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Immunomodulation of Labeo rohita juveniles due to dietarygelatinized and non-gelatinized starch

Vikas Kumar*, N.P. Sahu, A.K. Pal, Shivendra Kumar

Department of Fish Nutrition and Biochemistry, Central Institute of Fisheries Education, Seven Bungalow,

Versova, Andheri (W), Mumbai 400061, Maharashtra, India

Received 27 August 2006; revised 4 November 2006; accepted 17 November 2006

Available online 30 November 2006

Abstract

A 60 days experiment was conducted to study the effect of dietary gelatinized (G) and non-gelatinized (NG) starch on immu-

nomodulation ofLabeo rohita juveniles. Two hundred and thirty four juveniles (av. wt. 2.53 0.04) were randomly distributed insix treatment groups with each of three replicates. Six semi-purified diets containing NG and G corn starch, each at six levels of

inclusion (0, 20, 40, 60, 80, 100) were prepared viz., T1 (100% NG, 0% G starch), T2 (80% NG, 20% G starch), T3 (60% NG,

40% G starch), T4 (40% NG, 60% G starch), T5 (20% NG, 80% G starch) and T6 (0% NG, 100% G starch). After a feeding period

of 60 days, the juveniles were challenged withAeromonas hydrophila to study their immunomodulation due to feeding of G and NG

starch. RBC and haemoglobin content were significantly (P < 0.05) reduced due to bacterial challenge, but dietary starch (G/NGstarch) had no effect on it. G:NG starch ratio in the feed hadsignificant effect on total leukocyte count during pre- and post-challenge

periods. The leukocyte count concomitantly increased with the increased level of G starch in the diet. Highest albumin/globulin(A/G) ratio was recorded in T6 group (100% G starch) and lowest in T1 group (100% NG starch) group followed by T2 group

both in pre- and post-challenge periods. NBT, lysozyme activity, total protein and globulin content were highest in T2 group

(80% NG, 20% G starch) both in pre- and post-challenge periods. After challenge with A. hydrophila, the highest survival was

recorded in T2 group, whereas lowest survival was recorded in T6 group. Conclusively high level of G starch was found to be

immunosuppressive in Labeo rohita juveniles and NG:G starch ratio of 80:20 seems to be optimum for promoting growth and

protecting immunity in L. rohita juveniles.

2006 Elsevier Ltd. All rights reserved.

Keywords: Starch; Leukocyte count; NBT; Lysozyme activity; Serum protein; Serum cholesterol; Labeo rohita; Aeromonas hydrophila

1. Introduction

Recent advancement in immuno-nutrition studies reveal that some nutrients are linked to the immunological

status of fish [1e4]. This has drawn the attention of fish nutritionists to the immunoprotection of fish beside

the growth. Sustainable aquaculture depends on a perfect balance between growth and health condition of fish.

Stress associated with intensive aquaculture causes immuno-suppression in fish [5,6]. Hence, the nutrient contents

* Corresponding author: Tel.: 91 22 26361446x275; fax: 91 22 26361573.E-mail address: [email protected] (V. Kumar).

1050-4648/$ - see front matter 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.fsi.2006.11.008

www.elsevier.com/locate/fsi

Fish & Shellfish Immunology 23 (2007) 341e353
mailto:[email protected]:[email protected]://www.elsevier.com/locate/fsihttp://www.elsevier.com/locate/fsimailto:[email protected]

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of the ingredients should be critically considered before feed formulation so as to care for both growth and health

of the animal.

Unlike mammals fishes are metabolically weak to utilize much carbohydrate in their diet and hence excessive

intake of this nutrient may adversely affect the health [7e10]. Excess inclusions of carbohydrate in the diet causes

stress and hence reduces the growth rate due to poor feed utilization [11]. Excess carbohydrate in the diet also

causes increased blood glucose level and elevated hepatic glycogen deposition [11,2]. Dietary carbohydrate require-

ment of Labeo rohita is 26% [12]. However, there is a continuous effort to enhance the inclusion level of dietary

carbohydrate in the diets of carps for production of low cost feed and hence to reduce the production cost. Recently

it was reported that diet containing about 40% gelatinized starch was efficiently utililized by L. rohita juveniles for

growth without much detrimental effect on health [13]. But recently our detailed studies on this aspect reveals that

there exists an interesting relationship between the type of starch [gelatinized (G) or non-gelatinized (NG)] in the

diet and immunity status of L. rohita. Kumar et al. [2] observed that NG starch fed groups registered maximum

immunoprotection than their G starch counterparts, which was subsequently confirmed by Misra et al. [3]. On

the other hand feeding of G starch enhances the growth and nutrient digestibility in carps, L. rohita and Catla catla

[13,3].

This strongly indicates that a boundary has to be defined between the optimum inclusion of G and NG starch in the

diet to balance the growth and immunity ofL. rohita. Till now nutritionists only pay attention to maximum inclusion of

carbohydrate in the diet without considering its content of G or NG starch. Hence, knowledge on optimum G and NGstarch content of the diet with respect to their immunoprotection nature will help the fish nutritionist to recast their

feed formulation.

We found from various reports that the G starch supports best growth, whereas NG starch supports the nonspecific

immunity of fish. Hence, the main objective of this work was to optimize the ratio of G and NG starch in the diet of

L. rohita juveniles for the best growth and immunoprotection. The health status ofL. rohita juveniles was evaluated in

terms of immuno-haematological changes after challenge with Aeromonas hydrophila with special reference to the

pre- and post-challenge periods.

