effect of dietary garlic extract on growth, feeding ...jpg.inio.ac.ir/article-1-504-en.pdf · 29...

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Effect of dietary Garlic extract on growth, feeding parameters,

hematological indices and body composition

of Litopenaeus vannamei

Samadi, Leila1; Zanguee, Nasim

1; Mousavi, Seyed Mohammad

*1; Zakeri, Mohammad

1

1- Department of Fisheries, Faculty of Marine Natural Resources, Khorramshahr University of Marine

Science and Technology, Khorramshahr, Iran

Received: March 2016 Accepted: June 2016

© 2016 Journal of the Persian Gulf. All rights reserved.

Abstract In this study, Effects of garlic (Allium sativum) extract on growth indices and hemolymph

parameters of Litopenaeus vannamei with mean weight 18.21±0.29 g were studied for a period of

56 days. This study was conducted inside 300-liter polyethylene containers as four treatments each

with four replicates and each replicate testing 5 shrimps. A basic diet supplemented by 0 (control),

40, 80 and 100 ppm of garlic extract was provided for shrimps. Shrimp biometry was performed at

the end of experiment and hemolymph was collected by 2ml syringe. The results showed that garlic

extract positively affected daily feed intake, feed conversion efficiency, protein efficiency ratio,

molting rate (P<0.05), but no significant change in weight gain rate, specific growth rate, condition

factor and hepatosomatic index (P>0.05). Also, treated showed significant changes in body protein

(increased levels), fat and ash composition (decreased levels) compared with control group

(P<0.05). Total hemocytes count was not affected but differential hemocytes count and total plasma

protein was significantly changed treated shrimps compared with control shrimps (P<0.05). Based

on the results obtained from this study, garlic extract may be applied as a food supplementation in

shrimp aquaculture.

Keywords: Garlic extract, hemolymph, Growth, Litopenaeus vannamei

1. Introduction

The application of immunostimulants in shrimp

aquaculture is increasingly gaining interest as an

environmentally safe alternative to antibiotics and

chemotherapeutics (Song et al., 1997). Shrimp

possess an innate immune system, consisting of

cellular and humoral elements. Hemocytes play a

central role in the non-specific immune response of

shrimp, which rely mainly on phagocytosis,

melanization, encapsulation, cytotoxicity, and

clotting (Sritunyalucksana et al., 1999). Humoral

defense factors, such as clotting proteins, agglutinins,

hydrolytic enzymes, and antimicrobial peptides are

released upon lysis of hemocytes, which is induced

by lipopolysaccharides (LPS), peptidoglycans, and β-

1,3-glucans (Chisholm and Smith, 1995; Muta and

Iwanaga 1996; Destoumieux et al., 2000).

Diets containing immunostimulants are used in

aquaculture in order to increase resistance to stress

and diseases of cultured fishes and invertebrates by

Journal of the Persian Gulf

(Marine Science)/Vol. 7/No. 24/ June 2016/13/29-41

* Email: [email protected]

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http://jpg.inio.ac.ir/article-1-504-en.html

Mousavi et al. / Effect of dietary Garlic extract on growth…

30

alerting the immune system (Rendon and Balcazar,

2003; Donate et al., 2010).

Aquaculture represents one of the main food

providers in the world. In shrimp farming, Whiteleg

shrimp (Litopenaeus vannamei) is the primary

penaeid shrimp currently being cultured in Central

and South America (Burge et al., 2007; Chang-Che

and Jiann-Chu, 2008). However, during the past two

decades, worldwide commercial shrimp farming has

suffered outbreaks of diseases caused mainly by

Vibrio bacteria and viruses due to a deteriorated

pond environment (Lo et al., 2003).

The white shrimp, Litopenaeus vannamei , which

is naturally distributed along the Pacific coasts of

Central and South America has become the most

important cultured species worldwide due to

successful development of techniques for

manipulating brood stocks, spawning and seed

production (Tayag et al., 2010).

