The Use Of B-Glucan As An Immunostimulant To Increase The Non-Specific Iivimunity And The Performance Of Shrimp (Penaeus Monodon, Fab.) Under Artificial Stress Condition
THE USE OF P-GLUCAN AS AN IMMUNOSTIMULANT TO
INCREASE THE NON-SPECIFIC IMMUNITY
AND THE PERFORMANCE OF
SHRIMP (Perzaeus monodon, Fab.)
UNDER ARTIFICIAL STRESS CONDITION
BY
EMILIANA DHIAN ANGGERAHENI
I'OSTGIIADUATE PROGRAM
INSTITUT PERTANIAN BOGOR
2002
ABSTRACT
EMlLlANA DHlAN ANGGERAHENI.
The Use of (3-glucan as An
Immunostimulant to Increase the Non-Specific Immunity and the Performance of
Shrimp (Penaezts n~onodon,Fab.) Under Artificial Stress Condition. Under advisory
cotlimittee AHMAD MAAD WIRAWlDJAJA and DARNAS DANA.
Environmental stress has been a colnmon risk in shrimp culture ponds. This
stress could lead to the occurrence of diseases. The experiment was divided into three
trials. The objective of the first trial was to investigate the effect of oxygen
concentration. water salinity, and luniinous bacteria concentration on some
parameters of stress condition i.e. hemocyte count, survival rate, and number of
luminous bacteria (Kbrio splendidus biovar 1 ) in hemolymph. The result of this trial
could be used as a fit stressor in an advanced trial. The second trial was to investigate
the effect of P-glucan on some parameters of shrimp performance i.e. growth and feed
conversion ratio. The third trial had an objective to study the affectivity of (3-glucan
given in 1 and 2 months on some parameters of shrimp immune system i.e. hemocyte
count. phagocytic activity, clearance bacteria, and survival rate at low oxygen
concentration. The experiment was an advanced experiment using the worst condition
resulted by trial 1.
The result suggests that feed containing e-glucan 0.1% affected the immune
capacity of shrimp, such as clearance of bacteria in hepatopancreas and hemolymph,
total hemocyte and phagocytic activity of hemocyte. Its effect was better than the
others. But the feed containing P-glucan had no significant effect on the performance
of shri~npi.e. the growth and the feed conversion ratio. because the trial was
conducted in short time to give evidence of their effect on the performance of shrimp.
Shrimp given feed containing P-glucan 0.1% could clear Vibrio splendidzrs biovar I in
he~nolymph and hepatopancreas in less than 12 hours. Although the highest
hemocyte count was found in shrimp fed by P-glucan 0.5%, their phagocytic activity
had no significant difference with shrimp fed by P-glucan 0.1%. It indicated that the
content of hyaline cells (phagocytic cells) in shrimp hemocyte fed by P-glucan 0.1%
was higher than the other. Thus, the highest rate of phagocytic activity from the 7Ih up
to the 14"' day after bacteria immersion was found in shrimp hemocyte fed by IJglucan 0.1 %.
The prolonged periods with dosage P-glucan, with the aim of obtaining better
and faster productivity results, had no significant effect on the immune capacity of
shrimp. The best administration period of P-glucan for 30 days old culti~reof shrinip
was I month.
DECLARATION
I declare that thesis using title:
THE USE OF P-GLUCAN AS AN IMMUNOSTIMULANT TO lNCREASE
THE NON-SPECIFIC IMMUNITY AND THE PERFORMANCE OF
SHRIMF' (Perzaerrs ntortodort, Fab.)
UNDER ARTIFICIAL STRESS CONDITION
is my writing, as the results of my experiment. The thesis has been not published as
academic report at other universities or other institutions.
This thesis may not be reproduced in whole or part by photocopy or other
means without permission ofthe author.
E~nilianaDhian Anezeraheni
NRS : 99440lRIOLOGY
THE USE OF P-GLUCAN AS AN IMMUNOSTIMULANT TO
INCREASE THE NON-SPECIFIC IMMUNITY
AND THE PERFORMANCE OF
SHRIMP (Penae~csmonodon, Fab.)
UNDER ARTIFICIAL STRESS CONDITION
EMILIANA DHlAN ANGGERAHENI
A thesis submitted in partial fulfillment
of the requirements for the degree of
MASTER OF SCIENCE
in the
Department of B i o l o ~ y
POSI'CRADUATE PROGRAM
INSTITUT I'ERTANIAN BOGOR
2002
APPROVAL
Title of Thesis
: THE USE OF P-GIACAN AS AN IMMUNOSTIMULANT
TO INCREASE THE NON-SPECIFIC IIVIMUNITY AND
THE PERFORMANCE OF SHRIMP
(Penaeus monodon, Fab.)
UNDER ARTIFICIAL STRESS CONDITION
Name
: EMILIANA DHIAN ANGGERAHENI
No. Reg. Student
: 99440
Program
: Biology
Approved by,
I . Advisory Comtnittee
Dr. Ahmad Maad Wirawidiaia
Chairman
2. I-lead of Biology Department
Dr. Dedi Durvadi
Graduation date : Septcmbcr 28, 700 I
Dr. Darnas Dana. h4Sc.
Member
CURRICULUM VITAE
The writer, EMlLlANA DHlAN ANGGERAHENI, was born on January 5 ,
1971. in Semarang - Central Java. In September 1994, she graduated from the
Faculty of Fisheries, Aquaculture Department, Institut Pertanian Bogor.
From November 1994 until May 1996, she worked at PT. Birulaut
Khatulistiwa, the shrimp hatchery of PT. Dipasena Citra Darmaja in KaliandaLampung. Then, she worked at the shrimp hatchery of PT. Centralpertiwi Bahari in
Suak Sidomulyo - Lampung from June 1996 until February 1999.
In 1999, she continued her study in Institut Pertanian Bogor for the degree of
Master of Science in Biology Department and on September 28, 2001 she graduated
her study.
ACKNOWLEDGEMENTS
I gratefully acknowledge the financial support by the PT. Centralpertiwi
Baliari. I would like to express illy deep appreciation to Mr. Djoko M. Basoeki. Dr.
Sujint Tamniasart. and Mr. Johannes Kitono, the president director and directors of
the PT. Centralpertiwi Bahari. Thanks are due also to Mr. Wayan Agus Edhy. Mr.
Wahyudi, Mr. Cheawchan Ponpanitralsamee and Mr. Sarayuth for their expert advice;
Mr. Januar Pribadi for his laboratory services; Mr. lsman Haryanto, Mr. Risdianro
P.W, Ms. Maria Sisilia, and Ms. Eni for their attention; Dr. Dean Akiyama for his
invaluable discussion and attention at the final stage of my thesis.
I wish to express thanks to ~ n yparents, iny sisters, and mas Joko Sulistyo for
their loyal support throughout the study. Thanks also to the staff of PT Centralpertiwi
Bahari (Pondsite) Larnpung, especially Yogi Sukirman, who helped me in the wet
laboratory, Mr. Bastian Sitompul, Mr. Ilasan Solikin, Mr. Sutarto, all staff of
Microbiology laboratory, Mr. Dani Yukasano, Mr. Tonif, Mr. Kristianto, Mr.
Suherman, and all my friends at pond site.
Finally, 1 wish to thank to my supervisory committee, Dr. Ahmad hlaad
Wirawidjaja and Dr. Darnas Dana, MSc., my English Lecturer Ms. Nilawati, and my
Statistic advisor Mr. Ba~nbangSurnantri. Their dedication, concern and guidance
throughout my study are gratefully acknowledged.
TABLE O F CON'1'ENTS
PAGE
LIST OF TABLES ...... ................................ . ............,...........
vi
LIST OF FIGURES ............................. ..... . ......, ,..... .., ...........
vii
LIST OF APPENDICES.. ...... .... . ..... ..., .... .. . .... ... ........, ....., , ..
Vlll
...
INTRODUCTION
Justification and Problems.. ........................., ............. ...
Objective.. ........................................ .. .. .. .................
Hypotheses.. ................ ......... .... ... . . . . ... .. . ..... . .... .........
1
3
3
HISTORICAL REVIEW
.
.
Luminous Vlbrio... . .. .......................... . ................ .. ......
Disease Trigger.. ..... . ...... . ... .. .............. .. ... .. ... .... ...........,
Hemolymph.. .............. ........... .. . .... . . .... . . . . . . . , , .. .. . ......, . .
Shrin~pDefence Mechanism.. . .. .......... . ... . . .. . . . . ............. .. ..
P-glucan as An Immunostimulant ... ...... .. .. .. . . . . . . . .. ...............
4
5
5
6
7
METHODOLOGY
Times Lines ......... ................. . . .. .... . . .. .... . . .... . . . . . . .. ........
9
Materials.. ...... ..... ...... ... . .. . .. ......... . . . ... . . . . . . , .. . . ....... ., .. ..
9
Methods
9
Experimental Design
Trial 1: Artificial Stress Condition and Shrimp In~nii~nity
. .. . 12
Trial 2: p-glucan and Shrimp I'erformance.. .. ... . . . . . . .. ... ....
13
Trial 3: Administration Period of b-glucan and
Shrimp Immunity .. . . .... .. . . . . . . . . .. . . . . . ..... . . .. . . . , , ..
14
RESULTS AND DISCUSSION
Results
Identification of Vibrio Bacteria in Hemolymph
and Hepatopancreas.. ... ......... ...................... ...........
Trial 1: Artificial Stress Condition and Shrin~pIrmnuniry ....
Trial 2: P-glucan and Shrimp Perfornlance................... ..
Trial 3: Administration Period of P-glucan and
Shrimp Immunity.. ...................................... .
Discussion
Trial 1: Artificial Stress Condition and Shrimp Immunity..
Trial 2: p-elucan and Shrimp Perfornlance ............... ...
Trial 3: Administration Period of P-glucan
and Slvimp Iminunity .............................. ...
CONCLUSSION.. . ..... ........... ................... .. . .....................
BIBLIOGRAPHY.. . . . ......................... .............................. ...
APPENDICES. ........... ....... .... ..... ............... .......... ...... .......
LIST OF TABLES
PAGE
1. Mean number of the hemocyte count, survival rate of shrimp and
number of Vibrio splendidus biovar 1 colony (cfu) in hemoplymph
at 18 hours after stressing condition.. .................................
18
2. Mean number of growth and feed conversion ratio caused
..
by P-glucan adm~nlstration.............................................
21
3. Number of Vibrio splendidus biovar 1 colony in hepzropancreas
and hemolymph after challenge test.. ................................
22
4. Mean number of hemocyte count at the 0,71h. 14"', and 21'' day
after challenge test.. ......................................................
23
5. Mean number of phagocytic activity at the 0. 7"', 14u. and 21" day
after challenge test. .....................................................
24
6. Survival rate of shrimp.. ................................................
25
7. The nutrient content (% wet basis) of the trial diets.. ............
31
LIST OF FIGURES
PAGE
1. The water quality in each treatment.. ................ ......................
26
2. The relationship between phagocytic activity and hemocyte count ....
30
LIST OF APPENDICES
PAGE
1. The differential characteristics of the species and biovar of the
. .
Genus of fibrzo ..................................................
38
2. The analyses of variance for the hemocyte count in hemolyinph at
18 hours after stressing condition (Trial 1). ...................
40
3. The analyses of variance for survival rate of shrimp at
18 hours after stressing condition (Trial 1). ....................
41
4. The analyses of variance for number of luminous bacteria in
henlolymph at 18 hours after stressing (Trial 1)..............
42
5. The analyses of covariance for the growth of sluimp (Trial 2)
43
6. The analyses of variance for the feed conversion ratio (Trial 2)
44
7. The analyses of variance for the hemocyte count in hemolymph on
the 0 day after challenge test.. ..........................
45
8. The analyses of variance for the hemocyte count in hemolynlph on
the 7"' day after challenge test.. .....................
46
9. The analyses of variance for the hemocyte count in hemolymph on
the 14"' day after challenge test .......................
47
10. The analyses of variance for the heniocyte count in he~nolpmphon
the 21'' day after challenge test .........................
45
11. The analyses of variance for the phagocytic activity on
the 0 day after challenge test .........................
49
12. The analyses of variance for the pbagocytic activity on
the 7"' day after challenge test ....................
50
13. The analyses of variance for the phagocytic activity on
the 1411, day after challenge test ....................
14. The analyses of variance for the phagocytic activity on
the 21S1dayafter challenge test ......................
15. Tile analyses of variance for the phagocytic activity on
the 50"' day after challenge test ......................
