Iranian Journal of Fisheries Sciences 10(3)

Transcription

1 Irnin Journl of Fisheries Sciences 1(3) Assessment of digestive enzymes ctivity during the fry development of Rinow Trout, Oncorhynchus mykiss: from htching to primry stges fter yolk sc sorption Golchinfr F. 1 ; Zmni A. 2 * ; Hjimordloo A. 3 ; Mdni R. 1 Received: Jnury 21 Accepted: My 21 Astrct The im of this study ws to determine the ctivity of digestive enzymes during the fry development of Rinow trout (Oncorhynchus mykiss), which hs remrkle role in food digestion nd sorption in the first feeding. The ssessment of digestive enzymes ctivity of gstric (pepsin), pncretic (trypsin, chymotrypsin, α-mylse nd lipse) nd intestinl (lkline phosphtse) reveled tht enzymes were ville on the first dy fter htching (DAH) ut their ctivity showed no significnt difference from htching to dy 12 (P >.5). An incresed ctivity ws seen etween 12 nd 18 DAH nd this ctivity ws significntly higher thn the first 12 dys (P <.5). In the primry stges fter yolk sc sorption (fter 2 to 31 dys), enzymes ctivity showed n incresed profile; however none of them showed significnt difference etween 2 to 31 DAH (P >.5). These results could e used s sis for formulting suitle feeding nd lso selecting the est time for strting the feeding so tht optimum nutritionl vlues nd cost-effectiveness re chieved. Keywords: Digestive enzymes, Fry development, Rinow trout 1-Deprtement of Biotechnology of Rzi Vccine nd Serum Reserch Institute, P.O.BOX: Hesrk, Krj, Irn. 2-Nturl Resources nd Mrine Sciences Fculty of Trit Modres University, P.O.BOX: Nur, Irn. 3-Deprtement of Fisheries of Gorgn University of Agriculturl Sciences nd Nturl Resources, P.O.BOX: 386 Gorgn, Irn. *Corresponding uthor s emil: Zmniouzndn@yhoo.com

2 44 Golchinfr et l., Assessment of digestive enzymes ctivity during the fry.. Introduction The successful culturing of fish lrve hs een considered s n importnt trget in quculture wheres the fst development of this industry will increse the need for specilized feeds in future (Diz-Lopez et l., 1997; Kolkovski, 21; Rieiro et l., 28). Therefore, gret quot of current investigtions in fish nutrition is devoted to the improvement nd the development of rtificil diets for cultivted lrvl species (Moyno et l., 1996; Rieiro et l., 1999; Cr et l., 23; Lopez- Vsquez et l., 29). However, detiled knowledge is the necessity for relizing the lrvl digestive physiology (Cr et l., 27). In this respect, the nutritionl iochemistry nd physiology of fish lrve hve een investigted during the lst decde s impertive tools of knowing the nutritionl requisites, prticulrly the effectiveness of enzymes on food digestiility so tht digestive enzymes re still the focus of mny scientific ppers round the world (Buddington, 1985; Kuzmin, 1996; Kuzmin nd Skvortsov, 21; Zmonino-Infnte nd Chu, 21; Cruso et l., 28). The ssessment of digestive enzymes ctivity during the first weeks of lrvl life gives vlule informtion tht could e useful in determining the optimum moment nd etter djustment of nutritionl needs to etter understnd the nutritionl cpilities of young lrve (Lzo et l., 2; Kim et l., 21; Cr et l., 23; Moyno et l., 25; Fulk et l., 27; Suzer et l., 27; Alvrez-Gonzlez et l, 28; Pen et l., 29; Alvrez- Gonzlez et l., 21). Additionlly in recent studies, two sujects re led to lrvl nutritionl physiology: the ssessment of the presence nd level of ctivity of certin enzymes s n indictor of lrvl development nd lso, the potentil ility of fish lrve in digestion of compound diet, which cn e minly sed on ontogenic development of digestive enzyme ctivities (Cr et l., 23; Suzer et l., 27). Besides, the minimized mortlity could e discussed s necessry step during the lrviculture y improving culturl conditions (Kolkovski, 21). Rinow trout (Oncorhynchus mykiss) is n importnt commercil quculture species in Irn s production is incresed from 9 tones in 2 to 6263 tones in 28 (Anon., 28). Htching up to yolk sc reserves sorption is one of the most importnt stges of lrvl development ecuse high survivl in the mentioned phse cn ffect survivl nd growth in susequent stges (Gwlick et l., 2). The purpose of the present study is to determine the ctivities of min digestive enzymes in rinow trout fry from htching to erly stges fter yolk sc sorption s their importnce is distinct in the first feeding. Mterils nd methods The rinow trout eggs were otined from rood stock (weight = 13 ± 23g, totl length = 51 ± 5.5cm) from Kelrdsht Cold Wter Fisheries Center, Mzndrn, Irn nd incuted t 9-11ºC, ph = nd wter flow =.3 liter/sec. Incution period lsted out 32 dys. After htching, fry were cultured in incutors with the sme conditions except tht wter flow ws.5 liter/sec. At 15 nd 2 dys, 2/3 nd the whole yolk sc ws sored respectively nd feeding strted

