Blood parameters of Caspian brown trout (Salmo trutta caspius) fingerlings affected by dietary L-ascorbyl-2- polyphosphate

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1 Irnin Journl of Fisheries Sciences 15(3) Blood prmeters of Cspin rown trout (Slmo trutt cspius) fingerlings ffected y dietry L-scoryl-2- polyphosphte Downloded from jifro.ir t 17: on Sundy July 8th 2018 Rji Islmi H. 1* ; Ar N. 2 ; Assre R. 3 ; Rstrvn M.I. 4 ; Etekri R. 1 Received: Decemer 2014 Accepted: My 2015 Astrct This study ws imed to evlute the effect of L-scoryl-2-polyphosphte s dietry scoric cid source on lood prmeters of Cspin rown trout (Slmo trutt cspius), including red lood cell (RBC) count, white lood cell (WBC) nd WBC differentil. A totl numer of 600 Cspin rown trout (9.6±0.6 g) fingerlings were rndomly distriuted in triplictes mong five tretments ech contining 40 specimens. Experimentl diets were lso prepred y dding 0, 50, 100, 200 nd kg -1 L-Ascoryl-2-Polyphosphte to the sl diet. Feeding ws done for nine weeks in ech tretment. The survivl rte in ll tretments ws 100%. The results showed significnt increse in RBC, hemogloin, hemtocrit, WBC nd lymphocyte (p<0.05) y supplementing L-scoryl-2-polyphosphte compred to the control tretment. The fish fed y scoric cid kg -1 diet hd the mximum hemogloin nd hemtocrit in comprison with the other tretments. The current reserch showed tht dietry L-scoryl-2-polyphosphte influences the complete lood count of Cspin rown trout while fingerlings fed with the optimum mounts of scoric cid kg -1 diet for period of 9 weeks tril. Keywords: Ascoric cid, Growth, Hemtologicl prmeters, Slmo trutt cspius. 1-Deprtment of Fisheries, Science nd Reserch Brnch, Islmic Azd University, P.O. Box: , Tehrn, Irn. 2-Young Reserchers nd Elites clu, Science nd Reserch Brnch, Islmic Azd University, Tehrn, Irn. 3-Young Reserchers nd Elites Clu, North Tehrn Brnch, Islmic Azd University, Tehrn, Irn. 4 -Deprtment of Cultivtion, Coldwter Fishes Reserch Center (CFRC), Tonekon, Irn. * Corresponding Author's Emil Address: rji.h@sriu.c.ir

2 1168 Rji Islmi et l., Blood prmeters of Cspin rown trout (Slmo trutt cspius) Downloded from jifro.ir t 17: on Sundy July 8th 2018 Introduction Cspin rown trout (Slmo trutt cspius, Kessler, 1877) is n endngered ndromous species distriuted in southern region of the Cspin Se which migrtes to rivers which connected to the Cspin Se such s Sefid-Rud, Gorgn-Rud, nd Cheshme Kile rivers for reeding (Armntrout, 1980; Kii et l., 1999; Cod, 2000). Artificil propgtion nd relesing fingerlings to nturl wter is n pproch to prevent rown trout s extinction, ut chieving this gol is fced prolemtic from which feeding nd mortlity in fry nd fingerlings re the most importnt ones. Cspin rown trout is sensitive to stress nd disese like mny other species. Powerful defense mechnisms like specific nd non-specific immune re needed ginst pthogens to improve fish helth. Non-specific immune in fish is more importnt thn mmmlins (Mgndóttir, 2006). Hemtology, vlule tool for determintion of fish physiologicl condition nd lood prmeters, is commonly used s physiologicl index ginst internl nd externl chnges (Xioyun et l., 2009; Stheeshkumr et l., 2011). One of the solutions to improve helth, increse resistnce to stress, nd rise immune system efficiency is feed dditive supplementtion (Ai et l., 2004; Cerezuel et l., 2009) such s vitmin C. Feed qulity is n importnt fctor to fish helth preservtion. Immune system efficiency will e incresed y consuming immune stimulnt such s ntioxidnt like vitmin C, crotenoids nd other feed dditives (Mgndóttir, 2006; Orun et l., 2008). Results of different reserches hve shown tht vitmin C hs n effective Immunostimulnt for increses resistnce ginst stresses (Hrdie et l., 1991; Mulero et l., 1998; Kumri nd Shoo, 2005; Ai et l., 2006; Tewry nd Ptr, 2008). Vitmin C is esily otined from the gut nd distriutes widely through the ody (Secomes nd Ellis, 2012). Although, mny studies hve een done on feeding Cspin rown trout (Ser et l., 2005; Rmezni, 2009; Sotoudeh et l., 2011), few study hs een crried out on its lood prmeters. The present study ws conducted to exmine RBC nd WBC s chnges in Cspin rown trout fed y dietry supplementtion of L-scoryl-2- polyphosphte s n immunostimulnt. Mterils nd methods Diet preprtion The sl prcticl diet ws formulted (Tle 1) to contin out 49.60% crude protein nd 14.96% crude lipid, which is suitle for the growth of Cspin rown trout fingerlings (Ser et l., 2005). For prepring the experimentl diets, different levels (50, 100, 200 nd ) of scoric cid in the form of L-scoryl-2- polyphosphte (Tiger, Chin) were dded per kilogrm of diet nd the sl diet ws used s control.

