EFFECT OF ANESTHETICS ON STRESS AND THE INNATE IMMUNE SYSTEM OF GILTHEAD SEABREAM (SPARUS AURATA)

The Isreli Journl of Aquculture Bmidgeh 56(1), 24, 5-13 5 EFFECT OF ANESTHETICS ON STRESS AND THE INNATE IMMUNE SYSTEM OF GILTHEAD SEABREAM (SPARUS AURATA) Keren Bressler nd Benny Ron* Isrel Ocenogrphic nd Limnologicl Reserch, Ntionl Center for Mriculture, P.O. Box 1212, Eilt 88112, Isrel (Received 1.1.3, Accepted 1.11.3) Key words: nesthetics, enzocine, clove oil, innte immune system, Sprus urt, stress Astrct Anesthesi my depress the immune system in mmmls nd fish. In the present work, two nesthetics used in quculture, clove oil (.445 mm) nd enzocine (.225 mm), were tested to oserve their effects on the stress response nd innte immune system ctivity of gilthed serem (Sprus urt L.). Results showed tht oth nesthetics induced incresed lood glucose nd serum cortisol levels. In ddition, enzocine depressed lysozyme ctivity, production of rective oxygen species nd pinocytosis ctivity. These were not depressed y clove oil, suggesting tht clove oil is sfer nesthetic for serem s it does not cuse immunodepression in nesthetized fish. Introduction Anesthesi is iologicl stte induced y n externl gent which results in the prtil or complete loss of senstion or loss of voluntry neuromotor control through chemicl or nonchemicl mens (Summerfelt nd Smith, 199). Anesthetics re frequently used in fishery studies nd quculture to minimize stress response, preventing its negtive impct on performnce nd reducing physicl injury during hndling procedures (Wedemeyer, 1997). While nesthesi enefits the fish y minimizing the impct of greter stressors, it is lso inherently stressful nd its effectiveness depends on the procedure used (Iwm et l., 1989). Severe nesthesi my induce stress response in fish tht cn led to immunodepression nd incresed susceptiility to disese (Iwm et l. 1989; Thoms nd * Corresponding uthor. E-mil: ronenny@gri.huji.c.il

6 Bressler nd Ron Roertson, 1991). Different nesthetic gents cn cuse diverse chnges in physiologicl prmeters. Benzocine is common nesthesi, considered to e locl gent tht locks neurl N + chnnels, reducing trnsmission of nerve ction potentil (Crmichel, 1985). The prolem in using enzocine s n nesthetic is tht it produces typicl effects of stress such s hypoxi, hyperglycemi nd incresed lood lctte levels (Ross nd Ross, 1984). Clove oil hs received fvorle reviews s n lterntive nesthetic for vriety of fish species (Keene et l., 1998; Cho nd Heth, 2). Eugenol (2-methoxy-4-2-(2- propenyl)-phenol), the ctive component of clove oil, is otined from the uds, leves nd stem of the Eugeni cryophyllus plnt. Clove oil hs severl dvntges over other nesthetic gents in fishery reserch, ssessment studies nd quculture pplictions. It is n esily otinle nd inexpensive orgnic distillte, which is used s food dditive nd possesses ntifungl, ntivirl, nlgesic nd nticteril properties (Keene et l., 1998). Clove oil is orgnic, so no withdrwl period is required for fish intended for humn consumption. Another dvntge of clove oil is tht it does not pose chemicl helth hzrd to the user. The only known disdvntge of clove oil is its photosensitivity. The gol of this study ws to exmine the effect of two commonly used nesthetics, clove oil nd enzocine, on stress responses nd innte immune system ctivity of the serem (Sprus urt). Mterils nd Methods Experimentl fish nd rering conditions. Gilthed serem (Sprus urt), rered t the Ntionl Center for Mriculture (NCM) in Eilt, Isrel, were used in this