16. The analyses of variance for the s u ~ i v arate
l of shrimp
..................................
after challenge test
17. The steps of phagocytic activity ..................
INTRODUCTION
,Justification and Problems
Shrimp has a less developed immune system than fish or other vertebrates. It is
probable that shrimp is relatively more dependent on the non-specific immune processes,
such as phagocytosis, than vertebrates (Raa et al., 1992).
Thus, administration of
antibiotics and vaccines giving a specific immunity is an ineffective treatment (Anderson,
1992).
In~mu~lostimulant,a material that can elevate the non-specific defence
mechanism, i.e. P-glucan and lipopolysaccharide, may represent an alternative and
supplement to vaccines in the protection of farmed shrimp against diseases.
Lipopolysaccharide and P-glucan, present on the gram-negative bacterial and fungal
surface, can be recognized by shrimp henlolymph; thus, they.can increase the nonspecific in~nlunesystem of shrimp (Kondo et al., 1996; Vargas-Albores et al., 199s).
Although the im~nunostin~ulants
have numerous positive effects on the imnlune
system in shrimp, they have some limitations and possible negati\.e effects, especially in
case of over dosage.
Irnmunostiiuulants administration must notice the timing and
dosage of application (Anderson, 1992).
The timing of irnn~unostimulants application depends on the activating
characteristics of the ~naterialitself.
Some result of trials indicate that P-glucan has a
short duration cffect on irnnlune system. Thus. P-glucan must be given exactly at the
time as a preventive treatment before disease exposure (Galeotti, 199s).
The overdose or under dose of inlmunostimulants is an administration problem.
High doses of in~munostimulantscan suppress the defence mechanism, and low doses
may not be effective (Anderson, 1992). High dietary levels of P-glucan (>I .5 %) in feed
may impair nutrient digestibility because they increase the viscosity of the ingesta
(Galeotti, 1998; Volpatti et al., 1998).
Generally, the experiment on im~~~unostimulant
is conducted only to investigate
effectiveness of a material on animal immunity, and omit interaction of other factors in
environment that cannot be precisely controlled (Soderhall and Johansson, 1985, 1989;
Raa et al., 1992; Itami, 1994; Kondo et a/.,1996; Vargas-Albores el al., 1998; Clifford,
1999). The effect of imnlunostimulants on the interaction of disadvantageous conditions
of the environment causing stress has not been identified yet.
In fact, most diseases in aquaculture are caused by opportunistic pathogens i.e.
bacteria that cause disease when shrimp is weak or under stress. The phagocytic activity
is less affected by stress condition (Anderson, 1992). The stress is caused by temperature,
oxygen concentration, pH, salinity, etc, often exists under practical farming conditions
(Clifford, 1999).
Thus, this experinlent was done by combining some factors causing stress
condition i.e. oxygen concentration. salinity, and level of bacterial attack, to study the
ability of P-glucan to increase the non-specific iminunity of shrimp.
Objcctivc
The experiment was divided into three trials with some objectives as follows:
1. To study the effect of oxygen concentration, water salinity, and luminous
bacteria concentration on sotne parameters of stress condition i.e. hetnocyte
count, survival rate, and number of luminous bacteria (Vibrio splendidus
biovar 1) in hemolympll.
2. To study the efkct of P-glucan on some parameters of shrimp performance
i.e. growth and feed conversion ratio.
3. To study the effectiveness of 0-glucan given in 1 and 2 months on some
parameters of shrimp immune system i.e. hemocyte count, phagocytic
activity, clearance bacteria, and survival rate at low oxygen concentration.
The hypotheses of this study were as follows:
1. The lolv oxygen concentration, high water salinity, and high luminous bacteria
concentration could decrease hemocyte count, survival rate, and number of
luminous bacteria (Vibrio sl7lendidus biovar 1) in hemolympli of shrimp.
2. The administration of 0-glucan could increase the immune systeni and
performance of shrimp (Penaeus ~izorzodon Fab.) on disadvantageous
condition i.e. low oxygen concentration.
HISTORICAL REVIEW
Luminous Vibrio
The description information about genus Vibrio was reported by Baumann et al.
(1984).
Vibrios were straight or curve rods, which did not form endospores or
mycrocysts.
They were gram negative, facultative anaerobes capable of both
fermentative and respiratory metabolism. All were chemoorganothrophs;most grew in a
mineral medium containing D-glucose.
Most species were oxidase positive.
~ a +
stilllulated growth of all species of Vibrio. Species-of Vibrio vmy with respect to the
temperature at which growth occurred. All grew at 20°C and most did at 30°C; some
grew at 4°C and 45°C; none grew at 50°C. Many strains of marine Vibrio were able to
swarm on solid media. Until recently we. only know four species of Vibrio contained
lumi~lousstrain; Vibrio harveyi, Vibrio splendidus biovar I, Vibriojschery, and Vibrio
logei (Lavilla-Pitogo et al., 1990). She further explained some luminous bacteria have
the capacity of entering a symbiotic association with marine animals.
Lightner (1988) reported that vibriosis has become the most common bacterial
disease in sluimp culture, and in some tropical countries, the diseases occur throughout
the year. Rukyani and Sunarto (1998) observed that luminous bacteria caused disease on
shrimp. Vibrio spp were contagious disease agents as they were able to spread and infcct
through water, shrimp, feed, equipment, and hatchery operators.
Disease Trigger
The interaction of host, pathogen, and environment has become a common
relationship and could not be avoided in shrimp culture, a classic three-cycle system. But
disease could only occur when the system was an unequal interaction of host, pathogen,
and environment (Anderson, 1992).
Disease outbreak usually occurred on stressed shrimp during water exchange,
temperature and climate changes, larvae transportation, etc.
Direkbusarakom and
Danayadol (1998) reported that one of important states of the environment was the
presence of sufficient oxygen to maintain system vital to the health of an aquatic
ecosystem. Generally, the grade of oxygen concentration in a pond fluctuated; therefore
it has become a good indicator of water quality that affecting health of shrimp.
Hernolymph
Hemolymph or shrimp blood has become one of sonle shrimp health indicators
affected by stress condition i.e. oxygen concentration. Maynard (1960) reported that the
color of heniolymph was affected by hemocyanine (blood pigment). The hemocyanine
contained Cu and their tasks were to bind and to distribute the oxygen, and they play
important roles in blood osmotic.
The type of henlocytes (blood cells) in Crustacean was observed by Soderhall and
Johansson (1989). Based on the type of granules in helnocyte cytoplasn~,they reportcd
that crustacean generally contained three types of circulating hemocytes: hyaline cells (no
granules), semi granular cells (small granules), and granular cells (large granules).
Before this observation, the same category of hemocyte had been found in both Sicyor7iu
and Pet~aeusby Graves and Gary (1985). But they were called agranular cells, smallgranule cells, and large-granule cells.
Further they reported that the mean number of hemocytes per ml in Sicyo17ia and
Penaeus was similar. In both species, the proportion of agranular cells and the large
granule cells were very low, and the small granule cells composed approximately 75% of
all hemocytes.
Based on the phagocytic capability Soder!lall and Johansson (1989) noted that the
hyaline cells were typical phagocytic cells; the semi granular cells also had some
phagocytic capacity, but the granular cells had no phagocytic activity.
Shrimp Defence Mechanism
Soderhall and Johansson (1989) reported that arthropods and invertebrates in general
did not possess imn~unoglobulins. But with an open circulatory system, they had defence
and coagulation mechanism to entrap parasites and prevent blood loss upon wounding.
The cellular defence reaction in invertebrates was most often accompanied by
melanization involving prophenoloxidase (proPO) as an inactive pro-enzyme in
hemocyte. Some rllicrobial products could affect activation of prop0 to phenoloxidase.
The prophenolosidase-activating (ppA) system stimulated several cellular defence
reactions including phagocytosis. Both prophenoloxidase and inactive propllenoloxidaseactivating enzynle were stored in hemocyte, from where the): were released by
degranulation. Further they reported that the active enzyme, phenoloxidase, oxidized
pllenols to quinone. which then polymerized non-enzymatically to melanin.
An additional systcm to activate prophenoloxidase was reported by VargasAlbores ef al. (1998). They observed that the activation of prophenoloxidase involved
two steps.
The first was the degranulation that occurred when bacteria stimulated
hen~ocytes,and the second required the participation of Calciun~for the conversion of
inactive prophenoloxidase-activating enzyme to an active proteinase that, in turn,
transform prophenoloxidase to active-phenoloxidase.
Microbial products affected coagulation in hemolymph.
The mechanism has
become an essential defence response for crustacean to prevent loss of hemolymph
through a break in the exoskeleton and dissenliriation of bacteria throughout the body.
Vargas-Albores et al. (1998) observed that shrimp plasma had type C coagulation. It has
been named clotting protein. The clotting process occurred when prophenoloxidaseactivating enzyme was activated by the presence of calcium. Calcium triggering cellular
Transglutarninase (Tgase) enzyme was supplied by hyaline cells, on clotting protein to
build clot formation (like fibrin formation in vertebrates).
P-glucan as,An Imrnunostirnulant
Anderson (1992) defined that an imrnunostimulant is a chemical, drug, stressor, or
action that elevated the non-specific defence mechanism. Long-chain polysaccharide
extracted from yeast, P-glucan, has been known as one of some imniunos~imulantagcnts
stimulating non-specific defence mechanism in animals including shrimps. VargasAlbores e/ al. (1998) reported that P-glucan stimulated the propllenolosidase-activatillg
system conducted by serine protease enzyme (Soderhall and Johansson, 1989). In other
words, P-glucan is capable of stimulating shrimp hemocytes to release cellular
components.
The recognition of nlicrobial product in shrimp hemolymph has been reported by
Vargas-Albores ef cl. (1998). Two kinds of protein were involved in recognition of
microbial products in shrimp. The first group, lectins or hemaglutinins, was multivalent
sugar-binding agglutinins, which were able to react with bacterial lipopolysaccharide and
form some lipopolysaccharide-binding agglutinins (LPSBA).
The second group,
monovalent: was able to react with P-glucan, and therefore, it was called P-glucanbinding protein (BGBP). The existence of BGBP and LPSBA in shrimp plasma indicated
capability of shrimp immune system to detect, and then to bind P-glucan and
lipopolysaccharide given in order to trigger shrimp defence mechanism, especially in
2
bacterial attack.
METHODOLOGY
Time Lines
The experiment was conducted on January 22 up to June 30, 2001 at PT.
Centralpertiwi Bahari (Pondsite), in Lampung-Sumatera.
Materials
Shrimp Penaeus n2onodon Fab. (DOC: 50.and 30), Saccharonzyces cerevisiae Type
11 from SIGMA, Staphylococcus aureus (lo6 cfdml), isolate of Vibrio spp., Trypan Blue
solution, Saline solution, Triptych Soy brothlagar, Thiosulfate Citrate Bile Salt Sucrose
agar, Gelatin agar, Arginine, Ornithine, Lysine, Citrate agar, L-Arabinose, Sucrose,
Salicine, D-mannitol, D-sorbitol, Amylum, Kovacs solution, Sodium Chloride, DGlucose, Peptone agar, Trisodium Citrate, Citric Acid, EDTA, Chloride Acid, Ethanol,
Aquadest, Paraplast, Methanol, Giemsa stain, Gram stain. Xylol, H E stain, Entelan, Silk
screen 200 S, tank and aeration system, syringe Tuberculin 1 ml and needle, object glass,
cover glass, hemocytometer, petridisc, ose, Bunsen lamp. hotplate, stirrer, Becker glass,
Erlenilleyer glass, autoclave,n~icrotray. glass tubes,
DO and pH meter, disposable
pipette, microtome. and microscope.
Mcthods
Before running the experiment, extraction of P-glucan from cell \valls of
Srrcchcrronzyces cerevisiue was done by alkaline
-
acid nisthod (William el al., 1991).
Alkaline extract of P-glucan was conducted by dispersing and boiling 0.45 kg of dry
Saccharomyces cerevisiae in 3.5 L of 0.75 M (3%) Sodium hydroxide. The process of
separating residue from supernatant was conducted by keeping the mixture overnight and
discarding the dark brown supernatant. The NaON digestion was repeated twice. Then
the process of extraction was continued by acid digestion using Chloride acid 3.5 L of
2.45 M, 1.75 M, and 0.94 M. Separating process conducted at each step of acid digestion
was the same as alkaline digestion. The purification of P-glucan was continued by
bleaching process using 2 L of distilled water to gain white and flocculent residue. The
process was conducted ten times by boiling and separating process.