3 Irnin Journl of Fisheries Sciences 15(3) Downloded from jifro.ir t 17: on Sundy July 8th 2018 Tle 1: Ingredient nd chemicl proximte composition of experimentl diets suggested y Ser et l (2005) for Cspin rown trout, Slmo trutt cspius (% dry mtter). Ingredient % Fish mel Soyen mel 8.00 Whet mel 3.84 Fish oil 7.00 Dried cheese 3.00 Gmmrus powder 2.00 Minerl premix 2.00 Vitmin premix 2.00 Proximte nlysis (Averge) Crude protein % Crude lipid % Ash % Moisture % 5.64 Minerl premix (mg kg -1 diet): NF, 2 mg; KI, 0.8 mg; CoCl 2 6H 2 O (1%), 50 mg; CuSO 4 5H 2 O, 10 mg; FeSO 4 H 2 O, 80 mg; ZnSO 4 H 2 O, 50 mg; MnSO 4 H 2 O, 60 mg; MgSO 4 7H 2 O, 1; C (H 2 PO 3 ) 2 H 2 O, 3000 mg; NCl, 100 mg. Vitmin premix (mg or g kg -1 diet): thimin, 25 mg; rioflvin, 45 mg; pyridoxine HCL, 20 mg; vitmin B 12, 0.1 mg; vitmin K 3, 10 mg; inositol, 800 mg; pntothenic cid, 60 mg; nicin cid, ; folic cid, 20 mg, iotin, 1.20 mg; retinol cette, 32 mg; choleclciferol, 5 mg; lph-tocopherol, 120 mg; choline chloride, 2500 mg, ethoxyquin 150 mg, whet middling g. f L- 2APP, L-scoryl-2-polyphosphte (Tiger, Chin). L- 2APP ws seprtely supplemented with 0.0, 50.0, 100.0, nd mg scoric cid equivlent kg 1 diet. The nlyzed levels of dietry scoric cid were 9.8, 43.8, 89.4, nd mg kg 1 diets. Feed prepring ws done every three weeks due to prevent scoric cid degenertion. Feeds were stored t -12 C in freezer until used. Proximte nlysis of diets The mount of dry mtter ws evluted y drying n liquot of ech diet in mechnicl convection oven t 105 C for 16 h to otin constnt weight. Ash content ws determined y heting n liquot of the smples in muffle furnce t 550 C for 3 h nd weighing the remined mteril. Protein content ws lso determined y converting the nitrogen content (N 6.25) sed on the Kjeldhl method. Totl lipid content ws lso extrcted using chloroform-methnol (2:1 y vol.) mixture. All mesurements were done ccording to the AOAC (2000) nd the result ws expressed s g 100 g -1 diet. Ascoric cid content ws lso nlyzed sed on the Shiu nd Hsu (1999). Approximtely 3 5 g of grounded diet ws treted with 25 ml chloroform nd 100 ml distilled wter, shcked for 25 min, settled for 25 min, nd centrifuged for 5 min t 2739 g. One ml of the superntnt ws uffered y 0.2 M cetic cid uffer (ph=4.8) nd 0.2% DTT, kept in 37 C th for 2 h, nd centrifuged for 5 min

4 1170 Rji Islmi et l., Blood prmeters of Cspin rown trout (Slmo trutt cspius) Downloded from jifro.ir t 17: on Sundy July 8th 2018 t 2739 g. Afterwrds, 20 µl of superntnts were sieved through 0.22-pore size syringe filter nd sujected to scoric cid nlysis. The scoric cid content in the diets were determined y reverse-phse HPLC (KNAUER pump 1000, Germn) with n ODS column ( mm, Germn). Moile phse (flow rte 0.6 ml min 1 ) ws n queous solution of 0.05 M KH 2 PO 4 (djusted to ph 3 with phosphoric cid) nd the effluent ws monitored y UV-detector (254 nm wve length). Fish iossy Six hundred Cspin rown trout fingerlings hve een otined from the Coldwter Fishes Reserch Center (CFRC), Tonekon, Irn, nd trnsferred to locl fish frm in Dohezr Rod, Tonekon. After fsting for initil dpttion to the experimentl condition, the specimens were fed y rtificil feed (Behprvr Co., Krj, Irn). Fish were dpted with new conditions for 2 weeks nd fed with control diet. The verge weight of fish fingerlings t the eginning of the experiment ws 9.6±0.6 g. The wter ws supplied from spring with flow rte of 6 L s -1. The wter temperture, dissolved oxygen, ph, nd electricl conductivity were mesured every week. Wter temperture nd dissolved oxygen mesured y n oxygen meter WTW (Oxi 300i, Germny). Level of ph nd electricl conductivity (µs cm -1 ) were lso nlyzed y ph meter (Metrohm 827, Germny). During the experiment, the temperture rnged etween 9.6 to 10.2 C, the ph rnged etween , electricl conductivity rnged from to 252.8, nd the dissolved oxygen ws pproximtely 10.2 mg L -1. Accordingly, the wter condition ws pproprite for the growth of Cspin rown trout fingerlings. The fish were divided into 15 rcewys with dimensions of m. The experiments were designed in 5 tretments with different levels of scoric cid (0, 50, 100, 200 nd scoric cid per 1 kg feed) nd ech tretment ws set s 3 replictions. There were 40 fish in ech rcewy, nd they were fed to sturtion, 3 times dy, for period of nine weeks. Fish were fed quickly nd no feed remined in the wter. Determintion of growth nd lood prmeters Feeding ws stopped dy efore tests. Every 3 weeks, 5 fish were selected from ech tretment nd nesthetized using 250 ppm of Cloves oil (Soltni et l., 2001). Finl weight nd the growth prmeters including feed conversion rte (FCR) nd specific growth rte (SGR, % d -1 ) were clculted s follows:

5 Irnin Journl of Fisheries Sciences 15(3) Downloded from jifro.ir t 17: on Sundy July 8th 2018 Blood smples were collected y cutting the cudl peduncle nd 1 ml of lood ws poured into tue contining Ethylene dimine tetrcetic cid (EDTA) (Svoodov et l., 1991) nd moved to the lortory for further nlysis. Hemtocrit (Hct) nd Hemogloin (H) were ccording to the Vázquez nd Guerrero (2007) method. Red lood cell (RBC) nd white lood cell (WBC) counts were lso determined ccording to the methodology of Blxhll nd Disley (1973). Other lood indices including men corpusculr volume (MCV, femtoliter), men corpusculr hemogloin (MCH, picogrm), nd men corpusculr hemogloin concentrtion (MCHC, g dl -1 ) were clculted y the following formuls: 1. MCHC = 2. MCV = 3. MCH = Sttisticl nlyses The dt nlysis ws done using SPSS-16 softwre. The mens were sujected to the one-wy nlysis of vrinces (ANOVA) fter exmintion of normlity y Kolmogorov-Smirnov test. Where the differences occurred, Tukey s HSD test ws used to determine the difference points for growth prmeters nd LSD for hemtologicl prmeters, respectively. Results expressed s men ± stndrd error nd p-vlue less thn 0.05 ws ssigned s significnt difference. Results Proximte composition of experimentl diets The results of the proximte nlysis for ech experimentl diet were presented in Tle 2. No significnt difference ws found etween the tretments in the mount of moisture, protein, lipid, nd sh contents. Survivl nd growth rtes The survivl rte of fish in ll tretments ws %100 nd no symptom of scoric cid deficiency ws oserved in Cspin rown trout fingerlings during the 9 weeks of culture. The result showed significnt increse in growth of fish fed y 200 mg scoric cid kg -1 compred to fish fed y control diet (p<0.05). Tretments with 100, 200 nd scoric cid kg -1 showed no significnt differences in the fish growth fter 9 weeks experiment (Fig. 1). SGR hd significnt differences etween tretments (p<0.05). The lowest SGR ws recorded in the control group during ll smpling times. SGR in scoric cid kg -1 t third nd ninth weeks ws higher thn other tretments. The highest SGR were oserved in fish fed y scoric cid kg -1 t sixth week, while no significnt differences ws found in fish fed y 50, 100 nd scoric cid kg -1 (Tle 3).

6 1172 Rji Islmi et l., Blood prmeters of Cspin rown trout (Slmo trutt cspius) Downloded from jifro.ir t 17: on Sundy July 8th 2018 Tle 2: Proximte composition of experimentl diets (g 100 g -1 ). Moisture (g 100 g -1 ww ) Protein (g 100 g -1 dw ) Lipid (g 100 g -1 dw) Ash (g 100 g -1 dw) Ascoric cid (mg kg -1 dw) Control 5.23± ± ± ± mg AA 5.57± ± ± ± mg AA 5.56± ± ± ± AA 5.62± ± ± ± AA 5.64± ± ± ± Ascoric cid, Wet Weight; Dry Weight Finl Weight (g) Control 50 mg 100 mg 3rd week 6th week 9th week Time (week) Figure 1: Effects of Control grded supplementry scoric cid on finl weight of Cspin rown trout (Slmo trutt 50 mg cspius) during nine weeks. Different letters t the sme time indicte significnt 100 differences mg etween the tretments (p<0.05). Tle 3: Specific growth rte (SGR, % d -1 ) of Cspin rown trout (Slmo trutt cspius) fed diets with grded levels of scoric cid during 9 weeks experiment. 3 rd week 6 th week 9 th week Control 0.52± ± ± mg AA 0.60± ± ± mg AA 0.67± ± ±0.14 AA 0.71± ± ±0.28 c AA 0.64± ± ±0.06 Similr letters t the sme column show no significnt difference etween results for ech tretment (p<0.05). Ascoric cid

7 Irnin Journl of Fisheries Sciences 15(3) Downloded from jifro.ir t 17: on Sundy July 8th 2018 Control tretments hd the highest FCR compred to the other tretments contining scoric cid throughout the experiment (Tle 4). In contrst, the lowest FCR, 0.78±0.03, ws lso oserved on group of fish those fed 200 mg scoric cid kg -1 fter the third week, while it incresed in this tretment nd reched to 1.18±0.09 t the end of iossy. In the ninth week, the lowest FCRs were oserved on group of fish those fed with 400 nd scoric cid kg -1 diet (1.11±0.09 nd 1.18±1.44, respectively). Blood prmeters The results of this study showed tht nine weeks tretment of fish fed with scoric cid kg -1 cused significnt increse in RBC compred to those fed the control diet (p<0.05), however no significnt difference ws oserved in RBC fter nine weeks (Fig. 2). Cspin rown trout fingerlings fed with control diet hd the lowest hemogloin during the experiment (Fig. 3). The highest hemogloin ws found in fish fed with diet contining scoric cid kg -1 (18.00±1.52 g dl -1 ). Moreover, hemogloin in control, 50, 100 nd scoric cid kg -1 tretments ws lower thn scoric cid kg -1 tretment in the sixth week of the experiment. However, hemogloin of these tretments ws higher when compred to the control tretment. Hemtocrit ws lso significntly (p<0.05) different etween tretments (Fig. 4). The hemtocrit in tretment of scoric cid kg -1 ws higher thn other tretments, lthough there were no significnt differences etween tretments of 50, 100 nd scoric cid kg -1. Tle 4: Feed conversion rte (FCR) of Cspin rown trout (Slmo trutt cspius) fed diets with grded levels of scoric cid during 9 weeks. 3 rd week 6 th week 9 th week Control 1.69± ± ± mg AA 1.10± ± ± mg AA 1.06± ± ±0.35 AA 0.78±0.03 c 1.16± ±1.44 AA 1.00± ± ±0.09 Similr letters t the sme column show no significnt difference etween results for ech tretment (p<0.05). Ascoric cid

