Effect of Feeding on Digestive Enzyme Activity and Morphological Changes in the Liver and Pancreas of Pike-Perch (Sander lucioperca)

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1 The Isreli Journl of Aquculture - Bmidgeh 62(4), 2010, The IJA ppers exclusively s peerreviewed on-line Open Access journl t Sle of IJA ppers is strictly foridden. Effect of Feeding on Digestive Enzyme Activity nd Morphologicl Chnges in the Liver nd Pncres of Pike-Perch (Snder lucioperc) Mciej Kmszewski*, Łuksz Npor-Rutkowski, Teres Ostszewsk Division of Ichthyoiology nd Fisheries, Fculty of Animl Sciences, Wrsw University of Life Sciences, Ciszewskiego 8, Wrszw, Polnd (Received , Accepted ) Key words: pike-perch, histology, liver, pncres, digestive enzymes Astrct The present study evluted the effect of feeding on growth rte, histology of liver nd pncres, nd ctivity of some digestive enzymes in pike-perch (Snder lucioperc L). The fish were fed one of the following diets: Aglo Norse, csein-geltin, cod mel, or Artemi slin nuplii (control). On the lst dy of the experiment, fish fed the Artemi nd Aglo Norse diets hd the highest ody mss, length, nd survivl. Those fed the Aglo Norse diet showed glycogen nd lipid storge in heptocytes, nd hd the lrgest re of exocrine pncretic cells. They lso showed higher trypsin, lipse, nd mylse ctivity thn fish fed other diets. Fish fed the csein-geltin diet hd the lowest growth rte, smllest re of heptocytes nd exocrine pncretic cells, nd the lowest trypsin nd mylse ctivity. The group fed the cod mel diet hd the lowest survivl nd very low growth rte (similr to tht oserved in the csein-geltin group). These results indicte tht the csein-geltin nd cod mel diets did not meet the nutritionl requirements of pike-perch. * Corresponding uthor. Tel.: , fx: , e-mil: mciej_kmszewski@sggw.pl

2 226 Kmszewski et l. Introduction Aquculture is the most dynmiclly developing sector of niml production in the world. Further increse in production efficiency requires development of etter feeds, prticulrly improvement of protein qulity. Most economiclly importnt fish species re crnivorous nd require feeds with high protein content. Fishmel is the most commonly used source of high qulity protein ut its high cost nd incresed demnd hs creted the need to find lterntive protein sources. Therefore, soyen mel, csein, whet gluten mel, nd other products re eing evluted s lterntive protein sources. Aquculture of pike-perch (Snder lucioperc L.) is limited y high fish mortlity, cnnilism, nd poor uptke of rtificil diets (Ljunggren et l., 2003). However, reserch confirms tht pike-perch cn e rered on rtificil diets from the eginning of exogenous feeding under controlled conditions (Ostszewsk et l., 2005; Szkudlrek nd Zkęś, 2007). Usully, pikeperch lrve re first fed nturl food, nd rtificil diets re introduced lter. The pproprite time to chnge the diet is dys fter htching (Kestemont et l., 2007; Szkudlrek nd Zkęś, 2007). At tht time, lrve hve fully inflted swim ldder, the yolk is completely resored, nd the digestive trct is mture (Ostszewsk, 2005). Filures relted to feeding with rtificil diets my result from indequte nutrient content, poor uptke of feed, or inility of fish to digest nd sor nutrients. Therefore, stisfctory rtificil feeds tht could completely replce expensive nturl foods re eing sought (Ljunggren et l., 2003; Ostszewsk et l., 2005). Such feeds should contin optimum levels of ll nutrients to otin high survivl nd growth rtes, nd llow for correct development of fish. Poor uptke nd digestion of feeds in young fish hve often een explined y low ctivity of digestive enzymes or n undeveloped digestive trct (Hmz et l., 2007; Kestemont et l., 2007). The im of the present study ws to evlute the effect of vrious diets on survivl, growth rte, histology of liver nd pncres, nd ctivity of some digestive enzymes in pike-perch juveniles. Mterils nd Methods Experimentl design. The experiment ws crried out in the lortory of the Division of Ichthyoiology nd Fisheries t the Wrsw University of Life Sciences. Pike-perch juveniles (18-dys-old, 18.59±1.68 mm, 0.05±0.01 g) were rered in recirculting quri (20-l) t five individuls per liter. The system ws equipped with iologicl filter nd UV lmps. The light regime of 14 h light:10 h drk ws regulted y low intensity (100 lux) fluorescent tue t the wter surfce. The wter temperture ws 20.6±0.9 C nd ph 7.8±0.36. Concentrtions of nitrogen metolites did not exceed sfe levels: N-NH ws elow 0.1 mg/l nd N-NO 2 elow 0.01 mg/l. The tnks were clened dily nd ded individuls were removed. The experiment lsted 21 dys (from 18 to 39 dys post htching). The fish were divided into four feeding groups, with five replictes of ech tretment. The fish were fed six times dy (every two hours) from the first