2. Materials and methods

2.1. Preparation of the diet

In this experiment maize flour was used as carbohydrate source in the form of G and NG starch. Six isopro-

teinous and isocaloric semi-purified diets were prepared with NG and G starch each at six levels of inclusion

(0, 20, 40, 60, 80 and 100%) were prepared viz., T1 (100% NG, 0% G starch), T2 (80% NG, 20% G starch),

T3 (60% NG, 40% G starch), T4 (40% NG, 60% G starch), T5 (20% NG, 80% G starch) and T6 (0% NG,

100% G starch). All the ingredients were thoroughly mixed with water to make a dough except the vitamin min-

eral mix (EMIX PLUS, India). The dough was then kept for 1 h for proper conditioning followed by steaming for

5 min in a pressure cooker. After cooling, the vitamin mineral mix was mixed thoroughly and the dough was then

pressed through a hand pelletiser of 2 mm diameter to prepare the pellet. Finally the pellets were dried and stored

at 4

C until use (Table 1).

2.2. Experimental design

Labeo rohita juveniles, procured from Palgarh fish farm in Maharashtra, were acclimatized to the experimental

condition for 20 days. Two hundred thirty four juveniles of uniform size (avg. wt. 2.53 0.04 g) were randomlydistributed in six experimental groups with each of three replicates in a plastic tub of 150 l capacity

(80 57 42 cm) with a completely randomized design (CRD). Aeration was provided for 24 h in all the exper-imental tubs. Feed was given at 2.5% of the body weight for 60 days, twice a day at 8:00 and 18:00 h under normal

light regime (12:12 h light:dark cycle). Uneaten feed and faecal matters were siphoned out daily with about 75%

water exchange. The physio-chemical water parameters were maintained within the optimum range as required for

the L. rohita juveniles.

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2.3. Growth

Growth was monitored at 15 days intervals by collectively weighing each group of fish. Fishes were starved

overnight before taking the weight. The weight was taken in an electronic balance. The growth performance was

determined using the following formula:

%weight gain final weight initial weight 100=initial weight:

2.4. Sampling

At the end of the feeding trial of 60 days, the first sampling was carried out for the analysis of the different

blood parameters, respiratory burst and serum lysozyme activity. Three fish from each replicate with a total of

nine fish from each treatment were anaesthetised with clove oil (50 ml l1) and blood was collected from thecaudal vein using a syringe, which was previously rinsed with 2.7% EDTA solution. The blood was then trans-

ferred immediately to an eppendorf tube containing a pinch of dry EDTA powder and shaken gently and kept at

4 C. The blood was used for determination of haemoglobin content, total erythrocyte, leukocyte count and for

NBT assay. For serum, another six fish from each treatment were anaesthetised and blood was collected without

anticoagulant and allowed to clot for 2 h followed by collection of straw coloured serum with a micropipette and

stored at 20 C until use.

2.5. Challenge study

After 10 days of initial sampling, all the fish in the experimental groups were injected intraperitoneally with the

bacterial suspension of 0.2 ml (1.8 108 CFU ml

1). Mortality was observed for all the groups for 10 days. Sampling

Table 1

Composition of the experimental diets (% dry matter)

Ingredient % Inclusion

Caseina 30.57

Gelatinb 8.00

Corn Flour (NG or G)c 42.43

Cellulosed 7.00Sunflower oil: cod liver oil (2:1) 8.00

Carboxymethyl cellulosee 1.00

Vitamin mineral mixf (EMIX PLUS) 2.60Vitamin-Cg 0.10

Vitamin B complexh 0.10

Glycinei 0.20

BHTj 0.02

NG e Non gelatinized, G e Gelatinized.a Casein fat free, 79.5% CP (HiMedia Ltd, India).b Gelatin, 95.7% CP (HiMedia Ltd, India).c Procured from a local market.d Sd Fine Chemicals Ltd., India.e

Sd Fine Chemicals Ltd., India.f Composition of vitamin mineral mix (EMIX PLUS) (quantity/2.5 kg): vitamin A 5,500,000 IU; vitamin D3 1,100,000 IU; vitamin B2 2000 mg;

vitamin E 750 mg; vitamin K 1000 mg; vitamin B6 1000 mg; vitamin B12 6 mg; calcium pantothenate 2500 mg; nicotinamide 10 g; choline chlo-

ride 150 g; Mn 27,000 mg; I 1000 mg; Fe 7500 mg; Zn 5000 mg; Cu 2000 mg; Co 450 mg; Ca 500 g; P 300 g; L-lysine, 10 g; DL-methionine 10 g;

selenium 50 ppm.g Roche, India.h Composition of vitamin B complex (quantity/g). Thiamine mononitrate 20 mg; riboflavin 20 mg; pyridoxine hydrochloride 6 mg; vitamin B12

30 mcg; niaciamide 200 mg; Ca pantothenate 100 mg; folic acid 3 mg; biotin 200 mcg.i HiMedia Ltd, India.j Sd Fine Chemicals Ltd., India.

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of the surviving fish was carried out on the tenth day. A. hydrophila was confirmed after reisolating it from the dead

fish. Survival was calculated by using the following formula:

%survival Number of surviving fish after challenge

Number of fish injected with bacteria 100

2.5.1. Bacteria

Aeromonas hydrophila 018 was received from the Aquatic Animal Health and Management Division, Central

Institute of Fisheries Education (CIFE), Mumbai. A. hydrophila was grown on nutrient broth (HiMedia Ltd, India)

for 24 h at 30 C. The culture broth was centrifuged at 3000 g for 10 min. The supernatant was discarded andthe pellet was resuspended in sterile phosphate buffer saline (PBS, pH 7.4). The final bacterial concentration was

adjusted to 1.8 108 CFU ml1 by serial dilution.

