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Egy. J. Aquac., Vol.7, No. (2):33- 53 (2017) ISSN: 2090-7877

33

Efficacy of using Guava leaves ( Psidium Guajava ) as

nonspecific immune stimulant in Nile tilapia (Oreochromis

niloticus)

Gehan I.A.Shager

Fish Health and Diseases Depat; Central Laboratory for Aquaculture

Research, Abbassa, Agriculture Research Center, Ministry of Agriculture,

Egypt.

ABSTRACT

The aim of this study was to evaluate the effects of different

doses { 0, 0.5, 1 and 1.5 gm of Guava(Psidium Guajava) leaves

powder as immunostimulant on immune response of

Oreochromis niloticus and investigate antioxidant activity of

Guava in the blood of fish on 30 days. In this study, Oreochromis

niloticus were obtained from Central Laboratory for Aquaculture

Research (CLAR), Abbassa, Abu-Hammad, Sharkia, Egypt. Fish

were transported to a laboratory then the health status of the

experimental fish was inspected (with average body weight 52 ±

2 g / fish). All immune parameter ( lysozyme, NBT and nitric

oxide) were higher on 30 days of feeding of 1.0 gm of guava

powder.

In this experiment serum total protein and globulin showed

significant increase in the 4th week at groups feed 1% guava

powder when compared to control group Significant decreased of

serum albumin and A/G ratio was observed in all treated groups

and highest decrease were observed at 4th

week in 1%, compared

to control. All treatment showed significant decrease of creatinine

while first and second week show non-significant increase of urea

at 0.5 and 1.5% with significant decrease at 1% Guava

supplemented diet while fourth week revealed significant

increase at 0.5 and 1.5% with non-significant increase at 1%

compared with control.

Generally we can say that antioxidant enzymes (GST, SOD

& CAT) and MDA showed significant decrease than control

group.

All groups were challenged intraperitoneally injection of 0.2

(1× 108 cells / ml / fish) of 24 hours broth culture of virulent

Aeromonas hydrophila after four weeks of feeding experiment

and mortality rate percent were recorded.

Efficacy of using Guava leaves ( P. Guajava ) as nonspecific immune stimulant in O. niloticus

34

INTRODUCTION

Aquaculture fish production has increased drastically over the past few

decades to meet the increasing demand for animal protein food. However,

the highly demand for Nile tilapia, Oreochromis niloticus (O. niloticus) in

our Egyptian market was stimulating the development of intensive tilapia

culture due to its desirable characteristics for aquaculture such as rapid

growth, tolerance of wide range of water quality parameters, disease

resistance, good taste and high market value (Barcellos et.al., 1999).

Improper fish culture practices and the environmental conditions affect

the fish health, in turn resulting in production losses and infectious

disease.

In aquaculture, infectious diseases are mainly controlled by

chemotherapeutics and antibiotics. However, the use of antibiotics and

chemotherapy has recently been criticized because their usage has created

problems with drug-resistant bacteria, toxicity and accumulation both in

fish and environment. On the contrary, natural products such as plant

extracts might have beneficial effects while cause no problems (Citarasu

et.al., 2002 and Sagdic & Ozcan, 2003). Prevention of disease or

immuno stimulation of susceptible fish stock is much desirable. Hence,

immunostimulants are being concentrated more upon for disease control

measures. The use of natural immunostimulants seems to be the most

promising method of preventing fish diseases. Immunostimulants

enhances the innate immune systems, thus preventing infectious diseases

(Watanuki et.al., 2006). Several immunostimulants have been tested in

fish as some natural plants were rich sourcesof compounds including

volatile oils, saponins, phenolic compounds, tannins, alkaloids,

polypeptides and polysaccharides. These natural plant products were

reported to have such various activities as antistress, appetizer,

antimicrobials and immunostimulants (Citarasu et. al., 2002; Citarasu

et.al., 2003 and Kumar et.al., 2012).

Herbs and herbal products are incorporated in livestock feeds instead

of chemical products and antibiotics in order to stimulate the

effectiveness of feed nutrients which result in more rapid gain, higher

production and better feed efficiency. Herbs and the logically active

substances content stimulate body metabolism, improve digestion, have

bactericidal, immuno stimulant action and improved productivity of

poultry (Sabra and Mehta, 1990). Practically, reports have shown that

supplementing fish diets with various herbs have favorable effects on the

performance and health of reared birds (El–Gendi, 1996).

