Pre and post-challenge immuno-haematological changes in Labeo rohita juveniles fed gelatinised or non-gelatinised carbohydrate with n-3 PUFA

Fish & Shellfish Immunology 21 (2006) 346e356www.elsevier.com/locate/fsi

Pre- and post-challenge immuno-haematological changes inLabeo rohita juveniles fed gelatinised or non-gelatinised

carbohydrate with n-3 PUFA

Sougat Misra a, N.P. Sahu a,*, A.K. Pal a, Biji Xavier a,Shivendra Kumar a, S.C. Mukherjee b

a Department of Fish Nutrition and Biochemistry, Central Institute of Fisheries Education, Versova, Mumbai 400061, Indiab Department of Fish Pathology and Microbiology, Central Institute of Fisheries Education, Versova, Mumbai 400061, India

Received 7 October 2005; revised 15 December 2005; accepted 21 December 2005

Available online 10 March 2006

Abstract

The combined effect of dietary carbohydrate type and n-3 PUFA (EPA þ DHA) on pre- and post-challenge haemato-immunological responses in Labeo rohita juveniles was studied. Fish were fed for 67 days with six different test diets containing eithergelatinised (G) or non-gelatinised (NG) corn (43%) with three levels of n-3 PUFA (0.5%, 1.0% and 2.0%). During the pre-challenge period, significantly higher (P < 0.05) NBT, serum lysozyme activity, total protein and globulin content was recordedin the NG carbohydrate fed groups. Highest NBT value was recorded in the groups fed with 1.0% n-3 PUFA, whereas the highestserum lysozyme activity (P < 0.05) was recorded at either 0.5% or 2.0% n-3 PUFA fed groups in both the pre- and post-challengeperiod. Feeding of NG corn significantly increased the total leucocyte count, lysozyme activity, A/G ratio and decreased the totalerythrocyte count, haemoglobin, serum total protein and globulin content of L. rohita juveniles during the post-challenge period. Sim-ilarly, feeding of n-3 PUFA at any level significantly increased the immunological parameters like lysozyme activity or A/G ratio,whereas total leukocyte count increased due to feeding of either 0.5% or 1.0% n-3 PUFA. The NBT and albumin values remainedsimilar in both the pre- and post-challenge period. After challenge with Aeromonas hydrophila, the highest survival was recordedin the NG carbohydrate fed groups, whereas the lowest survival was recorded in the highest level of n-3 PUFA fed group irrespectiveof dietary carbohydrate type. Thus, a high level of G carbohydrate as well as n-3 PUFA is found to be immunosuppressive in L. rohitajuveniles. NG carbohydrate supplemented with 1.0% n-3 PUFA is found to be optimum to enhance the immunity in L. rohita juveniles.� 2006 Elsevier Ltd. All rights reserved.

Keywords: Carbohydrate; n-3 PUFA; NBT; Lysozyme activity; Serum protein; Labeo rohita; Aeromonas hydrophila

1. Introduction

Labeo rohita, an Indian Major Carp, is one of the most preferred species in the Indian subcontinent, which con-tributes about 35% of the total carp production [1]. Development of an economical artificial feed to accelerate the

* Corresponding author. Tel.: þ91 22 26361466; fax: þ91 22 26361573.

E-mail address: [email protected] (N.P. Sahu).

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

doi:10.1016/j.fsi.2005.12.010

347S. Misra et al. / Fish & Shellfish Immunology 21 (2006) 346e356

growth and to maintain the health status of this species is of prime importance for sustainable carp culture. Increasedutilisation of dietary carbohydrate has been tried by many researchers to reduce the feed cost considering the abundantavailability and low cost of carbohydrate sources. Requirement of dietary carbohydrate has been reported to be 26%for the Labeo rohita [2]. But, further study revealed that a dietary level of 40% gelatinised carbohydrate improved thegrowth without any detrimental effect on health [3]. Less work has been done on the health status of fish with respectto high dietary carbohydrate [4e7]. In rainbow trout, long term feeding with a high carbohydrate diet was found tohave no effect on the non-specific immunity [8]. Recently, it has been reported that feeding non-gelatinised carbohy-drate enhances the non-specific immunity of L. rohita compared to gelatinised carbohydrate [9].

Besides carbohydrate, long chain polyunsaturated fatty acids (PUFA) play an important role for normal growth andhealth status of fish [10e12]. The functional role of n-3 and n-6 PUFA in non-specific and specific humoral and cel-lular immunity is not clearly defined in fish [13e15]. The role of n-3 PUFA on the immunity of fish is of paramountinterest apart from its nutritional value. Long chain highly unsaturated fatty acid (HUFA) can be converted into eicos-anoids, which play an important role as mediator of the immune response. An adequate amount of n-3 fatty acid wasfound to be essential in maintaining the alternative complement activity in gilthead sea bream [16]. Besides, stabilityenhancement of the cell membrane mediated by n-3 HUFA is of special importance in fish for maintaining propermembrane function in the aquatic environment, which varies widely with temperature [17]. In general, cell membraneenriched with n-3 PUFA is also associated with decreased inflammatory response, improvements of growth rate, andeither increased or no change in specific immunity [18]. n-3 PUFA mediated enhancement of immune function is oftendose responsive. Dietary supplementation of high n-3 PUFA resulted in lowering of the antibody titre and survival ratein Atlantic salmon [19]. Low disease resistance and phagocytic capacity with low killing activity was reported inchannel catfish, when fed with a high level of n-3 PUFA [20]. Thus, it is evident that an optimum level of long chainn-3 PUFAs in the feed plays an important role in the immunity of fish.

