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J. Vet. Sci., Nov. (1), 1, 806-814 (2014)
Incorporation of Moringa oleifera leaf in Nile tilapia Oreochromis
niloticus diet and its effect on growth performance
and immune status
Ahmed, H. Sherif 1, Adel, M. El-Gamal 2 and Adel, E. Tolan 3
Agriculture Research Center
1Animal Health Research Institute, Kafr El-Sheikh branch, Fish diseases Dept., 2 Animal
Health Research Institute, Kafr El-Sheikh branch, Bacteriology Dept. and 3 central laboratory
for aquaculture research
ABSTRACT
This work was performed to evaluate Moringa oleifra (M. oleifera) leaf mealas
non-conventional protein source, growth promoter and immune enhancer for
Oreochromis niloticus (O. niloticus) fish. Six isocaloric 4320 Kcal/Kg and
isonitroginic diets 30% crud protein were formulated containing different levels of M.
oleifera leaf meal (0, 5%, 10%, 15%, 20% and 25%) and offered to fish for 6 week.
Growth parameters were estimated and the best substitution level was 20% and 25%
as they recorded significantly high Average daily gain ADG and feed conversion ratio
FCR. Blood analyses revealed that higher substitution level 40% and 50% had
adverse effect on immune status as they recoded the lowest RBCs and WBCs count
also, they had the lowest serum total protein and globulin indicating impacted
immune status. Liver enzyme AST and ALT were significantly raised with increased
substitution levels. So, obtained results recommended that addition level of M.
oleifera leaf meal must not exceed 15%.
Key word: Moringa oleifera, Oreochromis niloticus, growth performance and
immunity.
INTRODUCTION
Nile tilapia, O. niloticus is surface-feeding omnivore fish belong to the family
Cichlidae. They are the most popular fish for culture in Egypt. M. oleifera leaf (Family:
Moringaceae) is cultivated across the tropics and subtropics as the tree is native to
northeastern India (Jahn, 1986).The moringa (M. oleifera) leaf is a fast growing plant
widely available in tropics and subtropics with several economic-important industrial
and medicinal uses, as well as a native food in Southeast Asia. There is a need for new
and reliable protein for aquaculture. Plant proteins are cheap and readily available, but
have some antinutritional factors that limit their use as aquaculture feeds. Leaf extracts
show antioxidant and hypocholesterolaemic activities (Chumark et al., 2008). M.
oleifera leaf is a promising protein source for inclusion in fish diets at low levels
(Chiseva, 2006). The moringa plant (Moringa oleifera) has been the object of much
research due to its multiple uses and well-known bactericidal potential (Caceres et al.,
1991 and Suarez et al., 2005). It is rich in nutrients and, apart from a range of industrial
and medicinal applications, is used to purify water for human consumption. Not
surprisingly, as explained by (Makkar and Becker, 1997), the moringa is of economic
importance in the production of several commodities, such as oils, foods, condiments
and medicines. So, this work was performed to evaluate the corporation of Moringa
J. Vet. Sci., Nov. (1), 1, 806-814 (2014)
oleifera in O. niloticus diet and its possible impacts on immunity and growth
performance. MATERIALS & METHODS
Fish: 180 O. niloticus weighting 40±1.5 gram.
Aquaria: 18 glass aquarium 50x40x40 centimeter.
Feed: formulated ration purchased from Kafr El-Sheikh market.
Additives: Proximate composition of Dry M. oleifera leaf (%) moisture content 8.4,
crude protein 27.12, crude lipid 2.33, crude fibre 19.17, total ash 6.1 and nitrogen free
extract 36.88. M. oleifera leaf was purchased from local market and proximate
analyzed in nutrition laboratory in faculty of veterinary medicine, Kafr El-Sheikh
university.
