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Comparative Study of Biochemical and Non-specific Immunological

Parameters in Two Tilapia Species (Oreochromis aureusand

O. mossambicus)

Yaniv Palti1*

, Simon Tinman2, Avner Cnaani

1, Yaakov Avidar

3, Micha Ron1,

and Gideon Hulata1

1. Institute of Animal Science, Agricultural Research Organization, P.O. Box 6, Bet Dagan50250, Israel.

2. The Central Fish Health Laboratory, Nir David 19150, Israel.Fish_lab@netvision.net.il

3. Biochemistry Department, Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250,Israel.

Current Address: Dept. of Food Engineering and Biotechnology, The Technion, Haifa

32000, Israel.

ABSTRACT

Oreochromis aureus and O. mossambicus were compared in a preliminary study fordifferences in levels of blood biochemical and non-specific immunological parameters beforeand after exposure to acute stress, which was induced by air exposure for 10 minutes. Twoexperiments were conducted. In the first experiment comparisons were performed after twoweeks of acclimation (base-line level), after stress, and for stress response. The latter wascalculated for each fish by subtracting the level after stress exposure from the base line level.Significant differences (P < 0.01) were identified between species at the base-line level intotal plasma cholesterol and total protein levels. Glucose concentration was significantlydifferent after stress and in stress response. Significant differences between species were alsoidentified in respiratory burst activity of phagocytes after stress and in ceruloplasmin activityafter stress. No significant correlation was identified between body weight and each of theparameters tested, indicating that the differences detected in immunological parameters arenot related to the notable size difference between the two groups used in this study. Thesignificant differences in total cholesterol and protein were confirmed in the secondexperiment. Significant differences (P < 0.05) were also detected in levels of albumin,globulin, LDH, calcium, total bilirubin and triglycirides, which were measured by

autoanalyzer, and in alpha, beta2, and IgM globulins, %beta1 and %IgM, which wereseparated and measured by agarose gel electrophoresis. Wide variation was detected withinO. aureusin some of the parameters examined. A larger sample size should be used to learnif the differences are large enough to produce segregating O. aureus families for geneticanalysis of those parameters. The differences identified suggest that hybrid families from thetwo species can be used to construct a segregating population for genetic analysis ofimmunological traits and stress response in tilapia.

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INTRODUCTION

Fish diseases have become a major limiting factor in aquaculture. Current methods to controlinfectious diseases consist of hygiene, vaccination, drug therapy and eradication of infected

populations. Improving infectious disease resistance by genetic means is an attractivealternative because of its prospects for prolonged protection. The significant geneticvariation in disease resistance found in different fish species (reviewed by Chevassus andDorson 1990; Fjalestad et al. 1993; Wiegertjes et al. 1996) suggests the possibility of suchgenetic improvement.

Strain and species differences in disease resistance were previously demonstrated in fish(Parsons et al. 1986; Dorson et al. 1991; Ibarra et al. 1991, 1994; LaPatra et al. 1993, Palti etal. 1999). Strain differences in disease resistance in coho salmon were found to be associatedwith components of the non-specific immune system (Whithler and Evelyn 1990; Balfry etal. 1994). Recently, strain differences in non-specific immunity were also found in tilapia(Balfry et al. 1997a).

Variation in disease resistance has traditionally been measured by the rate of survival afterexposure to a pathogen. Such measurements can result in inaccurate estimation of geneticcomponents of immunity in animals (Gavora and Spencer 1983). The innate (non-specific)immunity is thought to have a major role in disease resistance of fish (e.g. Roed et al. 1993;Balfry et al. 1997a,b). Several parameters of the innate immune response, such as respiratoryburst activity, spontaneous haemolytic activity, lysozyme activity, complement concentration,and total IgM, were found to be associated with disease resistance in fish, and theirheritability estimates were mostly moderate (reviewed by Wiegertjes et al. 1996). The stronglink between stress and susceptibility to diseases in farm animals has long beenacknowledged. Parameters of high and low stress response (e.g. cortisol and glucose levels in

the blood) were also found to be associated with disease resistance in fish (Fevolden et al.1991, 1992, 1993). Levels of blood plasma ions and enzymes with important metabolicfunctions can give indication to the general health of the fish (e.g. Williams and Wootten1981; Asztalos and Nemcsok 1985; Heming and Paleczny 1987; Ellsaesser and Clem 1987;Waagbo et al. 1988).

Maita et al. (1998a) detected correlation between levels plasma lipids levels and resistance topathogen infection in yellowtail and rainbow trout. Levels of plasma lipids in fish areaffected by diet (Maita et al. 1998b) and by stress after exposure to low ambient dissolvedoxygen (Maita et al. 1998c). Those findings suggest that plasma cholesterol can be anindicator for fish health and innate immunity.