3 on dy 15. Fry were fed y hnd nd feeding rte ws 4.1 percent of ody weight, six times dily. The proximte composition of used commercil food ws crude protein (55 %), crude lipid (12 %), sh (2 %), crude fier (12 %), phosphorus (1.4 %), moisture (1 %) nd crude energy (54 kcl kg -1 ). After removing the morphologiclly norml smple fries on ice the 7-9 smple fry were rinsed in distilled wter nd dried of residul wter with pper toweling nd frozen t -2ºC until nlysis. After determintion of wet ody mss (men ± SD), frozen whole yolk sc fry were prtilly thwed t room temperture, weighed nd homogenized on ice in five volumes of.2m NCl (w/v) (Gwlick et l., 2) using homogenizer (DI 18 Disperser). The suspensions were centrifuged (5 rpm, 3min, 4ºC) nd superntnt stored t - 2ºC until nlysis. Concentrtion of solule protein in extrcts ws estimted y method of the Lowry et l. (1951) which ws lso used y Fuente-Betncourt et l. (29) using ovine serum lumin (1mg/ml) s stndrd. Fry extrcts were ssyed for the determintion of pepsin, trypsin, chymotrypsin, α-mylse, lipse nd lkline phosphtse. Pepsin ctivity ws mesured using the method detiled y Anson (1938) nd Alvrez-Gonzlez et l. (28) using hemogloin s sustrte. One unit of ctivity ws defined s 1 μmole of tyrosine relesed min -1 nd sornce ws mesured t 28 nm. Trypsin ctivity ws mesured ccording to the method of Erlnger et l. (1961) nd Chong et l. (22) using BAPNA (N-α-enzoyl-Lrginine-p-nitronilide hydrochloride) s sustrte. One unit of ctivity ws defined Irnin Journl of Fisheries Sciences, 1(3), s 1μ mole of p-nitroniline relesed min -1 t 41 nm. Chymotrypsin ctivity ws mesured using BTEE (N-enzoyl-Ltyrosine ethyl ester) s sustrte (Hummel, 1959) nd Degur et l. (23). One unit of ctivity ws defined s 1 μmole of N- enzoyl-dl-tyrosine relesed min -1 t 256 nm. The ctivity of α-mylse ws estimted using the Bernfeld (1951) nd Degur et l. (23) procedure nd strch s sustrte. One unit of ctivity ws defined s 1 μmole of mltose relesed min -1 nd sornce ws mesured t 54 nm. Lipse ctivity ws mesured using the titrtion method detiled y Worthington (1991) nd Zmni et l. (29) using olive oil-gum ric emulsion. One unit of ctivity ws defined s 1 μ mole of ftty cid relesed min -1. Alkline phosphtse ctivity ws ssyed following Wlter & Schutt (1974) nd Gwlick et l. (2) using 4-nitrophenyl phosphte s sustrte. One unit of ctivity ws defined s 1 μ mole of 4-nitrophenol relesed min -1 t 45 nm. The ctivity of different enzymes ws ssyed in reltion to solule protein in the extrcts (specific ctivity). Mesurements were crried out y UV/VS spectrophotometer (Ultro Spec 2 Phrmci Biotech). Concentrtion of the ssy products ws determined experimentlly sed on stndrd curve of sornce vs. product concentrtion using the sme volumes s the ssys. Men vlues of enzyme ctivities were compred mong the different stges of fry development from 1 to 58 dys with one wy ANOVA test. Results tht were significntly different were nlyzed y Duncn's multiple rnge test with.5 α. Also, the reltionship etween DAH nd wet ody mss ws estimted using