2.6. Haematological parameters

The haemoglobin percentage was determined by estimating cyanmethemoglobin using Drabkins fluid (Qualigens,

India). Five millilitres of Drabkins working solution was taken in a clean and dry test tube and 20 ml of blood was

added to it. The absorbance was measured using a spectrophotometer (MERCK, Nicolet, evolution 100) at a wave-

length of 540 nm. The final concentration was calculated by comparing with standard cyanmethemoglobin (Quali-

gens, India). Total erythrocytes and leukocytes were counted in a haemocytometer using erythrocyte and leukocyte

diluting fluids (Qualigens, India), respectively. Twenty microlitres of blood was mixed with 3980 ml of diluting fluid

in a clean glass test tube. The mixture was shaken well to suspend the cells uniformly in the solution. Then the cells

were counted using a haemocytometer.

The following formula was used to calculate the number of erythrocytes and leukocytes per ml of the blood

sample:

Number of cells ml1Number of cells counted dilution

Area counted depth of fluid

The mean cell volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concen-

tration (MCHC) were calculated as follows:

MCV Haematocrit Hct=RBC 10; MCH Hb=RBC 10; MCHC Hb=Hct 100

Differential count was done by using Abacus Hematology Analyzer (Fully Automated 3 Part Differential Counter,

Austria).

2.7. Respiratory burst activity

Respiratory burst activity of phagocytes was quantified by using the reduction of nitroblue tetrazolium (NBT) to

formazon as a measure of superoxide anion (O2) production as described by Secombes [14] and modified by Stasiak

and Baumann [15]. Fifty microlitres of blood was placed into the wells of U bottom microtitre plates and incubated

at 37 C for 1 h to facilitate adhesion of cells. Then the supernatant was removed and the loaded wells were washed

three times in PBS. After washing, 50 ml of 0.2% NBT was added and was incubated for further 1 h. The cells were

then fixed with 100% methanol for 2e3 min and again washed thrice with 30% methanol. The plates were then air

dried. Sixty microlitres of 2 N potassium hydroxide and 70 ml of dimethyl sulphoxide were added into each well

to dissolve the formazon blue precipitate formed. The OD of the turquoise blue coloured solution was then read in

an ELISA reader at 540 nm.


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2.8. Serum lysozyme activity

Serum lysozyme activity was carried out by using Ion-exchange chromatography kit (Bangalore Genei, India).

Serum samples were diluted with phosphate buffer (pH 7.4) to final concentration of 0.33 mg ml1. In a suitable cu-

vette, 3 ml of Micrococcus luteus (Bangalore Genei, India) suspension in phosphate buffer (A450 0.5e0.7) wastaken, to which 50 ml of diluted serum sample was added. The content of the cuvette was mixed well for 15 s and

reading was taken in a spectrophotometer at 450 nm exactly after 60 s of addition of serum sample. This absorbance

was compared with standard lysozyme of known activity following the same procedure as above. The activity was

expressed as U min1 mg1 protein of serum.

2.9. Serum total protein, albumin and globulin

Serum protein was estimated by Biuret method and BCG dye binding method [16] using the kit (total protein and

albumin kit, Qualigens Diagnostics, Glaxo Smithkline). Albumin was estimated by the bromocresol green binding

method [17]. The absorbance of the standard and test was measured against a blank in a spectrophotometer at

630 nm. Globulin was calculated by subtracting the albumin values from the total serum protein.

2.10. Serum cholesterol, triglyceride and very low density lipoprotein-cholesterol (VLDLC)

Serum total cholesterol was measured by method of Pelkonen et al. [18] by using commercially available kit from

SigmaeAldrich. In brief, 0.02 ml of serum was mixed with 2 ml of reaction solution (enzyme solution with colour

reagent). The absorbance of samples was measured at 540 nm against the reagent blank value.

Serum triglycerides was measured by the method described by Kaplan [19] by using commercially available kit

from SigmaeAldrich. In brief, 10 ml of serum was mixed with 1 ml of reaction solution. The absorbance of sample

was measured against the reaction solution. The increase in absorbance, measured at 540 nm, due to the formation of

the quinoneimine dye, was directly proportional to the triglyceride concentration in the sample. The increase in

absorbance was directly proportional to the glycerol concentration in the sample. True serum triglycerides were

calculated by subtracting the free glycerol concentration in the sample from total triglycerides.

Estimation of VLDLc was calculated by Sattyanarayanan [20]. The value of VLDLc was calculated by density

gradient centrifugation method based on the following formula:

VLDLc total cholesterol HDLLDL

The estimated values of VLDLc were expressed in mg/dl.

0

50

100

150

200

250

300

350

T1 T2 T3 T4 T5 T6

Treatments

Weightgain(%)

T1 (100NG)

T2 (80NG:20G)

T3 (60NG:40G)

T4 (40NG:60G)

T5 (20NG:80G)

T6 (100G)

b

a

b b

ab

a

Fig. 1. Effect of dietary treatment on growth (% weight gain) of Labeo rohita juveniles.


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2.11. Statistical analysis

The main effect of G and NG starch in the diet was tested by one way ANOVA. The comparison of any two mean

values was done by Duncans multiple range test (DMRT). The mean values for pre- and post-challenge parameters

were compared by Students t-test. All the statistical analysis was done by using the software programme SPSS

(version 14).