In this preliminary study we compared the levels of parameters of the innate immuneresponse and other blood plasma components in O. aureus and O. mossambicusto identifysignificant differences between the two species. Identification of differences between speciesin parameters of non-specific immunity and stress response is necessary for constructinghybrid families for genetic analysis of immunological traits in tilapia.

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MATERIALS AND METHODS

Fish

Two experiments were performed. In the first experiment we used 26 three years old O.aureusand O. mossambicusfrom the purebred stocks kept at the Department of Aquaculture,Agricultural Research Organization (A.R.O.), Bet Dagan, Israel. The O. aureus strainoriginated from the Mehadrin stock (Hulata et al. 1993). The O. mossambicus strainoriginated from a stock introduced to Israel from Natal, South Africa, in 1975 (Hulata 1988).Size range was 75330 g and 110315 g for O. aureusand O. mossambicus, respectively.Average (SD) was 163 g (83) for O. aureusand 209g (47) for O. mossambicus. The fishwere tagged individually and reared communally in two tanks. For the second experiment werandomly sampled 20 adult O. aureus and O. mossambicusfrom the ARO rearing tanks (10from each species). The fish were fed daily with a commercial pelleted tilapia feed, 30%protein (Zemach Mills, Israel).

Sampling Schedules

Each of the biochemical and immunological parameters recorded in the first experiment wasmeasured in blood samples taken after two weeks of acclimation period at the base linelevel. Two weeks later, fish were exposed to a 10 minutes air exposure stress. Watertemperature in the rearing tanks where the fish were kept during the experiment was 25C.Parameters were measured again from blood samples taken 4 hours after the stress exposure.Sample size was 13 fish from each species at the base line level. One fish was lost from eachgroup between sampling, and therefore, 12 fish were used from each species after stress.Blood samples for the second experiment were only taken from fish kept at normal rearing

conditions.

Assays to Measure Biochemical and Non-specific Immunological Responses:

Experiment I

Measurements were performed for glucose concentration, ceruloplasmin activity, lysozymeactivity, total protein and total cholesterol, and respiratory burst activity of blood cells. Theglucose concentration in fish blood is expected to increase four hours after stress exposure(Vijayan et al. 1997; Melamed et al. 1999). It was measured immediately after bleeding by akit of Haemo-Glukotest 20-800 R (Reflolux S, Boehringer Manheim). Ceruloplasmin is analpha globulin component of the blood plasma, involved in copper ion transport and oxygen

reduction. Its activity and concentration in the plasma is measured by spectrophotometry.Lysozyme is an important enzyme in the blood that actively lyses bacteria. We used an assaybased on the lysis ofMicrococcus lysodeikitusfor determining its activity. Lysozyme activityover time is measured by a spectrophotometric assay. Chicken egg white lysozyme was usedas a standard control (Ellis 1990). Total protein and total cholesterol were measuredaccording to established procedures (Doumas 1975; and Allain et al. 1974, respectively).Total protein was only measured at the base line level. Respiratory burst activity of

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phagocytes was measured by spectrophotometric assay of nitroblue tetrazolium (NBT)activity (Anderson and Siwicki 1995; Efthimiou 1996).

Experiment II

Measurements were performed at 30C using the Selective Autoanalyzers (Supra andProgress, Kone Inc., Finland) at the Kimron Veterinary Institute, Bet Dagan, Israel, accordingto established procedures. The components measured by the autoanalyzers are listed in Table2a. Globulin levels were determined indirectly by subtracting the measurement of albuminfrom total protein. Protein fraction levels (Table 2b) were determined by agarose gelelectrophoresis following the procedure described by Rehulka (1993). Purified IgM that wascontributed by Prof. Ramy Avtalion, Bar Ilan University, was used as a standard to identifythe IgM fraction

Statistical analysis

Student t-test analyses were performed to identify significant differences between O. aureusand O. mossambicusin each of the parameters at the base line level and after acute stress, andfor stress response. The latter was calculated for each fish by subtracting the level after stressexposure from the base line level. F-tests were used to identify significant differencesbetween variances of the two species for each of the parameters. Unequal variance t-test(Montgomery 1991) was used for parameters with significant variance differences.Correlations of body weight and different parameters were estimated to determine whetherbiochemical and immunological differences between the two species were caused by thenotable size difference between the two groups. Correlations were estimated within speciesand also for the pooled data from both species.

RESULTS

Significant differences (P < 0.01) were identified betweenO. aureusand O. mossambicusintotal plasma cholesterol and total protein at the base-line level (Table 1a), and in glucoseconcentration, NBT and ceruloplasmin activity after stress (Table 1b). Body weight was notcorrelated to the parameters tested (P > 0.1). The significant differences in total cholesteroland protein were confirmed in the second experiment (Table 2a). Significant differences (P