Garlic (Allium sativum) is a perennial bulb-

forming plant that belongs to the genus Allium and

family Liliaceae, which has been used for centuries

as a flavoring agent, traditional medicine and a

functional food to enhance physical and mental

health. Garlic effects have been studied in different

forms of extracts: aqueous, ethanol and dried powder

in 16 month-old rats (Shin and Kim, 2004). It

contains a variety of organosulfur compounds, such

as allicin, ajoene, S-allylcysteine, diallyl disulfide, S-

methylcysteine sulfoxide and S-allylcysteine (Chi et

al., 1982). Studies on garlic as an alternative to

growth promoters in livestock production were

conducted and its beneficial effects on growth,

digestibility and carcass traits have been reported

(Bampids et al., 2005; Tatara et al., 2008). Dietary

garlic as a growth promoter in Nile tilapia

(Oreochromis niloticus) improved body weight gain,

feed intake and feed efficiency (Diab et al., 2002;

Shalaby et al., 2006). Also, garlic has potential role

as prophylactic and therapeutic agent in aquaculture

(Amagase et al., 2001). Garlic can be used to control

the pathogens, especially bacteria and fungi and it

increases the welfare of fish (Corzo-Martinez et al.,

2007). Expression of innate immunity genes in

kuruma shrimp Marsupenaeus japonicus was studied

after in vivo stimulation with garlic extract (allicin)

by Tanekhy and Fall (2015). Also, efficacy of garlic

was studied on the survival, growth and haematology

of Penaeus monodon post larvae (Vidhya Malar and

Maria Charles, 2013). Labrador et al. (2016)

observed that the white leg shrimp fed the diet

containing 6% garlic powder obtained the highest

weight gain. Shrimps being fed by Artemia enriched

with 200mg garlic extract per kg food showed the

best growth, survival rates and length (Javadzadeh et

al., 2012).

The aim of this study was to examine the effects

of the dietary Garlic extract as a feed additive on

growth performance, feed utilization, biochemical

parameters of the body and some haematological

indices in Pacific white shrimp, Litopenaeus

vannamei.

2. Material and Methods

2.1. Preparation of garlic extract and the

experimental diets.

Fresh garlic was purchased from the local market

in Hamadan (Iran). Peeled fresh garlic was chopped

into small pieces, dried and ground finely and then

macerated in 95% ethyl alcohol incubated at room

temperature and after three days, filtered through

Whatman filter paper ( 42 micron) (Zargari, 1997).

The procedure was repeated three times to ensure

exhaustive extraction of the plant material. The

extract was pooled together, concentrated and the

solvent removed by evaporation under reduced

pressure in a rotary evaporator at 75ºC. The garlic

extract was kept in a refrigerator at 4°C before use.

Experimental diets were prepared by combining

powdered commercial shrimp pellet (21-beiza

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Journal of the Persian Gulf (Marine Science)/Vol. 7/No. 24/ June 2016/13/29-41

31

company, Iran) with plant extract and 2% gelatin to

produce four diets: (1) commercial diet as control

group, (2) 40 mg/kg of food, (3) 80 mg/kg of food,

(4) 100 mg/kg of food. After drying, the pellets were

stored in plastic packets at 4 °C until being used.

Composition of the basic diet used for white shrimp,

Litopenaeus vannamei in this study included of 33%

Crude protein, 8% Crude lipid, 4% Crude

carbohydrate,14% ash and 10% moisture.

2.2. Shrimp rearing

Ninety clinically healthy Pacific white shrimps

(Litopenaeus vannamei Boone) were obtained from a

commercial farm in Bushehr, South of Iran. Upon

arrival, they were acclimated to laboratory conditions

for 15 days in fiber glass-reinforced plastic (FRP)

tanks and fed by the commercial diet. The laboratory

conditions during the acclimation period were daily

monitored and maintained at the same level. There

were four treatments in four replicates. Each

replicate consisted of 5 shrimps with mean weight

18.21±0.29 g in a 300 liter circular FRP tank with

200 liter of water at a salinity of 29.42± 0.64 ppt.

Aeration was supplied by a single air-stone to

maintain the dissolved oxygen at 7.72 ± 0.18 mg l-1

.

Shrimp were fed thrice daily at 08:00, 11.00 and

18.00. Molts, feces and dead shrimps were daily

removed and 30% of water in each tank was

exchanged by new sea water (Javadzadeh et al.,

2012). During the experimental period, the water

temperature was maintained at 25.19 ± 0.087 °C and

pH 8.02 ± 0.051.