3. Results

3.1. Growth

The growth performance of fish is presented in Fig. 1. At the end of the experiment weight gain (%) of fish was

significantly higher in T2 group which was similar to T6 group.

3.2. Total erythrocyte count and haemoglobin content

The erythrocyte count (RBC), haemoglobin content (Hb), haematocrit (Hct), mean corpuscular volume (MCV),

mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) during the exper-

iment are given in Table 2. There was no significant difference (P > 0.05) in the total RBC, Hb, Hct, MCV, MCH andMCHC among the different groups before challenge with A. hydrophila. Significant reduction in total RBC and hae-

moglobin content in the post-challenge period was recorded in all the treatments except T2 (80% NG, 20% G starch).

There was significant reduction (P < 0.05) in haematocrit (Hct) and MCV value in the post-challenge period amongall the treatment groups but no change (P > 0.05) was found for the MCH and MCHC values.

3.3. Total leukocyte count and differential cell counts

G:NG starch ratio in the feed had significant effect on the total leukocyte count during pre- and post-challenge

periods (Table 3). As the G:NG starch ratio increased, there was an increase in the WBC count. The highest leukocytecount was observed in T5 group, which was similar to T4 and T6 groups. Lowest count was found in T1 group, which

was similar to T2 and T3 groups.

A general increase in leukocyte count was observed in post-challenge period than their pre-challenged counterparts

irrespective of G:NG starch ratio. The monocyte content was highest in T2 group, which was similar to T1 group dur-

ing both pre- and post-challenge periods. As the G:NG starch ratio increased from 20:80 (T2) to 40:60 (T3), the mono-

cyte content was decreased and remained similar with the rest of the groups. During the pre-challenge period, no

significant differences were observed with respect to lymphocyte and granulocyte counts among the treatment groups,

Table 2

Effects of experimental diets on the haematological parameters of L. rohita after challenged with Aeromonas hydrophila

T1 (0:100)a T2 (20:80)a T3 (40:60)a T4 (60:40)a T5 (80:20)a T6 (100:0)a

RBC (106 cells/mm3) Pre 2.13A 0.18 1.77 0.18 1.87A 0.19 1.91A 0.08 2.35A 0.18 1.99A 0.12Post 1.76B 0.07 1.61 0.13 1.67B 0.12 1.76B 0.04 1.80B 0.03 1.77B 0.06

Haemoglobin (gm%) Pre 9.27A 0.72 8.87 0.64 9.37A 0.77 9.00A 0.72 9.60A 0.65 9.07A 0.20Post 7.93B 0.64 7.60 0.60 7.93B 0.74 7.57B 0.76 8.37B 0.64 7.93B 0.09

Haematocrit (%) Pre 36.47A 1.03 35.47A 1.41 34.03A 0.89 37.30A 0.98 34.30A 1.85 35.27A 0.92Post 23.40B 0.68 22.80B 1.95 22.57B 0.70 21.07B 0.73 20.77B 1.33 21.70B 1.33

MCV (fL) Pre 174.28A 16.90 203.52A 17.30 186.71A 14.35 196.52A 11.49 147.54A 11.69 178.86A 12.06Post 132.83B 2.37 143.86B 19.91 137.18B 4.07 119.96B 6.48 115.53B 8.15 123.41B 11.62

MCH (pg) Pre 43.60 6.56 50.72 8.85 50.49 6.56 47.06 6.56 41.77 13.11 46.09 19.67Post 44.91 2.49 47.47 4.23 47.49 2.89 42.87 3.52 46.40 2.87 44.90 1.06

MCHC (gm/dl) Pre 25.54 2.63 24.93 0.86 27.58 2.44 24.23 2.40 28.13 2.38 25.73 0.59Post 33.89 2.49 33.48 2.17 35.31 3.83 36.05 3.91 40.60 4.03 36.89 2.68

Mean values in a column (pre and post) under each parameter bearing different superscript (A, B) differ significantly (P < 0.05).a The ratio in the parenthesis indicate G:NG.


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Table 3

Effects of experimental diets on the total differential counts, NBT (respiratory burst activity) and serum lysozyme activity of L. rohita after challeng

T1 (0:100)a T2 (20:80)a T3 (40:60)a T4 (60:40)a

WBC (103 cells/mm3) Pre 117.00cB 6.43 122.67bcB 4.67 125.67bcB 5.78 148.00aB 6.11 Post 135.50cA 2.36 140.671bA 5.24 142.27bA 6.30 155.33abA 5.49

Lymphocytes (%) Pre 77.43A 0.12 77.40A 0.30 78.50A 0.25 79.83 1.15 Post 74.20bcB 0.10 73.33cB 0.52 75.60abB 0.06 77.13a 1.33

Monocytes (%) Pre 9.97aB 0.54 10.03aB 0.43 8.63bB 0.30 8.43bB 0.48

Post 11.00

aA

0.50 11.73

aA

0.33 9.70

bA

0.23 9.33

bA

0.38 Granulocytes (%) Pre 12.60B 0.50 12.57 0.72 12.87B 0.50 11.73 1.48 Post 14.80A 0.47 14.93 0.84 14.70A 0.26 13.53 1.68

NBT (A540) Pre 0.13b 0.01 0.15a 0.01 0.12bB 0.01 0.11c 0.01

Post 0.14b 0.01 0.16a 0.01 0.14bcA 0.01 0.13cd 0.01 Lysozyme activity

(Unit/min/mg protein in serum)

Pre 859.02bB 6.56 903.60aB 8.85 832.79bcA 6.56 806.56cA 6.56 Post 1023.14bA 18.45 1122.34aA 17.43 955.83cB 8.61 915.68cB 14.44

Mean values in the same row with different superscript (a, b, c, d, e) differ significantly (P < 0.05).