study. Prior to initition of the experiment, sixty fish (22±15 g men weight) were rndomly distriuted into ech of four 9-liter flow-through tnks nd cclimted for 21 dys (4 ppt slinity, 124 l/h flow rte, 5.6 mg/l ertion, 24±.5 C, nturl photoperiod). During the cclimtion period, the fish were fed once dy t the rte of 1.2% ody weight with commercil dry pellets (Mtmor, M.P. Evtch, Isrel). Experimentl design. Three groups of fifteen fish, ech, were compred: control (no nesthetic), enzocine-nesthetized (22.5 µl/l enzocine) nd clove oil-nesthetized (44.5 µl/l clove oil). The nesthetic ws dministered y dissolving it in 1% ethnol (1:2 for the enzocine nd 1:5 for the clove oil) nd then in smll mount of se wter. The dissolved nesthetic ws shken nd poured into the tnk. The fish were exposed to the nesthesi until they reched nrcotic nesthesi stte (loss of reflex rectivity, slow operculr movements). Blood smples were tken from the live fish, then they were killed nd dissected. Smpling. After exposure to the nesthetic, one ml of lood ws collected from the cudl vein of ech specimen using 21-guge needle nd 1 ml syringe (in the stress exmintion, n = 15) within 5-7 minutes. Aliquots of 2 µl of the fresh lood smples were used for glucose nlysis. The rest of the lood smple ws llowed to clot t 4 C for 6 hours. Following centrifugtion, the serum ws removed nd frozen t -2 C until the cortisol level nd lysozyme ctivity were determined. Hed kidney leukocytes (in the immunologicl exmintion, n = 1) were isolted ccording to Steinhgen nd Hespe (1997). The hed kidney ws dissected out y ventrl incision nd trnsferred to 5 ml RPMI-164 medium supplement with 1 U/ml heprin (Sigm), 1 U/ml penicillin (Sigm),.1 g/l streptomycin (Sigm) nd 5% fetl clf serum (Sigm). Cells were suspended y forcing frgments of the orgn through nylon mesh (1 µm). The hed kidney suspension ws wshed three times, counted nd djusted to 4 x 1 6 in RPMI. Cell viility ws greter thn 95%, determined y the trypn lue exclusion test. Stress indictors. The percent hemtocrit ws determined y centrifuging the totl lood in heprinized microhemtocrit tues nd clculting the percent of erythrocytes. Smples of.2 ml fresh lood were used to mesure glucose levels y the One Touch Bsic glucose meter (Shnitmn, 1995). Serum cortisol levels were determined with tritium H 3 rdio

Effect of nesthetics on gilthed serem 7 immunossy (RIA). Cortisol in the smples competes with the rdioctive leled cortisol to ind with nti-cortisol ntiody. Rdioctivity levels were mesured in Betcounter (Redding et l., 1984). Lysozyme ctivity. The lysozyme ctivity ssy ws performed ccording to Ellis (199). Anlysis of the lysozyme level ws sed upon the lyses of the lysozyme-sensitive grm-positive cterium Micrococcus lysodeikiticus (Sigm). The cteri lysis ws mesured turimetriclly (ELISA reder, Spectr 2, SLT, Germny) t 492 nm,.5 nd 4.5 min fter the strt of the rection. The rection results were clculted s lysozyme ctivity units per 1 ml fish serum. Production of rective oxygen species (ROS). The ROS production ssy ws done ccording to Secomes (199). Three replictes of leukocytes from ech fish were incuted for two hours in 96 microtiter pltes with nitrolue tetrzelium (NBT; Sigm) nd phorool myristte cette (PMA; Sigm). During the rection, solule yellow NBT is reduced y O 2 to insolule lue formezn. The mount of lue formezn ws mesured fter it ws dissolved with KOH (Sigm) nd DMSO (Sigm). The opticl density (OD) ws red with spectrophotometer (ELISA reder, Spectr 2, SLT, Germny) t 62 nm nd 45 