Supplementation of high carbohydrate along with an optimum level of n-3 PUFA in the diet of fish can be of primeinterest in aquaculture practice to minimise the cost of the feed, and to maintain the health status of the farmed animal.The health status of L. rohita juveniles in this study was evaluated in terms of immuno-haematological changes afterchallenge with Aeromonas hydrophila, with special reference to the pre- and post-challenge period. To the best of ourknowledge, there are no reports available regarding the pre- and post-challenge changes of immuno-haematologicalparameters of fish when fed with high dietary carbohydrate along with different levels of n-3 HUFA. These data maybe helpful for dietary manipulation of similar species for maintaining the health status of fish, when fed with a highcarbohydrate diet.

2. Materials and methods

2.1. Preparation of diet

Maize flour either gelatinised or non-gelatinised were used as the source of carbohydrate to be studied in thisexperiment. Six isocaloric diets with two type of carbohydrates: non-gelatinised (NG) and gelatinised (G); three levelsof n-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA): 0.5%, 1.0% and 2% were prepared; viz.T1 (NG, 0.5% EPA and DHA), T2 (NG, 1.0% EPA and DHA), T3 (NG, 2.0% EPA and DHA), T4 (G, 0.5% EPA andDHA), T5 (G, 1.0% EPA and DHA), T6 (G, 2.0% EPA and DHA) (Table 1). All the ingredients were thoroughly mixedwith water to make a dough except the EPA and DHA concentrate (Maxepa, MERCK, India) and the vitamin mineralmix (EMIX PLUS, India) followed by steaming for 5 min in a pressure cooker. After cooling the dough, EPA andDHA concentrate and vitamin mineral mix were mixed properly and the dough was passed through a hand pelletiserof 2 mm diameter to prepare the pellet. Finally the pellets were dried and stored at 4 �C until use.

2.2. Experimental design

Labeo rohita juveniles, procured from Palgarh fish farm in Maharashtra, were acclimatised to the experimentalcondition for 15 days. Two hundred and sixteen juveniles of uniform size (8.07 � 0.12 g) were randomly distributedin six experimental groups with each of three replicates in a plastic tub of 150 l capacity (80 � 57 � 42 cm) followinga completely randomised design. Round the clock aeration was provided to all the experimental tubs. Feed was givenat 2.5% of body weight for 60 days twice daily at 10:00 and 18:00 h under a normal light regime (light/dark 12/12 h).

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Uneaten feed and faecal matter were siphoned out daily with about 80% water exchange with fresh bore well water.The temperature and the dissolved oxygen of the rearing medium were maintained at 24e27 �C and 5.6e6.8 mg l�1,respectively, throughout the experimental period.

2.3. Sampling

After completion of the feeding trial of 60 days, the first sampling was carried out for the analysis of the bloodparameters, respiratory burst and serum lysozyme activity. Two fish from each replicate with a total of six fishfrom each treatment were anaesthetised with clove oil (50 ml l�1) and blood was collected from the caudalvein using a syringe, which was previously rinsed with 2.7% EDTA solution. The blood was then transferred im-mediately to an Eppendorf tube containing a pinch of EDTA powder and shaken gently and kept at 4 �C. Theblood was used for determination of haemoglobin content, total erythrocyte and leukocyte count and for NBTassay. For serum, another six fish from each treatment were anaesthetised and blood was collected without anti-coagulant and allowed to clot for 2 h followed by collection of straw coloured serum with micropipette and storedat �20 �C until use.

2.4. Bacteria

Aeromonas hydrophila 018 was received from the Aquatic Animal Health and Management Division, CentralInstitute 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 wasadjusted to 1.8 � 108 CFU ml�1 by serial dilution.

Table 1

Composition of the experimental diets (% dry matter)

Ingredient % inclusion

0.5% EPA þ DHA 1.0% EPA þ DHA 2.0% EPA þ DHA

Caseina 30.0 30.0 30.0

Gelatinb 6.2 6.2 6.2

Corn flour (NG or G)c 43.0 43.0 43.0

Cellulosed 9.5 9.5 9.5

Sunflower oil 6.5 5.0 2.0

EPA-DHA concentratee 1.5 3.0 6.0

Carboxymethyl cellulosef 1.0 1.0 1.0

Vitamin þ mineral mixg 1.0 1.0 1.0

Vitamin Ch 0.5 0.5 0.5

Betaine hydrochloridei 0.275 0.275 0.275

Chromium oxidej 0.5 0.5 0.5

NG, non-gelatinised; G, gelatinised.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 Composition of EPA-DHA concentrate (quantity/capsule): EPA 180 mg, DHA 120 mg, MERCK, India.f Sd Fine Chemicals Ltd., India.g 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 chloride

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.h Roche, India.i Sd Fine Chemicals Ltd., India.j Sd Fine Chemicals Ltd., India.