Mean values in a column (pre and post) under each parameter bearing different superscript (A, B) differ significantly (P < 0.05).a The ratio in the parenthesis indicate G:NG.

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whereas post-challenged fish showed significantly higher (P < 0.05) lymphocyte and granulocyte (except T2, T4 andT6) count than their pre-challenged counterparts.

3.4. NBT assay

The production of superoxide due to NBT reduction in pre- and post-challenge periods was significantly (P < 0.05)influenced by G:NG starch ratio in the diet (Table 3). During pre- and post-challenge periods highest (P < 0.05) NBTvalue was observed in the T2 group containing minimum level of G starch (20%) and lowest in the T5 and T6 groups

containing more than 80% G starch. After challenge with A. hydrophila there was significant increase (P < 0.05) inNBT value in T3, T5 and T6 groups. However, T1 and T2 groups registered no change in NBT value after challenged

with A. hydrophila.

3.5. Serum lysozyme activity

Lysozyme activity gradually decreased with the increasing level of G starch in the diet. There was a significant

difference (P < 0.001) in the lysozyme activity among the various treatment groups in the pre- and post-challengeperiods (Table 3). Lysozyme activity was significantly higher (P < 0.05) in the T2 group during both the pre- andpost-challenge periods. Similarly, post-challenged fish showed significantly higher lysozyme activity (P < 0.05)than their pre-challenged counterparts in T1, T2, T3 and T4 groups and a reverse trend was observed in T5 and T6

groups (high G starch fed groups).

3.6. Serum protein, albumin (A), globulin (G) and albumin/globulin (A/G) ratio

A significant difference (P < 0.05) in the serum total protein, globulin and A/G ratio was found among the varioustreatment groups in the pre- and post-challenge periods (Table 4), whereas no significant difference (P > 0.05) wasobserved in the serum albumin content in any of the treatment groups in both the pre- and post-challenge periods

(Table 4). Serum total protein and globulin content of the post-challenge period was significantly lower (P < 0.05)than the pre-challenged fish of all the treatments.

Highest serum total protein and globulin content was recorded in the T2 group and lowest in T6 group (100% G

starch) and reverse trend was observed for A/G ratio during both the pre- and post-challenge periods. A/G ratio in the

post-challenge fish was significantly higher (P < 0.05) than the pre-challenge period.

3.7. Serum cholesterol, triglycerides and very low density lipoprotein (VLDL)

Serum cholesterol, triglycerides and VLDL during the experiment are given in Table 4. There was significant dif-

ference (P < 0.05) in the serum cholesterol, triglycerides and VLDL content in fish in both pre- and post-challenge

periods.A descending trend of serum cholesterol content was observed with respect to increasing level of G starch in the

diet in both periods. A general decrease of serum cholesterol content in post-challenge period was observed than the

pre-challenge period in the respective groups. An almost similar trend was observed for total triglyceride contents.

The VLDL content decreased with increasing level of G starch in both pre- and post-challenge periods but within

pre- and post-challenge periods the VLDL content was similar except T1 and T2 groups.

3.8. Survival

After challenge withA. hydrophila, the first mortality was recorded after 24 h. Mortality was recorded up to 10 days

after injection. The survival percentage is presented in (Fig. 2). The highest survival was recorded in the T2 group and

the lowest in the T6 group.


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Table 4

Effects of experimental diets on the serum protein and lipid profile of L. rohita after challenged with Aeromonas hydrophila

T1 (0:100)a T2 (20:80)a T3 (40:60)a T4 (60:40)a T

Total protein (gm %) Pre 4.07bA 0.03 4.71aA 0.06 3.73cA 0.13 3.65cA 0.05 Post 3.37bB 0.07 3.86aB 0.09 2.98cB 0.08 3.05cB 0.05

Albumin (A) (gm %) Pre 1.12 0.05 1.14 0.02 1.16 0.02 1.15 0.01 Post 1.15 0.03 1.19 0.07 1.11 0.07 1.20 0.01

Globulin (G) (gm %) Pre 2.95bA 0.02 3.58aA 0.08 2.57cA 0.15 2.49cA 0.04 Post 2.22bB 0.10 2.67aB 0.16 1.87cB 0.01 1.85cB 0.04

A/G ratio Pre 0.38

cdB

0.02 0.31

dB

0.01 0.45

bcB

0.03 0.46

bB

0.01 Post 0.52cdA 0.04 0.45dA 0.05 0.59bcA 0.03 0.65bA 0.01 Cholesterol (mg/dl) Pre 168.50aA 2.50 154.5bA 4.50 154.50bA 0.50 136.50cA 4.50 1

Post 131.00aB 1.00 118.00bB 3.00 110.0bB 4.00 113.00bB 2.00 1Triglycerides (mg/dl) Pre 580.00aA 8.00 468.50bA 7.50 427.00cA 9.00 382.00dA 8.50

Post 369.50aB 5.50 338.50bB 8.50 311.00cB 9.00 255.50dB 5.50 VLDL (mg/dl) Pre 114.40aA 7.20 94.90bA 0.80 85.50bc 3.50 77.20bcd 3.90

Post 76.50aB 4.50 69.00aB 3.00 72.00a 4.00 54.50b 4.50

Mean values in the same row with different superscript (a, b, c) differ significantly (P < 0.05).Mean values in a column (pre and post) under each parameter bearing different superscript (A, B) differ significantly (P < 0.05).a The ratio in the parenthesis indicate G:NG.