Gehan I. A. Shager (2017)

35

P. guajava or guava is a plant in the family Myrtaceae along with

clove, allspice and eucalyptus. Native totropical America, it is now

cultivated in many tropical and subtropical countries for its edible fruit

(Perez et.al., 2008). It has been used as an ingredient in many foodrecipes

and desserts. The leaves of guava are rich in in tannins, phenols,

triterpenes, flavonoids, essential oils, saponins, carotenoids, lectins,

vitamins, fiber and fatty acids. Much of guava's therapeutic activity is

attributed to these flavonoids in particular quercetin, saponins, tannins,

alkaloids anthraquinones, phlobatannins and cardiac glycosides.

Moreover, theses flavonoids had antibacterial activity. High amounts of

phenolic compounds in the leaves of Psidiumguajava showed antioxidant

activity (Haida et.al., 2011).Psidiumguajava is very rich in antioxidants

and vitamins as guava fruit is higher in vitamin C than citrus (80 mg of

vitamin C in 100 g of fruit) and contains appreciable amounts of vitamin

A and also high in lutein, zeaxanthine and lycopene (Hobert and Tietze,

1998).

The objective of the present study was to investigate the effect of dried

guava leaves supplementation on diet of Oreochromis niloticuson

immune response, antioxidant activity and biochemical parameters.

Material and Methods

Plant material and sample preparation:

Fresh leaves were purchased from herb shop at local market and the

identification was done according to (Arabshahi-Delouee&Urooj,

2007). Leaves were cleaned and dried at 70°C for 3 days and then ground

well. Finally dried herbal powders were stored at 4°C until use.

Feed formulation and diets:

The experimental diets were prepared by incorporating dried guava

leaves to the feeds, containing 0.5, 1 and 1.5 gm. powder of the guava.

Control diet was prepared using the same composition of ingredients,

except the powder. To prepare the diets, dry ingredients were first mixed

thoroughly and 1% binder was added. Sufficient water along with the

powder ingredients was then added to make a paste of each diet. The

paste was cold extruded and pelletized using a hand pelletizer. Finally, the

diets were dried and were sealed in plastic bags and stored at 4°C (Eloff,

1998 and Citarasu et.al., 2006) until fed.

Fish and experimental design:

Oreochromis niloticus were obtained from Central Laboratory for

Aquaculture Research (CLAR), Abbassa, Abu-Hammad, Sharkia, Egypt.

Fish were transported to a laboratory then the health status of the


36

experimental fish was inspected and both the fish and tank water were

disinfected. Before the initiation of the feeding trial, fish were acclimated

to experimental conditions in 600-L containers with recirculated water for

2 weeks by feeding the basal experimental diet at 3% body weight twice

daily without supplementation of the plant extracts. After the acclimation

period, the fish (mean initial weight of (52 ± 2gm) were randomly

distributed in 12 experimental aquaria (120 L) each stocked with 15 fish.

The aquaria were daily cleaned by siphoning out the fish faces and

uneaten food debris. Four treatments (A = 0, B = 0.5, C = 1 and D =

1.5gm powder kg _1 feed) were applied to each aquaria to evaluate the

effects of dried guava powder on the Oreochromis niloticus. Each

treatment was given in triplicate aquaria. The fish were fed twice a day at

9:30 and 16:30 hours at 3% of the body weight until the end of the

experiment. During the experimental period, the water quality parameters

were monitored every day and maintained at optimal levels by regular

water exchange (temperature, 24.5°C; dissolved oxygen, 7.2mg L _1;

salinity, 0.32 ppt; pH, 6.44 units; ammonia-nitrogen <0.21 mg L _1).

Immune analysis:

Lysozyme was estimated based on the turbidity measurements

according to Schaperclaus et.al., (1992). However, 10 µl of serum were

added in the cuvettes to 200 µl of Micrococcus suspension (35 mg of

Micrococcus dry powder/95 ml of 1/15 M phosphate buffer + 5.0 ml of

NaCl solution). The change in the extinction was measured at 546 nm, by

measuring the extinction immediately after adding the solution which

contained the lysozyme (start of reaction) and after a 20 minute

incubation of the preparation under investigation at 40˚C (end of

reaction). The lysozyme content is determined based on the calibration

curve and the extinction measured Respiratory burst (Rb) was assayed

according to the method of Siwicki (1989). Nitric oxide was assayed

spectrophotometrically (5010, Photometer, BM Co. Germany) according

to the method of Montgomery and Dymock (1961).