8 1174 Rji Islmi et l., Blood prmeters of Cspin rown trout (Slmo trutt cspius) Downloded from jifro.ir t 17: on Sundy July 8th 2018 RBC (No mm -3 ) 20x x x x x x10 6 8x10 6 6x10 6 4x10 6 2x rd week 6th week 9th week Time (Week) Figure 2: Effects of grded supplementry scoric cid on red lood cell (RBC) count of Cspin rown trout (Slmo trutt cspius) during nine weeks. Different letters indicte significnt differences etween the tretments (p<0.05). H (g dl -1 ) Control 50 mg 100 mg Control 50 mg 100 mg c c c c c 5 0 3rd week 6th week 9th week Time (Week) Figure 3: Effects of grded supplementry scoric cid on hemogloin (H) of Cspin rown trout (Slmo trutt cspius) during nine weeks. Different letters indicte significnt differences etween the tretments (p<0.05).

9 Irnin Journl of Fisheries Sciences 15(3) Downloded from jifro.ir t 17: on Sundy July 8th 2018 Hemtocrit (%) Control 50 mg 100 mg 3rd week 6th week 9th week Time (Week) Figure 4: Effects of grded supplementry scoric cid on hemtocrit of Cspin rown trout (Slmo trutt cspius) during nine weeks. Different letters indicte significnt differences etween the tretments (p<0.05). Evlution of MCH showed no significnt difference etween tretments t the third nd ninth week of experiment (Fig. 5). The fish fed contining 100 mg scoric cid kg -1 diet hd the highest mount (10.31±2.06 picogrm) t the sixth week. The results showed significnt differences in MCHC etween tretments t the third nd sixth weeks (Fig. 6). The highest MCHC ws oserved in 50 mg scoric cid kg -1, (33.95±0.80 g dl -1 ), nd 100 mg scoric cid kg -1, (33.28±0.25 g dl -1 ), t the third nd sixth weeks, respectively. There were no significnt differences etween fish fed y 50 nd 100 mg scoric cid kg -1 diet with those fed y 200 nd scoric cid kg -1 diet. Furthermore, no significnt differences were oserved in the ninth week. Control tretments hd the highest MCV compred to the diets contining dietry scoric cid t the third nd sixth week (Fig. 7). The highest MCV (36.96±4.40 femtoliter) ws oserved on fish fed scoric cid kg -1 diet t the ninth week, while it hd no significnt differences with other tretments. The lowest MCV (27.61±1.43 femtoliter) ws lso recorded on fish fed 50 mg scoric cid kg -1 diet fter the sixth week. There ws significnt difference in WBCs during 9 weeks of experiment (Fig. 8).

10 1176 Rji Islmi et l., Blood prmeters of Cspin rown trout (Slmo trutt cspius) Downloded from jifro.ir t 17: on Sundy July 8th 2018 MCH (picogrm) Control 50 mg 100 mg 3rd week 6th week 9th week Time (Week) Figure 5: Effects of grded supplementry scoric cid on men corpusculr hemogloin (MCH) of Cspin rown trout (Slmo trutt cspius) during nine weeks. Different letters indicte significnt differences etween the tretments (p<0.05). MCHC (g dl -1 ) Control 50 mg 100 mg rd week 6th week 9th week Time (Week) Figure 6: Effects of grded supplementry scoric cid on men corpusculr hemogloin concentrtion (MCHC) of Cspin rown trout (Slmo trutt cspius) during nine weeks. Different letters indicte significnt differences etween the tretments (p<0.05).

11 Irnin Journl of Fisheries Sciences 15(3) Control 50 mg 100 mg Downloded from jifro.ir t 17: on Sundy July 8th 2018 MCV (Femtoliter) rd week 6th week 9th week Time (week) Figure 7: Effects of grded supplementry scoric cid on men corpusculr volume (MCV) of Cspin rown trout (Slmo trutt cspius) during nine weeks. Different letters indicte significnt differences etween the tretments (p<0.05). WBC (No mm -3 ) Control 50 mg 100 mg c c rd week 6th week 9th week Time (week) Figure 8: Effects of grded supplementry scoric cid on white lood cell (WBC) count of Cspin rown trout (Slmo trutt cspius) during nine weeks. Different letters indicte significnt differences etween the tretments (p<0.05).