3 Digestive enzymes nd morphologicl chnges in pike-perch 227 dy of the experiment. The feeding rte ws 50% of the fish iomss during the first week, 15% during the second week, nd 10% during the third week. The following diets were used: Aglo Norse (Lrve Feed Ewos, Bergen, Norwy), two formulted diets, i.e., csein-geltin nd cod mel-geltin (Ostszewsk et l., 2005). The cod mel diet contined the sme components s the csein diet, except tht the csein ws replced with cod mel (Vereinigte Fischmehlwerke, Cuxhven, Germny). The control groups were fed Artemi slin nuplii d liitum. The protein nd lipid contents of the feeds re shown in Tle 1. Histologicl nd iochemicl nlyses. Ten fish from ech experimentl group were Tle 1. Protein nd lipid smpled weekly nd nesthetized with MS-222 contents (%) in diets. (tricine methnesulphonte, Sigm). Smples Diet Protein Lipid were tken in the morning efore food Artemi nuplii Aglo Norse Csein-geltin Cod mel-geltin distriution. Fish were weighed nd mesured to n ccurcy of 0.01 g nd 0.02 mm. Specific growth rte ws clculted s: SGR = (100 x [ln Wf - ln Wi ])/n, where Wf = finl ody weight, Wi = initil ody weight, nd n = experiment durtion (dys). Fish were preserved in Bouin's solution for histologicl nlysis. The preserved fish were emedded in prffin nd cut into 5 μm longitudinl sections using microtome (Leic RM2265, Leic Microsystems, Nussloch, Germny). The preprtions were stined with AB/PAS (lcin lue/schiff regent) nd H/E (hemtoxylin/eosin) ccording to Perse (1985). A Nikon Eclipse 90i microscope connected to Nikon Digitl Sight DS-U1 cmer (Nikon Corportion, Tokyo, Jpn) ws used for microscopic oservtions nd microgrphs. NIS-Elements AR 2.10 softwre (Nikon Corportion, Tokyo, Jpn) ws used for morphometric mesurements. Fifteen mesurements were tken in ech fish of the pncres exocrine cell re, the heptocyte re, the re occupied y lipids in the heptocyte cytoplsm, nd the dimeter of the heptocyte nuclei (15 x 10). Ten fish from ech experimentl group were pooled on the first nd lst dys of the experiment to otin 1 g smples (lrve were relieved of heds nd tils) to determine enzymtic ctivity. The smples were preserved in liquid nitrogen nd stored t -80 C. The mteril ws homogenized in pproprite uffers nd smples were centrifuged t 4 C for 15 min t 15,000 g. Enzymtic nlyses were repeted five times. Asornce ws mesured using n M501 Cmspec spectrophotometer (Cmspec Ltd., Swston, United Kingdom). Lipse ctivity ws mesured t 25 C using the specific sustrte p- nitrophenyl plmitte (p-npp), ccording to the method descried y Winkler nd Stuckmn (1979), nd expressed s μmol of p-nitrophenol (p-np) relesed from 1 g of fish extrct (U/g of fish) in one minute. The ctivity of α- mylse ws mesured ccording to Bernfeld (1955) using DNS (3,5- dinitroslicylic cid) t 30 C nd expressed s μmol of mltose relesed from