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4. Discussion

The gelatinization of starch improves the carbohydrate digestibility [2] and its utilization in fish [13]. In thepresent study, both partial and total replacement of dietary raw starch (non-gelatinized) by gelatinized starch

significantly influenced the blood parameters of L. rohita juveniles and consequently immune response too.

There was no variation in RBC count, haemoglobin content, Hct, MCV, MCH and MCHC among the treatment

groups either in pre- or post-challenge periods. Hence, increased availability of carbohydrate due to increased

gelatinization in the diet did not affect the blood parameters. This is supported by Kumar et al. [2], who

reported that increased availability of carbohydrate due to exogenous amylase or starch modification (gelatini-

zation) did not affect the RBC count and haemoglobin content in L. rohita juveniles. A significant decrease in

RBC count except T2 group in the post-challenge period may be the effect of A. hydrophila infection, which

can be correlated with the observation in Nile tilapia that showed decreased erythrocyte count after bacterial

inoculation [21]. Similarly, the post-challenge decrease in the haemoglobin content in this study is supported

by Foda [22], who reported decreased haemoglobin content in Atlantic salmon due to furunculosis, causedby Aeromonas salmonicida. Recently Misra et al. [3] also reported decreased RBC count and haemoglobin con-

tent in L. rohita juveniles after the challenge with A. hydrophila.

Leukocyte count is considered as an indicator of the health status of fish because of its role in nonspecific or innate

immunity [23]. In the present study, increased WBC count was observed due to increased G:NG starch ratio in the diet.

This may be due to metabolic stress mediated by the supplementation of more G starch. Hilton and Slinger [24] had

reported that gelatinization of starch causes quick absorption of glucose from the intestine and creates metabolic stress

to fish. Roberts [23] stated that in stress condition (due to infection, dietary imbalance etc.) the WBC count increases.

Leukocytes play an important role in the immune response of fish during inflammation [25]. The post-challenge

increase in leukocyte count irrespective of the G:NG starch ratio represents a possible increased inflammatory

response mediated by leukocyte against bacterial infection [3]. A general increase in monocyte count in post-challenge

period was observed compared to the pre-challenge period, which may be caused due to infection ofA. hydrophila.

McLeay [26] and Finn and Nielson [27] observed significant elevation in monocyte count in Oreochromis spp., 8

days after infected with complete Freuds adjuvant.

Phagocytes, after stimulation, are able to generate superoxide anion (O2) and its reactive derivatives (hydro-

gen peroxide and hydrogen radicals) during a period of intense oxygen consumption called respiratory burst

[28,25]. These reactive oxygen intermediates have been reported to have potent bactericidal activities against

fish bacterial pathogen [29,30]. In the present study the respiratory burst activity of phagocytes was measured

by reduction of nitroblue tetrazolium (NBT) by intracellular superoxide radicals produced by leukocytes. Sig-

nificantly increased NBT value in T1 group (100% NG starch) than T6 group (100% G starch) is supported

by observation of Kumar et al. [2] and Misra et al. [3]. Superoxide anion production, measured by NBT reduc-

tion, was maximum at 20:80 G:NG ratio (T2 group), which was significantly higher than all other treatment

groups during both pre- and post-challenge periods, suggesting the maximum production of superoxide anion,

and hence better immunity status of L. rohita juveniles. On the other hand, there was a gradual decrease in

0

20

40

60

80

100

T1 T2 T3 T4 T5 T6

Treatments

Su

rvival(%)

Fig. 2. Percentage survival of different groups after challenge with Aeromonas hydrophila.


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NBT value with the increasing level of G starch content in the diet. This indicates immunity decreases at higher

level of G starch in the diet.

Lysozyme plays an important role in nonspecific immune response and it has been found in mucus, serum and ova

of fish [31]. Innate immunity due to lysozyme is caused by lysis of bacterial cell wall and these stimulate the phago-

cytosis of bacteria [32]. In the present study, during both pre- and post-challenge periods, highest lysozyme activity

was recorded in T2 treatment. As the G:NG ratio increased from 40:60, the lysozyme activity was significantly

decreased. Misra et al. [3] also stated that NG starch fed groups showed higher lysozyme activity than the G starch

fed groups. Increased lysozyme activity in the post-challenge period is in agreement with Vladimirov[33] and Siwicki

and Studnicka [34], who found an enhanced serum lysozyme activity in carp, Cyprinus carpio, after challenge with

A. punctata. The concentration of total protein in blood serum is used as a basic index for health status of fish [35].

Among the serum protein, albumin and globulin are the major protein, which play a significant role in the immune

response. Globulin is important for its immunological responses, especially its gamma component. A significantly

higher serum globulin level in treatment T1 group fed with 100% NG starch than T6 group (fed with 100% G starch)

is in agreement with Kumar et al. [2] and Misra et al. [3] in L. rohita juveniles. There was a significant increase in the

serum globulin level in T2 group fed with diet containing G:NG starch (20:80) than T1 (100% NG starch), after which

the serum globulin content decreased as the G:NG starch ratio increased both during pre- and post-challenge periods.

This suggests an immunosuppressive action of high G starch content in the diet of L. rohita juveniles. In the present

study, the serum albumin content was similar to all the treatments, which is in agreement with the result of Kumar et al.[2] and Misra et al. [3]. Similar trend was also observed for serum total protein. This can be correlated with the result

of Hemre et al. [36] and Kumar et al. [2] that G starch fed groups registered lower serum protein level than their NG

counterparts. Hemre et al. [36] also reported a negative correlation between serum protein with dietary carbohydrate.