Experimental design: Experiment lasted for 6 weeks fed on isocaloric4320 Kcal/Kg
and isonitroginic diets 30% crud protein. M. oleifera leaf were incorporated in fish
diets at levels of 0=control, 5%=T1, 10%=T2, 15%=T3, 20%=T4 and 25%=T5. Every
treatment had 3 replicates and each replicate had 10 O. niloticus. Every seven days,
the fish in each aquarium were weighed and the amount of feed for O. niloticus was
corrected according to the new fish biomass as 3 % of live body weight from the diet
which was formulated requirements according to NRC (1993).
Growth parameters: Growth performance was calculated according to the following
equations:-
a-Average daily gain (ADG) = (W1-W0)/T Where, W0 and W1 were the initial and
final body weight per gram, and T is the number of days in the feeding experimental
period. (Castell and Tiews, 1980)
b-Total weight gain (TG) = Wt1-Wt0 Where wt1 is the final body weight (g) and
wt0 is the initial body weight (g) (Castell and Tiews, 1980).
c-Feed conversion ratio (FCR)= Feed intake (g)/ weight gain (g) Where, the weight
gain is (the biomass of fish at the start + the biomass of the dead fish- the biomass of
the fish at the end) (Tacon, 1987)
d-Relative Growth Rate (RGR %) = 100(final weight–initial weight)/Initial weight.
f-Survival rate (SR %)= (No. of fish at end / No. of fish at the start) ×100.
Haemogram determination in O. niloticus:
Blood samples were taken at the end of experiment period from tail vein. Blood
film was prepared according to the method described by Lucky (1977). Red blood
cell (RBCs) and White blood cell (WBCs) counts were counted by haemocytometer
according to Stoskopf (1993). Mean Corpuscular hemoglobin concentrations were
calculated according to the formula mentioned by Dacie and lewis (1975).
M.C.V. = (PCV / RBCs) x 10 as m/mm3.
M.C.H. = (HB content gm/100ml/ RBCs) x 10 as m/mm3.
M.C.H.C. = (HB content gm/100ml / PCV) x100 as %.
J. Vet. Sci., Nov. (1), 1, 806-814 (2014)
Biochemical analysis of experimental O. niloticus sera:
The activity of the liver enzymes, aspartate amino transaminase (AST) and alanine
amino transaminase (ALT) were determined according to (Reitman and Frankel,
1957) by using kits reagent supplied by Diamond Diagnostic Co.
The concentration of total protein (TP) and albumin (Alb) were performed
according to Weichselbaum (1946) and (Doumas et al., 1971) respectively were
measured by colorimetric methods, While, globulin concentrations (Glo) were
determined by subtracting the concentration of total protein from albumin
concentration.
Challenge test:-
A total number of 60 fish (10 fish from each treatment) were injected I/P with the
pathogenic A. hydrophila which kindly obtained from fish diseases department,
Animal Health Research Institute (0.3 ml of 108 cells/ml) according to Schaperclaus
et al. (1992). The injected fishes were kept under observation for 14 day to record the
mortality rate.
Mortality rate % = No. of death in specific period/ Total population during that
period x 100.
Statistical analysis was performed using the analysis of variance (ANOVA).
Duncan's Multiple Range Duncan (1955) was used to determine differences among
treatments mean at significance level of ≤ 0.05. All statistics were run on the
computer using the SAS program (SAS, 1998).
RESULTS
Data presented in table (1) showed that O. niloticus fed different level of M.
oleifera had significant improvement. T2 which M. oleifera leaf incorporated at 10%
in fish diet had the highest growth parameters as compared with control group and the
other treatments. Survival Rate (SR) showed insignificant results in all treatments as
compared with control.
Haemogram RBC, Hb, PCV, MCV, MCH and MCHC of O. niloticus in different
treatments represented in table (2) showed enhanced conditions indicating that
incorporation of M. oleifera leaf in O. niloticus diet had enhanced health status.
Addition level 10 % and 15 % had significant higher recorded blood indices.
