The 3rd International Seminar on New Pardigm and Innovation on Natural Sciences and Its

Application 2013

RESPONSE INTERLEUKIN-2 OF BROILER CHICKENS

AFTER FEEDING VIRGIN COCONUT OIL

Enny Yusuf Wachidah Yuniwarti

Jurusan Biologi Fakultas Sains dan Matematika UNDIP

enny_yusuf@yahoo.co.id

ABSTRACT

Interleukin-2 (IL-2) is a growth, survival, and differentiation factor to T lymphocyte, and plays a

major role in regulation of T cell response through its action on regulatory T cell. Because of its ability to

support proliferation of antigen stimulated T cells, IL-2 was originally called T cell growth factor. IL-2 is

produced mainly by CD4-Th lymphocyte, it functions as an autocrine. Virgin coconut oil (VCO) contains

various fatty acids to produce more energy which can increase the metabolism. An increase in metabolism

will cause cell function more efficiently and which can prevent from disease and accelerate the healing

process. Fatty acids mainly palmitic and miristic acids in VCO, which are required as a raw material for

phospholipid synthesis in lymphocyte. Lauric acid in VCO which can be converted into monolauric acid or

glyceromonolauric to increase lymphocyte sensitivity towards IL-2 receptor so that resulting in

lymphoproliferation, furthermore lymphocytes will produce IL-2. The research was done in commercial

broiler for five weeks. Employing RAL design in which the treatment factor consisted of 4 levels of VCO,

included 0, 5, 10 and 15 mL/kg feed. Sample and data collection were done at the end of research. Results of

this research indicated that VCO especially at the concentration of 10 mL VCO/kg feed increase the level

concentration of interleukin-2.

Key word: Interleukin-2, VCO, Broiler chicken.

INTRODUCTION

Interleukin-2 (IL-2) is produced by T lymphocyte mainly CD4-Th lymphocyte, it functions as an

autocrine. The constitutive expression of IL-2 receptors on regulatory T cells is consistent with their

requirement for IL-2 in order to survive. Interleukin-2 also promotes survival of cells by inducing the anti

apoptotic protein, IL-2 increases production of other effector cytokines[1]. Increased production of IL-2

further stimulates lymphocytic proliferation [2]. T lymphocyte plays an important role in the stimulation of

the immune system against certain diaseas and stressor [3]. Lymphocyte had an important role in the

protection of chicken against infection. The number of lymphocyte indicates the degree of immune status of

the chicken. The lower number of lymphocyte, the more easily of virus to cause desease due to the weakning

of the immune status [4]. Lymphocyte development requires energy derived from metabolism process [5],

and VCO can produce additional energy for metabolism by recirculating into liver through hepatic artery [6].

The lymphocyte-Th as a subset of T lymphocyte, will activate macrophage as cellular immunity response

against infection with intracellular pathogen [7].

Virgin coconut oil (VCO) is food supplement producible in Indonesia and safety proven for human

consumption, hence it is assumed to be safe for chicken. Nutritional value test showed VCO contains 51.23%

lauric acid, 17.13% myristic acid, 7.30% palmitic acid, 9.18 caprilic acid, 7.07% capric acid, 5.42% oleic

acid, 2.17 stearic acid and 0.51% caproic acid. 90% fatty acid in VCO is saturated fat and only 10% is

unsaturated fat [8]. Saturated fat in VCO especially palmitic and myristic acids are phospholipids component

The 3rd International Seminar on New Pardigm and Innovation on Natural Sciences and Its

Application 2013

of T cell [9], so that giving VCO especially 10 mL/kg of feed, able to increase the number of lymphocyte

[10]. Lymphocyte Th will activate macrophage as cellular immunity response against infection with

intracellular pathogen [7]. Some research showed that VCO was potential as antiviral and anti bacterial

agents [11]. Fatty acid in VCO is also potential as antivirus [12]. Interleukin-2 mediates its effect by binding

to IL-2 receptors, which are expressed by lymphocytes [13].

MATERIAL AND METHOD

Twenty broiler day old chick were used in the research. The cage used was collective cage for 10

chickens kept until they reached three weeks old, then they were moved to individual cage up to five weeks.

The cage was equipped with feed and water containers. Chickens were placed randomly in the cages. The

control feed used were manufactured BR1 pellet, while treatments of feed were mixed of control feed and

different level of VCO, namely 5, 10 and 15 mL of VCO/kg feed. VCO used was from factory so the quality

consistency was guaranteed. Feed and water were given ad libitum for four weeks.

Interleukin-2 was determined from blood by enzym linked imunosorbent assay (ELISA). The blood

was collected from the wing vena at the end of treatment and placed in 2 mL tube using EDTA as an

anticoagulant. Collect plasma by cetriuge for 15 minutes at 1000 g within 30 minutes o collection. Assay

immediately or aliquot and store the sample at -200 C. Centrifuge the sample again after thawing before the

assay. Reagents used in determination of this interleukin-2 is Wash Buffer. Dilute 20 ml of wash buffer

concentrate into deionized or distilled water to prepare 500 ml of wash buffer. Standard. Centrifuge the

standard vial at 6000 – 10000 rpm for 30s. Reconstitute the standard with 1.0 ml of sample diluent. This

reconstitution produces a stock solution of 12 pg/ml. Allow the standard to sif for minimum of 15 minutes

with gentle agitation prior to making serial dilutions. Undiluted standard serve as the high standard (12

pg/ml). The sample diluent serves as the zero standard (0 pg/ ml). Prepare fresh for each assay. Use within 4

hours and discard after use. Biotin-Antibody. Centrifuge the vial before opening. Dilute to the working

concentration using biotin-antibody diluent 1:100 respectively. HRP-Avidin. Centrifuge the vial before

opening. Dilute to the working concentration using HRP-avidin diluent 1:100 respectively. Assay procedure

begins by bringing all the reagen and sample to room temperature before use. It is recommended that all

samples, standards and control be assayed in duplicate. All the reagents should be added directly to the liquid

level in the well. The pipette should avoid contacting the inner wall of the well. Add 100 µL of standards,