Total serum protein and globulin content was significantly reduced after challenge with A. hydrophila. This reduction

may be due to vascular leaking of serum protein [37,38] along with impaired synthesis and nonspecific proteolysis of

serum protein [38].

Highest survival percentage was registered in the T2 group. After challenge with A. hydrophila, the survival of

L. rohita juveniles decreased as the G:NG ratio increased from 40:60. This study shows that high G starch in the

diet had a negative influence on the survival of L. rohita juveniles. Higher mortality in the T6 group fed with

100% G starch could be due to reduced immunomodulatory effect of G starch, which also registered lower leukocyte

count, NBT and lysozyme value in the post-challenge period. Misra et al. [3] also reported that higher mortality in theG starch fed groups than NG starch fed group in L. rohita juveniles.

From the above experiment it reveals that some of the haematological parameters like WBC count, NBT value and

lysozyme activity changes significantly due to variation in G:NG starch ratio in the diet ofL. rohita juveniles. Excess

feeding of G starch causes a metabolic stress and hence lowers the immunological status of the fish. RBC and hae-

moglobin content significantly reduced due to bacterial challenge, however dietary starch content had no effect on

it. But a clear trend of decreasing NBT, lysozyme activity, total serum protein and globulin content with respect to

increasing G starch content of the diet, suggests a negative impact of G starch on immunity on carp. The immunity

status was also reflected as higher survival rate on NG starch fed groups.

All the immunological parameters were in favour of T2 group. Higher growth was also recorded in T2 group (80%

NG, 20% G starch), which was similar to T6 group fed with 100% G starch (Fig. 1). Peres and Oliva-Teles [39]

reported that, both partial and total replacement of dietary raw starch by G starch significantly improved feed

efficiency; however, the total replacement of raw starch by G starch induced a significant reduction in growth rate.

In rainbow trout fed a diet containing 30% starch, it was observed that growth and feed efficiency were improved

by increasing G starch but reached a plateau at around 40% [40].

From the above results it concludes that NG:G starch ratio of 80:20 seems to be optimum for promoting growth and

protecting immunity in L. rohita. This prima facie report may be useful for the nutritionists to recast their feed

formulation for different species.

Acknowledgment

The authors are grateful to the Director, Central Institute of Fisheries Education, Mumbai for providing facilities

for carrying out the work. The first author is grateful to Central Institute of Fisheries Education, Mumbai for awarding

the institutional fellowship.


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References

[1] Priya K, Mukherjee SC, Pal AK, Sahu NP. Effects of dietary lipids histological changes in hepatic tissues of Catla catla fingerlings. Indian

Journal of Veterinary Pathology 2004;28:121e4.

[2] Kumar S, Sahu NP, Pal AK, Choudhury D, Yengkokpam S, Mukherjee SC. Effect of dietary carbohydrate on haematology, respiratory burst

activity and histological changes in L. rohita juveniles. Fish Shellfish Immunol 2005;19:331e44.

[3] Misra S, Sahu NP, Pal AK, Xavier B, Kumar S, Mukherjee SC. Pre- and post-challenge immuno-haematological changes in Labeo rohitajuveniles fed gelatinised or non-gelatinised carbohydrate with n-3 PUFA. Fish Shellfish Immunol 2006;21:346e56.

[4] Yengkokpam S, Sahu NP, Pal AK, Mukherjee SC, Debnath D. Haematological and hepatic changes in Catla catla fingerlings in relation to

dietary sources and levels of gelatinized carbohydrate. Acta ichthyologica et piscatoria 2005;35:87e92.

[5] Barcellos LJG, Nicolaiewsky S, De Souza SMG, Lulhier F. The effects of stocking density and social interaction on acute stress response in

Nile tilapia Oreochromis niloticus (L.) fingerlings. Aquacult Res 1999;30:887e92.

[6] Cruz EM, Ridha M. Production of the tilapia, Oreochromis spilurus (Gunther), stocked at different densities in sea cages. Aquaculture

1991;99:95e105.

[7] Jauncey K. The effect of varying dietary protein level on the growth, food conversion, protein utilization and body composition of juvenile

tilapias (Sarotherodon mossambicus). Aquaculture 1982;27:43e54.

[8] Roberts RJ, Bullock AM. The nutritional pathology of fishes. In: Halver JE, editor. Fish nutrition. London: Academic Press; 1989. p. 423e73.

[9] Roberts RJ. Nutritional pathology of teleosts. In: Roberts RJ, editor. Fish pathology. London: Bailliere Tindall; 1989. p. 337e62.

[10] Lall SP. Salmonid nutrition and feed production. In: Cook RH, Pennel W, editors. Proceedings of the special session on salmonid aquacul-

ture. Los Angeles, CA: World Aquaculture Society; 1991. p. 107e23.

[11] Hemre GI, Mommsen TP, Krogdahl A. Carbohydrates in fish nutrition: effects on growth, glucose metabolism and hepatic enzymes. Aqua-cult Nutr 2002;8:175e94.

[12] Sen PR, Rao NGS, Ghosh SR, Rout M. Observation on the protein and carbohydrate requirement of carps. Aquaculture 1978;13:245e55.

[13] Mohapatra M, Sahu NP, Chaudhari A. Utilization of gelatinized carbohydrate in diets in Labeo rohita fry. Aquacult Nutr 2002;8:1e8.