nm. Pinocytosis ctivity. The pinocytosis ssy ws done ccording to Mthews et l. (199). The leukocytes were incuted in microtiter pltes for two hours together with neutrl red (Sigm), wshed twice nd dissolved with cid lcohol. The OD of the neutrl red relesed into the superntnt ws mesured spectrophotometriclly t 492 nm (ELISA reder, Spectr 2, SLT, Germny) Sttisticl nlysis. All ssys were performed in triplicte nd the men±stndrd error (SEM) ws clculted for ech group (no. fish = 1 for immune prmeters nd 15 for stress prmeters). A one-wy nlysis of vrince (ANOVA) with Newmn-Keuls multiple rnge test ws performed to discover differences etween mens. Differences were considered sttisticlly significnt when p<.5. Results Anesthesi. In preliminry experiments (dt not shown), oth enzocine nd clove oil (t the sme dosge) cused rpid nesthesi in the fish, which lost senstion nd equilirium no more thn 8 minutes fter the nesthesi ws induced. The fish recovered from the nesthesi no more thn 9 seconds fter they were trnsferred to new tnk. No mortlity ws oserved in either group. In the control group, three fish died within 72 hours fter smpling. Blood glucose concentrtion. The lood glucose concentrtion of the nesthetized fish ws significntly higher thn tht of the control fish with oth nesthetics (Fig. 1). There ws no significnt difference in glucose concentrtion etween fish nesthetized with clove oil nd those nesthetized with enzocine. Hemtocrit. The hemtocrit level of the control fish ws significntly higher thn tht of the nesthetized fish (Fig. 2). There ws no significnt difference in hemtocrit levels of fish nesthetized with clove oil or enzocine. Serum cortisol concentrtion. Serum cortisol levels of the nesthetized fish were significntly higher thn tht of the control fish with oth nesthetics (Fig. 3). The cortisol level of fish nesthetized with enzocine ws significntly higher thn tht of fish nesthetized with clove oil. Serum lysozyme ctivity. The serum lysozyme ctivity (mesured in unit/ml/min) ws significntly reduced y enzocine (Fig. 4). There ws no significnt difference etween the control nd the clove oil nesthetized fish. Respirtory urst ctivity. The respirtory urst ctivity of serem hed-kidney leucocytes ws significntly reduced y enzocine (Fig. 5). There ws no significnt difference etween the control nd the clove oil nesthetized fish. Pinocytosis ctivity. The pinocytosis ctivity of serem hed-kidney leucocytes ws significntly reduced y enzocine (Fig. 6). There ws no significnt difference etween the control nd the clove oil nesthetized fish.

8 Bressler nd Ron 1 Glucose (mg/dl) 75 5 n=16 25 Control Clove oil Benzocine Tretments Fig. 1. Blood glucose levels of nesthetized nd control gilthed serem. Dt represent mens±sd. Different letters denote significnt difference etween groups. Hemtocrit (%) 4 3 2 n=14 1 Control Clove oil Benzocine Tretments Fig. 2. Hemtocrit levels of nesthetized nd control gilthed serem. Dt represent mens±sd. Different letters denote significnt difference etween groups.

Effect of nesthetics on gilthed serem 9 75 c Cortisol (ng/ml) 5 25 Control Clove oil Benzocine Tretments Fig. 3. Serum cortisol concentrtion of nesthetized nd control gilthed serem. Dt represent mens±sd. Different letters denote significnt difference etween groups. 1 Lysozyme ctivity (unit/ml/min) 75 5 25 Control Clove oil Benzocine Tretments Fig. 4. Serum lysozyme ctivity of nesthetized nd control gilthed serem. Dt represent mens±sd. Different letters denote significnt difference etween groups.