The colorless
process was continued by adding 1.5. L of ethanol to the residue and the process was
conducted tluee times by boiling and separating process. Before filtering the particle of
P-glucan by using fine silkscreen, the washing process using 2 L of distilled water was
conducted three times by boiling and separating process. Then the particle of P-glucan
was dried by freeze-lyophilize process.
Mixing of P-glucan in feed was condxted by using albumin as a binder. The
com~nercialfeed was mixed into the mixture of P-glucan and albumin, and then the blend
of this feed was baked in an oven in the temperature of 60°C for 2 hours to get the
nloisture 5 - 8.
Isolate of lulllinous bacteria used in this experiment came from hatchery of PT.
Ce~itralpertiwiBahari. The bacteria have beconle a prob!cm in mortality of shrimp in
hatchery. Type of luminous bacteria used for challenge test and type of bacteria in
shrimp hernolymph and hepatopancreas were identified by Baumann el CII., (1984)
method, including perfonnance and biochemistry of bacteria.
The number of bacteria in shrimp hemolymph and hepatopancreas, to examine the
clearance process, was counted by using disc-counting method (Thompson et al., 1997).
One gram of shrimp hepatopancreas was mixed into 9 ml of saline solution. The blend
was spread into petridisc containing Thiosulfate Citrate Bile Salt Sucrose agar and held
12 hours before counting the bacteria. Counting bacteria in hemolymph was conducted
by the same method. The shrimp hemolymph 0.02 ml was drawn from thc ventral
abdominal artery of shrimp and then was spread into petridisc containing Thiosulfate
Citrate Bile Salt Sucrose agar.
The number of hemocyte was counted by Blaxhall and Daisley (1973) abbreviation.
Hemolymph 0.1 ml was drawn from the ventral abdominal artery of shrimp into syringe
containing 0.1 ml Trypan Blue solution. To make a homogenized fluid, the mixture was
shaken carefully. One drop of the solution was added to a hemocytometer and the
number of cell per n1m3 was counted.
n~~
of counted-cell x diluting factor x 100 x 10
Total of h e n ~ o c ~ t e / m=Number
Number of counted-block 1 mn1 x 4
The ability of phagocytosis by hemocyte was evaluated by Anderson and Siwicki
(1995) method. Hen~olympll0.1 ml was drawn from the ventral abdominal artery of
shrimp into syringe containing 0.1 ml anticoagulant (0.14 M Sodium chloride, 0.1 M
Glucose, 30 mM Trisodium citrate, 26 n1M Citric acid, and 10 inM EDTA pH 4.6).
uureu.5
Then, the hcmolymph was transferred into microtray and 0.1 ml S/c~~~lzylococc~i.s
(lo6 cfulml) was added. The mixturc was mixed and incubated for 20 minutes. Every 5
luinutes the suspension was spread on glass slide, stained with Giemsa, and then
observed under the light microscope. Phagocytic index was counted based on percentage
of phagocyte cells showing phagocytosis activity.
Phagocytic index (%)
=
Number of cell showing phagocytosis activity
I
x 100
I
Number of cell hemocyte in glass slide
The survival rate, growth, and rate of feed conversion ratio were counted by using
formula Zonneveld el al., 1991:
I
Survival Rate (%) = Number of final population
loo
Number of initial population
I
Growth = Average of final growth - Average of initial growth
Feed Conversion Ratio = Total given feed - total uneaten feed
Expcrinlental Design
Trial 1: Artificial Stress Condition and SIlrimp Immunity
The objcctive of this trial was to study the effect of oxygen concentration, water
salinity, and luminous bacteria concentration on solne parameters i.e. hemocpte count,
survival rate, and nunlber of luminous bacteria in hemolymph. The result of this trial
could be used as a fit stressor in advanced trial. .
The trial was conducted with 3 replications and used factorial design.
statistical model was factorial 2 x 2 x 2.
The
The first factor I stressor was oxygen
concentration which had levels of 3 and 6 ppm, the second factor I stressor was water
salinity which had levels of 20 and 70 ppt, and the third factor / stressor was luminous
bacteria concentration with levels of 0 and 10' cfu/ml. Statistical analysis for survival
rate data used the arcsine transformation method (Steel and Torrie, 1991).
One hundred and forty-four healthy shrimps (DOC: 50) were randomly divided
and put into 24 tanks: 6 shrimpsltank. The fattors were given in three steps; the first
stressor was given until the shrinip attained weak condition, then the secolid stressor was
given in 10 minutes (Baticados ct al., 1986), finally the third stressor was given until the
shrimp attained weak condition.
The temperature during the experimental period was maintained at 25
-
27 'C.
Oxygen concentration was made and maintained by regulating aeration. To avoid the
negative effect of feed in the environment. shrimp was not fed.
After shrimp attained weak condition, three shrimps from each tank were selected
to cxanline the number of lun~inousbacteria in shrimp hemolymph, total hemocyte, and
survival rate of shrimp.
Trial 2: P-glucan and Shrimp Performance
.Ihc
. objective of this trial was to study the effect of P-glucan on some parameters
of shrimp performance i.e. growth and feed conversion ratio. The trial was conducted
with 3 replications and used randomized coluplete design. The treatments in this trial
were the dozes of P-glucan mixed in feed: 0%, 0.1%, and 0.5%. Statistical analyze for
growth data used the analyzes of covariance method (Steel and Torrie, 1991).
Three hundred healthy shrimps (DOC: 30) were randomly divided and put into 30
tanks: 10 shrimpsltank.
Before dividing, shrimp weight was counted.
To avoid
cannibalism, approximately, the shrimp weight in each tank was similar. The trial needed
30 days and feed for treatment was given adlibitum everyday.
After 30 days, shrimp weight and uneaten feed were counted to investigate shrimp
growth and feed conversion ratio. Uneaten feed was collected every 6 hours and was
baked in an oven with temperature 60°C.
Trial 3: Administration Period of P-glucan and Shrimp Immunity
The objective of this trial was to study the effectivity of 0-glucan given in 1 and 2
months on some parameters of shrimp immune system i.e. hemocyte count, phagocytic
activity, clearance of bacteria, and survival rate at a low oxygen concentration. The
experiment was an advanced experinieilt using the worst condition resulted by trial 1.
Tlie experiment was conducted with 3 replicatioils and used factorial design. The
statistical lnodel was factorial 3 x 2 x 2. The first factor was doze of P-glucan mixed in
feed which had levels 0%, 0.1% and O.j%, the second hctor was administration period of
0-giucan which had levels I aiid 2 months, and the third factor was luminous bactcria
concentration which had levels 0 and 10' cfd~iil.Tlie number of luminous bacteria in
hernolymph and hepatopaticreas was a~ialyzedby description method. Statistical alinlyze
for phagocytic activity and survival rate data used the arcsine transformation method
(Steel and Torrie, 1991).
Tluee hundred healthy shrimps (DOC: 30) were randomly divided and put into 30
tanks: 10 shrimpltank. Before dividing, shrimp weight was counted.
To avoid
cannibalism, approximately, the shrimp weight in each tank was similar. Based on the
result of the stressor test, shrimp was kept in low water concentration (3 ppm). The water
in each tank was changed every 3 days and the temperature during the experimental
period was maintained at 25 -27
OC.
Besides, the stopping of aeration for about 6 hours
everyday, the stocking of low oxygen water, to water exchange process, was conducted to
maintain the low oxygen concentration in tank.
The feed for treatment given everyday was adlibitum. The commercial feed was
used as a continued feed given at the treatment using 1-month administration period of Pglucan.
Immersion of luminous bacteria (challenge test) was conducted when shrimp had
been kept and had been given P-glucan dozes for 30 days. The process of challenge test
in this experiment was the same as process conducted in the stressor test.
After shrimp attained weak condition caused by challenge test, three shrimps from
each tank were selected to examine the number of lulninous bacteria in hemolymph and
hepatopancreas, hemocyte count, and ~hagocyticactivity of shrimp. Then, to investigate
bacteria clearance, the nu~nberof luminous bacteria in hemolymph and hepatopancreas
was examined in the 6"', 1211',and 24"' hours after challenge test.
Continued examination of hemocyte count was conducted in the 711', 14'11, and 21''
day after challenge test. Continued examination of phagocytic activity was conducted in
the 711', 14", 21L', and 30''' day after challenge test. The survival rate of shrimp was
examined in the 30"' day after challenge test.
,
As additional examination, the water quality in each treatment, including total
ammonia nitrogen, nitrite, nitrate, and alkalinity concentration, was examined weekly. In
order to complete the data, the bacteria found in shrimp hemolymph and hepatopancreas
was identified.
RESULTS AND DISCUSSION
Results
Identification of Vibrio Bacteria in Hemolymph and Hepatopancreas
The characteristic and biovar of Vibrio from henlolymph and hepatopancreas
were reported (Appendix I). Based on the Baumann et al. (1984) method these yellow,
green, and luminous bacteria indicated the characteristic of Vibrio alginolyticus, Vibrio
parahaemolyticus, and Vibrio splendidus biovar 1.
Trial 1: Artificial Stress Condition and Shrimp Immunity
Hemocyte count of shrimp was affected by individual and interaction of the
factors in treatment (Table 1).
The significant effects (Pi0.05) on hemocyte count
occurred as the result of individual factors i.e. water salinity and Vibrio splendidus biovar
1 concentration and as the result of interaction factor involving oxygen, water salinity and
Vibrio splendidus biovar 1 concentration.
Although interaction among factors gave significant effect on hemocyte count, the
interaction between water salinity factor and ~ i b r i osplendidus biovar 1 concentration
factor gave the biggest effect. Qxygen factor only gave significant effect on hemocyte
count when it was interacted with water salinity factor and Vibrio splendidus biovar 1
conce~~tration
factor.
The highest llemocyte count occurred at oxygen concentration G ~ P I I I water
,
salinity 20 ppt, and Vibrio s1~iendidu.sbiovar I concentration 10' ciidnll.
Table 1. Mean number of the hemocyte count, su:vival rate of shrimp and number of
Vibrio splendidus biovar 1 colony (cfu) in hemolymph at 18 hours after
stressing condition.
Oxygen
@pm)
3
Salinity
@PO
20
70
Note *: Significant at P < 0.05
Vibrio
splendidus
biovar I
(~fU/llll)
Mean of parameters
Heinocyte
count
(cellln~mj)
Survival
Rate (%)
0
448.00
82.98
-
10'
526.67*
78.68
3.89
0
221.OO*
82.98
-
Numb. Of
lum. Bac. In
hemolymph
(cfidml)
As an individual factor, Vibrio splendidus biovar 1 and oxygen concentration
affected the survival rate of shrimp significantly (P < 0.05). The lowest survival rate
occurred at low oxygen concentration (3 p p n ~ ) and Vibrio splendidus biovar 1
concentration lo7 cfdml.
Significantly, the number of Vibrio splendidus biovar 1 in hemolymph was
affected by water salinity. The highest concentration of Vibrio splendidus biovar 1 in
hemolymph was found at water salinity 70 ppt.
Trial 2: P-glucan and Shrimp Performance
The result of trial 2 indicated that the growth and feed conversion ratio of shrimp
were not affected by the treatment.. All treatment had no significant effect on the
performance of shrimp (P < 0.05) (Table 2).
Trial 3: Administration Period of p-glucan and Shrimp Immunity
One of some results of trial 3 indicated that the most rapid bacteria clearance in
hemolymph occused in shrimp fed by feed containing P-glucan 0.1%, in less than 12
hours after challenge test. The clearance of bacteria in hepatopancreas for all treatment
occurred in less than 12 hours after the challenge test (Table 3).
Star~sticalanalyze for hemocyte count indicated as individual factor, P-glucan and
Vibrio sl)lei7cf1dzrsbiovar 1 concentration gave significant effect on heliiocyte count in the
7"' day afier challenge test. The highest hemecyte count occurred in levcl of P-glucan
0.5%. The level of Tfibrio sl,lendidus biovar 1 lo7 cfidml gave the same result.
In 14"' day, as individual factor, P-glucan and administration period gave
significant effect on hemocyte count. The level of P-glucan 0%, 0.1%, and 0.5% could
affect significantly, but the biggest effect was given by 0-glucan 0.5%. The same result
was given by the level of 1-month administration period.
Besides as individual factor, factor of P-glucan and administration period,
together with Vibrio splendidus biovar 1 factor affected the hemocyte count of shrimp on
21'' day. The highest hemocyte count occurred in shrimp fed by feed containing
P-
glucan 0.5% given in 1 month and it was given under no bacteria condition (Table 4).
