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 immuno- stimulant 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
Efficacy of using Guava leaves ( P. Guajava ) as nonspecific immune stimulant in O. niloticus
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.,
Gehan I. A. Shager (2017)
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
Efficacy of using Guava leaves ( P. Guajava ) as nonspecific immune stimulant in O. niloticus
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
Gehan I. A. Shager (2017)
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
Efficacy of using Guava leaves ( P. Guajava ) as nonspecific immune stimulant in O. niloticus
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
Gehan I. A. Shager (2017)
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
Efficacy of using Guava leaves ( P. Guajava ) as nonspecific immune stimulant in O. niloticus
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
Gehan I. A. Shager (2017)
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
Efficacy of using Guava leaves ( P. Guajava ) as nonspecific immune stimulant in O. niloticus
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
Gehan I. A. Shager (2017)
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
Efficacy of using Guava leaves ( P. Guajava ) as nonspecific immune stimulant in O. niloticus
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
Egy. J. Aquac., Vol.7, No. (2):33- 53 (2017) ISSN: 2090-7877
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
Egy. J. Aquac., Vol.7, No. (2):33- 53 (2017) ISSN: 2090-7877
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
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