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2.5. Challenge study

After 7 days of initial sampling, all the fish in the experimental groups were injected intraperitoneally with thebacterial suspension of 0.2 ml (1.8 � 107 CFU ml�1). Mortality was observed for all the groups for 10 days. Samplingof the surviving fish was carried out on the 10th day. Aeromonas hydrophila was confirmed after reisolating it from thedead fish. Survival was calculated using the following formula:

Relative % survival¼ Number of surviving fish after challenge

Number of fish injected with bacteria� 100

2.6. Haematological parameters

The haemoglobin percentage was determined by estimating cyanmethemoglobin using Drabkin’s fluid (Qualigens,India). Five millilitres of Drabkin’s working solution was taken in a clean and dry test tube and 20 ml of blood wereadded 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 erythrocyte and leucocyte were counted in a haemocytometer using erythrocyte and leucocyte diluting fluids(Qualigens, India), respectively. Twenty microlitres of blood were mixed with 3980 ml of diluting fluid in a clean glasstest tube. The mixture was shaken well to suspend the cells uniformly in the solution. Then the cells were countedusing a haemocytometer. The following formula was used to calculate the number of erythrocytes and leucocytesper ml of the blood sample:

Number of cells ml�1 ¼ Number of cells counted� dilution

Area counted� depth of fluid

2.7. Respiratory burst activity

The respiratory burst activity of the phagocytes was measured by nitroblue tetrazolium (NBT) assay following themethod of Secombes [21] subsequently modified by Stasiack and Baumann [22]. Fifty microlitres of blood wereplaced into the wells of ‘U’ bottom microtitre plates and incubated at 37 �C for 1 h to allow adhesion of cells.Then the supernatant was removed and the wells were washed three times with PBS. After washing, 50 ml of 0.2%NBT were added and incubated for a further 1 h. The cells were then fixed with 100% methanol for 2e3 min andwashed three times with 30% methanol. The plates were air-dried and 60 ml of 2 N potassium hydroxide and 70 mlof dimethyl sulphoxide were added to each well. The OD was recorded in an ELISA reader at 540 nm.

2.8. Serum total protein, albumin and globulin

Serum protein was estimated by the Biuret and BCG dye binding method [23] using a kit (total protein and albuminkit, Qualigens Diagnostics, Glaxo Smithkline). Albumin was estimated by the bromocresol green binding method[24]. 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.

2.9. Serum lysozyme activity

Serum lysozyme activity was measured using an ion exchange chromatography kit (Bangalore Genei, India).Serum samples were diluted with phosphate buffer (pH 7.4) to a final concentration of 0.33 mg ml�1 protein. In a suitablecuvette, 3 ml of Micrococcus luteus (Bangalore Genei, India) suspension in phosphate buffer (A450 ¼ 0.5e0.7) wastaken, to which 50 ml of diluted serum sample were added. The content of the cuvette was mixed well for 15 s anda reading was taken in a spectrophotometer at 450 nm exactly 60 s after the addition of the serum sample. This

350 S. Misra et al. / Fish & Shellfish Immunology 21 (2006) 346e356

absorbance was compared with standard lysozyme of known activity following the same procedure as above. Thelysozyme activity was expressed as U min�1 per mg protein of serum.

2.10. Statistical analysis

The main effect of carbohydrate type and n-3 PUFA and their interaction was done by two-way ANOVA. Thecomparison of any two mean values was done by Duncan’s multiple range test (DMRT). The mean values forpre- and post-challenge parameters were compared by Student’s t-test. All the statistical analysis was done usingthe software programme SPSS (version 11).

3. Results

3.1. Total erythrocyte count and haemoglobin content

Total erythrocyte count and haemoglobin content of L. rohita juveniles during the experiment are shown in Table 2.There was a significant difference (P < 0.05) in the total erythrocyte count among the different treatment groups be-fore the challenge with Aeromonas hydrophila. Dietary carbohydrate type had no effect on the total erythrocyte counteither at the pre- or post-challenge period. Significant reduction in erythrocyte count in the post-challenge period wasrecorded in the NG carbohydrate fed groups. Significantly lower (P < 0.05) erythrocyte count was recorded in thegroups fed the lowest level of n-3 PUFA compared to 1.0% and 2.0% during the pre-challenge period. Erythrocytecount in the post-challenge period was not affected by dietary n-3 PUFA. A significant interaction was recorded inNG carbohydrate � n-3 PUFA in the pre- and post-challenge period. The erythrocyte count of the G carbohydratefed group either at 1.0% or 2.0% n-3 PUFA was significantly reduced in the post-challenge period.

Like the total erythrocyte count, haemoglobin content was not affected either at the pre- or post-challenge perioddue to dietary carbohydrate type. However, a significant decrease (P < 0.05) in haemoglobin content was found in thepost-challenge period compared to the pre-challenge period irrespective of the type of carbohydrate fed to the juve-niles. A similar trend was reflected for dietary n-3 PUFA during the pre- and post-challenge period. A significantinteraction of carbohydrate � n-3 PUFA on haemoglobin content was recorded both in the pre- and post-challengeperiod. Haemoglobin content of all the G carbohydrate fed groups at any level of n-3 PUFA in the pre-challenge periodwas significantly higher than the post-challenge period.