Physiological analysis:

Weekly during feeding trial, three fish from each aquarium were taken

for physiological investigation. Fish were an anesthetized using buffer

tricaine methane sulfonate (20mg/L), and blood was collected from

the caudal vein with a sterile syringe and divided equally among

three clean and dry tubes. The first part was centrifuged at 3,000 g

for 15 min and the serum was stored at −20◦C for further assays.

Total protein content was determined color metrically according to

Henry, (1964), albumin was measured according to Doumas et.al.,


37

(1971); globulin and A/G ratio determined according to Henry (1964).

Urea was determined according to Patton and Crouch (1977).

Creatinine was determined calorimetrically according to Henry (1974)

Antioxidant activity:

Liver samples were collected from the euthanized fish for antioxidant

enzymes assay. The liver tissue was homogenized in 9 volumes of 20 mM

phosphate buffer (pH 7.4) containing ethylene diamine tetra acetic acid

(EDTA) and 0.1% Triton X-100. The homogenates were centrifuged at

600 ×g for 10 minutes and the supernatants were collected in clean

Epindroff tube for antioxidant enzymes assay. Superoxide dismutase

activity (SOD) was measured according to (Kakkar et.al., 1984), MDA,

Catalase activity (CAT) was measured according to (luck, 1974) and

Glutathione-peroxides activity (Habig et.al., 1974).

Experimental challenge:

On day 30 after feeding, 30 fish from each treatment were injected

intraperitoneally (Schaperclaus et.al., 1992) with 100 lL of live A.

hydrophila (1 × 108 cells / ml / fish) (Harikrishnan et.al., 2003).

Mortality of the challenged fish was monitored up to 10 days.

Statistical analysis:

All experimental data were analysed using one way analysis of

variance (ANOVA) using the soft ware SPSS version 17 (SPSS Inc.,

Chicago, IL, USA). Differences between means were determined and

compared using Duncan’s test at a significance level of 5%.

RESULTS AND DISCUSSION

Effect of Guava on immunity of Nile tilapia:

All immune parameter {lysozyme, NBT(nitroblue tetrazolium) and

nitric oxide} results revealed that Guava leaves powder enhance the

immune response of fish from 1st week till 4

th week compared to normal

control group. It was noticed that immune response of fish increase at 4th

week in groups feed 1% guava powder (Fig.1, 2 & 3). Lysozyme activity

is an important index of innate immune response of fish. It is well

documented that fish lysozyme possess lytic activity against bacteria. It is

also known to be opsonic in nature and activates the complement system

and phagocytes. Lysozyme is an important defence molecule of the non-

specific immunity and plays a significant role in mediating protection

against bacterial invasion (Saurabh & Sahoo 2008) also it was found that

significant increase in serum lysozyme activity in all groups fed leaves

extracts of Achyranthes aspera compared with the control group. This

may be due to the ability of the leaves extracts to modulate lysozyme


38

activity of the fish also It was reported that it increase plasma lysozyme

activity level in Nile tilapia fed Chinese medicinal herbs (Astragalus

membranaceus and Lonicera japonica) alone or in combination after first

7 days of feeding (Ardo et.al., 2008). Also it was notiged that increase of

lysozyme in shrimp serum and hepatopancrease fed with Guava leave

than control ( Yin et.al., 2014). It was reported that dietary administration

of guava increase NBT and nitric oxide at group feed 1%guava so NBT

& NO assay were mostly used to measure the oxidative radical production

by leukocytes in the defense against pathogens (Cook et al., 2003; Sahoo

et.al., 2005). Verlhac and Gabaudan (1994) showed that respiratory

burst activity of salmonid stimulated were significantly enhanced by

vitamin C treatment. (Lin and Shiau, 2005). A significantly higher

respiratory burst activity was observed in the group fed leaf (P. guajava

and M. indica) extracts, which suggest that the extracts enhanced the

formation of reactive oxygen species to fight against the bacteria

pathogen and stimulate the non specific immune function compared with

the control. In this study, the respiratory burst activity of phagocytes was

quantified by the NBT assay, which measures the quantity of intracellular

superoxide radicals produced by leucocytes (Siwicki & Studnicka,

2006). Giri et.al., ( 2015) showed evaluatation of the effects of Psidium

guajava (guava) leaves on the growth and immune response of the fish

species Labeorohita.