4 46 Golchinfr et l., Assessment of digestive enzymes ctivity during the fry.. exponentil regression nlysis (Cr et l., 23; Fulk et l., 27; Zmni et l., 29). All mesurements were crried out in triplicte nd results were given s men ± SD. Results As demonstrted in figure 1, wet ody mss ws mesured during the experimentl period from 1 to 31 dys, t 1, 15, 2 nd 31 dys it ws 74.2 ±1.9, 13.2 ± 1.8, ± 2.1 nd ± 2.3 mg respectively. At the first dy, yolk sc formed the min prt of the fry ody ut sored grdully from1 to 31 dys. Wet Body Mss (mg) y = e.632x R 2 = Dys After Htching Figure 1: The reltionship etween DAH nd wet ody mss during the growth of O. mykiss fry (Wet ody mss (mg) indicted s men ± SD from the first dy till to 31 th ). As showed t figure 2, trypsin, lipse nd lkline phosphtse ctivities indicted n incresing trend in the first 12 dys without significnt difference (P >.5). From 12 to 18 dys, there ws n incresed ctivity nd more significnt difference thn the first 12 dys (P <.5). After 18 dys, trypsin nd lipse decresed up to 25 dys then incresed from 25 to 31 dys while lkline phosphtse decresed up to 2 dys then incresed from 2 to 31 dys without significnt difference (P >.5).

5 Irnin Journl of Fisheries Sciences, 1(3), Trypsin Chymotrypsin c c c Specific Activity (U mg protein -1 ) Pepsin Amylse Lipse Alkline phosphtse /3 yolk sc is sored nd feeding is strted Yolk sc is sored completely DAH Figure 2: Men vlue (±SD for replicte (n = 3) pooled fry) of specific ctivity of digestive enzymes in O.mykiss fry during the experimentl period. Different superscripts denote significnt differences (P <.5) within DAH. Sttisticl nlyses re sujected to the one wy ANOVA test y spss11.5 softwre.

6 48 Golchinfr et l., Assessment of digestive enzymes ctivity during the fry.. Chymotrypsin ctivity demonstrted n incresing grde in the first 12 dys ut no difference sttisticlly (P >.5) nd showed incresed slope with more significnt difference in its ctivity during 12 to 18 dys compred to the first 12 dys (P <.5). After 18 dys, chymotrypsin ctivity decresed initilly up to 2 dys nd then incresed to 31 dys without significnt difference (P >.5). The difference in chymotrypsin ctivity ws more significnt during 2 to 31 thn the first 2 dys (P >.5). Pepsin ctivity reveled n incresing profile during 31 dys without significnt difference in the first 12 dys (P >.5), while fter 12 to 31 dys there ws more significnt difference thn the first 12 dys (P >.5). Amylse ctivity showed constnt trend in the first 12 dys without significnt difference (P >.5) ut incresed significntly from 12 to 18 dys thn the first 12 dys (P <.5). After 18 dys, it decresed up to 25 dys nd then incresed up to 31 dys without significnt difference (P >.5). Amylse ctivity showed more significnt difference from 18 to 31 dys thn 12 to 18 dys (P <.5) ut etween 18 to 31 dys nd the first 12 dys there ws no significnt difference (P >.5). All ssessments in enzyme ctivities showed no significnt difference fter 12 to 18 dys (P >.5). Discussion Studies hve expressed tht etween the ge nd growth in fish lrve there is close correltion tht cn e relted to formtion nd differentition of ody orgns such s stomch, intestine, liver nd other orgns (Govoni et l., 1986; Diz-Lopez et l., 1997: Cr et l., 23; Chu et l., 24; Fulk et l., 27; Zmni et l., 29). In rinow trout high correltion ws oserved etween the wet ody mss nd the DAH where the estimted vlues were y = e.632x, r 2 = In ddition, the ssessments in different fish lrve reveled tht formtion nd differentition of ody orgns cn ffect some physiologicl chnges such s enzymes secretion (Kuzmin nd Gelmn, 1998; Cr et l., 23; Fulk et l., 27). The presence of some min digestive enzymes in fish lrve in primry stges fter htching hs een shown clerly in species such s, Acipenser fulvescens (Buddington, 1985), Acipenser trnsmontnus L. (Gwlick et l., 1995), Sprus urt (Moyno et l., 1996), Sole seneglensis kup (Mrtinez et l., 1999), Oreochromis niloticus L. (Tengjroenkul et l., 21), Diplodus puntzzo (Suzer et l., 27), Rchycentron cndum (Fulk et l., 27) Prlrx mcultofscitus (Pen et l., 29), Slmo trutt cspius (Zmni et l., 29) nd Prlrx mcultofscitus (Alvrez-Gonzlez et l., 21). The present considertion showed enzyme existence in the rinow trout fry 1 dy fter htching. After 1 DAH, further development of such ctivities seems to e ffected minly y the progressive ppernce of the digestive orgns nd y the response to feeding s oserved in some species such s Cspin rown trout (Mojzi Amiri et l., 25; Zmni et l, 29). These typicl results in ptterns show succession of incresing nd decresing with time s oserved in