Table 2

Dietary carbohydrate type and n-3 PUFA level on RBC (�106 cells ml�1), WBC (�103 cells ml�1), haemoglobin content (g/dl) of different exper-

imental groups

Treatment RBC WBC Haemoglobin

Pre challenge Post challenge Pre challenge Post challenge Pre challenge Post challenge

Carbohydrate type (CHO)

NG 1.32 � 0.04B 1.14 � 0.03A 61.28 � 1.06A 71.05 � 1.13bB 7.38 � 0.12B 6.31 � 0.15A

G 1.33 � 0.05 1.16 � 0.03 60.10 � 0.99A 67.67 � 1.06aB 7.53 � 0.51B 6.24 � 0.22A

Fatty acids level (FA)

0.5% 1.09 � 0.03a 1.12 � 0.05 59.65 � 0.88aA 70.56 � 0.84B 6.85 � 0.06aB 5.96 � 0.27A

1.0% 1.42 � 0.02bB 1.13 � 0.02A 57.55 � 0.96aA 71.42 � 1.26B 7.59 � 0.25bB 6.35 � 0.15A

2.0% 1.47 � 0.04bB 1.19 � 0.04A 64.87 � 0.84b 66.10 � 1.08 7.91 � 0.11bB 6.52 � 0.17A

CHO � FA

NG � 0.5 1.14 � 0.05a 1.11 � 0.02 60.89 � 1.39aA 71.86 � 0.94cB 6.83 � 0.12a 6.26 � 0.31

NG � 1.0 1.48 � 0.04cB 1.10 � 0.02A 57.85 � 1.51aA 73.57 � 0.48cB 7.55 � 0.51b 6.22 � 0.29

NG � 2.0 1.35 � 0.06b 1.19 � 0.08 65.10 � 1.37b 67.73 � 0.37b 7.74 � 0.19bB 6.45 � 0.38A

G � 0.5 1.05 � 0.02a 1.13 � 0.11 58.40 � 0.94aA 69.26 � 0.14bB 6.86 � 0.06aB 5.66 � 0.40A

G � 1.0 1.45 � 0.02cB 1.16 � 0.03A 57.25 � 1.30aA 69.26 � 0.23aB 7.64 � 0.09bB 6.48 � 0.13A

G � 2.0 1.49 � 0.03cB 1.18 � 0.02A 64.63 � 1.09b 64.47 � 1.20a 8.07 � 0.07bB 6.59 � 0.04A

Mean values (n ¼ 6) in a column under each category bearing different superscript (lower case) vary significantly (P < 0.05). Mean values (n ¼ 6)

in a row under each parameter bearing different superscript (capital) vary significantly (P < 0.05). NG, non-gelatinised; G, gelatinised.

351S. Misra et al. / Fish & Shellfish Immunology 21 (2006) 346e356

3.2. Total leukocyte count

Dietary carbohydrate had no effect on the total leucocyte count during pre-challenge period. However, there wasa significant reduction in the total leukocyte count in the G carbohydrate fed groups than the NG carbohydrate fedgroups during the post-challenge period. But a universal increase in leukocyte count was observed in post-challengedfish than their pre-challenged counterparts either due to feeding of NG or G carbohydrate. The highest level of dietaryn-3PUFA (2.0%) increased the leukocyte count in the pre-challenge period only. The post-challenge period showedincreased numbers of leukocyte than the pre-challenge period either at 0.5% or 1.0% dietary PUFA. Similarly, a higherleukocyte count was recorded in the post-challenge period, either fed with G or NG carbohydrate at 0.5% or 1.0% n-3PUFA than their pre-challenge counterparts.

3.3. NBT assay

The respiratory burst activity (NBT reduction) of neutrophils of L. rohita juveniles was higher in NG carbohydratefed groups than G carbohydrate fed groups during the pre-challenge period (Table 3). However, the type of carbohy-drate had no effect on NBT value during the post-challenge period. No variation in NBT value was observed in pre-and post-challenged juveniles either due to feeding of G or NG carbohydrates.

The highest NBT value was recorded in those groups fed with 1.0% n-3 PUFA either in the pre- or post-challengeperiod. No significant changes in the pre- or post-challenge NBT value were recorded due to feeding of n-3 PUFA.

3.4. Serum lysozyme activity

There was a significant difference (P < 0.001) in lysozyme activity among the various treatment groups in the pre-and post-challenge period (Table 3). Lysozyme activity was significantly higher (P < 0.05) in NG carbohydrate fedgroups than the G carbohydrate fed groups during both the pre- and post-challenge period. Similarly, post-challengedfish showed significantly higher lysozyme activity (P < 0.05) than the pre-challenged fish either fed with G or NGcarbohydrates. Dietary n-3 PUFA at each level showed a significantly (P < 0.05) higher lysozyme activity in thepost-challenge period than its pre-challenge value. A combination of NG carbohydrate either with 1.0% or 2.0%n-3 PUFA had higher lysozyme activity in the post-challenge period than in the pre-challenge period.