4 228 Kmszewski et l. 1 g of fish extrct (U/g of fish) in one minute. Trypsin ctivity ws evluted ccording to Erlnger et l. (1961) using the specific sustrte BAPNA (α-n- Benzoyl-DL-rginine-4-nitronilide hydrochloride) t 25 C nd expressed s μmol of p-nitroniline relesed from 1 g of fish extrct (U/g of fish) in one minute. Sttisticl nlysis. Results were sujected to sttisticl nlysis using Sttgrphics Plus 4.1 nd Sttistic 8.0 softwre. Arithmetic mens nd stndrd devitions were clculted for survivl, totl ody length nd mss, morphometric mesurements, nd enzymtic ctivity for ech feeding group. Significnces of differences were clculted using one-wy ANOVA nd LSD post-hoc test. Results Totl ody length, weight, growth, nd survivl. The highest increse in verge ody length nd weight ws oserved in pike-perch fed the control diet (Fig. 1). On the lst dy of the experiment their length ws 43.24±1.94 mm nd mss ws 0.61±0.08 g. Fish fed the Aglo Norse diet were slightly smller (42.59±2.65 mm nd 0.58±0.11 g) ut not significntly different. The length nd mss in the csein (26.24±2.51 mm; 0.11±0.03 g) nd cod mel (28.47±2.63 mm; 0.14±0.04 g) groups significntly differed from the control nd Aglo Norse groups dy 14 dy 21 dy 0,8 0,7 7 dy 14 dy 21 dy 40 0,6 Averge totl ody length (mm) Averge totl ody weight (g) 0,5 0,4 0,3 0,2 0,1 c c 0 Art An Cs Mc Art An Cs Mc Art An Cs Mc Diets 0,0 Art An Cs Mc Art An Cs Mc Art An Cs Mc Fig. 1. Averge totl ody () length nd () weight of pike-perch fed Artemi nuplii (Art), Aglo Norse commercil feed (An), csein-geltin diet (Cs), or cod melgeltin diet (Mc); mens±sd, n = 10. Vlues with different superscripts significntly differ t p<0.05. Diets The SGR ws highest in the control nd Aglo Norse groups (11.96±0.61%/dy nd 11.68±0.92%/dy, respectively) nd did not significntly differ from ech other. The SGR in fish fed the cod mel

5 Digestive enzymes nd morphologicl chnges in pike-perch 229 (4.9±1.29%/dy) nd csein (3.81±1.4%/dy) diets significntly differed from ech other nd from the control nd Aglo Norse diets. Survivl ws highest in the control group (65.5±14.8%), followed y the Aglo Norse (62.3±7.7 %) nd csein (43.9±18.1%) groups. Survivl in the cod mel group (35.4±16.4%) significntly differed from survivl in the control nd Aglo Norse groups. Liver. At the eginning nd end of the experiment, the liver ws visile s lrge compct orgn, ventrlly situted. Heptocytes showed centrlly locted lrge nuclei with distinct nucleoli (Fig. 2). The lrgest heptocytes were oserved in fish fed the control diet, the smllest in those fed the csein, while the highest heptocyte nucleus dimeter occurred in the csein group Fig. 2. Histologicl imge of liver of pike-perch fed: (A) Artemi nuplii, (B) Aglo Norse commercil feed, (C) csein-geltin diet, or (D) cod mel-geltin diet; H/E stining. Scle r = 10 µm. nd the most undnt lipid storge ws found in the control fish (Tle 2, Fig. 2A). Glycogen grins were seen in the heptocytes (the PAS-positive

6 230 Kmszewski et l. res), nd the mount of glycogen ws similr in ll groups except the csein group. At the end of the experiment, ll morphometric liver prmeters (heptocyte re, dimeter of heptocyte nuclei, cytoplsm re occupied y lipids) were significntly higher thn t the eginning of the experiment. Activity of digestive enzymes. Amylse ctivity incresed with time in ll dietry groups. The highest mylse ctivity ws oserved in fish fed the Aglo Norse diet while the lowest ws in the csein group. Trypsin ctivity incresed in ll groups except the csein group. The highest lipse ctivity ws oserved in the Aglo Norse group. Tle 2. Pncres cell re, heptocyte mesurements, nd enzyme ctivity in pike-perch fed different diets for 21 dys (mens±sd, n = 150). Initil Artemi Aglo Norse Csein-geltin Cod mel-geltin Pncres cell re (µm 2 ) 60.53± ± ± ±17.57 d 85.09±18.62 c Heptocyte mesurements Are (µm 2 ) 59.58± ± ± ±16.42 c ±23.63 Nucleus dimeter (µm) 3.54± ± ± ± ±0.71 Cytoplsm lipid (µm 2 ) 2.93± ± ± ±3.82 c 8.42±3.45 c Enzyme ctivity (U/g) Amylse 0.129± ± ± ± ±0.057 Trypsin 0.192± ± ± ±0.043 c 0.273±0.044 Lipse 0.017± ±0.003 c 0.034± ± ±0.003 c Different superscripts indicte significnt differences t p<0.05. Exocrine pncres. On the first nd lst dy of the experiment, the pncres ws smll orgn situted ehind the liver, etween the nterior intestine nd the stomch. Exocrine pncretic cells showed lrge centrlly locted nuclei with distinct nucleoli (Fig. 3). PAS-positive cidophilous proenzyme grnules were present in the cytoplsm. The pncretic ducts nd lood vessel network were well developed. Adipocytes were oserved in the pncres of fish fed the Aglo Norse diet, ut were less numerous in control fish. The lrgest exocrine pncres cells occurred in fish fed the Aglo Norse diet, while the smllest were in the csein group. The most undnt proenzyme grnules were oserved in the exocrine pncretic cells of fish fed the Aglo Norse diet nd Artemi nuplii, ut they were considerly less undnt in the csein nd cod mel groups.