7 other fish lrv (Moyno et l., 1996; Mrtinez et l., 1999). Nevertheless, severl uthors hve descried pttern chrcterized y grdul decrese of specific ctivities of the some digestive enzymes, (e.g. mylse) result tht my e explined y the progressive increse in solule proteins tking plce in growing lrve s the ody tissues frction ecomes stedily more importnt. In the present study, n increse ws oserved in enzyme ctivities t 15 DAH, which my reflect the response to feeding with significnt difference from the ctivity from 1 to 12 DAH (P <.5), since the synthesis of the min digestive enzymes in lrvl nd juvenile fishes is gretly dependent on the food (Suzer et l., 26). Pepsin secreted vi stomch indicted to come long with incresing DAH, s seen in other fish lrv (Buddington, 1985; Suzer et l., 27; Zmni et l., 29). The sence of pepsin in the digestive trct of teleosts during erly stges of life is thought to e compensted y micropinocytosis nd intrcellulr digestion of proteins in the posterior intestine (Cr et l., 23). From 12 DAH up to 31 DAH, pepsin ctivity showed reltively incresed profile whic could proly e explined y the development of orgns such s gstric glnds s oserved in others (Cr et l., 23; Alvrez-Gonzlez et l., 28; Zmni et l., 29). Alkline phosphtse hs een detected during lrvl development in different fish (Cr et l., 23; Suzer et l., 27; Alvrez- Gonzlez et l., 28; Zmni et l., 29). According to Lojd et l., (1979), lkline phosphtse is found primrily in cell memrnes where ctive trnsport occurs nd its increse in the intestine of Irnin Journl of Fisheries Sciences, 1(3), fish lrv should indicte more functionl development of entrocytes. The presence of mylse ctivity hs een reported in lrve of severl fishes including Acipenser fulvescens (Buddington, 1985), Sprus urt (Moyno et l., 1996), D.srgus (Cr et l., 23) nd Pgrus pgrus, L. (Suzer et l., 27) nd Cyprinus crpio vr. Jin (Jing et l., 29). Initil high levels of mylse could e etter explined s result of progrmmed gene expression, s suggested y Zmonino-infnte nd Chu (21). However, mylse hd constnt profile in rinow trout fry from 1 to 12 DAH (2/3 yolk sc ws sored) ut the presence of certin mount of crohydrte in the formulted diet given to fry t 15 DAH, could led to enzyme secretion. Lipse hs een detected during lrvl development in fishes including D.srgus, A.fulvescens, S.seneglensis, D.puntzzo, Cyprinus crpio (vr. Jin) nd Slmo Cspius. The mjor lipse in fishes ppers to e non-specific nd ile slt-dependent lipse (Gjellesvik et l., 1992). Oozeki & iley (1995) recommended the existence of two types of lipse in fish lrve, one relted to yolk sc sorption (which is rich in phospholipids) nd the other, whose ctivity is developed lter, relted to digestion of exogenous lipids. In the present study, lipse ws lso ffected y mjor events such s the strt of feeding (15 DAH). Trypsin nd chymotrypsin elong to lkline proteses with considerle role in food digestion (Joling, 1995). The pttern of trypsin nd chymotrypsin showed pek t the 15 DAH which cn reflect the response to feeding. There is some indiction tht the