1 Bressler nd Ron.3.2 O.D. n=1 n=9.1 n=1 Control Clove oil Benzocine Tretments Fig. 5. Respirtory urst of hed-kidney leukocytes from nesthetized nd control gilthed serem. Dt represent mens±sd. Different letters denote significnt difference etween groups..4.3 O.D..2 n=1 n=1.1 n=1 Control Clove oil Benzocine Tretments Fig. 6. Pinocytosis ctivity of hed-kidney leukocytes from nesthetized nd control gilthed serem. Dt represent mens±sd. Different letters denote significnt difference etween groups.

Effect of nesthetics on gilthed serem 11 Discussion Previous studies hve shown tht nesthetics cn reduce fish stress responses when suitly dministrted, preventing the negtive impct of stress on fish performnce nd reducing physicl injury during hndling (Roertson et l., 1988; Wedemeyer, 1997). Indeed, in the present study there ws no mortlity in the nesthetized fish fter lood smpling in comprison with 2% mortlity in the control fish. On the other hnd, some nesthetics my induce stress response in fish when exposure time is too long (Iwm et l., 1989; Thoms nd Roertson, 1991). Blood glucose nd serum cortisol concentrtions re commonly used s stress indictors in fish studies ecuse they hve proven to e relile endocrine nd secondry indictors for mny stressors to fish nd re esily mesurle prmeters (Schreck, 1981). Severl studies demonstrted the influence of nesthetics on the mgnitude of corticosteroid nd hyperglycemic responses of fish to stress (Iwm et l., 1989; Ortuno et l., 22). In the present study, enzocine nd clove oil induced stress response in the fish, indicted y the increse in lood glucose level nd serum cortisol concentrtion. The rise of serum cortisol in this study is coincident with the increse in lood glucose. This wellknown pttern of hyperglycemi fter stress hs een shown to result from ctecholmine nd corticosteroids relesed into the lood nd hs een reported in other reserch (Anderson et l., 1991; Ortuno et l., 22). The glucose vlue nd serum cortisol concentrtion of the control fish in this study were typicl for this species s well s other sprids (Rotllnt nd Tort, 1997; Ortuno et l., 22). Presumly, the intensity of the smpling stress ws elow the threshold required to induce corticosteroid nd glucose stress responses (Thoms nd Roertson, 1991). Incresing the durtion of the smpling time to 15-2 minutes elicited stress responses in gilthed serem nd severl-fold increses in serum cortisol nd lood glucose levels in our preliminry experiment (dt not shown). In the present study, the nesthesi did not cuse n elevtion of the percent hemtocrit. Similr results were reported in nother study using similr concentrtion of clove oil nesthesi in serem (Tort et l., 22). Anesthetic gents re known to hve immunodepression effects y depressing phgocytic functions such s leucocyte recruitment, ttchment, chemotctic motility, engulfment nd intercellulr killing in mmmls nd fish (Brdosi et l., 1992; Ortuno et l., 22; Puig et l., 22). Severl reserchers hve showed tht lysozyme nd phgocytic ctivity cn e used s immune indictors (Demers nd Byne, 1997; Ortuno et l., 22c). Other studies hve shown correltion etween n elevted plsm level of cortisol, immunodepression nd incresed susceptiility to disese in cultured fish (Thoms nd Lewis, 1987; Mul et l., 1989). A study on mice showed tht stress induced rise in plsm corticosterone nd inotropes nd might e involved in modifying the distriution of neutrophils nd lymphocytes (Puig et l., 22). Another fish study found correltion etween modifiction of the glucose concentrtion nd complement ctivity. This study lso found correltion etween modifiction of the cortisol level nd phgocytosis. These studies suggest tht drenline hs stronger effect on the complement system nd cortisol hs stronger effect on the phgocytic function (Ortuno et l., 22c). On the other hnd, nother study suggested tht the immunodepression cused y the nesthesi is not cused y stress signls, ut y direct interction etween the nesthetic gent nd immune components, or through the nervous system (Ortuno et l., 22). In the present study, there ws no immunodepressive effect of nesthesi with clove oil. On the other hnd, oth humorl nd cellulr immune responses were significntly depressed y enzocine. Our results re consistent with those reported for serem y Ortuno et l. (22). Our reserch showed tht nesthesi my ffect fish stress responses nd therefore might e unsuitle for some reserch purposes. In quculture, the use of clove oil s n nesthetic gent is preferred to the use of