12 1178 Rji Islmi et l., Blood prmeters of Cspin rown trout (Slmo trutt cspius) Downloded from jifro.ir t 17: on Sundy July 8th 2018 However, no significnt differences were oserved in the sixth week of the experiment. WBCs in Cspin rown trout fed the control diet were the lowest in comprison with other diets group. The highest numer of WBCs ws ± found in fish fed y scoric cid kg -1 diet. Evlution of lymphocyte showed significnt prolifertion in lymphocyte mong different tretments y increse of dietry scoric cid level (Fig. 9). However, no significnt differences ws found in lymphocyte of fish fed with kg -1 scoric cid diet fter six nd nine weeks of feeding tril except control tretment. Neutrophil count decresed in the sixth nd ninth week with other Lymphocyte (%) Control 50 mg 100 mg supplemented scoric cid tretment fter six nd nine weeks of feeding tril. Neutrophil count decresed significntly y supplementing scoric cid to the diet during the experiment (Fig. 10). The highest numer of neutrophil otined in control tretment (31.00±1.20 %), which ws significntly different with tretment contining scoric cid kg -1 diet t the third week. Furthermore, there ws significnt difference etween control tretment with those supplemented y scoric cid t the sixth nd ninth weeks. There ws no significnt difference in monocytes nd sophiles counts etween tretments throughout the experiment rd week 6th week 9th week Time (week) Figure 9: Effects of grded supplementry scoric cid on lymphocyte of Cspin rown trout (Slmo trutt cspius) during nine weeks. Different letters indicte significnt differences etween the tretments (P<0.05).

13 Irnin Journl of Fisheries Sciences 15(3) Downloded from jifro.ir t 17: on Sundy July 8th 2018 Neutrophil (%) rd week 6th week 9th week Time (week) Figure 10: Effects of grded supplementry scoric cid on neutrophil of Cspin rown trout (Slmo trutt cspius) during nine weeks. Different letters indicte significnt differences etween the tretments (p<0.05). Discussion The Cspin rown trout is n economiclly vlule species which the recruitment ctivities ffects their popultion in the southern re of the Cspin Se. This study showed tht scoric cid s n essentil component of the diet incresed the growing rte of Cspin rown trout fingerlings. Ascoric cid is n essentil coenzyme in tyrosine mino cid oxidtion, phenyllnine (Brnder nd Pugh, 1977), lysine, proline nd methionine. Therefore, more growth of Cspin rown trout fingerlings in fish fed y scoric cid concentrtions could e confirmed y synthesis of more protein in muscle tissue (Ar et l., 2013). The higher SGR of Cspin rown trout fingerlings in this study cn e due to the lower initil weight (9.6±0.6 g) nd Control 50 mg 100 mg their potentil to use supplementry fctors like scoric cid in nturl metolisms nd weight gin (Iiyo et l., 2007; Adunni Adewolu nd Aro 2009). Confirming the previous studies, this study showed tht scoric cid cn ffect FCR (Ai et l., 2006; Adunni Adewolu nd Aro, 2009). The lowest FCR in Cspin rown trout ws found in scoric cid tretment, lthough it hd no significnt difference with scoric cid tretment. Indeed, it is vitl to use principles nd tools to prevent fish ginst diseses nd culture them helthy. White cells ct s defense rrier ginst pthogens nd unwnted conditions. Any chnges in lood cells numer cn e n pproprite index for evluting fish immune conditions (Stoskopf, 1993; Hruec et l., 2001).

14 1180 Rji Islmi et l., Blood prmeters of Cspin rown trout (Slmo trutt cspius) Downloded from jifro.ir t 17: on Sundy July 8th 2018 There re different fctors cusing improvement or wekness of defense system in fish which the most importnt one is feeding conditions (Houston et l., 1996; Kori-Sikpere et l., 2005; Orun nd Tls, 2008). Ascoric cid is known to perform numerous iochemicl nd physiologicl functions, especilly in teleost fish. Ascoric cid is n importnt nutrient in fish diets. In mny types of fish, the iosynthesis of scoric cid does not occur. Accordingly, vitmin C must e dded s supplementry into the feed. The fish requirement is round scoric cid kg -1, lthough it is relted to mny fctors like stress influence, growth rte, size of fish, temperture of wter nd other nutrients in the diet. The result of the present study showed tht use of scoric cid kg -1 efore creting stress condition rings out improvement of fish immune system nd their resistnce to stress nd diseses. Different species of fish need different levels of scoric cid in their diet. For instnce, rinow trout (Oncorhynchus mykiss) is one of the most common species which ws studied, ssocited with requirements for vitmin C. It is noted tht n mount of 100 mg kg -1 dry weight would e enough for rinow trout. In trout with musculr injury, the norml dose of 500 mg kg -1 dry weight should e given five times to cope with this sitution. Also, high level of vitmin C is dded to the rtio would mke the rinow trout fce n infectious disese (Hlver, 2002). Excess dose of vitmin C will e resist rinow trout injected with Virio nguillrum (Nvrre nd Hlver, 1989). The sme result hs een otined for virl diseses to rinow trout with excessive mount of scorte-2-phosphte (Anggwti- Styudhy et l., 1989). However, it is stted y Hlver (2002) tht dose of kg -1 of dry rtion would e sufficient, in the cse of rinow trout. The required level of scoric cid in Yellowtil (Seriol dumerili), Europen se ss (Dicentrrchus lrx) nd common crp (Cyprinus crpio) is 122 mg kg -1 (Msumoto et l., 1991), 20 mg kg -1 (Merchie et l., 1996) nd 270 mg kg -1 (Gouillou-Coustns et l., 1998), respectively. By implementing hemtologicl techniques, including evlution of erythrocyte count, hemogloin concentrtion, hemtocrit vlue nd leukocyte count, vlule knowledge for fishery iologists in fish helth ssessment hs een provided (Blxhll, 1972; Tls et l., 2012). Results of this study emphsize on the previous findings out significnt effects of scoric cid on RBCs (Clementi et l., 1997; Domezin et l., 1997). RBC, H nd Hct of fish in control group were less thn other tretments. There were lso significnt differences etween control group nd fish fed with scoric cid kg -1 in third, sixth nd ninth weeks of smpling. There is no significnt difference etween control group nd