Statistical analyze for phagocytic activity indicated that as an individual factor
P-
glucan gave significant effect on phagocytic activity in the 7"' up to 3oth day after
challenge test.
Although feed containing P-glucan gave better result in phagocytic
activity than other, both levels of P-glucan in feed (0.1% and 0.5%) had no significant
difference (Table 5).
Statistical analyze for the survival rate of sluimp indicated that all treatment save
the same effect. But, s!witnp fed by feed containing P-glucan gave survival rate higher
than shrimp fed by no P-glucan feed (Table 6).
The water quality in each treatment indicated the same result (Figure 1).
Table 2. Mean number of growth and feed conversion ratio caused P-glucan administration
Mean of parameters
Treatment
Growth
Feed Conversion Ratio
Glucan 0 %
5.00
1.19
Glucan 0.1 %
4.98
1.12
Glucan 0.5%
5.31
1.15
Table 3. Number of l/iDrio splendidus biovar 1 colony in hepatopancreas and hemolymph after challenge test
Number of Vibrio splendidzls biovar I ( c f u h l )
Treatment
Hemolyniph
Hepatopancreas
The 24"'
hour
100
50
0
150
100
0
0
225
150
50
0
The 12"'
hour
The 24"'
hour
'The 1''
hour
1500
420
0
0
300
1500
505
0
0
975
750
0
0
-
The 6"'
hour
The 12"'
hour
l'he G"'
hour
The 1
hour
Table 4. Mean number of hemocyte count at tllc 0. 7"', 14th. and 2ISLday after
7
challenge test
Treatment
B
2
0.5
Note
Meall of hemocvte count (cell/mni.')
The 0 day The 71h day The 14"' day The 21" day
7166
6966
8000
7316
7833
8216
8600
8400
C
0
1 o7
1
0
1o7
7900
8100
7550
9616
13850
11666
9183
10366
2
0
10:
7566
7000
7900
9333
11400
9166
9500
6716
1
0
1o7
8!00
8000
10916
14516
14933
14350
12700*
1 1033"*
2
0
1o7
7666
8166
10900
11233
13800
12766
11366"*
1 1066"'
1
*: Significant at P < 0.05
A: % p-glucan in feed
B: Administration period (month)
C: Vibrio splendidrr.: biovar I (cfulml)
Table 5. Mean number of phagocytic activity at the 0, 71h, 141h,21s', and 30"' day after
challenge test
Note
*:
Significant
at P < 0.05
A: % P-glucan in feed
B: Administration period (month)
C: Vibrio splendidus biovar I (cfuiml)
Table 6 . Survival rate of shrimp
B: ~dlninistrationperiod (month)
C: l'ibrio splendidus biovar I (cfulml)
i
TAN
parameter
.
~-~~~
~~~~
I
i
NlTRlT
NITRAT
ALK
!
PH
I
parameters
.
I I
I
I!
1 (I
I
--
-.-.
@CONTROL =CONTROL U G L U C . 0.1%
U G L U C . 0.1%
P G L U C . 0.1%
CIGLUC. 0.1%
B G L U C . 0.5%
O G L U C . 0.5%
I G L U C . 0.5%
H G L U C . 0.5%
..
BACTERIA
N O BACTERIA
- 1 MONTH -BACTERIA
- 1 M O N T H - N O BACTERIA
- 2 MONTHS -BACTERIA
- 2 M O N T H S - N O BACTERIA
- 1 M O N T H - BACTERIA
- 1 M O N i H - N O BACTERIA
- 2 MONTHS -BACTERIA
- 2 M O N T H S - N O BACTERIA
-...-..-.._---d..
Figure 1 . The water quality in each treatment
~~
-. .
-
~
Trial 1: Artificial Stress Condition and Shrimp Immunity
The number of hemocyte in he~nolympli indicated their ability to respond
some unadvantages condition e.g. stress condition. Anderson and Siwicki (1995)
reported that the highest heinocyte count wandering in hernolymph, .the biggest
respond could be given to hold the stress condition
111
(Anderson and Siwicki, 1995).
this trial, the stress condition caused by concentration 10' cfu/ml of Vibrio
splendidus biovar I gave the biggest effect on helnocyte count.
Besides affecting the hemocyte count, this concentration gave the worst effect
on survival rate of shrimp.
Besides the highest concentration of Vibrio splendidus biovar I , the worst
effect on survival rate of shrimp was caused by low oxygen concentration (3 ppm).
Direkbusarakom and Danayadol (1998) reported that one of ilnportant states of the
environlnent was the presence of sufficient oxygen to maintain a system vital to the
health of an aquatic ecosystein. and the critical concentration for shrinip ponds was
3.7 pp111.
Salinity 70 ppt gave effect on the introduction of bacteria in shrimp body. It
occurred easily when the oxygen concentration was low enough and especially wlieti
the carapace of shrimp was soft. The low oxygen concentration increased the shrimp
metabolism, thus to fulfill the osygen consumption, the shrimp lnlust more active to
take oxygen in water. At tlie same time, high concentration of bacteria in water
would enter tlie shrimp body. Balicados el crl. (1986) observed that a hypet osmotic
condition could be caused by high salinity leading to a chronic soli-shell syndroine
that could arkct tlie defence meclianisni of'sliriiiil~.
All results in trial 1 indicated that shrimp im~nunitycould be triggered by a
stress condition involving stressor factors such as low oxygen concentration (3 ppm),
IGrio .sp/endiidr(s biovar I with concentralion 20' cfu/inl, and high salinity (70 ppi).
Therefore, the interaction of these factors could bc a challenge test for the other trial.
The challenge test was a test to give a challenge to investigate the animal capability
after given a treatment.
Trial 3: P-glucan and Sl~rirnpPerformance
Feed containing P-glucan had no effect on the performance of shrimp i.e. the
shrimp growth and the feed conversion ratio. It indicated that no additional nutritious
materials affecting the growth cells of shrimp body. It also indicated that adding
P-
gluca~idid not change the shrimp respond to the feed.
A proximate analysis conducted to analyze the contain of protein, lipid, ash,
fibber? and nitrogen free materials indicated that all feed had no different level at their
nutrient content (Table 7).
Trial 3: Administration Period of P-glucan and Shrimp Imniunity
Martin and arenda (1985) reported that gram positive and ~iegativebacteria in
hemolymph could be cleared from hemalymph rapidly by gills in circulation process.
In the first time, bacteria entered the shrimp body and before it was distributed to
hemolymph the bacteria would stay in hepatopancreas a li~tlewhile. In this trial,
shrimp fed by P-glucan O,1% cleared Vibrio sp1ri~dicili.s biovav 1 i n their
hepatopancreas and Iiemolymph more quickly than the otiisrs (Table 3). It indicated
that p-glucan O. I % \\,as a good bacleria-clearable substance.
'[.he infection caused by Vibrio .s,~ie~ldicili.r
I~iovar I co~itiii~ted
to the 7"' day
after challenge test. Increasing of hemoc!.te couiii and phagocytic activity at thc 7"'
day after challenge test \\,as the effect of P-glucan and bacteria factor individually
(Table 4 and 5 ) .
Anderson and Siwicki (1995) reported that the increasing of
heniocyie count in hemoly~npliwas caused by infection, stress. and blood disease.
The infection caused an inflammation; a non-specitic characteristic triggered by some
factors e.g. parasite and bacteria (Person et al., 1987; Itami et a/.,1996).
The tendency of increasing and decreasing of hemocyte count and phagocytic
activity was similar (Figure 2). It indicated that the biggest composition of hemocyte
was hyaline cells i.e. phagocytic cells (Soderhall and Johansson, 1989).
Figure 2. The relationship between phagocytic activity and hemocyte count
FA: Pliagocytic activity, HC: He~nocytecount, -0, -7, -14, -21, -30: the 0: 7'h, 1411',
21s', and 30"' day after challenge test, A: p-glucan 0% + 7 months + 0 cfulml K
splendidzrs biovar 1, B : p-glucan 0% + 2 months + lo7 cfulml V. splendidzrs biovar 1,
C: P-glucan 0.1% + 1 months + 0 cfulinl V. splendidzrs biovar 1, D: P-glucan 0.1% +
I months + lo7 cfulml T'. splendidus biovar 1, E: P-glucan 0.1 % + 2 lnonrhs + 0
cfi~lml V. splendidzrs biovar 1, F: P-gluca~i 0.1% + 2 months i- lo7 cfulml V.
splendidzts biovar 1, G : P-glucan 0.5% + 1 months + 0 cfulml V. sp1endidz1.r biovar I,
H : P-glucan 0.5% + 1 months + 10' cfulml V. sp1endid;rs biovar 1, 1 : P-glucan 0.5%
+ 2 ~iionths+ 0 cful1111I/. splendidus biovar I, .I : P-glucan 0.5% + 2 monihs + lo7
cfuiml I/ sl~ie!7did1i.s
biovar 1.
31
Table 7. The nutrient content (% wet basis) of the trial diets.
Feed containing
glucan 0%
Crude Protein
1
Crude Lipid
Crude Fibber
Moisture
Nitrogen-Free
Materials
P-
Feed containing Pylucan 0.1%
Feed containing Pglt~can0.5%
The phagocytic cells could phagocyte a peculiar substance or bacteria that
entered the host.
The process of phagocytosis involved some mechanism of
attachment. engulfing, degranulation, and lyscsbacleria (Gudkovs, 1988). T h e cells
which running phagocytic activity could be observed by light niicroscope.
The
process could be differed with the accuinulation of bacteria on the cells by observing
the characteristic of color absorption.
Although the highest hemocyte count was found in shrimp fed by 6-glucan
O.j%, the phagocytic activity in shrimp fed by P-glucan 0.5% had no a significant
difference with the phagocytic activity in shrimp fed by P-glucan 0.1% (Table 4 and
5). It indicated that the composition of hyaline cells in hemocyte of shrimp fed by
P-
glucan 0.1% was higher than in shrimp fed by P-glucan 0.5%. Thus, shrimp fed by
P-
glucan 0.1% was more resistant than the others because of their ability in phagocytic
activity.
The p-glucan in feed could be digested by P-I, 3-glucanase enzymes in
crustacean digestion system (Dall and Moriarty, 1983) before it \\,as distributed to
he~nolymphsystem. Recognition protein in hemocyte plasnia could recognize the
characteristic of P-glucan and lipopolysaccharide (Vargas-Albores e1 al., 1998).
Then, P-glucan activated prophenoloxidase enzyme in he~nocyteto plienoloxydase.
The activated plienoloxydase triggered the coagulation system in order t o close the
l v o ~ ~ n dThe
. process to activate phenoloxydase caused releasing of
a free
(radical material that could trigger the step of degranulation in phagocytosis caused the
clcal-ance of bacteria.
Figure 2 showed that the increasing of phagocytic activity was caused by
adding P-glucan in feed. Fecd containing P-glucan could increase the phagocytic
activity higher than commercial feed that had no P-glucan inside.
All treatnlenrs did not give significant difference on the survival rate of
shrimp. However, the survival rate of shrimp fed by P-glucan was higher than the
other (Table 6).
Galeotti (1998) reported that prolonged im~nunostiniulantadministration, in
doze and time, did not give a better effect. In this trial, (3-glucan adtiiinistration in 2
months did not give a better result than the administration in 1 month, because of the
effective time.
CONCLUSSJON
The general conclusion of all trial was the low oxygen and high bacteria
concentration gave a bad effect on shrimp defence mechanism.
The high water
salinity affected the entering of bacteria in shrimp body.
P-glucan extracted from Saccharon~ycescerevisiae by alkali-acid method
(William el al., 1991) could increase some parameters of the non-specific immune
syste~iiof shrimp that was kept in low oxygen concentration (3 ppm), but they had no
effect on the performance of shrimp, because the trial was conducted in short time to
give evidence of their effect on the performance of shrimp. Feed containing P-glucan
0.1% gave a better effect than P-glucan 0.5% in their role to increase the non-specific
immune system of shrimp by increasing phagocytic activity.
The administration of P-glucan in 2 months did not give a better result than the
ad~ninistrationof P-glucan in I month. Thus. the best administration period of
glucan for 30 days old culture of shrimp was I month.
P-
Andcrson, D.P. 1992. Iinmunostimulants. Adjuvant, and Vaccine Carriers in
Fish: Applications to Aquaculture. .Ann. Rev. Fish Dis.. 2: 281307.
and Siwicki. 1995. Basic Hematology and Serology for Fish
Health Programs, p. 185-202. Inshariff, M.. R.P. Subasinghe and
J. Arthur (ed.).
Diseases in Aquaculture 11. Fish Healrh Secr.