Fig. (1): Effect of different treatments of Guava leaves powder on Lysozyme values

0

1

2

3

4

Control Guava0.5%

Guava 1% Guava1.5%

Lysozyme

1st week

2nd week

4th week

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39

Fig. (2): Effect of different treatments of Guava leaves powder on NBT values

Fig. (3): Effect of different treatments of Guava leaves powder on Nitric oxid

Effect of Guava on blood parameter of experimental fish:

In this experiment serum total protein and globulin showed significant

increase in the 4th week at groups feed 1% guava powder when compared

to control group (Fig. 4&5). Significant decreased of serum albumin and

A/G ratio was observed in all treated groups and highest decrease were

observed at 4th

week in 1%, compared to control (Fig. 6&7).The total

protein in the serum can be divided into two groups, albumin and

globulin. They are the major proteins, which plays an important role in

the immune response of fish (Kumar et.al., 2007). A rise in the total

protein, albumin and globulin levels is thought to be associated with a

0

2

4

6

8

10

12

Control Guava0.5%

Guava 1% Guava1.5%

NBT

1st week

2nd week

4th week

0

0.05

0.1

0.15

0.2

0.25

0.3

Control Guava0.5%

Guava 1% Guava1.5%

Nitric oxid

1st week

2nd week

4th week


40

stronger non-specific immune response in fish (Wiegertjes et.al., 1996).

The leaf extracts used showed significantly enhanced serum total protein,

albumin and globulin contents in the treatment groups compared with the

control. This is consistent with the findings of Kumar et.al., (2013),

Basha et.al., (2013) and Sahu et.al., (2007) in L. rohita Similarly, a

significant increase in total protein, albumin and globulin was recorded in

C. carpio fed diets containing 0.5% and 1% Chinese herbal medicine

(Yuan et.al., 2007). However, a significantly lower albumin-globulin

(A/G) ratio observed indicates the presence of more amounts of globulin

in the treatment groups compared with control. All treatment showed

significant decrease of creatinine while first and second week show non

significant increase of urea at 0.5 and 1.5% with significant decrease at

1% Guava supplemented diet while fourth week revealed significant

increase at 0.5 and 1.5% with non significant increase at 1% compared

with control (Fig. 8&9). There's no significant (P < 0.05) difference in

urea and creatinine among all the treatments (Asmaa Abd El-Naby,

2014). Cuvelier et.al., (1994) showed total lipid, urea, creatinine, AST

and ALT were significantly decreased by increasing levels of S. officinal.

Sage has been observed to have excellent properties in inhibiting lipid

peroxidation and this activity is attributed principally to the presence of

phenolic compound such as carnosic acid, carnosol and rosmarinic acid.

.

Fig.(4): Effect of different treatments of Guava leaves powder on Total protein

0

2

4

6

8

Control Guava0.5%

Guava 1% Guava1.5%

Total protein

1st week

2nd week

4th week


41

Fig.(5): Effect of different treatments of Guava leaves powder on Globulin

Fig.(6): Effect of different treatments of Guava leaves powder on Albumin

0

0.5

1

1.5

2

2.5

Control Guava 0.5% Guava 1% Guava 1.5%

Globulin

0

0.5

1

1.5

2

2.5

3

3.5

4

Control Guava 0.5% Guava 1% Guava 1.5%

Albumin


42

Fig. (7): Effect of different treatments of Guava leaves powder on A/G ratio

Fig.(8): Effect of different treatments of Guava leaves powder on Creatinine

0

1

2

3

Control Guava0.5%

Guava 1% Guava1.5%

A/G ratio

0

0.1

0.2

0.3

0.4

0.5

0.6

Control Guava0.5%

Guava1%

Guava1.5%

Creatinine

1st week

2nd week

4th week


43

Fig.(9): Effect of different treatments of Guava leaves powder on Urea

Effect of Guava on antioxidant enzymes:

At the feeding trials treatment 0.5% showed significant decrease GST

at all period also 1 and 1.5% at 1st and 2

nd weeks while 1 and 1.5% at 4

th

week showed non-significant decrease (Fig.10). SOD all treatments

showed significant decrease except 1% at 1st week and 1.5% at 4

th week

non-significant decrease (Fig.11) and catalase activity showed significant

decrease in all treatment except 1% at 1st week (Fig.12) while all

treatments of MDA revealed significant decrease compared to control

(Fig.13). Generally we can say that antioxidant enzymes (GST, SOD &

CAT) and MDA showed significant decrease than control.