Table 3

Dietary carbohydrate type and n-3 PUFA level on NBT (A540) and serum lysozyme (unit min�1 per mg serum protein) activity of different

experimental groups

Treatment NBT Lysozyme activity

Pre challenge Post challenge Pre challenge Post challenge

Carbohydrate type (CHO)

NG 0.129 � 0.006b 0.138 � 0.006 725.74 � 30.70bA 949.28 � 29.33bB

G 0.107 � 0.004a 0.121 � 0.003 655.45 � 34.56aA 848.09 � 41.73aB

Fatty acids level (FA)

0.5% 0.108 � 0.006a 0.128 � 0.006a 736.74 � 21.55bA 898.36 � 40.37abB

1.0% 0.134 � 0.009b 0.141 � 0.007b 730.45 � 44.07bA 989.34 � 40.17bB

2.0% 0.113 � 0.005a 0.120 � 0.004a 604.59 � 21.62aA 809.02 � 44.86aB

CHO � FA

NG � 0.5 0.124 � 0.008bc 0.135 � 0.012ab 735.16 � 20.57bc 961.75 � 56.12b

NG � 1.0 0.143 � 0.009c 0.155 � 0.006b 801.88 � 16.15cA 1008.20 � 43.63bB

NG � 2.0 0.120 � 0.006bc 0.126 � 0.007a 640.16 � 15.57abA 879.23 � 44.87abB

G � 0.5 0.092 � 0.002aA 0.121 � 0.008aB 738.31 � 48.57bc 834.97 � 49.15ab

G � 1.0 0.124 � 0.007bc 0.128 � 0.003a 659.02 � 34.42abA 970.49 � 71.11bB

G � 2.0 0.106 � 0.007a 0.113 � 0.001a 569.02 � 5.59a 738.79 � 69.78a

Mean values (n ¼ 6) in a column under each category bearing different superscript (lower case) vary significantly (P < 0.05). Mean values (n ¼ 6) in

a row under each parameter bearing different superscript (capital) vary significantly (P < 0.05). NG, non-gelatinised; G, gelatinised.

352 S. Misra et al. / Fish & Shellfish Immunology 21 (2006) 346e356

3.5. Serum protein, albumin, globulin and A/G ratio

There were significant differences in the serum total protein and globulin content among the different experimentalgroups before and after the challenge study (Table 4). NG carbohydrate fed groups showed significantly higher totalprotein and globulin content in both the pre- and post-challenge period. A significant decrease (P < 0.05) in the serumtotal protein and globulin content was recorded after challenge with Aeromonas hydrophila either fed with G or NGcarbohydrate. Globulin content of the post-challenged fish was significantly (P < 0.05) lower than the pre-challengedfish either fed with G or NG carbohydrate with 1.0% or 2.0% n-3 PUFA.

No changes in the serum albumin content were recorded either pre- or post-challenge studies. The A/G ratio wassignificantly (P < 0.05) increased in the post-challenge period than the pre-challenged period irrespective of the typeof carbohydrate fed. NG carbohydrate fed groups recorded a significantly lower A/G ratio after challenge. A similartrend was also observed for all levels of n-3 PUFA in the diet. During the post-challenge period, a significantly(P < 0.05) higher A/G ratio was recorded in the groups fed 2.0% n-3 PUFA irrespective of dietary carbohydrate type.

3.6. Survival

After injection with Aeromonas hydrophila, the first mortality was recorded after 12 h. Mortality was recorded upto 7 days after injection. The relative % survival is presented in Fig. 1. The highest survival was recorded in the T2

group and the lowest survival in the T6 group. NG carbohydrate fed groups showed significantly higher % survival.Significantly lower survival was recorded for the 2.0% n-3 PUFA fed groups irrespective of dietary carbohydrate type.

4. Discussion

The present study indicates that dietary carbohydrate type and n-3 PUFA affect the blood parameters of L. rohitajuveniles and consequently the immune response too. The erythrocyte count among the various treatment groupsvaried significantly before the challenge study. Erythrocyte number was significantly higher in fish fed 1.0% and2.0% n-3 PUFA, which could be related to a higher oxygen requirement due to a higher peroxisomal b-oxidation[25]. A significant decrease in erythrocyte count in the post-challenge period can be correlated with the observationin Nile tilapia that showed decreased erythrocyte numbers after bacterial inoculation [26]. Anaemia in diseased fishinfected with Aeromonas hydrophla is a gross sign [27] and decrease in the erythrocyte count in the present study maycorrelate to this. Dietary carbohydrate type had no effect on haemoglobin content in L. rohita juveniles, which is inagreement with Kumar et al. [9]. Like erythrocytes, the haemoglobin content was higher in the 1.0% and 2.0% n-3PUFA fed groups corresponding to the higher erythrocyte content of these groups during the pre-challenge period.The post-challenge decrease in the haemoglobin content in this study is in agreement with Foda [28] in Atlanticsalmon due to furunculosis, caused by Aeromonas salmonicida.

Leucocytes play an important role in non-specific or innate immunity and their count can be considered as anindicator of the health status of fish. A significantly higher WBC count was recorded at the highest level (2.0%) ofdietary n-3 PUFA. This may be due to the metabolic stress mediated by supplementation of a high level of n-3PUFA. Leucocytes play an important role in the immune response of fish during inflammation [14]. In the presentstudy, post-challenge increase in the leukocyte count irrespective of dietary carbohydrate as well as n-3 PUFA levelrepresents a possible increased inflammatory response mediated by leucocytes against bacterial infection [29].

Both macrophages and neutrophilic granulocytes are characterised by having high phagocytic activity against theinvading microorganisms, acting as the first line of defence to eliminate infectious agents. During phagocytosis, stim-ulation of the cell membrane triggers the production of oxygen free radicals by generating superoxide anion (O2

�) andits derivatives such as hydrogen peroxide (H2O2) and hydroxyl free radicals (OH

�). This process is known as respira-

tory burst. These reactive oxygen intermediates have been reported to have potent bactericidal activities against fishbacterial pathogens [30,31]. Increased respiratory burst activity in the NG carbohydrate fed groups before challenge inthis study is supported by the observation of Kumar et al. [9] in L. rohita. In the same study, lowering of the NBT valuein the G carbohydrate fed groups is consistent with the present experiment. Superoxide anion production, measured byNBT reduction, was maximum at 1.0% n-3 PUFA supplemented groups, suggesting the maximum enhancement ofimmunity at this level in L. rohita juveniles.