12 Bressler nd Ron enzocine ecuse it does not pose chemicl helth hzrd to people who hndle the fish or to consumers nor does it cuse ny significnt immunodepression effect on the fish. References Anderson D.E., Reid S.D., Moon T.W. nd S.F. Perry, 1991. Metolic effects ssocited with chroniclly elevted cortisol in rinow trout (Oncorhynchus mykiss). Cn. J. Fish Aqut. Sci., 48:1811-1817. Brdosi L., Brdosi A. nd H.J. Gius, 1992. Chnges of expression of endogenous sugr receptors y polymorphonucler leukocytes fter prolonged nesthesi surgery. Cn. J. Anesthesi, 39:143-15. Crmichel F.J., 1985. Generl nesthetics nd locl nesthetics. pp. 265-289. In: H. Kolnt, W.H.A. Roschlu, E.M. Sellers (eds.). Principles of Medicl Phrmcology. Univ. Toronto, Toronto. Cho G.K nd D.D. Heth, 2. Comprison of tricine methnesulphonte (MS 222) nd clove oil nesthesi effects on the physiology of juvenile chinook slmon Oncorhynchus tshwytsch (Wlum). Aquculture Res., 31:537-546. Demers N.E. nd C.J. Byne, 1997. The immedite effects of stress on hormones nd plsm lysozyme in rinow trout. Dev. Comp. Immunol., 21:363-373. Ellis A., 199. Lysozyme ssys. pp. 11-13. In: J.S. Stolen, T.C. Fletcher, D.P. Anderson, B.S. Roerson, W.B. vn Muiswinkel (eds.). Techniques in Fish Immunology 1. SOS Pulictions, Fir Hven, New Jersey. Iwm G.K., McGeer J.C. nd M.P. Pwluk, 1989. The effects of five fish nesthetics on cid-se lnce, hemtocrit, lood gses, cortisol nd drenline in rinow trout. Cn. J. Zool., 67:265-273. Keene J.L., Nokes D.L.G., Mocci R.D. nd C.G. Soto, 1998. The efficcy of clove oil s n nesthetic for rinow trout, Oncorhynchus mykiss (Wlum). Aquculture Res., 29:89-11. Mthews E.S., Wrriner J.E. nd B.A. Weeks, 199. Assys of immune function in fish mcrophges. Techniques used s indictors of environmentl stress. pp. 155-163. In: J.S. Stolen, T.C. Fletcher, D.P. Anderson, B.S. Roerson, W.B. vn Muiswinkel (eds.). Techniques in Fish Immunology 1. SOS Pulictions, Fir Hven, New Jersey. Mul A.G., Tripp R.A., Keetteri S.L. nd C.B. Schreck, 1989. Stress lters immune function nd disese resistnce in chinook slmon (Oncorhynchus tshwytsch). J. Endocrinol., 12:135-142. Ortuno J., Esten M.A. nd J. Messeguer, 22. Effects of four nesthetics on the innte immune response of gilthed serem (Sprus urt L.). Fish Shellfish Immunol., 12:49-59. Ortuno J., Esten M.A. nd J. Mesesguer, 22. Lck of effect of comining different stressors on innte immune responses of serem (Sprus urt L.). Vet. Immunol. Immunopthol., 84:17-27. Ortuno J., Esten M.A. nd J. Mesesguer, 22c. Effects of phenoxyethnol on the innte immune system of gilthed serem (Sprus urt L.) exposed to crowding stress. Vet. Immunol. Immunopthol., 89:29-36. Puig N.R., Ferrero P., By M.L., Hidlgo G., Vlenti J., Amerio N. nd G. Elen, 22. Effects of sevoflurne generl nesthesi: immunologicl studies in mice. Int. Immunopthol., 2:95-14. Redding J.M., Schreck C.B., Birks E.K. nd R.D. Ewing, 1984. Cortisol nd its effects on plsm thyroid hormones nd electrolyte concentrtions during sewter cclimtion in yerling coho slmon Oncorhynchus kisutch. Gen. Comp. Endocrinol., 56:146-155. Roertson L., Thoms P. nd C.R. Arnold, 1988. Plsm cortisol nd secondry stress responses of cultured red drum (Scienops ocelltus) to severl trnsporttion procedures. Aquculture, 68:115-13. Ross L.G. nd B. Ross, 1984. Anesthetic nd Sedtive Techniques for Fish. Stirling Institute of Aquculture, Univ. Stirling. Rotllnt J. nd L. Tort, 1997. Cortisol nd glucose responses fter cute stress y net hndling in the sprid red porgy previously sujected to crowding stress. J. Fish Biol., 51:21-28.