Asian Fish Soc., Manila.
Baticados,
INCREASE THE NON-SPECIFIC IMMUNITY
AND THE PERFORMANCE OF
SHRIMP (Perzaeus monodon, Fab.)
UNDER ARTIFICIAL STRESS CONDITION
BY
EMILIANA DHIAN ANGGERAHENI
I'OSTGIIADUATE PROGRAM
INSTITUT PERTANIAN BOGOR
2002
ABSTRACT
EMlLlANA DHlAN ANGGERAHENI.
The Use of (3-glucan as An
Immunostimulant to Increase the Non-Specific Immunity and the Performance of
Shrimp (Penaezts n~onodon,Fab.) Under Artificial Stress Condition. Under advisory
cotlimittee AHMAD MAAD WIRAWlDJAJA and DARNAS DANA.
Environmental stress has been a colnmon risk in shrimp culture ponds. This
stress could lead to the occurrence of diseases. The experiment was divided into three
trials. The objective of the first trial was to investigate the effect of oxygen
concentration. water salinity, and luniinous bacteria concentration on some
parameters of stress condition i.e. hemocyte count, survival rate, and number of
luminous bacteria (Kbrio splendidus biovar 1 ) in hemolymph. The result of this trial
could be used as a fit stressor in an advanced trial. The second trial was to investigate
the effect of P-glucan on some parameters of shrimp performance i.e. growth and feed
conversion ratio. The third trial had an objective to study the affectivity of (3-glucan
given in 1 and 2 months on some parameters of shrimp immune system i.e. hemocyte
count. phagocytic activity, clearance bacteria, and survival rate at low oxygen
concentration. The experiment was an advanced experiment using the worst condition
resulted by trial 1.
The result suggests that feed containing e-glucan 0.1% affected the immune
capacity of shrimp, such as clearance of bacteria in hepatopancreas and hemolymph,
total hemocyte and phagocytic activity of hemocyte. Its effect was better than the
others. But the feed containing P-glucan had no significant effect on the performance
of shri~npi.e. the growth and the feed conversion ratio. because the trial was
conducted in short time to give evidence of their effect on the performance of shrimp.
Shrimp given feed containing P-glucan 0.1% could clear Vibrio splendidzrs biovar I in
he~nolymph and hepatopancreas in less than 12 hours. Although the highest
hemocyte count was found in shrimp fed by P-glucan 0.5%, their phagocytic activity
had no significant difference with shrimp fed by P-glucan 0.1%. It indicated that the
content of hyaline cells (phagocytic cells) in shrimp hemocyte fed by P-glucan 0.1%
was higher than the other. Thus, the highest rate of phagocytic activity from the 7Ih up
to the 14"' day after bacteria immersion was found in shrimp hemocyte fed by IJglucan 0.1 %.
The prolonged periods with dosage P-glucan, with the aim of obtaining better
and faster productivity results, had no significant effect on the immune capacity of
shrimp. The best administration period of P-glucan for 30 days old culti~reof shrinip
was I month.
DECLARATION
I declare that thesis using title:
THE USE OF P-GLUCAN AS AN IMMUNOSTIMULANT TO lNCREASE
THE NON-SPECIFIC IMMUNITY AND THE PERFORMANCE OF
SHRIMF' (Perzaerrs ntortodort, Fab.)
UNDER ARTIFICIAL STRESS CONDITION
is my writing, as the results of my experiment. The thesis has been not published as
academic report at other universities or other institutions.
This thesis may not be reproduced in whole or part by photocopy or other
means without permission ofthe author.
E~nilianaDhian Anezeraheni
NRS : 99440lRIOLOGY
THE USE OF P-GLUCAN AS AN IMMUNOSTIMULANT TO
INCREASE THE NON-SPECIFIC IMMUNITY
AND THE PERFORMANCE OF
SHRIMP (Penae~csmonodon, Fab.)
UNDER ARTIFICIAL STRESS CONDITION
EMILIANA DHlAN ANGGERAHENI
A thesis submitted in partial fulfillment
of the requirements for the degree of
MASTER OF SCIENCE
in the
Department of B i o l o ~ y
POSI'CRADUATE PROGRAM
INSTITUT I'ERTANIAN BOGOR
2002
APPROVAL
Title of Thesis
: THE USE OF P-GIACAN AS AN IMMUNOSTIMULANT
TO INCREASE THE NON-SPECIFIC IIVIMUNITY AND
THE PERFORMANCE OF SHRIMP
(Penaeus monodon, Fab.)
UNDER ARTIFICIAL STRESS CONDITION
Name
: EMILIANA DHIAN ANGGERAHENI
No. Reg. Student
: 99440
Program
: Biology
Approved by,
I . Advisory Comtnittee
Dr. Ahmad Maad Wirawidiaia
Chairman
2. I-lead of Biology Department
Dr. Dedi Durvadi
Graduation date : Septcmbcr 28, 700 I
Dr. Darnas Dana. h4Sc.
Member
CURRICULUM VITAE
The writer, EMlLlANA DHlAN ANGGERAHENI, was born on January 5 ,
1971. in Semarang - Central Java. In September 1994, she graduated from the
Faculty of Fisheries, Aquaculture Department, Institut Pertanian Bogor.
From November 1994 until May 1996, she worked at PT. Birulaut
Khatulistiwa, the shrimp hatchery of PT. Dipasena Citra Darmaja in KaliandaLampung. Then, she worked at the shrimp hatchery of PT. Centralpertiwi Bahari in
Suak Sidomulyo - Lampung from June 1996 until February 1999.
In 1999, she continued her study in Institut Pertanian Bogor for the degree of
Master of Science in Biology Department and on September 28, 2001 she graduated
her study.
ACKNOWLEDGEMENTS
I gratefully acknowledge the financial support by the PT. Centralpertiwi
Baliari. I would like to express illy deep appreciation to Mr. Djoko M. Basoeki. Dr.
Sujint Tamniasart. and Mr. Johannes Kitono, the president director and directors of
the PT. Centralpertiwi Bahari. Thanks are due also to Mr. Wayan Agus Edhy. Mr.
Wahyudi, Mr. Cheawchan Ponpanitralsamee and Mr. Sarayuth for their expert advice;
Mr. Januar Pribadi for his laboratory services; Mr. lsman Haryanto, Mr. Risdianro
P.W, Ms. Maria Sisilia, and Ms. Eni for their attention; Dr. Dean Akiyama for his
invaluable discussion and attention at the final stage of my thesis.
I wish to express thanks to ~ n yparents, iny sisters, and mas Joko Sulistyo for
their loyal support throughout the study. Thanks also to the staff of PT Centralpertiwi
Bahari (Pondsite) Larnpung, especially Yogi Sukirman, who helped me in the wet
laboratory, Mr. Bastian Sitompul, Mr. Ilasan Solikin, Mr. Sutarto, all staff of
Microbiology laboratory, Mr. Dani Yukasano, Mr. Tonif, Mr. Kristianto, Mr.
Suherman, and all my friends at pond site.
Finally, 1 wish to thank to my supervisory committee, Dr. Ahmad hlaad
Wirawidjaja and Dr. Darnas Dana, MSc., my English Lecturer Ms. Nilawati, and my
Statistic advisor Mr. Ba~nbangSurnantri. Their dedication, concern and guidance
throughout my study are gratefully acknowledged.
TABLE O F CON'1'ENTS
PAGE
LIST OF TABLES ...... ................................ . ............,...........
vi
LIST OF FIGURES ............................. ..... . ......, ,..... .., ...........
vii
LIST OF APPENDICES.. ...... .... . ..... ..., .... .. . .... ... ........, ....., , ..
Vlll
...
INTRODUCTION
Justification and Problems.. ........................., ............. ...
Objective.. ........................................ .. .. .. .................
Hypotheses.. ................ ......... .... ... . . . . ... .. . ..... . .... .........
1
3
3
HISTORICAL REVIEW
.
.
Luminous Vlbrio... . .. .......................... . ................ .. ......
Disease Trigger.. ..... . ...... . ... .. .............. .. ... .. ... .... ...........,
Hemolymph.. .............. ........... .. . .... . . .... . . . . . . . , , .. .. . ......, . .
Shrin~pDefence Mechanism.. . .. .......... . ... . . .. . . . . ............. .. ..
P-glucan as An Immunostimulant ... ...... .. .. .. . . . . . . . .. ...............
4
5
5
6
7
METHODOLOGY
Times Lines ......... ................. . . .. .... . . .. .... . . .... . . . . . . .. ........
9
Materials.. ...... ..... ...... ... . .. . .. ......... . . . ... . . . . . . , .. . . ....... ., .. ..
9
Methods
9
Experimental Design
Trial 1: Artificial Stress Condition and Shrimp In~nii~nity
. .. . 12
Trial 2: p-glucan and Shrimp I'erformance.. .. ... . . . . . . .. ... ....
13
Trial 3: Administration Period of b-glucan and
Shrimp Immunity .. . . .... .. . . . . . . . . .. . . . . . ..... . . .. . . . , , ..
14
RESULTS AND DISCUSSION
Results
Identification of Vibrio Bacteria in Hemolymph
and Hepatopancreas.. ... ......... ...................... ...........
Trial 1: Artificial Stress Condition and Shrin~pIrmnuniry ....
Trial 2: P-glucan and Shrimp Perfornlance................... ..
Trial 3: Administration Period of P-glucan and
Shrimp Immunity.. ...................................... .
Discussion
Trial 1: Artificial Stress Condition and Shrimp Immunity..
Trial 2: p-elucan and Shrimp Perfornlance ............... ...
Trial 3: Administration Period of P-glucan
and Slvimp Iminunity .............................. ...
CONCLUSSION.. . ..... ........... ................... .. . .....................
BIBLIOGRAPHY.. . . . ......................... .............................. ...
APPENDICES. ........... ....... .... ..... ............... .......... ...... .......
LIST OF TABLES
PAGE
1. Mean number of the hemocyte count, survival rate of shrimp and
number of Vibrio splendidus biovar 1 colony (cfu) in hemoplymph
at 18 hours after stressing condition.. .................................
18
2. Mean number of growth and feed conversion ratio caused
..
by P-glucan adm~nlstration.............................................
21
3. Number of Vibrio splendidus biovar 1 colony in hepzropancreas
and hemolymph after challenge test.. ................................
22
4. Mean number of hemocyte count at the 0,71h. 14"', and 21'' day
after challenge test.. ......................................................
23
5. Mean number of phagocytic activity at the 0. 7"', 14u. and 21" day
after challenge test. .....................................................
24
6. Survival rate of shrimp.. ................................................
25
7. The nutrient content (% wet basis) of the trial diets.. ............
31
LIST OF FIGURES
PAGE
1. The water quality in each treatment.. ................ ......................
26
2. The relationship between phagocytic activity and hemocyte count ....
30
LIST OF APPENDICES
PAGE
1. The differential characteristics of the species and biovar of the
. .
Genus of fibrzo ..................................................
38
2. The analyses of variance for the hemocyte count in hemolyinph at
18 hours after stressing condition (Trial 1). ...................
40
3. The analyses of variance for survival rate of shrimp at
18 hours after stressing condition (Trial 1). ....................
41
4. The analyses of variance for number of luminous bacteria in
henlolymph at 18 hours after stressing (Trial 1)..............
42
5. The analyses of covariance for the growth of sluimp (Trial 2)
43
6. The analyses of variance for the feed conversion ratio (Trial 2)
44
7. The analyses of variance for the hemocyte count in hemolymph on
the 0 day after challenge test.. ..........................
45
8. The analyses of variance for the hemocyte count in hemolynlph on
the 7"' day after challenge test.. .....................
46
9. The analyses of variance for the hemocyte count in hemolymph on
the 14"' day after challenge test .......................
47
10. The analyses of variance for the heniocyte count in he~nolpmphon
the 21'' day after challenge test .........................
45
11. The analyses of variance for the phagocytic activity on
the 0 day after challenge test .........................
49
12. The analyses of variance for the pbagocytic activity on
the 7"' day after challenge test ....................
50
13. The analyses of variance for the phagocytic activity on
the 1411, day after challenge test ....................
14. The analyses of variance for the phagocytic activity on
the 21S1dayafter challenge test ......................
15. Tile analyses of variance for the phagocytic activity on
the 50"' day after challenge test ......................
