 CONCLUSION

The conclusions of this study are:

1. There was an effect of tadpole serum (Rana

catesbeiana) on the total leucocyte increase of

white rats (Rattus norvegicus) with DMBA-

induced (Dimetylbenz-α-anthracene).

2. There was an effect of tadpole serum (Rana

catesbeiana) on the increase of white rat type

leukocytes (Rattus norvegicus) with DMBA-

induced (Dimetylbenz-α-anthracene).

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The Effect of Tadoples (Rana catesbeiana) Serum on Total and Differential Leukocyte in Rats (Rattus norvegicus) That Have Been Induced

With Dimetylbenz-Î

s-anthracene as Animal MODEL of Skin Cancer

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