2.3. Growth and nutritional parameter

At the end of the feeding trial, total weight and

length of shrimp were measured and hepatopancreas

was collected. The relative weight gain rate (WG%)

= 100× (Wt - W0)/ W0 ×100; Molting rate (MR,

%.day-1

)= (Nm / Ns) / T, Specific growth rate (SGR)

= ln Wt-ln W0 ÷ T × 100, feed intake (FId)= 100 × F/

(T×(Wt+ W0)/2), food conversion efficiency

(FCEd)= 100× (Wt- W0)/F , Protein Efficiency Ratio

(PER)= (Wt- W0)/P , hepatosomatic index (HIS) =

Liver weight/ fish body weight, Condition Factor

(CF%)= (Wt× 100)/ (L)3 and daily feeding rate

(DFR, %) = feed intake/((W0+Wt)/2 days of the

experiment)×100 were calculated where, Wt and W0

are the final and initial wet body weight of the

shrimp, respectively; Nm, molting frequency; Ns, the

number of shrimps per aquarium, respectively; T,

the time of the experiment lasted; F, the total food

consumed in dry weight; P, the total Protein

consumed in dry weight; L, the total length of each

shrimp (Wang et al., 2004).

2.4. Hematological Parameters (Total Haemocyte

count (THC) and differential Haemocyte count

(DHC))

Half a ml of haemolymph was collected from

second swimming leg by using a 21-gauge

hypodermic needle and by 2-ml syringe. Each

syringe was pre-filled with 0.3 ml of anticoagulant

(10 mM Tris–HCl, 250 mM sucrose, 100 mM

sodium citrate, pH 7.6). Anticoagulant was added to

make an equal volume ratio of hemolymph to

anticoagulant. A volume of 50 ml anticoagulated

hemolymph was fixed with an equal volume of

neutral buffered formalin (10%) for 30 min to

measure the total haemocyte count (THC) and

differential hemocyte count (DHC). The remaining

anticoagulated hemolymph was centrifuged at 300×g

for 10 min at 4°C to separate the hemocytes from

plasma. The plasma total protein was measured by

photometric method by using trading diagnostic kit

(Thomas, 1998).

Total hemocytes were counted using a

hemocytometer (Boeco, Germany) and light

microscope at 100×. Fixed hemolymph was smeared

on a slide and stained with Giemsa solution (10%)

for 10 min. The differential hemocytes include

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Hyaline, Semi-granular and Granular cells were

characterized according to Tsing et al. (1989) and

100 cells from each smear were counted under a light

microscope.

2.5. Body biochemical composition analysis

The experimental diets were sampled before

adding and after the Garlic extract for analysis of

crude protein, fat and ash. At the end of the trial,

shrimp per each tank were randomly sampled and

meat yields (%) were evaluated after peeling the

shell and removing the head. The meat was

homogenized for the analysis of moisture, crude

protein and crude ash. The chemical composition

was determined following procedures (AOAC,

1984): crude protein (Nx6.25) by the Kjeldahl

method (Kjeltec 2100 Distillation Unit, Foss Tecator,

Hoganas, Sweden), crude fat using an ether-

extraction method, moisture by oven drying at

105[degrees]C for 24 h, ash using a muffle furnace at

550[degrees]C for 4 h.

2.6. Statistical Analysis

Results were analyzed using SPSS (version 16.0)

software. Data was normalized by Kolmogorov-

Smirnov Test. One way ANOVA and Tukey's

multiple range tests were used to determine the

significance of differences between groups. All the

results were expressed as means ± standard error (M

± SE) and significant differences were expressed at a

significance level of P<0.05.

3. Results

3.1. Growth parameter

At the end of the 56 days feeding trial, molting rate

was high in shrimp fed by 80 and 100 mg kg-1 Garlic

extract and lowest in shrimp fed by diet 40 mg kg-1

and group fed by the diet without Garlic extract

supplementation (p < 0.05) (Table 1).

There were no significant increase in the weight

gain rate, SGR, HIS and CF among treatments but the

results presented in Table 1, indicated a trend of

increased weight gain rate and SGR in group 80 mg

kg-1garlic extract supplementation. Also, the shrimp

fed by 100 mg kg-1garlic extract had higher HIS than

shrimp fed by other concentrations of garlic extract

and the shrimp fed by 40 mg kg-1garlic extract had

higher CF than shrimp fed by other concentrations.

Table 1: Growth parameters and feeding rate of white leg shrimp fed by different experimental diets

supplemented by different level of garlic extract.