8 41 Golchinfr et l., Assessment of digestive enzymes ctivity during the fry.. ctivities of digestive enzymes re low t first feeding nd increse during the lrvl development. Kwi nd Iked (1973) oserved tht ctivities of mylse, mltse nd trypsin incresed in ulk ssys of lrve of incresing ge fter yolk sorption. In contrst, Hjelmelnd et l. (1983) used more sensitive nd specific rdio-immuno ssy etween htching to yolk sc sorption nd showed tht trypsin nd trypsinogen ctivities decresed to low levels for 14 dys, nd then incresed. There my e two explntions for the increse in crohydrolytic nd proteolytic ctivities in fish lrve fter yolk sorption nd first feeding. First, the inherent enzymes ville in the food my hve cused n increse nd second, enzymes production y the liver, pncres nd mucosl epithelium my hve stimulted in response to initil food consumption nd incresed rtion size. Results of enzymtic ssessments reveled the existence of n incresed profile in enzyme ctivity etween 15 to 18 DAH nd there ws sttisticlly significnt difference from the ctivity from 1 to 12 DAH (P <.5). In conclusion, enzyme ssessments revel tht rinow trout fry cn receive food erlier thn 15 DAH (etween 12 to 15 DAH). Also, the results of our study indicte the est time for strt of feeding so tht useless feeding is inhiited nd suitle growth is chieved. More reserches re necessry on digestive physiology (e.g. histochemicl study of digestive enzymes), in order to delinete t exctly wht ge the fry is le to digest nd sor exogenous nutrients. One of the most importnt discussions in feeding is pying ttention to physiologic chrcteriztions nd understnding digestive enzymes ctivity from the htching to yolk sc sorption. Acknowledgments The uthors would like to thnk the stff of Kelrdsht Cold Wter Fisheries Center for their help in collections of eggs nd fries. We re lso grteful for the most efficient technicl ssistnce given y the personnel of the iotechnology deprtment of Rzi Vccine nd Serum Reserch Institute for the ssy of enzymtic ctivity. References Alvrez-Gonzlez, C. A., Moyno- Lopez, F. J., Civer-Cerecedo, R., Crrsco-Chvez, V., Ortiz- Glindo, J. L. nd Dums, S., 28. Development of digestive enzyme ctivities in lrve of spotted snd ss Prlrx mcultofscitus: I. Biochemicl nlysis. Fish Physiology nd Biochemistry, 34, Alvrez-Gonzlez, C. A., Moyno- Lopez, F. J., Civer-Cerecedo, R., Crrsco-Chvez, V., Ortiz- Glindo, J. L., Nolsco-Sori, H., Tovr-Rmırez, D. et l., 21. Development of digestive enzyme ctivity in lrve of spotted snd ss Prlrx mcultofscitus II: Electrophoretic nlysis. Fish Physiology nd Biochemistry, 36, Anonymous, 28. Irnin Fisheries Orgniztion Annul Report. 56 P. (in Persin). Anson, M. L., The estimtion of pepsin, trypsin, ppin nd cthepsin