Data in table (3) represented WBCs and differential leuckocytic count of O.
niloticus in different fed groups reflected that immune status of experimental O.
niloticus showed improving with M. oleifera leaf addition to O. niloticus diet.
Serum Total protein, albumin, globulin and albumin / globulin ratio represented in
table (4) showed that treatments 2 and 3 had significant higher level 24.2, 24.6 and
14.5, 15.1 respectively as compared with control group indicating improved immune
status.
J. Vet. Sci., Nov. (1), 1, 806-814 (2014)
Table (4) presented liver enzymes AST and ALT activities in O. niloticus fed
different levels of M. oleifera leaf which showed that treatments 4 (20%) and 5 (25%)
had significantly higher levels of AST and ALT activities compared to control (19.5
and 11.8 U/L) and other treatments indicating impacts of M. oleifera leaf addition on
liver.
Table (5) presented the effect of different level of M. oleifera on the mortality rate
of O. niloticus. Lower mortality rate were recorded among fish fed 5, 10, and 15 % of
M. oleifera and was 60% and control group was10 %. While higher mortality rate had
recorded in fish fed diet had incorporation levels 20 and 25 %.
DISCUSSION
Feed utilization and growth parameters improved significantly with the addition of
Moringa oleifera leaf until level 15% then decline along with increasing the addition
level. These findings agreed with those obtained by Ozovehe and Nzeh (2013) who
stated that M. oleifera leaf meal could substitute fish meal up to 10% level in Clarias
gariepinus diets without any negative effects on the growth and feed efficiency.
Moreover, Afung et al.(2003) reported that leaf meal protein at low levels of
substitution (less than 50%) in fish diets were able to support growth and solvent –
extracted of M. oleifera leaf meal could replace till 30% of fish meal of O. niloticus
diets. Azaza et al. (2009) agree with our findings as they mentioned that dietary
protein source from plant origins did not affect growth and survival of fish with
increased inclusion of sustainable. The decrease in growth parameters in O. niloticus
with corporation level more than 15% could be explained by the presence of
antinutrients in M. oleifera leaf meal such as phenol, tannins, phytates and saponins
had an impact on lowering the growth performance with higher substitution of fish
meal (Richter et al., 2003). Also, the decrease in growth rate with higher corporation
level could be due to reduction of level of protein and amino acids in the diets having
higher substitution levels, from the optimum level for growth and feed utilization
(Russel et al., 1983). Moreover, dry plant materials with low digestible property
inhibit utilization of other nutrients contained in diet of fish (Dongmeza et al., 2010).
Declined results of heamogram represented in table (2) with higher level of
addition agreed with those obtained with Ozovehe and Nzeh (2013) who claimed that
the decrease in RBC may be ascribed to the higher concentration of antimetabolite
especially tannin in the diets containing more M. oleifera leaf meal. Also, Knox et al.
(1975) mentioned that tannins have been reported to negatively impact feed intake.
However our results could be explained that low feed intake in higher addition levels
had decreased the protein intake which cause reduction of RBCs and blood protein
synthesis. Robert et al. (2000) supported our results as they stated that the reduction
in the Hb concentration could imply that diets having higher substitutions contained
low quality protein.
Results concerning WBCs and lymphocytes (table, 3) showed higher level in
addition level 5, 10 and 15% and had returned to control level with higher addition.
These findings could be explained by improvement of immune status along with
improved feed utilization. However, decreased level after addition of higher levels of
M. oleifera could be attributed to the fact that M. oleifera contained anti nutritional
materials that cause sensitization of immune system but did not cause its destruction.
High WBC count is usually associated with microbial infection or the presence of
J. Vet. Sci., Nov. (1), 1, 806-814 (2014)
foreign body or antigen in the circulating system (Oyawoye and Ogunkunle 1998).
Also, Douglass and Janes (2010) demonstrated that the amount of M. oleifera leaf
meal incorporated in fish diet has implication in immune responses and the ability of
the animal to fight infection.