Blank, or sample per well. Cover with the adhesive strip. Incubate for 2 hours at 370C. Remove the liquid o

each well, don’t wash. Add 100 µL of biotin-antibody working solution to each well incubate for 1 hour at

370C. Biotin antibody working solution may appear cloudy. Warm up to room temperature and mix gently

until solution appears uniform. Aspirate each well and wash, repeating the process three times for a total of

three washes. Wash: fill each well with wash buffer (200μL) and let it stand or 2 minutes, then remove the

liquid by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper

towel. Complete removal of liquid at each step is essential to good performance. Added 100μL of hrp-avidin

working solution to each well. Cover the microtiter plate with a new adhesive strip. Incubate or 1 hour at

370C. repeat the aspiration and wash five time as step before. Add 90 μL of tbm substrate to each well.

Incubate for 10-30 minutes at 370C. keeping the plate away from drafts and other temperature luctuation in

the dark. Add 50 μL of stop solution to each well when the first four well containing the highest

concentration of standards develop obvious blue color. I color change does not appear uniform, gently tap

the plate to ensure thorough mixing. Determine the optical density of each well within 30 minutes using

microplate reader or ELISA reader, set to 450 nm [13].

This research applied complete random design in which factor treatment used four levels of VCO, 0,

5, 10, 15 ml/kg feed. Chickens were divided into four treatment groups and repeated in five experiment units.

Treatment was done within five weeks. The obtained data was then analyzed using ANOVA and continued

with LSD test [14].

The 3rd International Seminar on New Pardigm and Innovation on Natural Sciences and Its

Application 2013

RESULT AND DISCUSSION

Results of this research indicated that VCO especially at the concentration of 10 mL

VCO/kg feed increase the level concentration of Interleukin-2 (Figure 1). Statistical analysis with

complete random design showed significant difference (P<0.05) between all treatments with VCO and at the

concentration of 10 mL VCO/kg feed of VCO (Figure 2). Increasing in the level concentration of

interleukin-2 can be explain that interleukin-2 is produced by T lymphocyte mainly CD4-Th

lymphocyte. Gliceromonolaurat from lauric acid in VCO can be converted into monolauric acid or

glyceromonolauric to increase lymphocyte sensitivity towards IL-2 receptor so that resulting in

lymphoproliferation [15].

Interleukin-2 is one of the lymphokines secreted by T helper cells upon activation mediated by T-cell

receptor (TCR) and accessory molecules. The ability to express IL-2 is correlated with T-lineage

commitment and is regulated during T cell development and differentiation. Inducibility of IL-2 gene is

controlled at each transition and each differentiation process of T-cell development [16]. Chicken CD4-Th

lymphocyte cells did not proliferate in vitro in the absence of recombinant chicken IL-2 [17]. Another study

showed that IL-2 stimulates the immune system and can lead to large increases in the number of CD4+ -T-

helper cells. Interleukin-2 is a lymphocyte growth factor that is required for T cells to progress from G, into

S phase of the cell cycle. According to the current paradigm, the messages provided by increased

intracellular ionized calcium and protein kinase C activation result in IL-2 gene activation [18]. This

research showed that there is interaction between IL-2 and Th lymphocyte, with the result that VCO will

increase the production of IL-2 by increasing the number of CD4-Th lymphocyte. Increasing in the

number of CD4-Th lymphocytes can be explained through three pathways. The first pathways fatty

acids mainly palmitic and miristic acids in VCO, which are required as a raw material for

phospholipid synthesis in lymphocyte, the second pathway is related to all synthesis processes

which required energy and VCO can provide such energy more readily in higher amount because

medium fatty acid chain and monoglyceride molecule will directly enter into sistemic circulation to

use the systemic effect and action. Recirculation into liver through hepatic artery would release

additional energy which explained the additional energy from coconut oil. The third pathway is

related to VCO, especially lauric acid which can be converted into monolauric acid or

glyceromonolauric to increase CD4-Th lymphocyte sensitivity towards IL-2 receptor so that

resulting in lymphoproliferation.

Fig 2. Level IL-2 after feeding various doses of VCO

Fig 1. ELISA test result of IL-2

The 3rd International Seminar on New Pardigm and Innovation on Natural Sciences and Its

Application 2013

CONCLUSION

Virgin Coconut oil is able to increase the production of Interleukin-2

ACKNOWLEDGMENT I am very grateful to Prof. Charles Rangga Tabbu, MSc, Ph.D, Prof. Dr. drh. Wayan Tunas Artama and

Prof.drh. Widya Asmara, MS. Ph.D for their very helpful advice.

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