Effect of nesthetics on gilthed serem 13 Schreck C.B., 1981. Stress nd compenstion in teleosten fishes: responses to socil nd physicl fctors. pp. 295-321. In: A.D. Pickering (ed.). Stress in Fish. Acdemic Press, London. Secomes C.J., 199. Isoltion of slmonid mcrophges nd nlysis of their killing ctivity. pp. 137-154. In: J.S. Stolen, T.C. Fletcher, D.P. Anderson, B.S. Roerson, W.B. vn Muiswinkel (eds.). Techniques in Fish Immunology 1. SOS Pul., Fir Hven, New Jersey. Shnitmn S., 1995. Influence of hndling nd mmoni stress on the susceptiility of Sprus urt L. to the dinoflgellte ectoprsite Amyloodinium ocelltum (Brown). M.Sc. Thesis, Tel Aviv University. Steinhgen D. nd K. Hespe, 1997. Crp coccidosis: ctivity of phgocytic cells from common crp infected with goussi crpelli. Dis. Aqut. Org., 31:155-159. Summerfelt R.C nd L.S. Smith, 199. Anesthesi, surgery nd relted techniques. pp. 213-272. In: C.B. Schreck nd P.B. Moyle (eds.). Method for Fish Biology. Am. Fish. Soc., Bethesd, Mrylnd. Thoms P. nd D.H. Lewis, 1987. Effect of cortisol on immunity in red drum Scienops ocelltus. J. Fish Biol., 31(Suppl. A):123-127. Thoms P. nd L. Roertson, 1991. Plsm cortisol nd glucose stress responses of red drum (Scienops ocelltus) to hndling nd shllow wter stressors nd nesthesi with MS-222, quinldine sulphte nd metomidte. Aquculture, 96:69-86. Tort L., Puigcerver M. nd F. Pdros, 22. Cortisol nd hemtologicl response in se rem nd trout sujected to the nesthetics clove oil nd 2-phenoxyethnol. Aqucult. Res., 33:97-91. Wedemeyer G.A., 1997. Effects of rering conditions on the helth nd physiologicl qulity of fish in intensive culture. pp. 35-72. In: G.K. Iwm, A.D. Pickering, J.P. Sumpter, C.B. Schreck (eds.). Fish Stress nd Helth in Aquculture. Cmridge University Press, Cmridge. ERRATUM In the rticle "Defining energy nd protein requirements of gilthed serem (Sprus urt) to optimize feeds nd feeding regimes" y Ingrid Luptsch, George Wm. Kissil nd Dvid Skln, pulished in the Decemer 23 issue 55(4), the recommended supplies of digestile energy for gilthed serem shown in Tle 3 refer to tempertures of 2 C nd 26 C s indicted in the text nd column hedings, nd not s in the tle cption. We regret this editoril error.