16. The analyses of variance for the s u ~ i v arate
l of shrimp
..................................
after challenge test
17. The steps of phagocytic activity ..................
INTRODUCTION
,Justification and Problems
Shrimp has a less developed immune system than fish or other vertebrates. It is
probable that shrimp is relatively more dependent on the non-specific immune processes,
such as phagocytosis, than vertebrates (Raa et al., 1992).
Thus, administration of
antibiotics and vaccines giving a specific immunity is an ineffective treatment (Anderson,
1992).
In~mu~lostimulant,a material that can elevate the non-specific defence
mechanism, i.e. P-glucan and lipopolysaccharide, may represent an alternative and
supplement to vaccines in the protection of farmed shrimp against diseases.
Lipopolysaccharide and P-glucan, present on the gram-negative bacterial and fungal
surface, can be recognized by shrimp henlolymph; thus, they.can increase the nonspecific in~nlunesystem of shrimp (Kondo et al., 1996; Vargas-Albores et al., 199s).
Although the im~nunostin~ulants
have numerous positive effects on the imnlune
system in shrimp, they have some limitations and possible negati\.e effects, especially in
case of over dosage.
Irnmunostiiuulants administration must notice the timing and
dosage of application (Anderson, 1992).
The timing of irnn~unostimulants application depends on the activating
characteristics of the ~naterialitself.
Some result of trials indicate that P-glucan has a
short duration cffect on irnnlune system. Thus. P-glucan must be given exactly at the
time as a preventive treatment before disease exposure (Galeotti, 199s).
The overdose or under dose of inlmunostimulants is an administration problem.
High doses of in~munostimulantscan suppress the defence mechanism, and low doses
may not be effective (Anderson, 1992). High dietary levels of P-glucan (>I .5 %) in feed
may impair nutrient digestibility because they increase the viscosity of the ingesta
(Galeotti, 1998; Volpatti et al., 1998).
Generally, the experiment on im~~~unostimulant
is conducted only to investigate
effectiveness of a material on animal immunity, and omit interaction of other factors in
environment that cannot be precisely controlled (Soderhall and Johansson, 1985, 1989;
Raa et al., 1992; Itami, 1994; Kondo et a/.,1996; Vargas-Albores el al., 1998; Clifford,
1999). The effect of imnlunostimulants on the interaction of disadvantageous conditions
of the environment causing stress has not been identified yet.
In fact, most diseases in aquaculture are caused by opportunistic pathogens i.e.
bacteria that cause disease when shrimp is weak or under stress. The phagocytic activity
is less affected by stress condition (Anderson, 1992). The stress is caused by temperature,
oxygen concentration, pH, salinity, etc, often exists under practical farming conditions
(Clifford, 1999).
Thus, this experinlent was done by combining some factors causing stress
condition i.e. oxygen concentration. salinity, and level of bacterial attack, to study the
ability of P-glucan to increase the non-specific iminunity of shrimp.
Objcctivc
The experiment was divided into three trials with some objectives as follows:
1. To study the effect of oxygen concentration, water salinity, and luminous
bacteria concentration on sotne parameters of stress condition i.e. hetnocyte
count, survival rate, and number of luminous bacteria (Vibrio splendidus
biovar 1) in hemolympll.
2. To study the efkct of P-glucan on some parameters of shrimp performance
i.e. growth and feed conversion ratio.
3. To study the effectiveness of 0-glucan given in 1 and 2 months on some
parameters of shrimp immune system i.e. hemocyte count, phagocytic
activity, clearance bacteria, and survival rate at low oxygen concentration.
The hypotheses of this study were as follows:
1. The lolv oxygen concentration, high water salinity, and high luminous bacteria
concentration could decrease hemocyte count, survival rate, and number of
luminous bacteria (Vibrio sl7lendidus biovar 1) in hemolympli of shrimp.
2. The administration of 0-glucan could increase the immune systeni and
performance of shrimp (Penaeus ~izorzodon Fab.) on disadvantageous
condition i.e. low oxygen concentration.
HISTORICAL REVIEW
Luminous Vibrio
The description information about genus Vibrio was reported by Baumann et al.
(1984).
Vibrios were straight or curve rods, which did not form endospores or
mycrocysts.
They were gram negative, facultative anaerobes capable of both
fermentative and respiratory metabolism. All were chemoorganothrophs;most grew in a
mineral medium containing D-glucose.
Most species were oxidase positive.
~ a +
stilllulated growth of all species of Vibrio. Species-of Vibrio vmy with respect to the
temperature at which growth occurred. All grew at 20°C and most did at 30°C; some
grew at 4°C and 45°C; none grew at 50°C. Many strains of marine Vibrio were able to
swarm on solid media. Until recently we. only know four species of Vibrio contained
lumi~lousstrain; Vibrio harveyi, Vibrio splendidus biovar I, Vibriojschery, and Vibrio
logei (Lavilla-Pitogo et al., 1990). She further explained some luminous bacteria have
the capacity of entering a symbiotic association with marine animals.
Lightner (1988) reported that vibriosis has become the most common bacterial
disease in sluimp culture, and in some tropical countries, the diseases occur throughout
the year. Rukyani and Sunarto (1998) observed that luminous bacteria caused disease on
shrimp. Vibrio spp were contagious disease agents as they were able to spread and infcct
through water, shrimp, feed, equipment, and hatchery operators.
Disease Trigger
The interaction of host, pathogen, and environment has become a common
relationship and could not be avoided in shrimp culture, a classic three-cycle system. But
disease could only occur when the system was an unequal interaction of host, pathogen,
and environment (Anderson, 1992).
Disease outbreak usually occurred on stressed shrimp during water exchange,
temperature and climate changes, larvae transportation, etc.
Direkbusarakom and
Danayadol (1998) reported that one of important states of the environment was the
presence of sufficient oxygen to maintain system vital to the health of an aquatic
ecosystem. Generally, the grade of oxygen concentration in a pond fluctuated; therefore
it has become a good indicator of water quality that affecting health of shrimp.
Hernolymph
Hemolymph or shrimp blood has become one of sonle shrimp health indicators
affected by stress condition i.e. oxygen concentration. Maynard (1960) reported that the
color of heniolymph was affected by hemocyanine (blood pigment). The hemocyanine
contained Cu and their tasks were to bind and to distribute the oxygen, and they play
important roles in blood osmotic.
The type of henlocytes (blood cells) in Crustacean was observed by Soderhall and
Johansson (1989). Based on the type of granules in helnocyte cytoplasn~,they reportcd
that crustacean generally contained three types of circulating hemocytes: hyaline cells (no
granules), semi granular cells (small granules), and granular cells (large granules).
Before this observation, the same category of hemocyte had been found in both Sicyor7iu
and Pet~aeusby Graves and Gary (1985). But they were called agranular cells, smallgranule cells, and large-granule cells.
Further they reported that the mean number of hemocytes per ml in Sicyo17ia and
Penaeus was similar. In both species, the proportion of agranular cells and the large
granule cells were very low, and the small granule cells composed approximately 75% of
all hemocytes.
Based on the phagocytic capability Soder!lall and Johansson (1989) noted that the
hyaline cells were typical phagocytic cells; the semi granular cells also had some
phagocytic capacity, but the granular cells had no phagocytic activity.
Shrimp Defence Mechanism
Soderhall and Johansson (1989) reported that arthropods and invertebrates in general
did not possess imn~unoglobulins. But with an open circulatory system, they had defence
and coagulation mechanism to entrap parasites and prevent blood loss upon wounding.
The cellular defence reaction in invertebrates was most often accompanied by
melanization involving prophenoloxidase (proPO) as an inactive pro-enzyme in
hemocyte. Some rllicrobial products could affect activation of prop0 to phenoloxidase.
The prophenolosidase-activating (ppA) system stimulated several cellular defence
reactions including phagocytosis. Both prophenoloxidase and inactive propllenoloxidaseactivating enzynle were stored in hemocyte, from where the): were released by
degranulation. Further they reported that the active enzyme, phenoloxidase, oxidized
pllenols to quinone. which then polymerized non-enzymatically to melanin.
An additional systcm to activate prophenoloxidase was reported by VargasAlbores ef al. (1998). They observed that the activation of prophenoloxidase involved
two steps.
The first was the degranulation that occurred when bacteria stimulated
hen~ocytes,and the second required the participation of Calciun~for the conversion of
inactive prophenoloxidase-activating enzyme to an active proteinase that, in turn,
transform prophenoloxidase to active-phenoloxidase.
Microbial products affected coagulation in hemolymph.
The mechanism has
become an essential defence response for crustacean to prevent loss of hemolymph
through a break in the exoskeleton and dissenliriation of bacteria throughout the body.
Vargas-Albores et al. (1998) observed that shrimp plasma had type C coagulation. It has
been named clotting protein. The clotting process occurred when prophenoloxidaseactivating enzyme was activated by the presence of calcium. Calcium triggering cellular
Transglutarninase (Tgase) enzyme was supplied by hyaline cells, on clotting protein to
build clot formation (like fibrin formation in vertebrates).
P-glucan as,An Imrnunostirnulant
Anderson (1992) defined that an imrnunostimulant is a chemical, drug, stressor, or
action that elevated the non-specific defence mechanism. Long-chain polysaccharide
extracted from yeast, P-glucan, has been known as one of some imniunos~imulantagcnts
stimulating non-specific defence mechanism in animals including shrimps. VargasAlbores e/ al. (1998) reported that P-glucan stimulated the propllenolosidase-activatillg
system conducted by serine protease enzyme (Soderhall and Johansson, 1989). In other
words, P-glucan is capable of stimulating shrimp hemocytes to release cellular
components.
The recognition of nlicrobial product in shrimp hemolymph has been reported by
Vargas-Albores ef cl. (1998). Two kinds of protein were involved in recognition of
microbial products in shrimp. The first group, lectins or hemaglutinins, was multivalent
sugar-binding agglutinins, which were able to react with bacterial lipopolysaccharide and
form some lipopolysaccharide-binding agglutinins (LPSBA).
The second group,
monovalent: was able to react with P-glucan, and therefore, it was called P-glucanbinding protein (BGBP). The existence of BGBP and LPSBA in shrimp plasma indicated
capability of shrimp immune system to detect, and then to bind P-glucan and
lipopolysaccharide given in order to trigger shrimp defence mechanism, especially in
2
bacterial attack.
METHODOLOGY
Time Lines
The experiment was conducted on January 22 up to June 30, 2001 at PT.
Centralpertiwi Bahari (Pondsite), in Lampung-Sumatera.
Materials
Shrimp Penaeus n2onodon Fab. (DOC: 50.and 30), Saccharonzyces cerevisiae Type
11 from SIGMA, Staphylococcus aureus (lo6 cfdml), isolate of Vibrio spp., Trypan Blue
solution, Saline solution, Triptych Soy brothlagar, Thiosulfate Citrate Bile Salt Sucrose
agar, Gelatin agar, Arginine, Ornithine, Lysine, Citrate agar, L-Arabinose, Sucrose,
Salicine, D-mannitol, D-sorbitol, Amylum, Kovacs solution, Sodium Chloride, DGlucose, Peptone agar, Trisodium Citrate, Citric Acid, EDTA, Chloride Acid, Ethanol,
Aquadest, Paraplast, Methanol, Giemsa stain, Gram stain. Xylol, H E stain, Entelan, Silk
screen 200 S, tank and aeration system, syringe Tuberculin 1 ml and needle, object glass,
cover glass, hemocytometer, petridisc, ose, Bunsen lamp. hotplate, stirrer, Becker glass,
Erlenilleyer glass, autoclave,n~icrotray. glass tubes,
DO and pH meter, disposable
pipette, microtome. and microscope.
Mcthods
Before running the experiment, extraction of P-glucan from cell \valls of
Srrcchcrronzyces cerevisiue was done by alkaline
-
acid nisthod (William el al., 1991).
Alkaline extract of P-glucan was conducted by dispersing and boiling 0.45 kg of dry
Saccharomyces cerevisiae in 3.5 L of 0.75 M (3%) Sodium hydroxide. The process of
separating residue from supernatant was conducted by keeping the mixture overnight and
discarding the dark brown supernatant. The NaON digestion was repeated twice. Then
the process of extraction was continued by acid digestion using Chloride acid 3.5 L of
2.45 M, 1.75 M, and 0.94 M. Separating process conducted at each step of acid digestion
was the same as alkaline digestion. The purification of P-glucan was continued by
bleaching process using 2 L of distilled water to gain white and flocculent residue. The
process was conducted ten times by boiling and separating process.