353S. Misra et al. / Fish & Shellfish Immunology 21 (2006) 346e356

Fish serum lysozyme is believed to be leucocyte origin [32,33]. Lysozyme is known to act as an important innatedefence mediator against parasitic, bacterial and viral infections and in response to infection, its activity is found toincrease in fish blood [34]. Lysozyme plays an important role in innate immunity by lysis of the bacterial cell wall andthus stimulating the phagocytosis of bacteria [35]. An earlier study [8] indicated that dietary carbohydrate did notaffect the lysozyme activity, but in both the pre- and post-challenge period, a higher lysozyme activity was recordedin the NG carbohydrate fed groups in the present study. An increase in the post-challenge lysozyme activity was foundirrespective of the dietary carbohydrate type and n-3 PUFA level in the diet. This is in agreement with the observationin carp, Cyprinus carpio, which showed an enhanced serum lysozyme activity after challenge with Aeromonas punc-tata [36,37]. A similar observation was also observed in Atlantic salmon experimentally challenged with Aeromonassalmonicida [38]. A significant increase in the leukocyte count after challenge can correlate with the above observa-tion. Many substances and bacterial preparations mediate phagocyte activation with a corresponding increase in thesynthesis of non-specific defence factors [39e41]. Increase in the c-type lysozyme m-RNA in head kidney, spleenand ovary in Japanese flounder during experimental infection with Edwardsiella tarda suggested the augmentationof lysozyme synthesis [42], which supports the results of present study. In the present study, an increased serumlysozyme level in the infected fish is indicative of an activation of the non-specific immune system.

Among the serum proteins, albumin and globulin are the major proteins, which play a significant role in theimmune response. Globulins like gamma globulin are absolutely essential for maintaining a healthy immune systemand contain all the immunoglobulin in the blood. A higher serum globulin level in the pre-challenge period is in agree-ment with Kumar et al. [9] who reported a higher globulin level due to NG carbohydrate feeding in L. rohita juveniles.The lowest globulin level at 2.0% n-3 PUFA supplemented groups both in the pre- and post-challenge period suggestsan immunosuppressive action of high n-3 PUFA. In the present study, the serum albumin content was similar in all thetreatments, which is in agreement with the result of Kumar et al. [9]. Serum protein was higher in NG carbohydrate fedgroups supplemented with 1.0% n-3 PUFA. Gelatinised carbohydrate fed groups registered lower serum proteinlevels, in agreement with Hemre et al. [43] and Kumar et al. [9]. Hemre et al. [43] reported a negative correlationof serum protein with dietary carbohydrate. After challenge study, reduction of serum protein may be due to vascularleaking of serum protein because of increased permeability [44,45] along with impaired synthesis and non-specificproteolysis of serum protein [46].

After challenge with Aeromonas hydrophila, the highest mortality was observed in the group fed with G carbohy-drate with 2.0% n-3 PUFA. This may be due to increased susceptibility of juveniles to infection due to stress caused byexcess carbohydrate along with excess n-3 PUFA. In Atlantic salmon, reduced antibody titres and low survival were

Table 4

Dietary carbohydrate type and n-3 PUFA level on serum total protein (g dl�1), albumin (g dl�1), globulin (g dl�1) and albumin to globulin ratio of

different experimental groups

Treatment Total protein Albumin (A) Globulin (G) A/G ratio

Pre challenge Post challenge Pre challenge Post challenge Pre challenge Post challenge Pre challenge Post challenge

Carbohydrate type (CHO)

NG 4.09 � 0.03bB 3.71 � 0.14bA 1.14 � 0.02 1.19 � 0.01 2.95 � 0.03bB 2.52 � 0.14bA 0.39 � 0.01A 0.48 � 0.03aB

G 3.83 � 0.07aB 3.39 � 0.06aA 1.12 � 0.02 1.18 � 0.01 2.71 � 0.07aB 2.21 � 0.06aA 0.42 � 0.01A 0.53 � 0.02bB

Fatty acids level (FA)

0.5% 4.16 � 0.04b 3.71 � 0.11b 1.17 � 0.02 1.20 � 0.01 2.99 � 0.04b 2.52 � 0.10b 0.39 � 0.01A 0.48 � 0.02aB

1.0% 3.95 � 0.08aB 3.64 � 0.18bA 1.11 � 0.01 1.17 � 0.01 2.84 � 0.09abB 2.47 � 0.18bA 0.39 � 0.02A 0.48 � 0.03aB

2.0% 3.77 � 0.07aB 3.29 � 0.03aA 1.12 � 0.03 1.19 � 0.01 2.65 � 0.07aB 2.10 � 0.04aA 0.43 � 0.02A 0.56 � 0.02bB

CHO � FA

NG � 0.5 4.15 � 0.07bc 3.89 � 0.05b 1.18 � 0.02 1.21 � 0.03 2.96 � 0.07b 2.67 � 0.03b 0.40 � 0.01A 0.454 � 0.01aB