9 with hemogloin. Journl of Generl Physiology, 22, Bernfeld, P., Amylses α nd β. In Methods in Enzymology, Vol.1 (Colowick, P. & Kpln, N.O., eds), pp: New York: Acdemic Press. Buddington, R. K., Digestive secretions of lke sturgeon, Acipenser fulvescens, during erly development. Journl of Fish Biology, 26, Chu,C., Ronnestd,I.,Grngier,V. nd Zmonino Infnte,J. L.,24. Expression nd ctivities of pncretic enzymes in developing se ss lrve (Dicentrrchus lrx) in reltion to intct nd hydrolyzed dietry protein; involvement of cholecystokinin.aquculture,238(1-4), Cr, J. B., Moyno, F. J., Crdens, S., Fernndez-Diz, C. nd Yuffer, M., 23. Assessment of digestive enzyme ctivities during lrvl development of white rem. Journl of Fish Biology, 63, Cr, B, Moyno, F.J., Zmonino, J.L. nd Fuvel, C., 27. Trypsin nd chymotrypsin s indictors of nutritionl sttus of post-wened se ss lrve. Journl of Fish Biology, 7, Cruso, G., G. Denro, M. nd Genovese, L., 28. Temporl chnges in digestive enzyme ctivities in the gstrointestinl trct of Europen eel (Anguill nguill) following feeding. Mrine nd Freshwter Behviour nd Physiology, 41(4), Chong, A.S.C., Hshim, R., Chow-Yng, L., nd Ali, A.B. 22. Prtil Irnin Journl of Fisheries Sciences, 1(3), chrcteriztion nd ctivities of proteses from the digestive trct of discus fish (Symphysodon equifscit). Aquculture, 23, Degur, S., Juncey, K. nd Agius, C., 23. Enzyme ctivities nd ph vritions in the digestive trct of gilthed se rem. Journl of Fish Biology, 62, Diz-Lopez, M., Moyno, F.J., Grci- Crreno, F.L.,Alrcon, F.J. nd Srsquete,M.C.,1997. Sustrte SDS-PAGE determintion of protese ctivity through lrvl development in se rem. Aquculture Interntionl, 5, Erlnger, B., Kokowsky, N. nd Cohen, W., The preprtion nd properties of two new chromogenic sustrtes of trypsin. Archive Biochemistry nd Biophysics, 95, Fulk, C.K., Benninghoff, A.D. And Holt, G.J., 27. Ontogeny of the gstrointestinl trct nd selected digestive enzymes in coi Rchycentron cndum (L.). Journl of Fish Biology, 7, Fuente-Betncourt, D.LG.,Grci- Crreno, F., Del Toro, N.M.A. nd Cordov-Muruet, J.H., 29. Effect of ph nd temperture on jumo squid proteins. Journl of Food Biochemistry, 33, Gwlick, A., Teh, S. L., Hung, S. S. O., Hinton, D. E. nd De L Noue, J., Histologicl nd histochemicl chnges in the digestive trct of white sturgeon lrve during ontogeny. Fish Physiology nd Biochemistry, 14,

10 412 Golchinfr et l., Assessment of digestive enzymes ctivity during the fry.. Gwlick, A., Prent, B., Horn, M.H., Ross, N., Opstd, I. nd Torrissen, O.J., 2. Activity of digestive enzymes in yolk-sc lrve of Atlntic hliut (Hippoglossus hippoglossus): indiction of rediness for first feeding. Aquculture, 184(3-4), Gjellesvik, D.R., Lomrdo, D. nd Wlther, B.T., Pncretic ile slt dependent lipse from cod (Gdus morhu): purifiction nd properties. Biochemic And Biophysic Act, 1124, Govoni, J.J, Boehlert, G.W. nd Wtne,Y., Physiology of digestion in fish lrve. Environmentl Biology of Fishes, 16 (1-3), Hjelmelnd, K., Huse, I., Jorgensen, T., Molvik, G. nd R, J., Trypsin nd trypsinogen s indices of growth nd survivl potentil of cod (Gdus morhu L.) lrve. Flodevigen Rpportserie, 3, Hummel, B. C. W., A modified spectrophotometric determintion of chymotrypsin, trypsin nd thromin. Cndin Journl of Biochemistry nd Physiology, 37, Jing,W.D., Feng, L., Liu, Y., Jing, J. nd Zhou, X.Q., 29. Growth, digestive cpcity nd intestinl microflor of juvenile Jin crp (Cyprinus crpio vr. Jin) fed grded levels of dietry inositol. Aquculture Reserch, 4, Joling, M., Digestion nd sorption. In:Joling, M.(Ed),Environmentl Biology of Fishes, Chpter6.Chpmn & Hll, London Englnd. Pp, Kwi, S. nd Iked, S., Studies enzymes of fishes IY. Development of the digestive enzymes of crp nd lck se rem fter htching. Bulletin of the Jpnese Society of Scientific Fisheries, 39, Kim, B.G., Divkrn, S., Brown, C.L nd Ostrowski, A. C., 21. Comprtive digestive enzyme ontogeny in two mrine lrvl fishes: Pcific thredfin (Polydctylus sexfilis) nd luefin trevlly (Crnx melmpygus). Fish Physiology nd Biochemistry, 24, Kolkovski, S., 21. Digestive enzymes in fish lrve nd juveniles-implictions nd ppliction to formulted diets. Aquculture, 2, Kuzmin, V. V Influnce of ge on digestive enzymes ctivity in some freshwter teleosts. Aquculture, 148, Kuzmin,V. V. nd Gelmn, A. G., Trits in the development of the digestive function in fish. Journl of Ichthyology, 39 (1), Kuzmin, V. V. nd Skvortsov, E. G., 21. Activity of proteolytic enzymes of potentil prey of predtory fish influence of nturl nd nthropogenic fctors. Journl of Ichthyology, 41(3), Lzo, J. P., Holt, G. J. nd Arnold, C. R., 2. Ontogeny of pncretic enzymes in lrvl red drum, Scienops ocelltus. Aquculture Nutrition, 6, Lojd, Z., Gossru, R. nd Schieler,T. H., Enzyme Histochemistry: A Lortory Mnul.NewYork:Springer-Verlg.