Parameters Treatments

Control 1

(40 mg/kg)

2

(80 mg/kg)

3

(100 mg/kg)

Molting rate 1.04± 0.08b 1.12±0.08 b 1.75±0.17 a 1.74±0.05 a

weight gain rate (g) 6.26±0.48 7.45±0.61 8.88±0.67 7.44±0.91

Specific growth rate (%) 0.51±0.03 0.51±0.11 0.7±0.07 0.6±0.06

Hepatosomatic index 3.30±0.16 3.35±0.3 3.45±0.34 3.52±0.19

Condition Factor 0.67±0.02 0.69±0.01 0.69±0.02 0.68±0.008

Daily feeding rate (g) 2.71±0.07ab 2.92±0.01 a 2.62±0.05 b 2.59±0.05 b

food conversion efficiency 0.19±0.01 b 0.2±0.02 ab 0.27±0.02 a 0.23±0.02 ab

Protein Efficiency Ratio 0.56±0.16 b 0.62±0.06 ab 0.81±0.13 a 0.66±0.2 ab

* Different letters show significant differences between treatments (p<0.05). All data were presented in Mean ±SE.

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3.2. Feeding parameter

At the end of the 56 days feeding trial, DFI was

high in shrimps fed by 40 mg kg -1garlic extract and

lowest rate for shrimp fed by the diet with 100 mg kg

-1 garlic extract supplementation (p < 0.05). The Feed

conversion efficiency in Group 80 mg kg-1garlic

extract was significantly higher than shrimp fed by

other concentrations. The PER followed the same

pattern as the Feed conversion efficiency (Table 1).

3.3. Hematological Related Parameters

The THCs and DHCs of shrimp fed by diets

containing garlic extract and control diet for 56 days

are shown in Fig. 1. No significant differences in the

THCs of shrimp were recorded and the THCs of

shrimp fed by control and garlic extract diets

including of 40, 80 and 100 mg kg-1 garlic extract

were (8.75 ± 1.96) ×103, (8957.7±1.36) ×103,

(13431.1±2.68) ×103, and (15005.7± 4.01) ×103cells

ml_1, respectively (Fig. 1A). Also, the Granular Cells

were the most affected cell types by garlic extract

treatments, with the highest value in the group fed by

100 mg kg -1 at 56 days from the start of feeding (Fig,

1B). The hyaline cell in Group 80 mg kg-1garlic

extract was significantly lower than shrimp fed by 40

mg kg -1garlic extract and control diet. In addition,

semi-granular cell in Group 80 mg kg-1garlic extract

was significantly lower than shrimp fed by other diets.

Moreover, the shrimp fed by 100 mg kg-1garlic

extract had higher total plasma protein than shrimp

fed by other diets.

Fig. 1: Total hemocyte counts (A) , Hyaline cell (B) Semi-granular cell (C) and Granular cell (D) of Pacific white shrimp, Litopenaeus

vannamei, fed by diet without Garlic extract as the control and diets with different concentrations of Garlic extract of 40, 80 and 100 (mg

kg diet)_1 for 56 days. Data (mean ±SE) with different letters significantly differ (p < 0.05) among treatments.

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3.4. Body composition Analysis

The results of body composition analysis including

total protein, fat, ash and moisture from muscle tissues

samples are given in Table 2.

The significant highest protein content of 18.92 ±

0.47 % in muscle tissue was observed in the shrimp

fed by 80 mg kg-1garlic extract than shrimp fed by

other diets. Lipid content in muscle tissue were found

to be significantly lowered for shrimp fed by 40, 80

and 100 mg kg -1 garlic extract and highest for shrimp

fed by diet without garlic extract supplementation

(p < 0.05). Additionally, the ash content in muscle

tissue were determined to be significantly increased in

40, 80 and 100 mg kg -1 garlic extract and highest for

shrimp fed by diet without garlic extract

supplementation (p < 0.05).

Table 2: Body biochemical composition of white leg shrimp fed by different experimental diets

supplemented by different level of garlic extract

Parameters Treatments

Control 1 2 3

Crude protein(%) 16.53±0.43b 15.93±0.59 b 18.92±0.47 a 16.59±0.59 b

Crude lipid (%) 0.48±0.06 a 0.23±0.03 b 0.23±0.03 b 0.28±0.02 b

Ash (%) 4.74±0.14 ab 5.48±0.38 a 4.41±0.08 b 5.47±0.27 a

Moisture (%) 77.00±0.52 77.17±0.29 77.01±0.71 76.68±0.12

* Different letters show significant differences between treatments (p<0.05). All data were presented in Mean ±SE.