NG � 1.0 4.17 � 0.04cB 3.94 � 0.10bA 1.15 � 0.04 1.17 � 0.02 3.02 � 0.04bB 2.77 � 0.12bA 0.38 � 0.01 0.423 � 0.03a

NG � 2.0 4.02 � 0.05bcB 3.29 � 0.05aA 1.11 � 0.02 1.18 � 0.03 2.92 � 0.05bB 2.10 � 0.07aA 0.38 � 0.01A 0.564 � 0.03aB

G � 0.5 3.89 � 0.16ab 3.54 � 0.10a 1.11 � 0.02 1.18 � 0.01 2.77 � 0.17ab 2.36 � 0.11a 0.41 � 0.03 0.501 � 0.03ab

G � 1.0 3.86 � 0.12ab 3.34 � 0.10a 1.13 � 0.04 1.16 � 0.02 2.73 � 0.12abB 2.17 � 0.08aA 0.42 � 0.02A 0.536 � 0.01bB

G � 2.0 3.69 � 0.07aB 3.29 � 0.05aA 1.11 � 0.04 1.2 � 0.01 2.57 � 0.06aB 2.09 � 0.06aA 0.43 � 0.02A 0.575 � 0.02bB

Mean values (n ¼ 6) in a column under each category bearing different superscript (lower case) vary significantly (P < 0.05). Mean values (n ¼ 6) in

a row under each parameter bearing different superscript (capital) vary significantly (P < 0.05). NG, non-gelatinised; G, gelatinised.

354 S. Misra et al. / Fish & Shellfish Immunology 21 (2006) 346e356

recorded when fed with high n-3 PUFA containing diets [19]. Channel catfish fed a diet containing high n-3 fatty acidcontent had decreased survival following a challenge with Edwardsiella ictaluri, compared to those fed a diet withhigh n-6 fatty acid content [47]. In channel catfish, low disease resistance and immune functions such as phagocyticcapacity and killing activity was observed when fed diets high in n-3 PUFAs [20]. In this study, lower mortality wasrecorded in the T1 and T2 groups in spite of supplementation of high carbohydrate, suggesting the acquisition of non-specific immunity due to feeding of NG carbohydrate as reported in L. rohita [9]. On the other hand, a higher mortalityin the G carbohydrate fed groups could be due to the reduced immunomodulatory effect of G carbohydrate as reflectedin the significantly lower (P < 0.05) leukocyte count and lysozyme value in the post-challenge period. NG carbo-hydrate supplemented with 1.0% n-3 PUFA seems to be optimum to stimulate the immune system of L. rohitajuveniles.

From the above results, it is concluded that feeding of NG corn significantly increased total leucocyte count,lysozyme activity, A/G ratio and decreased total erythrocyte count, haemoglobin, serum total protein and globulincontent of L. rohita juveniles during the post-challenge period. Similarly, feeding of n-3 PUFA at any level signifi-cantly increased the immunological parameters like lysozyme activity or A/G ratio, whereas total leucocyte countincreased due to feeding of either 0.5% or 1.0% n-3 PUFA. The NBT and albumin values remained similar in bothperiods. Feeding of G carbohydrate at 43% level was found to suppress the immunity of L. rohita juveniles. However,feeding of a similar amount of NG carbohydrate (43%) along with 1.0% n-3 PUFA enhances the immunity as reflectedby high survival followed by immunological parameters in the post-challenge study.

Acknowledgements

The authors are grateful to the Director, Central Institute of Fisheries Education, Mumbai, for providing facilitiesfor carrying out the work. The first author is grateful to the Indian Council of Agricultural Research for awarding theJunior Research fellowship.

References

[1] FAO. Yearbook on fishery statistics. Rome (Italy); 2001.

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

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

[4] Landolt ML. The relationship between diet and immune response of fish. Aquaculture 1989;79:193e206.

[5] Lall SP. Disease control through nutrition. In: Proceedings of the aquaculture international congress and exposition. Vancouver (BC):

Parilion Corporation; 1988. p. 607e12.

0

20

10

30

50

70

90

40

60

80

100

T1 T2 T3 T4 T5 T6

Treatments

Relative %

su

rvival

cb

d

c c

a

T1 - NG + 0.5% EPA & DHAT2 - NG + 1.0% EPA & DHAT3 - NG + 2.0% EPA & DHAT4 - G + 0.5% EPA & DHAT5 - G + 1.0% EPA & DHAT6 - G + 2.0% EPA & DHA

Fig. 1. Survival of different experimental groups (mean � SE) after challenge with Aeromonas hydrophila (P < 0.05), n ¼ 36.

355S. Misra et al. / Fish & Shellfish Immunology 21 (2006) 346e356

[6] Blazer VS. Nutrition and disease resistance in fish. Annu Rev Fish Dis 1992;2:309e23.

[7] Waagbø R, Glette J, Sandnes K, Hemre GI. Influence of dietary carbohydrate on blood chemistry, immunity and disease resistance in

Atlantic salmon, Salmo salar L. J Fish Dis 1994;17:245e58.

[8] Page GI, Hayworth KM, Wade RR, Harris AM, Bureau DP. Non-specific immunity parameters and the formation of advanced glycosylation

end-products (AGE) in rainbow trout, Oncorhynchus mykiss (Walbaum), fed high levels of dietary carbohydrates. Aquac Res 1999;30:

287e97.

[9] 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.