15 Irnin Journl of Fisheries Sciences 15(3) Downloded from jifro.ir t 17: on Sundy July 8th 2018 fish fed y 50 nd 100 mg scoric cid kg -1, nd etween fish treted with 200 nd kg -1 scoric cid. MCH nd MCHC in fish fed with scoric cid kg -1 were significntly more thn control group just in the sixth week. In ddition, no significnt differences were oserved in other times nd in MCV. Since MCV nd MCHC re rtio etween RBC, H nd Hct, the results were predictle. The increse of H nd MCHC indictes tht vitmin C cn e effective in hemogloin concentrtion of fish erythrocyte. WBC nd rtio of different kinds of WBC re other importnt indexes for showing the stte of helth nd immune system in nimls (Shly et l., 2006; Ates et l., 2008; Tls et l., 2012). WBC in fish fed y diet supplemented with scoric cid kg -1 ws significntly more thn the control group. In greement with Kumri nd Shoo, (2005) lso demonstrted tht supplementtion of 2000 mg scoric cid kg -1 in ctfish diet improves growth performnce nd the immune system. Supplementry scoric cid in fish feed increses the production of ntiody ginst cteri nd improves phgocytosis nd lysosome ctivity which hve importnt roles to reduce stress (Hrdie et l., 1991; Henrique et l., 1998; Shiu nd Hsu, 2002; Tls et l., 2014). Lymphocytes re the most common type of WBCs tht re used ginst virl diseses (Groff nd Zinkl, 1999). The mount of lymphocyte in qutic nimls like mmmlins is ffected y stress nd hormones (Secomes nd Ellis, 2012). It hs een proven tht vitmin C regulted the lymphocyte B ctivity (Bergsten et l., 1995). Significnt increse of lymphocyte level in fish, treted y using kg -1 scoric cid in comprison with the control group, showed tht cultured rown trout defense ility ginst viruses hd incresed. Incidence of virl diseses is widespred prolem tht my thret Brown trout stocks in Cspin Se. Ascoric cid cn improve fish defense ility y incresing lymphocyte production, especilly when it is used for long time. Incresing trend in lymphocyte production over time shows positive effects of scoric cid on immune system improvement, chronic stress reduction, fish dpttion to culture environment nd increses ody resistnce to diseses in Cspin rown trout (Grci et l., 2007; Innocent et l., 2011). In the present study, neutrophil numer in fish feed diet supplemented with scoric cid ws significntly lower thn control group. Also with incresing scoric cid level nd time, more decrese ws oserved in neutrophil numer. Neutrophil hs phgocytes property nd increses immunity ginst cteril diseses. Different fctors such s pthogens, stress nd fish species could lso influence on the numer of neutrophil (Stoskopf, 1993; Tls et l., 2008; Orun et l., 2011). Norml culture

16 1182 Rji Islmi et l., Blood prmeters of Cspin rown trout (Slmo trutt cspius) Downloded from jifro.ir t 17: on Sundy July 8th 2018 condition nd lck of stress cuse reduction in neutrophil numer (Dvis et l., 2008). In the present study, neutrophil percentge decresed over the time which could e relted to improvement of fish immune system nd their esier dpttion to the culture condition. Enhnced innte immunity y increse of lysosome ctivity could e nother reson for neutrophil reduction over the experiment. In conclusion, the current reserch showed tht dietry L-scoryl-2- polyphosphte influences the complete lood count of Cspin rown trout fingerlings fter 9-week feeding tril with the optimum mounts y feeding scoric cid kg -1 diet. References Ai, Q.H., Mi, K.S., Zhng, C.X., Xu, W., Dun, Q.Y., Tn, B.P. nd Liufu, Z.G., Effects of dietry vitmin C on growth nd immune response of Jpnese sess, Lteolrx jponicus. Aquculture, 242, Ai, Q., Mi, K., Tn, B., Xu, W., Zhng, W., M, H. nd Liufu, Zh., Effects of dietry vitmin C on survivl, growth, nd immunity of lrge yellow croker, Pseudoscien croce. Aquculture, 261, Adunni Adewolu, M. nd Aro, O.O., Growth, feed utiliztion nd hemtology of Clris griepinus (Burchell, 1822) fingerlings fed diets contining different levels of vitmin C. Americn Journl of Applied Science Puliction, 6: Anggwti-Styudhy, A.M., Grnt, B.F. nd Hlver, J.E., Effects of L-scoryl phosphtes (AsPP) on growth nd immunoresistnce of rinow trout to infectious hemtopoietic necrosis (IHN) virus. Proceeding Third Interntionl Symposium on Feeding nd Nutrition in Fish, To, Jpn. Ar, N., Rji Islmi, H. nd Shmsei Mehrjn, M., Effect of dietry vitmin C on survivl nd growth prmeters of Cspin rown trout (Slmo trutt cspius) fingerlings. Journl of Fisheries, 66, (In Persin). Armntrout, N.B., The freshwter fishes of Irn. Ph.D. Thesis Oregon Stte University. Oregon, USA, 427 P. AOAC Assocition of Officil Anlyticl Chemists, Officil Methods of Anlysis, Githersurg, Mrylnd, USA. Ates, B., Orun, I., Tls, Z.S., Durmz, G. nd Yilmz, I., Effects of sodium selenite on some iochemicl nd hemtologicl prmeters of rinow trout (Oncorhynchus mykiss Wlum, 1792) exposed to P 2+ nd Cu 2+. Fish Physiology nd Biochemistry, 34, Bergsten, P., Yu-Kehrl, J. nd Levine, M., Ascoric cid trnsport nd distriution in humn B lymphocytes. Archive of