Data concerning serum total proteins, albumin and globulin showed enhancement
significantly (p<0.05) with lower addition levels (5, 10 and 15%) as compared with
higher incorporation levels. These findings could be due to impaction of high level
incorporation on hepatic tissues. As there was significant increase (p<0.05) in the
activities of serum enzymes AST and ALT in all experimental groups in comparing
with the control group. Elevated AST and ALT activities in fish fed higher levels of
M. oleifera leaf meal diet and maybe due to hepatic cellular damage leading to their
leakage into circulation (Ozovehe and Nzeh, 2013).
From data concerning challenge test (table, 5) it was obvious that incorporation of
M. oleifera leaf 5, 10 and 15 % had the lowest mortality rate comparing with other
groups. This could be explained by the results of blood analyses which showed
increased WBCs and serum protein indicating enhancement of immune status in these
groups. In the same trend the immune status of Clarias gariepinus had improved with
incorporation of M. oleifera leaf meal to level 10 % and increase the level of
incorporation has implication in immune responses and the ability of the animal to
fight infection Douglass and Janes, 2010 and Ozovehe and Nzeh, 2013).
In conclusion, the results obtained from this study showed that M. oleifera leaf
meal could be incorporated up to 15% level in O. niloticus diets as it enhance feed
utilization and immune status.
Table (1): The effect of adding M. oleifera growth performance, feed utilization
and survival rate of O. niloticus.
Item
IW
FW
TG
ADG
FI
FCR
RGR
SR
Control
(0)
40.5
±0.29 a
58
±0.6 c
17.5
±0.4 c
0.42
±0.0 c
44.1
±0.32 a
2.5
±0.04 a
43.2
±0.8 c
83.3
±0.3 ab
T1
(5%)
40.2
±0.5 a
68.9
±0.25 a
28.7
±0.4 a
0.68
±0.0 a
44
±0.53 a
1.53
±0.04 d
71.4
±1.8 a
93
±0.3 a
T2
(10%)
38.77
±0.18 b
68
±0.7 a
29.4
±1.4 a
0.7
±0.02 a
42.5
±0.17 b
1.44
±0.05 d
75.6
±2.4 a
90
±0.6 ab
T3
(15%)
40.4
±0.3 a
63.9
±1 b
23.5
±1 b
0.56
±0.02 b
44.1
±0.33 a
1.88
±0.1 c
58.3
±2.7 b
90
±0.6 ab
T4
(20%)
39.7
±0.56 ab
58.6
±0.5 c
18.9
±0.2 c
0.45
±0.01 c
43.2
±0.6 ab
2.29
±0.05 b
47.6
±1 c
77
±0.3 b
T5
(25%)
39.9
±0.4 ab
57.3
±0.5 c
17.4
±0.6 c
0.42
±0.01 c
43.5
±0.43 ab
2.5a
±0.1
43.5
±1.8 c
87
±0.3 ab
Group with different letter within the same column are significantly different at P≤ 0.05.
IW= Intial Weight, FW= Final Weight, ADG= Average Daily Gain, TG= Total weight Gain, FCR=
Food Conversion Ratio, FI= Feed Intake, RGR=Relative Growth Rate and SR=Survival Rate.
J. Vet. Sci., Nov. (1), 1, 806-814 (2014)
Table (2): The effect of adding M. oleifera haemogram of O. niloticus.