[14] Secombes CJ. Isolation of salmonid macrophage and analysis of their killing ability. In: Stolen JS, Fletcher TC, Anderson DP, Roberson BS,

Van WB, Winkel M, editors. Techniques in fish immunology. New Jersey: SOS Publication; 1990. p. 137e52.

[15] Stasiack AS, Bauman CP. Neutrophil activity as a potent indicator for concomitant analysis. Fish Shellfish Immunol 1996;5:9.

[16] Reinhold JG. Manual determination of serum total protein, albumin and globulin fractions by Biuret method. In: Reiner M, editor. Standard

method of clinical chemistry. New York: Academic Press; 1953. p. 88.

[17] Doumas BT, Watson W, Biggs HG. Albumin standards and measurement of serum albumin with bromocresol green. Clin Chim Acta

1971;31:87e96.

[18] Palkonen R, Foegelholm R, Nikkila EA. Increase in serum cholesterol during Phenytoin treatment. Br Med J 1975;4:85e7.

[19] Kaplan MM. Clinical and laboratory assessment of thyroid abnormalities. Med Clin North Am 1985;5:69e71.

[20] Sattyanarayanan U. Textbook of biochemistry [1st ed. March 1999]. revised ed. Press Book and Allied Private Limited; 2001. p. 238e41.

[21] Ranzani-Paiva MJT, Ishikawa CM, Eiras AC, Silveira VR. Effects of an experimental challenge with Mycobacterium marinum on the blood

parameters of Nile Tilapia, Oreochromis niloticus (Linnaeus, 1757). Braz Arch Biol Tech 2004;47:945e53.

[22] Foda A. Changes in hematocrit and hemoglobin in Atlantic salmon (Salmo salar) as a result of furunculosis disease. J Fisheries Res Board of

Canada 1973;30:467e8.

[23] Roberts RJ. The pathophysiology and systemic pathology of teleosts. In: Roberts RJ, editor. Fish pathology. London: Bailliere Tindal; 1978.

p. 55e91.

[24] Hilton JW, Slinger SJ. Effect of wheat bran replacement of wheat midlings in extrusion processed (floating) diets on the growth of juvenile

rainbow trout (Salmo gairdneri). Aquaculture 1983;35:201e10.

[25] Secombes CJ. The nonspecific immune system: cellular defences. In: Iwama G, Nakanishi T, editors. The fish immune system, organism,

pathogen and environment. Toronto: Academic Press; 1996. p. 63e103.

[26] McLeay DJ. Effects of a 12-hr and 25-day exposure to kraft pulp mill effluent on the blood and tissues of juvenile coho salmon (Oncorhyn-

chus kisutch). J Fish Res Board Can 1973;30:395e400.

[27] Finn JP, Nielson NO. The inflammatory response of rainbow trout. J Fish Biol 1971;3:463e78.

[28] Secombes CJ, Fletcher TC. The role of phagocytes in protective mechanism of fish. Annu Rev Fish Dis 1992;2:53e

71.[29] Hardie LJ, Ellis AE, Scomber CJ. In vitro activation of rainbow trout macrophages stimulates inhibition of Renibacterium salmoninarum

growth concomitant with augmented generation of respiratory products. Dis Aquat Org 1996;25:175e83.

[30] Itou T, Iida T, Kawatsu H. The importance of hydrogen peroxide in phagocytic bactericidal activity of Japanese eel neutrophils. Fish Pathol

1997;32:121e5.

[31] Murray CK, Fletcher TC. The immunohistochemical localization of lysozyme in plaice (Pleuronectes platessa L.) tissues. J Fish Biol

1976;9:32e4.

[32] Ellis AE. Lysozyme assays. In: Stolen JS, Fletcher TC, Anderson BS, Van Muiswinkel WB, editors. Techniques in fish immunology. Fair

Haven (NJ, USA): SOS Publications; 1990. p. 101e3.

[33] Vladimirov VL. Immunity in fish. Bull Off Int Epizoot 1968;69:1365e72.

[34] Siwicki A, Studnica M. The phagocytic ability of neutrophils and serum lysozyme activity in experimentally infected carp, Cyprinus carpio.

J Fish Biol 1987;31(A):57e60.

[35] Mulcahy MF. Serum protein changes associated with ulcerative dermal necrosis (UDN) in the trout Salmo trutta L. J Fish Biol 1971;3:

199e201.


7/30/2019 1-s2.0-S1050464806002233-main

13/13

[36] Hemre GI, Waagbo R, Hjeltnes B, Aksnes A. Effect of gelatinized wheat and maize in diets for large Atlantic salmon (Salmo salar L.) on

glycogen retention, plasma glucose and fish health. Aquacult Nutr 1996;2:33e9.

[37] Green JH. Tissue fluids and lymph. In: Basic clinical physiology. UK: Oxford University Press; 1978. p. 49e52.

[38] Ellis AE, Hastings TS, Munro ALS. The role ofAeromonas salmonicida extracellular products in the pathology of furunculosis. J Fish Dis

1981;4:41e52.

[39] Peres MH, Oliva-Teles A. Utilization of raw and gelatinized starch by European sea bass (Dicentrarchus labrax) juveniles. Aquaculture

2002;205:287e99.

[40] Jeong KS, Takeuchi T, Okamoto N, Watanabe T. The effect of dietary gelatinized ratios at different dietary energy levels on growth and

characteristics of blood in rainbow trout fingerlings. Nippon Suisan Gakkaishi 1992;58:937e44.


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