11 Lopez-Vsquez,K., Cstro-Perez, C. A. nd Vl, A. L., 29. Digestive enzymes of eight Amzonin teleosts with different feeding hits. Journl of Fish Biology, 74, Lowry, O. H., Roserough, N. J., Frr, A. L nd Rndll, R. J., Protein mesurement with the folin phenol regent. Journl of Biologicl Chemistry, 193, Mrtinez, I., Moyno, F. J., Fernndez- Diz, C nd Yufer, M., Digestive enzyme ctivity during lrvl development of the Senegl sole (Sole seneglensis). Fish Physiology nd Biochemistry, 21, Mojzi Amiri, B., Bhrekzemi, M., Pousti, I. nd Vilki, A. S., 25. A histologicl study on the development of the digestive trct of Slmo trutt cspius (Kessleri) from htching to prr stge. Irnin Journl of Fisheries Science, 5 (1), Moyno F. J., Brros A. M., Prieto, A., Cñvte J. P. nd Cārdens S., 25. Evlución de l ontogeni de enzims digestivs en lrvs de hurt, Pgrus urig (Pisces: Spride). Revist Aqutic Sciences, 22, Moyno, F. J., Diz, M., Alrcon, F. J. nd Srsquete, M. C., Chrcteriztion of digestive enzyme ctivity during lrvl development of gilthed serem (Sprus urt). Fish Physiology nd Biochemistry, 15, Oozeki, Y. nd Biley, K. M., Ontogenetic development of digestive enzymes ctivities in lrvl wlleye pollock, Thergr chlcogrm. Mrine Biology, 122, Irnin Journl of Fisheries Sciences, 1(3), Pen, R., Dums, S. nd Rodriguez- Jrmillo, C., 29. Development nd distriution of intestinl enzymtic ctivity in Prlrx mcultofscitus lrve fed with live prey. Aquculture Reserch, 4, Rieiro, L., Couto, A., Olmedo, M., Alvrez-Blzquez, B., Linres, F. nd Vlente, L. M. P., 28. Digestive enzyme ctivity t different developmentl stges of lckspot serem, Pgellus ogrveo. Aquculture Reserch, 39, Rieiro, L., Zmonino Infnte, J. L., Chu, C. nd Dinis, M. T., Development of digestive enzymes in lrve of Sole seneglensis, Kup Aquculture, 179, Suzer, G., Aktülün, S., Çon, D., Kmci, H.O., Sk, Ş., Fırt, K. nd Alpz, A., 27. Digestive enzyme ctivities in lrve of shrpsnout se rem (Diplodus puntzzo), Comprtive Biochemistry nd Physiology, 148A, Suzer, G., Kmci, H.O., Çon, D., Sk, Ş., Firt, K., Özkr, B. nd Özkr, A., 27. Digestive enzyme ctivity of the red porgy (Pgrus pgrus, L.) during lrvl development under culture conditions. Aquculture Reserch, 38, Suzer, G., Firt, K. nd Sk, S., 26. Ontogenic development of the digestive enzymes in common pndor, Pgellus erythrinus, L. lrve, Aquculture Reserch, 37(15), Tengjroenkul, B., Bonnie, J., Smith, S. A. nd Chtreewongsin, U., 21. Ontogenic development of the

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