Item
RBC
X106
Hb
g/dl
PCV
%
MCV
m/mm3
MCH
m/mm3
MCHC
g/dl
Control
(0)
1.8
±0.02 cd
5.15
±0.06 d
16.5
±0.17 e
92.47
±0.2 f
29
±0.01 f
31.9
±0.1 e
T1
(5%)
1.89
±0.02 ab
6.6
±0.08 b
22.8
±0.6 b
120.8
±1.5 c
35
±0.01 b
34.5
±0.2 c
T2
(10%)
1.96
±0.04 a
7.29
±0.12 a
27
±0.7 a
137.6
±2.4 a
37.1
±0.0 a
37
±0.05 a
T3
(15%)
1.87abc
±0.06
6.37
±0.2 b
23
±1.2 b
123.5
±2.5 b
34
±0.0 c
36.3
±0.1 b
T4
(20%)
1.81
±0.01 bc
5.8
±0.1 c
20.2
±0.3 c
111.3
±1.2 d
32
±0.01 d
34.7
±0.03 c
T5
(25%)
1.73
±0.01 d
5.37
±0.1 d
18.2
±0.2 d
104.9
±0.5 e
31
±0.01 e
33.8
±0.04 d
Group with different letter within the same column are significantly different at P≤ 0.05.
Table (3): The effect of adding M. o leifera WBCs and differential leuckocytic
count of O. niloticus.
Item
WBCs
x103
Hetero%
Mono%
Esino%
Baso%
Lymph%
Control
(0)
7.26±0.04 b
39±2.6 a
3±0.0 c
2.67±0.3 ab
1±0.01 a
50.3±0.3 c
T1
(5%)
7.55±0.02 ab
35.7±0.9 b
4.33±0.6 ab
2.33±0.3 ab
1±0.0a
56.7±0.9 a
T2
(10%)
7.9±0.1 a
33.7±0.3 b
4.7±0.6 a
2.67±0.3 ab
1±0.01a
58±0.6 a
T3
(15%)
7.9±0.2 a
39±0.6 a
3.67±0.6 bc
2.33±0.3 ab
1±0.01a
54±0.6 b
T4
(20%)
7.33±0.3 b
41.7±0.6 a
3.3±0.6 c
3±0.0 a
1±0.01a
51±0.6 c
T5
(25%)
7.23±0.1 b
41.7±1.5 a
3±0.0 c
2±0.0 b
1± 0.01a
52.3±1.5 bc
Group with different letter within the same column are significantly different at P≤ 0.05.
J. Vet. Sci., Nov. (1), 1, 806-814 (2014)
Table (4): The effect of adding M. oleifera serum Total protein and liver enzymes
of O. niloticus.
Item
Total Protein
(g/100ml)
Albumin
(g/100ml)
GLobuline
(g/100ml)
A/G
Ratio
AST
(U/L)
ALT
(U/L)
Control
(0)
4.2±0.03 c
2.8±0.03 c
1.36±0.01 c
2.06±0.1a
19.5±0.3 d
11.8±0.4 e
T1
(5%)
4.9±0.1 b
3.1±0.1 b
1.86±0.2 b
1.67±0.05c
21.3±0.3 c
13±0.3 d
T2
(10%)
5.8±0.15 a
3.5±0.06 a
2.27±0.1 a
1.54±0.04c
22.6±0.6 b
13.8±0.1 bc
T3
(15%)
5.6±0.4 a
3.5±0.1 a
2.12±0.1 a
1.65±0.03c
22.7±0.27 b
13.4±0.1 cd
T4
(20%)
4.2±0.06 c
2.9±0.03 bc
1.37±0.02 c
2.12±0.01a
24.2±0.5 a
14.5±0.0 3 ab
T5
(25%)
4.2±0.6 c
2.8±0.02 bc
1.54±0.02 c
1.8±0.06b
24.6±0.2 a
15.1±0.06 a
Group with different letter within the same column are significantly different at P≤ 0.05.
Table (5): Effect of M. oleifera on mortality rate of O. niloticus challenged
against A. hydrophilla infection.
Item
Control (0)
T1 (5%)
T2 (10%)
T3 (15%)
T4 (20%)
T5 (25%)
Total
number
10
10
10
10
10
10
Mortality
10
6
6
6
8
10
Mortality
rate (%)
100
60
60
60
80
100
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