Superoxide dismutase is a class of enzymes that catalase the

dismutation of superoxide into hydrogen peroxide (H2O2), and catalase

convert the H2O2 to oxygen and water. They are an important antioxidant

defense system in nearly all cells and offers a protection against the

damaging effects of free radicals induced by oxidative stress. This

oxidative stress can alter the antioxidant defense mechanism when fish

are exposed to unfavorable condition like high or low temperature,

toxicant, pathogen, etc., which may lead to cellular disruption, DNA

damage, liver impairment and or immune suppression (Martınez-

Alvarez et.al., 2005; Patra et.al., 2008). However, antioxidant

containing compound can quench this harmful effect of free radicals by

giving out their own electrons to stop the chain of reaction. Some studies

had demonstrated that the antioxidant enzymes can serve as stress and

immune–response biomarkers, quantified by the enzyme activity to

evaluate the health of animal including fish (Sagstad et.al., 2007; Tovar-

0

5

10

15

20

25

Control Guava0.5%

Guava1%

Guava1.5%

Urea

1st week

2nd week

4th week


44

Ramıorez et.al., 2010; Tancredo et.al., 2015). They are also said to

scavenge free radicals generated by external stimuli such as pathogen,

toxicant, etc. (Johnson 2002). the lower antioxidant enzymes activities

recorded in the treatment groups is an indication of the leaf extracts

capability to scavenge or neutralize the free radicals generated due to

oxidative stress induced by pathogenic organism. This scavenging effects

can be attributed to the phenolics, a secondary metabolite present in the

leaf extracts which are powerful antioxidant agent (Liu et.al., 2008;

Fawole et.al., 2013). Similar observation was made by Mamdouh and

Abdel-Raheim (2003), who observed a significant reduction in SOD

activities of rats fed garlic oil. MDA revealed that best result in

concentration 1% of livelong attempt, also (5th

and 6th

) A better than B

trials. The accumulation of MDA in tissues or biological fluids is

indicative of the extent of free radical generation, oxidative stress and

tissue damage(Gutteridge, 1995). Increase in MDA concentrations have

been related to the amount of stress and well correlated with lipid

membrane damage and deterioration of membrane integrity (Ekmekci &

Terzioglu, 2005).

Fig.(10): Effect of different treatments of Guava leaves powder on GST ng/ml

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Control Guava0.5%

Guava1%

Guava1.5%

GST ng/ml

1st week

2nd week

4th week


45

Fig.(11): Effect of different treatments of Guava leaves powder on SOD µ/L

Fig.(12): Effect of different treatments of Guava leaves powder on CAT ng/ml

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Control Guava0.5%

Guava1%

Guava1.5%

SOD µ/L

1st week

2nd week

4th week

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Control Guava0.5%

Guava1%

Guava1.5%

CAT ng/ml

1st week

2nd week

4th week


46

Fig.(13): Effect of different treatments of Guava leaves powder on MDA nmol/l

Bacterial challenge test:

Fish mortalities due to the experimentally infection with A. hydrophila

in fish fed with basal diet,0, 0.5, 1 and 1.5% guava powder, it was 70,

30, 20 and 30 % respectively during 14 dayes after inoculation (table 1).

Fawole et.al., (2016) and Sahu et.al., (2007) showed after challenges

with A. hydrophila, the highest percentage survival was recorded in the

group fed mango followed by guava leaf extracts and least in the control

group. This is due to the enhancement of the nonspecific immunity of the

fish by the leaf extracts, reveal that the supplementation of the leaf

extracts had a positive influence on the survival of L. rohita fingerlings by

resisting the A. hydrophila infection. Similar results were also reported

after feeding L. rohita fingerlings with M. indica kernel and challenged

with A. hydrophila. Basha et.al., (2013) reported that L. rohita fed dietary

andrographolide (EC 50%) showed significantly enhanced resistance

against A. hydrophila infection. Similarly, Pachanawan et.al., (2008)

reported an increased survival in Oreochromis niloticus fed P. guajava

after experimentally infected with A. hydrophila.

CONCLUSION

In this study, the guava (P. guajava) leaves powder enhance the non-

specific immunity and increase resistance of Oreochromis niloticus

against Aermonous hydrophila infection.