4. Discussion

Garlic (Allium sativum) has been reported that

have various beneficial effects including antistress,

growth promotion, appetite stimulation,

immunostimulation and antimicrobial properties in

finfish and shrimp larviculture (Aly et al., 2010; Diab

et al., 2008; Guo et al., 2011a; Millet et al., 2011;

Nya and Austin, 2009; Peyghan et al., 2008; Sahu et

al., 2007; Sasmal et al., 2005; Shalaby et al., 2006;

Soko and Barker, 2005; Vaseeharan et al., 2011; Xie

et al., 2009). The present study was carried out to

understand the role of this plant extract on growth

promotion, appetite stimulation, immunostimulation

and biochemical constituents in body of Litopenaeus

vannamei. In the present study, the dietary garlic

extract supplementation led to enhanced trend on

weight gain rate and SGR, but this increase was not

significant. Khalil et al, (2001) mentioned that garlic

contains allicin, which promotes the performance of

the intestinal flora, thereby improving digestion and

enhancing the utilization of energy, leading to

growth improvement. Results of this study are

comparable with the studies of Diab et al. (2002),

Farahi et al. (2010), Nya and Austin (2009) and

Shalaby et al. (2006), where significant growth rates

were observed in treated fish over the controls. The

crustacean hepatopancreas is a vital organ involved

in excretion, molting, lipid and carbohydrate

metabolism and diverse metabolic activities,

including synthesis and secretion of digestive

enzymes, absorption of nutrients, synthesis of plasma

proteins such as hemocyanin and lipoproteins

(Yepiz-Plascencia et al., 2000) and storage of energy

reserves (Gibson and Barker, 1979). In crustacean,

relative weight of hepatopancreas, which plays a key

role in food assimilation (Dhall and Moriaty, 1984)

and probably manifests the provision for energy

utilization for growth and metabolism. Sagi and

Ra'anan (1988) found that the relative size of

hepatopancreas was highly correlated with the

morphotypic stage of development and its relative

energy expenditure in growth and sexual activity.

The increase trend of HIS in experimental shrimp fed

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with garlic supplementation diets may be a

consequence of the effect of chemical component in

garlic extract on stimulation of the liver to produce

and secrete the major pancreatic enzymes, enhance

activities of pancreatic digestive enzymes and

reserve energy in hepatopancerace (Platel and

Srinivasan, 2004). In general, the growth curve of

shrimp is similar to other crustaceans, i.e. a ladder-

style with much fast growth during each molting

process. After molting, the size of crustacean rarely

increases until the next molting (Dall et al.,

1990).Wang et al. (2004) suggested that the growth

of shrimp was correlated to feed, digestion and

absorption of food, while the molting was related

with the secretion of hormone. So there may be two

distinct regulations of these two physical activities.

In this study, higher MF resulted in faster growth in

shrimps; while for shrimps with poor growth, the MF

was relatively low. Thus, there was a clear

correlation between growth and molting. It seems

that garlic extract at level of 80 ppm can increase

quality of basic diet.

Feed efficiency ratio (FER) and PER are used as

quality indicators for shrimp diet and amino acid

balance. So, these parameters are used to assess

protein utilization and turnover. Feed quality,

stocking density and water quality are the main

factors affecting productivity for semi- intensive and

intensive culture of penaeid shrimps (Cruz-Suárez et

al., 1993). Wanapat et al. (2008) found significantly

higher digestibility, absorption and retention of

Nitrogen in garlic powder supplemented groups

compared to control group. Our present finding also

accords with these results that shrimp fed diet with

garlic extract has higher Nitrogen utilization

(PER,%) than control group.

The effects of garlic supplementation on protein

metabolism have not been fully clarified. It is

believed that garlic involved in hormonal secretion,

may affect whole-body protein metabolism due to

hormonal regulation by stimulating hormone

secretion (noradrenaline and adrenaline), or it may

affect protein metabolism by enhancing protein

anabolism (Srivastava and Pathak, 2012). Oi et al.

(2001) reported that protein anabolism occurs in rats

fed the high protein diet supplemented with garlic.

Garlic has depressed the hepatic activities of

lipogenic and cholesterogenic enzymes such as malic

enzyme, fatty acid synthase, glucose-6 phosphate

dehydrogenase which are led to decreasing of lipid

levels of body (Yeh and Liu, 2001) which is

comparable with results obtained from current study.