17 Irnin Journl of Fisheries Sciences 15(3) Downloded from jifro.ir t 17: on Sundy July 8th 2018 Biochemistry nd Physiology, 317, Blxhll, P.C., The hemtologicl ssessment of the helth of the freshwter fish. A review of selected literture. Journl of Fish Biology, 4, Blxhll, P.C. nd Disley, K.W., Routine hemtologicl methods for use with fish lood. Journl of Fish Biology, 5, Brnder, G.C. nd D. M., Pugh Veterinry Applied Phrmcology nd Therpeutics, Third Edition. The English lnguge Book Society nd Billiere Tindll, London, UK. Cerezuel, R., Cuest, A., Meseguer, J. nd Ángeles Esten, M., Effects of dietry vitmin D 3 dministrtion on innte immune prmeters of serem (Sprus urt L.). Fish nd Shellfish Immunology, 26, Clementi, M.E., Ctldi, E., Cpo, C., Peteru Zzelli, R. nd Girdin, B., Purifiction nd chrcteriztion of the hemogloin components from Adritic sturgeon, Acipenser nccrri. Proceedings of 3rd Interntionl symposium of sturgeon, Itly. Cod, B.W., Criteri for ssessing the conservtion sttus of tx (s pplied to Irnin freshwter fishes). Biologi, 55, Dvis, A.K., Mney, D.L. nd Merz, J.C., The use of leukocyte profiles to mesure stress in vertertes: review for ecologists. Functionl Ecology, 22, Domezin, A., Grci, O., Gllego, M., Domezin, J. nd Snz, A., Evolution during growth of the iometry nd the lood constnts of the sturgeon, Acipenser nccrii. Proceedings of 3rd Interntionl Symposium of sturgeons, Itly. Grci, F., Pilrski, F., Onk, E.M., de Mores, F.R. nd Mrtins, M.L., Hemtology of Pirctus mesopotmicus fed diets supplemented with vitmins C nd E, chllenged y Aeromons hydrophil. Aquculture, 271, Gouillou-Coustns, M.F., Bergot, P. nd Kushik, S.J., Dietry scoric cid needs of common crp (Cyprinus crpio) lrve. Aquculture, 161, Groff, J.M. nd Zinkl, J.G., Hemtology nd clinicl chemistry of Cyprinid fish. Common crp nd goldfish. Veterinry Clinics of North Americ: Exotic Animl Prctice, 2, Hlver, J.E., The vitmins. In: Hlver, J.E. nd Hrdy, R.W. (Eds), Fish Nutrition, 3rd edition. Acdemic Press Inc, Sn Diego, CA, USA. Hrdie, L.J., Fletcher, T.C. nd Secomes, C.J., The effect of dietry vitmin C on the immune response of the Atlntic slmon (Slmo slr L.). Aquculture, 95, Henrique, M.M.F., Gomes, E.F., Gouilou-Coustns, M.F., Oliv- Teles, A. nd Dvies, S.J., Influence of supplementtion of

18 1184 Rji Islmi et l., Blood prmeters of Cspin rown trout (Slmo trutt cspius) Downloded from jifro.ir t 17: on Sundy July 8th 2018 prcticl diets with vitmin C on growth nd response to hypoxic stress of serem, Sprus urt. Aquculture, 161, Houston, A.H., Doric, N. nd Khurnng, R., The nture of hemtologicl response in fish. Fish Physiology nd Biochemistry, 15, Hruec, T.C., Smith, S.A. nd Roertson, J.L., Age relted in hemtology nd chemistry vlues of hyrid striped ss chrysops Morone sxtilis. Veterinry nd Clinicl Pthology, 30, Iiyo, L. M. O., J. O. Atteh, J. S. Omotosho, nd C. T. Mdu, Vitmin C (scoric cid) requirements of Heterornchus longifilis fingerlings. Africn Journl Biotechnology, 6(13): Innocent, B.X., Fthim, S.A. nd Sivgurunthn, A., Hemtology of Cirrhinus mrigl fed with vitmin C supplemented diet nd post chllenged y Aphnomyces invdens. Journl of Applied Phrmceuticl Science, 1, Kii, B.H., Adoli, A. nd Nderi, M., Sttus of fish fun in south Cspin sin of Irn. Zoology in the Middle Est, 16, Kori-Sikpere, O., Ake, J.E.G. nd Idoge, E., Hemtologicl chrcteristics of the Africn snkehed, Prchcnn oscur. Africn Journl of Biotechnology, 4, Kumri, J. nd Shoo, P.K., High dietry vitmin C ffects growth, non-specific immune responses nd disese resistnce in Asin ctfish, Clris trchus. Moleculr nd Cellulr Biochemistry, 280, Mgndóttir, B., Innte immunity of fish (overview). Fish nd Shellfish Immunology, 20(2), Msumoto, T., Hidetuyo, H. nd Shimeno, S., Ascoric cid's role in quculture nutrition. Proceedings of the Aquculture Feed Processing nd Nutrition Workshop. Americn Soyen Assocition, Indonesi. Merchie, G., Lvens, P., Storch, V., Uel, U., De Nelis, H., Leenheer, A. nd Soregeloos, P., Influence of dietry vitmin C dosge on turot (Scophthlmus mxmus) nd Europen se ss (Dicentrchus lrex) nursery stges. Comprtive Biochemistry nd Physiology, 11, Mulero, V., Esten, M.A., Munoz, J. nd Meseguer, J., Dietry intke of levmisole enhnces the immune response nd disese resistnce of the mrine teleosts gilthed serem (Sprus urt L.). Fish nd Shellfish Immunology, 8, Nvrre, O. nd Hlver, J., Disese resistnce nd humorl ntiody production in rinow trout fed high levels of vitmin C. Aquculture, 79,