[10] Sargent JR, Bell JG, Bell MV, Henderson RJ, Tocher DJ. The metabolism of phospholipids and polyunsaturated fatty acids in fish. In:

Lahlou B, Vitiello P, editors. Aquaculture: fundamental and applied research. Coastal and estuarine studies, vol. 43. Washington (DC):

American Geophysical Union; 1993. p. 103e24.

[11] Sargent JR, Bell JG, Bell MV, Henderson RJ, Tocher DR. Requirement criteria for essential fatty acids. Symposium of European Inland

Fisheries Advisory Commission. J Appl Ichthyol 1995;11:183e98.

[12] Sargent JR, Bell JG, McEvoy LA, Tocher D, Estevez A. Recent developments in the essential fatty acid nutrition of fish. Aquaculture

1999;177:191e9.

[13] Bell JG, Farndale BM, Dick JR, Sargent JR. Modification of membrane fatty acid composition, eicosanoid production, and phospholipase-A

activity in Atlantic salmon (Salmo salar) gill and kidney by dietary lipid. Lipids 1996;31:1163e71.

[14] 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.

[15] Lall SP. Dietary lipids, immune function and pathogenesis of disease in fish. The Eighth International Symposium on Feeding and Nutrition

in Fish, 1e4 June 1998, Las Palmas de Gran Canaria, Spain; 1998. p. 169.

[16] Montero D, Tort L, Izquierdo MS, Robaina L, Vergara JM. Depletion of serum alternative complement pathway activity in gilthead seabream

caused by alpha-tocopherol and n-3 HUFA dietary deficiencies. Fish Physiol Biochem 1998;18:399e407.

[17] Klinger RC, Blazer VS, Echevarria C. Effects of dietary lipid on the hematology of channel catfish, Ictalurus punctatus. Aquaculture

1999;147:225e33.

[18] Korver DR, Klasing KC. Dietary fish oil alters specific and inflammatory immune responses in chicks. Nutrition 1997;127(20):39e46.

[19] Erdal JI, Evensen O, Kaurstad OK, Lillehaug A, Solbakken R, Thorud K. Relationship between diet and immune response in Atlantic salmon

(Salmo salar L.) feeding various levels of ascorbic acid and omega-3 fatty acids. Aquaculture 1991;98:363e79.

[20] Fracalossi DM, Lovell RT. Dietary lipid sources influence responses of channel catfish (Ictalurus punctatus) to challenge with the pathogen

Edwardsiella ictaluri. Aquaculture 1994;119:287e98.

[21] Secombes CJ. Isolation of salmonid macrophage and analysis of their killing activity. In: Stolen JSTC, Fletcher DP, Anderson BS, Van

Muiswinkel WB, editors. Techniques in fish immunology. Fair Haven (NJ): SOS Publication; 1990. p. 137e52.

[22] Stasiack AS, Bauman CP. Neutrophil activity as a potent indicator of concomitant analysis. Fish Shellfish Immunol 1996;5:37e9.

[23] 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.

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

1971;31:87e96.

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

[26] 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.

[27] Miyazaki T, Kaige N. A histopathological study on motile aeromonad disease in Crucian carp. Fish Pathol 1985;21:181e5.

[28] 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(3):467e8.

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

p. 55e91.

[30] 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.

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

1997;32:121e5.

[32] Lie ØEvensen ØSørensen A, Frøysadal E. Study on lysozyme activity in some fish species. Dis Aquat Org 1989;6:1e5.

[33] Cecchini S, Terova G, Caricato G, Saroglia M. Lysosome activity in embryos and larvae of sea bass (Dicentrarchus labrax L.), spawned

by broodstocks fed with vitamin C enriched diets. Bull Eur Assoc Fish Pathol 2000;20:120e4.

[34] Alexander JB, Ingram GA. Noncellular nonspecific defense mechanisms of fish. Annu Rev Fish Dis 1992;2:249e79.

[35] 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.

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

[37] 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.

[38] Møyner K, Røed KH, Sevtdal S, Heum M. Changes in nonspecific immune parameters in Atlantic salmon, Salmo salar L., induced by

Aeromonas salmonicida infection. Fish Shellfish Immunol 1993;3:253e65.

[39] Chung S, Secombes CJ. Activation of rainbow trout macrophages. J Fish Biol 1987;31(A):51e6.

[40] Secombes CJ, Fletcher TC. The role of phagocytes in protective mechanism of fish. Annu Rev Fish Dis 1992;2:53e71.

356 S. Misra et al. / Fish & Shellfish Immunology 21 (2006) 346e356

[41] Ellis AE. Immunity to bacteria in fish. Fish Shellfish Immunol 1999;4:291e308.

[42] Hilima JI, Hirono I, Aoki TS. Characterisation and expression of c-type lysozyme cDNA from Japanese flounder (Paralichthys olivaceous).

Mol Mar Biol Biotechnol 1997;6:339e45.

[43] 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.

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

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

1981;4:41e52.

[46] Ellis AE. Serodiagonistics and vaccines. In: International symposium on fish biologics. Leetown, W, Va, USA. Develop Biol Standard

1981;49. p. 337e52.

[47] Li MH, Wise DJ, Johnson MR, Robinson EH. Dietary menhaden oil reduced resistance of channel catfish (Ictalurus punctatus) to Edward-

siella ictaluri. Aquaculture 1994;128:335e44.