Recently some studies revealed that water-soluble

organosulfur compounds, especially S-allyl cysteine

(SAC) and diallyl-di-sulfide (DADS), are also potent

inhibitors of cholesterol synthesis (Yeh and Liu,

2001; Gebhardt and Beck, 1996). The different

composition and quantity of sulfur components of

different garlic preparations used in various studies

could be a reason for different findings. The mineral

components are required for some important

functions, including shell or bone building, blood

clotting, muscle function and neuromascular

transmission (Lovell, 1989; Coote et. al., 1996).

Davis et al. 1992 reported that quality of feed can

affect growth, survival and mineral component in

body of P. vannamei. Increase of carcass ash content

may be a result of food accessibility and mineral

absorption by the animal (Tacon et al., 2002).

Crustaceans mainly rely on innate immunity to

protect themselves against pathogen infections and

other external factors that continually threaten their

lives (Sarathi, et al., 2007).

Haemocytes play important roles in the immune

response and based on the presence of cytoplasmic

granules can be classified into three types: hyaline

cells (HCs), semi-granular cells, and granular cells

(GCs). HCs are involved in phagocytosis, whereas

semi-granular cells and GCs are involved in the

encapsulation and release of the prophenoloxidase

(proPO) system (Jiravanichpaisal et al.,2006).

In the present study, the dietary garlic extract

supplementation led to enhanced trend on THC. But,

this increase trend was not significant. In many

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crustaceans, the sheet-like haematopoietic tissue

covers the dorsal and dorsolateral sides of the

stomach and is surrounded by connective tissue.

Cells, believed to be haematopoietic cells, of

different morphology are organized and densely

packed in small lobules and some of these

morphological cell types are also found in the

interlobular spaces (Johansson et al., 2000). Molting,

development of organs, reproductive status,

nutritional condition and disease have been shown to

influence haemocyte abundance ( Cheng and Chen,

2001). Tayag et al. (2010) expressed this fact that L.

vannamei that received the extract via injection or

immersion, all has been increased haemocyte count

which suggests a proliferation of haemocyte in

hematopoiesis. In the present study, shrimps which

were fed by the garlic extract-containing diets

increased haemocyte count.

The immune response is a physiological response

and is normally regulated to maintain homeostasis.

El-Desouky et al. (2012) expressed that small

granular hemocytes were the most affected cell type

by Z.officinalis and C. dactylon in Macrobrachium

rosenbergii. Decreasing trend in semi granular and

hyaline cells can be attributed to long term feeding

period by diets containing garlic extract and its

negative effect on hemostasis by storing immune

factors (hemocytes) in hemopoetics tissues. It is

notified that long term feeding by diets containing

garlic extract may affect hemostasis of white leg

shrimp and decreased hyaline and semi granular cells

in the current study. Also, increase in granular cells

and decrease in semi granular cells may be related to

effect of garlic extract (Soltani, 2008). Granular cells

contain antibacterial peptides that introduced as the

first defense of host in innate immune system

(Soltani, 2008).

In aquaculture, immunostimulants can stimulate

the innate immune system by activation or increasing

the activity of granulocytes and macrophages and

increasing the number of phagocytes (Sakai, 1999;

Yin et al., 2006, 2009). They can also increase

protein synthesis to produce more of the molecules

involved in innate metabolism system such as

lysozyme, alkaline phosphatase and nitrogen

monoxide (Xie et al., 2008; Rao et al., 2006; Liu et

al., 2008). Blood serum protein is a biochemical

system, precisely reflecting the condition of the

organism and the changes happening to it under

influence of internal and external factors. Total

protein in serum was significantly high with shrimp

fed on diet containing garlic extract which agrees

with the results of Hussein et al (2001) who showed

that serum total protein level was elevated in male

albino rats after administration of garlic oil. Increase

the total protein level in hyperlipidemic rats treated

with Allium sativum oil could be attributed to the

increase in the immunoglobulin level and total

globulin concentration (Hussein, 1996). High serum

protein levels have been reported due to improve

liver and other organs functions which synthesized

plasma protein (Metwally, 2009).

Conclusions

Finally, based on the results obtained from this

study, garlic extract may be applied as a food

supplementation in shrimp aquaculture, but using

this extract should be done after supplementary

examination with other levels of extract and at

different stages of shrimp life.

Acknowledgements

The authors acknowledge the financial support

and supplying of research facilities of Khorramshahr

University of Marine Science and Technology.

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