The colorless
process was continued by adding 1.5. L of ethanol to the residue and the process was
conducted tluee times by boiling and separating process. Before filtering the particle of
P-glucan by using fine silkscreen, the washing process using 2 L of distilled water was
conducted three times by boiling and separating process. Then the particle of P-glucan
was dried by freeze-lyophilize process.
Mixing of P-glucan in feed was condxted by using albumin as a binder. The
com~nercialfeed was mixed into the mixture of P-glucan and albumin, and then the blend
of this feed was baked in an oven in the temperature of 60°C for 2 hours to get the
nloisture 5 - 8.
Isolate of lulllinous bacteria used in this experiment came from hatchery of PT.
Ce~itralpertiwiBahari. The bacteria have beconle a prob!cm in mortality of shrimp in
hatchery. Type of luminous bacteria used for challenge test and type of bacteria in
shrimp hernolymph and hepatopancreas were identified by Baumann el CII., (1984)
method, including perfonnance and biochemistry of bacteria.
The number of bacteria in shrimp hemolymph and hepatopancreas, to examine the
clearance process, was counted by using disc-counting method (Thompson et al., 1997).
One gram of shrimp hepatopancreas was mixed into 9 ml of saline solution. The blend
was spread into petridisc containing Thiosulfate Citrate Bile Salt Sucrose agar and held
12 hours before counting the bacteria. Counting bacteria in hemolymph was conducted
by the same method. The shrimp hemolymph 0.02 ml was drawn from thc ventral
abdominal artery of shrimp and then was spread into petridisc containing Thiosulfate
Citrate Bile Salt Sucrose agar.
The number of hemocyte was counted by Blaxhall and Daisley (1973) abbreviation.
Hemolymph 0.1 ml was drawn from the ventral abdominal artery of shrimp into syringe
containing 0.1 ml Trypan Blue solution. To make a homogenized fluid, the mixture was
shaken carefully. One drop of the solution was added to a hemocytometer and the
number of cell per n1m3 was counted.
n~~
of counted-cell x diluting factor x 100 x 10
Total of h e n ~ o c ~ t e / m=Number
Number of counted-block 1 mn1 x 4
The ability of phagocytosis by hemocyte was evaluated by Anderson and Siwicki
(1995) method. Hen~olympll0.1 ml was drawn from the ventral abdominal artery of
shrimp into syringe containing 0.1 ml anticoagulant (0.14 M Sodium chloride, 0.1 M
Glucose, 30 mM Trisodium citrate, 26 n1M Citric acid, and 10 inM EDTA pH 4.6).
uureu.5
Then, the hcmolymph was transferred into microtray and 0.1 ml S/c~~~lzylococc~i.s
(lo6 cfulml) was added. The mixturc was mixed and incubated for 20 minutes. Every 5
luinutes the suspension was spread on glass slide, stained with Giemsa, and then
observed under the light microscope. Phagocytic index was counted based on percentage
of phagocyte cells showing phagocytosis activity.
Phagocytic index (%)
=
Number of cell showing phagocytosis activity
I
x 100
I
Number of cell hemocyte in glass slide
The survival rate, growth, and rate of feed conversion ratio were counted by using
formula Zonneveld el al., 1991:
I
Survival Rate (%) = Number of final population
loo
Number of initial population
I
Growth = Average of final growth - Average of initial growth
Feed Conversion Ratio = Total given feed - total uneaten feed
Expcrinlental Design
Trial 1: Artificial Stress Condition and SIlrimp Immunity
The objcctive of this trial was to study the effect of oxygen concentration, water
salinity, and luminous bacteria concentration on solne parameters i.e. hemocpte count,
survival rate, and nunlber of luminous bacteria in hemolymph. The result of this trial
could be used as a fit stressor in advanced trial. .
The trial was conducted with 3 replications and used factorial design.
statistical model was factorial 2 x 2 x 2.
The
The first factor I stressor was oxygen
concentration which had levels of 3 and 6 ppm, the second factor I stressor was water
salinity which had levels of 20 and 70 ppt, and the third factor / stressor was luminous
bacteria concentration with levels of 0 and 10' cfu/ml. Statistical analysis for survival
rate data used the arcsine transformation method (Steel and Torrie, 1991).
One hundred and forty-four healthy shrimps (DOC: 50) were randomly divided
and put into 24 tanks: 6 shrimpsltank. The fattors were given in three steps; the first
stressor was given until the shrinip attained weak condition, then the secolid stressor was
given in 10 minutes (Baticados ct al., 1986), finally the third stressor was given until the
shrimp attained weak condition.
The temperature during the experimental period was maintained at 25
-
27 'C.
Oxygen concentration was made and maintained by regulating aeration. To avoid the
negative effect of feed in the environment. shrimp was not fed.
After shrimp attained weak condition, three shrimps from each tank were selected
to cxanline the number of lun~inousbacteria in shrimp hemolymph, total hemocyte, and
survival rate of shrimp.
Trial 2: P-glucan and Shrimp Performance
.Ihc
. objective of this trial was to study the effect of P-glucan on some parameters
of shrimp performance i.e. growth and feed conversion ratio. The trial was conducted
with 3 replications and used randomized coluplete design. The treatments in this trial
were the dozes of P-glucan mixed in feed: 0%, 0.1%, and 0.5%. Statistical analyze for
growth data used the analyzes of covariance method (Steel and Torrie, 1991).
Three hundred healthy shrimps (DOC: 30) were randomly divided and put into 30
tanks: 10 shrimpsltank.
Before dividing, shrimp weight was counted.
To avoid
cannibalism, approximately, the shrimp weight in each tank was similar. The trial needed
30 days and feed for treatment was given adlibitum everyday.
After 30 days, shrimp weight and uneaten feed were counted to investigate shrimp
growth and feed conversion ratio. Uneaten feed was collected every 6 hours and was
baked in an oven with temperature 60°C.
Trial 3: Administration Period of P-glucan and Shrimp Immunity
The objective of this trial was to study the effectivity of 0-glucan given in 1 and 2
months on some parameters of shrimp immune system i.e. hemocyte count, phagocytic
activity, clearance of bacteria, and survival rate at a low oxygen concentration. The
experiment was an advanced experinieilt using the worst condition resulted by trial 1.
Tlie experiment was conducted with 3 replicatioils and used factorial design. The
statistical lnodel was factorial 3 x 2 x 2. The first factor was doze of P-glucan mixed in
feed which had levels 0%, 0.1% and O.j%, the second hctor was administration period of
0-giucan which had levels I aiid 2 months, and the third factor was luminous bactcria
concentration which had levels 0 and 10' cfd~iil.Tlie number of luminous bacteria in
hernolymph and hepatopaticreas was a~ialyzedby description method. Statistical alinlyze
for phagocytic activity and survival rate data used the arcsine transformation method
(Steel and Torrie, 1991).
Tluee hundred healthy shrimps (DOC: 30) were randomly divided and put into 30
tanks: 10 shrimpltank. Before dividing, shrimp weight was counted.
To avoid
cannibalism, approximately, the shrimp weight in each tank was similar. Based on the
result of the stressor test, shrimp was kept in low water concentration (3 ppm). The water
in each tank was changed every 3 days and the temperature during the experimental
period was maintained at 25 -27
OC.
Besides, the stopping of aeration for about 6 hours
everyday, the stocking of low oxygen water, to water exchange process, was conducted to
maintain the low oxygen concentration in tank.
The feed for treatment given everyday was adlibitum. The commercial feed was
used as a continued feed given at the treatment using 1-month administration period of Pglucan.
Immersion of luminous bacteria (challenge test) was conducted when shrimp had
been kept and had been given P-glucan dozes for 30 days. The process of challenge test
in this experiment was the same as process conducted in the stressor test.
After shrimp attained weak condition caused by challenge test, three shrimps from
each tank were selected to examine the number of lulninous bacteria in hemolymph and
hepatopancreas, hemocyte count, and ~hagocyticactivity of shrimp. Then, to investigate
bacteria clearance, the nu~nberof luminous bacteria in hemolymph and hepatopancreas
was examined in the 6"', 1211',and 24"' hours after challenge test.
Continued examination of hemocyte count was conducted in the 711', 14'11, and 21''
day after challenge test. Continued examination of phagocytic activity was conducted in
the 711', 14", 21L', and 30''' day after challenge test. The survival rate of shrimp was
examined in the 30"' day after challenge test.
,
As additional examination, the water quality in each treatment, including total
ammonia nitrogen, nitrite, nitrate, and alkalinity concentration, was examined weekly. In
order to complete the data, the bacteria found in shrimp hemolymph and hepatopancreas
was identified.
RESULTS AND DISCUSSION
Results
Identification of Vibrio Bacteria in Hemolymph and Hepatopancreas
The characteristic and biovar of Vibrio from henlolymph and hepatopancreas
were reported (Appendix I). Based on the Baumann et al. (1984) method these yellow,
green, and luminous bacteria indicated the characteristic of Vibrio alginolyticus, Vibrio
parahaemolyticus, and Vibrio splendidus biovar 1.
Trial 1: Artificial Stress Condition and Shrimp Immunity
Hemocyte count of shrimp was affected by individual and interaction of the
factors in treatment (Table 1).
The significant effects (Pi0.05) on hemocyte count
occurred as the result of individual factors i.e. water salinity and Vibrio splendidus biovar
1 concentration and as the result of interaction factor involving oxygen, water salinity and
Vibrio splendidus biovar 1 concentration.
Although interaction among factors gave significant effect on hemocyte count, the
interaction between water salinity factor and ~ i b r i osplendidus biovar 1 concentration
factor gave the biggest effect. Qxygen factor only gave significant effect on hemocyte
count when it was interacted with water salinity factor and Vibrio splendidus biovar 1
conce~~tration
factor.
The highest llemocyte count occurred at oxygen concentration G ~ P I I I water
,
salinity 20 ppt, and Vibrio s1~iendidu.sbiovar I concentration 10' ciidnll.
Table 1. Mean number of the hemocyte count, su:vival rate of shrimp and number of
Vibrio splendidus biovar 1 colony (cfu) in hemolymph at 18 hours after
stressing condition.
Oxygen
@pm)
3
Salinity
@PO
20
70
Note *: Significant at P < 0.05
Vibrio
splendidus
biovar I
(~fU/llll)
Mean of parameters
Heinocyte
count
(cellln~mj)
Survival
Rate (%)
0
448.00
82.98
-
10'
526.67*
78.68
3.89
0
221.OO*
82.98
-
Numb. Of
lum. Bac. In
hemolymph
(cfidml)
As an individual factor, Vibrio splendidus biovar 1 and oxygen concentration
affected the survival rate of shrimp significantly (P < 0.05). The lowest survival rate
occurred at low oxygen concentration (3 p p n ~ ) and Vibrio splendidus biovar 1
concentration lo7 cfdml.
Significantly, the number of Vibrio splendidus biovar 1 in hemolymph was
affected by water salinity. The highest concentration of Vibrio splendidus biovar 1 in
hemolymph was found at water salinity 70 ppt.
Trial 2: P-glucan and Shrimp Performance
The result of trial 2 indicated that the growth and feed conversion ratio of shrimp
were not affected by the treatment.. All treatment had no significant effect on the
performance of shrimp (P < 0.05) (Table 2).
Trial 3: Administration Period of p-glucan and Shrimp Immunity
One of some results of trial 3 indicated that the most rapid bacteria clearance in
hemolymph occused in shrimp fed by feed containing P-glucan 0.1%, in less than 12
hours after challenge test. The clearance of bacteria in hepatopancreas for all treatment
occurred in less than 12 hours after the challenge test (Table 3).
Star~sticalanalyze for hemocyte count indicated as individual factor, P-glucan and
Vibrio sl)lei7cf1dzrsbiovar 1 concentration gave significant effect on heliiocyte count in the
7"' day afier challenge test. The highest hemecyte count occurred in levcl of P-glucan
0.5%. The level of Tfibrio sl,lendidus biovar 1 lo7 cfidml gave the same result.
In 14"' day, as individual factor, P-glucan and administration period gave
significant effect on hemocyte count. The level of P-glucan 0%, 0.1%, and 0.5% could
affect significantly, but the biggest effect was given by 0-glucan 0.5%. The same result
was given by the level of 1-month administration period.
Besides as individual factor, factor of P-glucan and administration period,
together with Vibrio splendidus biovar 1 factor affected the hemocyte count of shrimp on
21'' day. The highest hemocyte count occurred in shrimp fed by feed containing
P-
glucan 0.5% given in 1 month and it was given under no bacteria condition (Table 4).
Statistical analyze for phagocytic activity indicated that as an individual factor
P-
glucan gave significant effect on phagocytic activity in the 7"' up to 3oth day after
challenge test.