19 Irnin Journl of Fisheries Sciences 15(3) Downloded from jifro.ir t 17: on Sundy July 8th 2018 Orun, I., Tls, Z.S., Ozdemir, I. Alkn, A. nd Erdogn, K., Antioxidtive role of selenium on some tissues of (Cd 2+, Cr 3+ ) induced rinow trout. Ecotoxicology nd Environmentl Sfety, 71, Orun, I. nd Tls, Z.S., Antioxidtive role of sodium selenite ginst the toxic effect of hevy metls (Cd 2+, Cr 3+ ) on some iochemicl nd hemtologicl prmeters in the lood of rinow trout (Oncorhynchus mykiss Wlum, 1792). Fresenius Environmentl Bulletin, 17(9), Orun, I., Tls Z.S. nd Alkn, A., Modulting effect of selenium on gills of fish exposed to hevy metls. Fresenius Environmentl Bulletin, 20, Rmezni, H., Effects of different protein nd energy levels on growth performnce of Cspin rown trout, Slmo trutt cspius (Kessler, 1877). Journl of Fisheries nd Aqutic Sciences, 4, Ser, A., Aedin kinri, A.M. nd Hyti F., Effects of dietry protein nd energy levels on growth nd ody composition of Cspin rown trout (Slmo trutt cspius). Journl of Mrine science nd Technology, 4, (In Persin) Stheeshkumr, P., Annthn, G., Senthil Kumr, D. nd Jgdeesn, L., Hemtology nd iochemicl prmeters of different feeding ehvior of teleost fishes from Vellr estury, Indi. Comprtive Clinicl Pthology, 21(6), Secomes, C.J. nd Ellis, A.E., The immunology of teleosts. In: Roerts, R.J. (ed) Fish Pthology, Fourth Edition. Wiley-Blckwell, Oxford, UK. Shly, A.M., Khtt, Y.A. nd Adel Rhmn, A.M., Effects of grlic (Allium stivum) nd chlormphenicol on growth performnce, physiologicl prmeters nd survivl of Nile tilpi (Oreochromis niloticus). Journl of Venomous Animl nd Toxins Including Tropicl Disese, 12, Shiu, S.Y. nd Hsu, T.S., Quntifiction of vitmin C requirement for juvenile hyrid tilpi, Oreochromis niloticus Oreochromis ureus, with L-scoryl-2 monophosphte-n nd L-scoryl-2-monophosphte-Mg. Aquculture, 175, Shiu, S.Y. nd Hsu, C.Y., Vitmin E spring effect y dietry vitmin C in juvenile hyrid tilpi, Oreochromis niloticus O. ureus. Aquculture, 210, Soltni, M., Omideigi, R., Rezvni, S., Mehri, M.R. nd Chitsz, H., Study of nesthetic effects induced y clove flower (Eugin cryophillt) on rinow trout (Oncorhynchus mykiss) under vrious qulity condition. Journl of the Veterinry Medicine, 56, (In Persin)

20 1186 Rji Islmi et l., Blood prmeters of Cspin rown trout (Slmo trutt cspius) Downloded from jifro.ir t 17: on Sundy July 8th 2018 Sotoudeh, E., Aedin kenri, A. nd Hii Rezei, M., Growth response, ody composition nd ftty cid profile of Cspin rown trout (Slmo trutt cspius) juvenile fed diets contining different levels of soyen phosphtidylcholine. Aquculture Interntionl, 19, Stoskopf, M.A., Fish medicine. Sunders Compny, USA. 882P. Svoodov, Z., Prvd, D. nd Plkov, J., Unified methods of hemtologicl exmintion of fish. Reserch institute of fish culture nd hydroiology. Vodnny, Czech Repulic, 31P. Tls, Z.S., Orun, I., Ozdemir, I., Erdogn, K., Alkn, A. nd Yılmz, I., Antioxidtive role of selenium ginst the toxic effect of hevy metls (Cd 2+, Cr 3+ ) on liver of rinow trout (Oncorhynchus mykiss Wlum, 1792). Fish Physiology nd Biochemistry, 34, Tls, Z.S., Dundr, S.P., Gulhn, M.F., Orun, I. nd Kkoolki, S., Effects of propolis on some lood prmeters nd enzymes in crp exposed to rsenic. Irnin Journl of Fisheries Sciences, 11(2), Tls, Z.S., Mehmet Fut Gulhn, M.F., Erdogn, K. nd Orun, I., Reserching of ntioxidnt effects of propolis in crp (Cyprinus crpio) exposed to rsenic: iochemicl nd histopthologic findings. Diseses of Aqutic Orgnisms, 108, Tewry, A. nd Ptr, B.C., Use of vitmin C s n immunostimulnt. Effect on growth, nutritionl qulity, nd immune response of Leo rohit (Hm.). Fish Physiology nd Biochemistry, 34, Vázquez, G.R. nd Guerrero, G.A., Chrcteriztion of lood cells nd hemtologicl prmeters in Cichlsom dimerus (Teleostei, Perciformes). Tissue Cell, 39, Xioyun, Z., Mingyun, L., Khlid, A. nd Weinmin, W., Comprtive of hemtology nd serum iochemistry of cultured nd wild Dojoloch Misgurnus nguillicdtus. Fish Physiology nd Biochemistry, 33,