7 Digestive enzymes nd morphologicl chnges in pike-perch 231 Fig. 3. Histologicl imge of exocrine pncres of pike perch fed: (A) Artemi nuplii, (B) Aglo Norse commercil feed, (C) csein-geltin diet, or (D) cod melgeltin diet; AB/PAS stining. Scle rs = 10 µm. Discussion Results indicte tht juvenile pike-perch cn e rered on rtificil feed lone from dy 19 post htching. At tht time, the digestive system is sufficiently mture to digest rtificil feed nd sor nutrients (Ostszewsk, 2005; Kestemont et l., 2007). The est results were otined with the Aglo Norse diet, confirming erlier dt (Ljunggren et l., 2003; Ostszewsk et l., 2005; Ostszewsk nd Borut, 2006). Fish fed the csein or cod mel diets hd considerly lower survivl, ody length, nd mss, suggesting tht these diets re inpproprite for pike-perch feeding. The SGR of pike-perch fed Artemi nuplii nd Aglo Norse ws stisfctory nd comprle with results otined y Ostszewsk nd Borut (2006) ut higher thn the 7.3%/dy otined y Ljunggren et l. (2003) nd lower thn the %/dy otined y Kestemont et l. (2007). Histologicl imge is relile indictor of the nutritionl condition of fish, especilly chnges in the liver structure. Heptocyte lipid deposits indicte

8 232 Kmszewski et l. good nutritionl condition nd pproprite energy stores (Cllero et l., 1999). In the present study, heptocytes of fish fed Artemi nuplii showed the highest surfce re of lipid vcuoles nd no chnges indicting ftty degenertion (stetosis). The higher lipid content in the heptocyte cytoplsm of fish fed Artemi nuplii might hve resulted from the difference in ftty cid profile etween the nturl food of pike-perch juveniles (i.e., freshwter invertertes) nd Artemi nuplii (Ostszewsk nd Borut, 2006). The size of heptocyte nuclei my e n indictor of nutritionl condition in fish (Strüssmnn nd Tkshim, 1990) nd reflect metolic ctivity of heptocytes (Segner nd Bruneck, 1988). In the present study, fish fed the csein diet hd the lrgest heptocyte nuclei. The size of heptocyte nuclei correltes with the cytoplsm glycogen content (Strüssmnn nd Tkshim, 1990). Glycogen stored in heptocytes is the first energy component to e moilized y fish fced with strvtion (Green nd McCormick 1999). Therefore, the lrge heptocyte nuclei oserved in fish fed the csein diet might hve een relted to metolism of glycogen stores (Strüssmnn nd Tkshim, 1990), source of energy during food limittion (Souz et l. 2001). Fish fed the csein diet hd lower heptocyte re, cytoplsm lipid re, nd numer of glycogen grins thn fish fed Artemi nuplii or Aglo Norse. Smll heptocytes indicte limited heptic nutrient storge (Przyył et l., 2006). Reduction in heptocyte cytoplsm re is oserved in strving fish (Souz et l., 2001; Ostszewsk et l., 2006) nd reduced numer nd size of lipid vcuoles is n importnt nd sensitive indictor of fish mlnutrition or strvtion (Chen et l. 2007). The heptocyte nucleus decreses in strving fish fter nutrient stores re utilized (Strüssmnn nd Tkshim, 1990; Souz et l., 2001). In the present study, no groups showed reduction in heptocyte nuclei size. The slow growth, reduced heptocyte re, nd smll cytoplsm lipid content in fish fed the csein diet indicte tht this diet did not meet the nutritionl requirements of the fish. Exocrine pncres structure is nother good indictor of the nutritionl condition of fish. Strvtion induces chnges in this orgn, resulting in tissue degenertion nd ltertion in exocrine pncres cell structure (Crespo et l., 2001). Strved lrve nd juveniles of strd hliut (Prlichthys olivceus) show reduced pncres volume nd locl necrosis of exocrine cells (Gwk et l., 1999). In the present study, the smllest pncretic cell re ws oserved in fish fed the csein diet. This group lso hd low numer of grnules contining digestive proenzymes, possily indicting indequte feeding or strvtion (Gisert et l., 2004; Ostszewsk et l., 2006). The pncres of fish fed Aglo Norse showed undnt dipose cells, which were lso found in the control group. Excessive dietry lipid contents my result in ft deposition in the pncres (Ostszewsk et l., 2005). Both groups hd undnt proenzyme grnules in the exocrine pncretic cells. Proenzyme grnules re precursors of exocrine pncres enzymes (Gisert et l., 2004). Digestive enzyme ctivity nd chnges lso my reflect the nutritionl condition of fish (Drossou et l. 2006). Nutrient sorption nd digestive enzyme ctivity in pike-perch depend on the dietry nutrient contents nd the