0

0.05

0.1

0.15

0.2

0.25

0.3

Control Guava0.5%

Guava1%

Guava1.5%

MDA nmol/l

1st week

2nd week

4th week


47

Table(1): Mortality rate in treated Oreochromis niloticus due to challenge with Aermonous hydrophila

Groups N

o.

of

incu

late

d f

ish

1st D

ay

2n

d D

ay

3th

Day

4th

Day

5th

Day

6th

Day

7th

Da

y

8th

Day

9th

Day

10

th D

ay

11

th D

ay

12

th D

ay

13

th D

ay

14

th D

ay

No

. M

ort

ali

ty f

ish

%of

Mort

ali

ty r

ate

Control 30 3 8 7 1 2 - - - - - - - - - 21 70

0.5% 30 3 3 1 - 1 1 - - - - - - - - 9 30

1% 30 - 1 2 2 1 - - - - - - - - - 6 20

1.5% 30 2 2 3 - 2 - - - - - - - - - 9 30


48

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الجوافة على اإلستجابة المناعية غير المتخصصة فى أوراقكفاءة إستخدام

أسماك البلطى النيلى

يهان إبراهيم عبد البر شجرچ

-أبو حماد -األسماك المعمل المركزى لبحوث الثروة السمكية بالعباسةقسم صحة وأمراض

جمهورية مصر العربية-مركز البحوث الزراعية -شرقية

الملخص العربى

من }جرام 1.5و 1، 0.5، 0الهدف من هذا العمل هو دراسة آثار الجرعات المختلفة }

النشاط المضاد من ومحاولة للتحقيق ق الجوافة لإلستدالل على األداء المناعياورأمسحوق

يوما. 30 لمدةلألكسدة من الجوافة في دم أسماك البلطى النيلى

الثروة تم الحصول على أسماك البلطى النيلى من أحواض مزرعة المعمل المركزي لبحوث

فحص الحالة الصحية لألسماك مع مالحظة وتم نقل األسماك إلى المعمل ،السمكية بالعباسة

جم / سمكة(. 2± 52) السمكة جسموزن متوسط وزنها وكان تسجيل و

وذلك أثبتت الدراسة أن إضافة مسحوق أوراق الجوافة له تأثير إيجابى على رفع المناعة

أعلى زيادة في األسبوع وكان وأكسيد النيتريك(، واألكسجين النشط )الليزوزيم، من خالل قياس

٪.1المجموعة التى تم تغذيتها بمسحوق الجوافة الرابع حيث سجلت مع

أظهرت التجربة زيادة معنوية فى نسبة البروتين الكلي والجلوبيولين في الدم وذلك في

٪ 1األسبوع الرابع من تغذية المجموعات بالعلف المضاف إليه مسحوق أوراق الجوافة بنسبة

معنوي في نسبة الزالل في الدم ونسبة ، ولوحظ إنخفاض ضابطةال عند مقارنتها بمجموعة

التى تم تغذيتها بالعلف في جميع المجموعات المعاملة فى األسبوع الرابع الزالل / الجلوبيولين

، وأظهرت جميع بالمجموعة الضابطةمقارنة ٪، 1المضاف إليه مسحوق أوراق الجوافة بنسبة

ول والثاني ، وأظهرت زيادة معنوية األ اضا معنويا في الكرياتينين في األسبوعيننخفالمعامالت إ

.بالمجموعة الضابطة٪ مقارنة 1نخفاض معنوي عند مع إ٪ 1.5و 0.5في اليوريا عند

)جلوتاسيون ترانس فيراز، وسوبر أوكسيد لوحظ إنخفاض معنوى لإلنزيمات المضادة لألكسدة

.المجموعة الضابطةعن ديسميوتاز، وكاتاالز( ومالوناندهايد

اإلصطناعيه بالحقن البروتونى لألسماك التى تم تغذيتها باألعالف المعالجة ريت العدوى وقد أج

يدروفيال الممرضة مع يروموناس هاإل بمسحوق أوراق الجوافة لمدة أربعة أسابيع بميكروب

في المئة. نفوقمعدل اليوما بعد الحقن مع تسجيل 14إبقائها تحت المالحظة لمدة

efficacy of using guava leaves ( psidium guajava ) as ...2)/3efficacy_of_using_guava_leaves... ·...

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