Although feed containing P-glucan gave better result in phagocytic
activity than other, both levels of P-glucan in feed (0.1% and 0.5%) had no significant
difference (Table 5).
Statistical analyze for the survival rate of sluimp indicated that all treatment save
the same effect. But, s!witnp fed by feed containing P-glucan gave survival rate higher
than shrimp fed by no P-glucan feed (Table 6).
The water quality in each treatment indicated the same result (Figure 1).
Table 2. Mean number of growth and feed conversion ratio caused P-glucan administration
Mean of parameters
Treatment
Growth
Feed Conversion Ratio
Glucan 0 %
5.00
1.19
Glucan 0.1 %
4.98
1.12
Glucan 0.5%
5.31
1.15
Table 3. Number of l/iDrio splendidus biovar 1 colony in hepatopancreas and hemolymph after challenge test
Number of Vibrio splendidzls biovar I ( c f u h l )
Treatment
Hemolyniph
Hepatopancreas
The 24"'
hour
100
50
0
150
100
0
0
225
150
50
0
The 12"'
hour
The 24"'
hour
'The 1''
hour
1500
420
0
0
300
1500
505
0
0
975
750
0
0
-
The 6"'
hour
The 12"'
hour
l'he G"'
hour
The 1
hour
Table 4. Mean number of hemocyte count at tllc 0. 7"', 14th. and 2ISLday after
7
challenge test
Treatment
B
2
0.5
Note
Meall of hemocvte count (cell/mni.')
The 0 day The 71h day The 14"' day The 21" day
7166
6966
8000
7316
7833
8216
8600
8400
C
0
1 o7
1
0
1o7
7900
8100
7550
9616
13850
11666
9183
10366
2
0
10:
7566
7000
7900
9333
11400
9166
9500
6716
1
0
1o7
8!00
8000
10916
14516
14933
14350
12700*
1 1033"*
2
0
1o7
7666
8166
10900
11233
13800
12766
11366"*
1 1066"'
1
*: Significant at P < 0.05
A: % p-glucan in feed
B: Administration period (month)
C: Vibrio splendidrr.: biovar I (cfulml)
Table 5. Mean number of phagocytic activity at the 0, 71h, 141h,21s', and 30"' day after
challenge test
Note
*:
Significant
at P < 0.05
A: % P-glucan in feed
B: Administration period (month)
C: Vibrio splendidus biovar I (cfuiml)
Table 6 . Survival rate of shrimp
B: ~dlninistrationperiod (month)
C: l'ibrio splendidus biovar I (cfulml)
i
TAN
parameter
.
~-~~~
~~~~
I
i
NlTRlT
NITRAT
ALK
!
PH
I
parameters
.
I I
I
I!
1 (I
I
--
-.-.
@CONTROL =CONTROL U G L U C . 0.1%
U G L U C . 0.1%
P G L U C . 0.1%
CIGLUC. 0.1%
B G L U C . 0.5%
O G L U C . 0.5%
I G L U C . 0.5%
H G L U C . 0.5%
..
BACTERIA
N O BACTERIA
- 1 MONTH -BACTERIA
- 1 M O N T H - N O BACTERIA
- 2 MONTHS -BACTERIA
- 2 M O N T H S - N O BACTERIA
- 1 M O N T H - BACTERIA
- 1 M O N i H - N O BACTERIA
- 2 MONTHS -BACTERIA
- 2 M O N T H S - N O BACTERIA
-...-..-.._---d..
Figure 1 . The water quality in each treatment
~~
-. .
-
~
Trial 1: Artificial Stress Condition and Shrimp Immunity
The number of hemocyte in he~nolympli indicated their ability to respond
some unadvantages condition e.g. stress condition. Anderson and Siwicki (1995)
reported that the highest heinocyte count wandering in hernolymph, .the biggest
respond could be given to hold the stress condition
111
(Anderson and Siwicki, 1995).
this trial, the stress condition caused by concentration 10' cfu/ml of Vibrio
splendidus biovar I gave the biggest effect on helnocyte count.
Besides affecting the hemocyte count, this concentration gave the worst effect
on survival rate of shrimp.
Besides the highest concentration of Vibrio splendidus biovar I , the worst
effect on survival rate of shrimp was caused by low oxygen concentration (3 ppm).
Direkbusarakom and Danayadol (1998) reported that one of ilnportant states of the
environlnent was the presence of sufficient oxygen to maintain a system vital to the
health of an aquatic ecosystein. and the critical concentration for shrinip ponds was
3.7 pp111.
Salinity 70 ppt gave effect on the introduction of bacteria in shrimp body. It
occurred easily when the oxygen concentration was low enough and especially wlieti
the carapace of shrimp was soft. The low oxygen concentration increased the shrimp
metabolism, thus to fulfill the osygen consumption, the shrimp lnlust more active to
take oxygen in water. At tlie same time, high concentration of bacteria in water
would enter tlie shrimp body. Balicados el crl. (1986) observed that a hypet osmotic
condition could be caused by high salinity leading to a chronic soli-shell syndroine
that could arkct tlie defence meclianisni of'sliriiiil~.
All results in trial 1 indicated that shrimp im~nunitycould be triggered by a
stress condition involving stressor factors such as low oxygen concentration (3 ppm),
IGrio .sp/endiidr(s biovar I with concentralion 20' cfu/inl, and high salinity (70 ppi).
Therefore, the interaction of these factors could bc a challenge test for the other trial.
The challenge test was a test to give a challenge to investigate the animal capability
after given a treatment.
Trial 3: P-glucan and Sl~rirnpPerformance
Feed containing P-glucan had no effect on the performance of shrimp i.e. the
shrimp growth and the feed conversion ratio. It indicated that no additional nutritious
materials affecting the growth cells of shrimp body. It also indicated that adding
P-
gluca~idid not change the shrimp respond to the feed.
A proximate analysis conducted to analyze the contain of protein, lipid, ash,
fibber? and nitrogen free materials indicated that all feed had no different level at their
nutrient content (Table 7).
Trial 3: Administration Period of P-glucan and Shrimp Imniunity
Martin and arenda (1985) reported that gram positive and ~iegativebacteria in
hemolymph could be cleared from hemalymph rapidly by gills in circulation process.
In the first time, bacteria entered the shrimp body and before it was distributed to
hemolymph the bacteria would stay in hepatopancreas a li~tlewhile. In this trial,
shrimp fed by P-glucan O,1% cleared Vibrio sp1ri~dicili.s biovav 1 i n their
hepatopancreas and Iiemolymph more quickly than the otiisrs (Table 3). It indicated
that p-glucan O. I % \\,as a good bacleria-clearable substance.
'[.he infection caused by Vibrio .s,~ie~ldicili.r
I~iovar I co~itiii~ted
to the 7"' day
after challenge test. Increasing of hemoc!.te couiii and phagocytic activity at thc 7"'
day after challenge test \\,as the effect of P-glucan and bacteria factor individually
(Table 4 and 5 ) .
Anderson and Siwicki (1995) reported that the increasing of
heniocyie count in hemoly~npliwas caused by infection, stress. and blood disease.
The infection caused an inflammation; a non-specitic characteristic triggered by some
factors e.g. parasite and bacteria (Person et al., 1987; Itami et a/.,1996).
The tendency of increasing and decreasing of hemocyte count and phagocytic
activity was similar (Figure 2). It indicated that the biggest composition of hemocyte
was hyaline cells i.e. phagocytic cells (Soderhall and Johansson, 1989).
Figure 2. The relationship between phagocytic activity and hemocyte count
FA: Pliagocytic activity, HC: He~nocytecount, -0, -7, -14, -21, -30: the 0: 7'h, 1411',
21s', and 30"' day after challenge test, A: p-glucan 0% + 7 months + 0 cfulml K
splendidzrs biovar 1, B : p-glucan 0% + 2 months + lo7 cfulml V. splendidzrs biovar 1,
C: P-glucan 0.1% + 1 months + 0 cfulinl V. splendidzrs biovar 1, D: P-glucan 0.1% +
I months + lo7 cfulml T'. splendidus biovar 1, E: P-glucan 0.1 % + 2 lnonrhs + 0
cfi~lml V. splendidzrs biovar 1, F: P-gluca~i 0.1% + 2 months i- lo7 cfulml V.
splendidzts biovar 1, G : P-glucan 0.5% + 1 months + 0 cfulml V. sp1endidz1.r biovar I,
H : P-glucan 0.5% + 1 months + 10' cfulml V. sp1endid;rs biovar 1, 1 : P-glucan 0.5%
+ 2 ~iionths+ 0 cful1111I/. splendidus biovar I, .I : P-glucan 0.5% + 2 monihs + lo7
cfuiml I/ sl~ie!7did1i.s
biovar 1.
31
Table 7. The nutrient content (% wet basis) of the trial diets.
Feed containing
glucan 0%
Crude Protein
1
Crude Lipid
Crude Fibber
Moisture
Nitrogen-Free
Materials
P-
Feed containing Pylucan 0.1%
Feed containing Pglt~can0.5%
The phagocytic cells could phagocyte a peculiar substance or bacteria that
entered the host.
The process of phagocytosis involved some mechanism of
attachment. engulfing, degranulation, and lyscsbacleria (Gudkovs, 1988). T h e cells
which running phagocytic activity could be observed by light niicroscope.
The
process could be differed with the accuinulation of bacteria on the cells by observing
the characteristic of color absorption.
Although the highest hemocyte count was found in shrimp fed by 6-glucan
O.j%, the phagocytic activity in shrimp fed by P-glucan 0.5% had no a significant
difference with the phagocytic activity in shrimp fed by P-glucan 0.1% (Table 4 and
5). It indicated that the composition of hyaline cells in hemocyte of shrimp fed by
P-
glucan 0.1% was higher than in shrimp fed by P-glucan 0.5%. Thus, shrimp fed by
P-
glucan 0.1% was more resistant than the others because of their ability in phagocytic
activity.
The p-glucan in feed could be digested by P-I, 3-glucanase enzymes in
crustacean digestion system (Dall and Moriarty, 1983) before it \\,as distributed to
he~nolymphsystem. Recognition protein in hemocyte plasnia could recognize the
characteristic of P-glucan and lipopolysaccharide (Vargas-Albores e1 al., 1998).
Then, P-glucan activated prophenoloxidase enzyme in he~nocyteto plienoloxydase.
The activated plienoloxydase triggered the coagulation system in order t o close the
l v o ~ ~ n dThe
. process to activate phenoloxydase caused releasing of
a free
(radical material that could trigger the step of degranulation in phagocytosis caused the
clcal-ance of bacteria.
Figure 2 showed that the increasing of phagocytic activity was caused by
adding P-glucan in feed. Fecd containing P-glucan could increase the phagocytic
activity higher than commercial feed that had no P-glucan inside.
All treatnlenrs did not give significant difference on the survival rate of
shrimp. However, the survival rate of shrimp fed by P-glucan was higher than the
other (Table 6).
Galeotti (1998) reported that prolonged im~nunostiniulantadministration, in
doze and time, did not give a better effect. In this trial, (3-glucan adtiiinistration in 2
months did not give a better result than the administration in 1 month, because of the
effective time.
CONCLUSSJON
The general conclusion of all trial was the low oxygen and high bacteria
concentration gave a bad effect on shrimp defence mechanism.
The high water
salinity affected the entering of bacteria in shrimp body.
P-glucan extracted from Saccharon~ycescerevisiae by alkali-acid method
(William el al., 1991) could increase some parameters of the non-specific immune
syste~iiof shrimp that was kept in low oxygen concentration (3 ppm), but they had no
effect on the performance of shrimp, because the trial was conducted in short time to
give evidence of their effect on the performance of shrimp. Feed containing P-glucan
0.1% gave a better effect than P-glucan 0.5% in their role to increase the non-specific
immune system of shrimp by increasing phagocytic activity.
The administration of P-glucan in 2 months did not give a better result than the
ad~ninistrationof P-glucan in I month. Thus. the best administration period of
glucan for 30 days old culture of shrimp was I month.
P-
Andcrson, D.P. 1992. Iinmunostimulants. Adjuvant, and Vaccine Carriers in
Fish: Applications to Aquaculture. .Ann. Rev. Fish Dis.. 2: 281307.
and Siwicki. 1995. Basic Hematology and Serology for Fish
Health Programs, p. 185-202. Inshariff, M.. R.P. Subasinghe and
J. Arthur (ed.).
Diseases in Aquaculture 11. Fish Healrh Secr.
Asian Fish Soc., Manila.
Baticados,