9 Digestive enzymes nd morphologicl chnges in pike-perch 233 timing of diet chnges (Hmz et l., 2008). The highest mylse, trypsin, nd lipse ctivity ws oserved in fish fed Aglo Norse. Trypsin ctivity in this group ws significntly higher thn in control fish, indicting tht the protein content nd composition of diet my ffect trypsin ctivity (Zmonino Infnte nd Chu, 2007). Trypsin nd mylse secretion increse in red drum (Scienops ocelltus) lrve fed high lipid diet (Buchet et l., 2000). Similrly in the present study, higher trypsin ctivity occurred in fish fed Aglo Norse which contined higher lipid content thn the cod mel diet (which hd higher protein content thn Aglo Norse). Fish fed Aglo Norse, with the highest lipid content, lso hd the highest lipse ctivity. Similrly, the highest lipse ctivity ws oserved in sterlet (Acipenser ruthenus) fed high lipid feed (Npor-Rutkowski et l., 2009). Pike-perch fed the csein diet hd the lowest trypsin ctivity, similr to the level t the eginning of the experiment. Trypsin nd mylse ctivity dropped in strved strd hliut (Gwk et l., 1999) nd strved Nile tilpi lrve (Oreochromis niloticus; Drossou et l., 2006). Pike-perch fed the csein diet hd slightly (ut not significntly) lower α-mylse ctivity thn other groups. Amylse ctivity decresed followed y drop in protese nd lipse ctivity in strved Adritic sturgeon (Acipenser nccrii; Furne et l. (2008). Activity of these enzymes nd morphometric mesurements of the heptocytes nd exocrine pncretic cells indicte tht the csein diet does not meet the nutritionl requirements of juvenile pike-perch. Likewise, csein is indequte s unique protein source for yellow perch (Perc flvescens) due to its low rginine content (Brown et l., 1996). The low lipse ctivity in fish fed Artemi nuplii confirms erlier oservtions tht this food is not good source of lipids for pike-perch (Ostszewsk nd Borut, 2006). Artemi nuplii contin too little highly unsturted ftty cids (HUFA; Ostszewsk nd Borut, 2006). Ftty cids, prticulrly n-3 (18:3, 20:5, 22:6) nd n-6 (18:2 nd 20:4), re essentil for correct growth nd development of fish nd cnnot e synthesized y the orgnism. Juvenile pike-perch fed Artemi nuplii enriched with HUFA nd vitmin C show etter growth, survivl, lower frequency of skeletl mlformtions, nd higher tolernce to slinity stress thn those fed unsupplemented Artemi nuplii (Kestemont et l., 2007). Pike-perch show low ility to utilize lipids nd other non-protein diet components s energy sources, explining the high protein demnd of this species (Schulz et. l. 2007). Diets contining low lipid (~10%) nd high protein (>40%) contents re recommended for yellow perch (Perc flvescens) nd perch (Perc fluvitilis) juveniles (Brown et l., 1996; Kestemont et l., 2001). Results of the present study, however, indicte tht high lipid diets re more pproprite for pike-perch fed n rtificil diet from 19 dys post htch. A high lipid level is eneficil for fst growing juveniles tht hve much higher metolic rte thn dults (Schulz et l., 2008). Digestiility of ft y percid fishes my e ffected y the qulity nd quntity of dietry crohydrtes (Schulz et l., 2008). An pproprite lnce

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