Journal of Chemical Health Risks

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1 JCHR (2019) 9(1), 1-10 Journl of Chemicl Helth Risks ORIGINAL ARTICLE Hemtologicl, Histopthologicl nd Growth Performnce Studies on Alino Rts Fed on Mystus gulio Fillet with Formulted Cerel Mosummth Hosn Ar 1, Kykod Md Rezul Krim 1, 2, Niro Kumr Sh 1, 3, A.B.M. Nzmul Islm 1, Plsh Kumr Dhr *1 1 Chemistry Discipline, Khuln University, Khuln-9208, Khuln, Bngldesh 2 Fculty of Chemicl nd Nturl Resources Engineering, University Mlysi Phng, Leuhry Tun Rzk, Kuntn, Phng, Mlysi 3 Deprtment of Chemistry nd Biochemistry, Auurn University, Auurn, Alm 36849, United Sttes (Received: 3 June 2018 Accepted: 10 Novemer 2018) KEYWORDS Growth performnce; Toxicologicl effect; Mystus gulio; Non Tengr ABSTRACT: The growth nd development of humn ody is dependent on the protein, crohydrte, vitmins nd minerls. Fishes re one of the mjor sources of protein, vitmins nd minerls for humn. This study ws conducted to know the effect of Mystus gulio fish fillet on growth performnce of lino rts fter feeding different concentrtions of fish fillet s protein source with formulted cerel for 32 dys. Hemtologicl nd histopthologicl nlysis were performed etween control group nd exmined groups of lino rts. It ws noticed tht the ody weight of ech experimentl lino rt ws incresing grdully with incresing fish fillet up to 15% nd eyond this the vlue ws decresed. Moreover, the highest protein efficiency rtio (PER) nd clorie efficiency rtio (CER) were oserved 2.53% nd 13.02% respectively for 15% supplied fish fillet. The gined in ody weight (GBW) of lino rts in ech experimentl group depended minly on supplied fish protein concentrtion ut not on formulted cerel. According to hemtologicl nd histopthologicl studies, no toxicologicl effect ws oserved etween control group nd experimentl groups of rts treted with different fish protein concentrtion for 32 consecutive dys. Supplement of 15% fish fillet with formulted cerel ws required to produce mximum nutritive vlues nd the Mystus gulio could e considered s protein rich humn diet. INTRODUCTION For humn nd ll nimls, protein is n essentil constituent of diet nd source of essentil nd non-essentil mino cids. Without ingesting the essentil mino cids, deth is inevitle. Indequte protein or mino cid results in muscle loss, impired immune system nd poor hir cot qulity [1]. However, high dietry protein intke is ssocited with incresed risk of kidney disese [2, 3], incresed one loss [4] nd my increse formtion of clcium oxlte crystls [5] in humn ody. Foods with high protein concentrtion nd imlnce of insulin sensitivity my put the humn t risk for other disese relted to excess nitrogen metolism or moiliztion of clcium [6] in ody tissue. Any toxicologicl effect ssocited with these tissues nd lood cn e investigted through hemtology nd histopthologicl studies. Hemtology is the study of lood, lood forming *Corresponding uthor: plshdhr@ku.c.d (P. Kumr Dhr) DOI: /jchr

2 tissues nd reltive sustnces concerned in lood production, conservtion nd destruction of lood nd their disorders. Histopthology refers to the microscopic exmintion of tissue in order to study the mnifesttions of disese [7-9]. Diet contining lnce protein is essentil for mintining sound helth. Fishes re the principle source of proteins nd minerls (oth micro nd mcro minerls) [10]. These re known to ply importnt roles in physiologicl processes nd in certin diseses. Nutritionl studies hve proved tht fish protein is rnked in the sme clss of chicken portions ut re superior to milk, eef protein nd egg. Trditionlly the South Asin people consume fishes of fresh wter nd ner shore rckish wter origin [11]. However, nture hs endowed Bngldesh lvish sources of oth mrine nd fresh wter fishes. The people of Bngldesh, Indi nd Srilnk re highly dependent on fish protein due to the vilility nd low price. Mystus gulio, loclly known s Non Tengr, is rckish wter fish ville in South-Western costl re of Bngldesh. This species is preferred y the people of our country for its delicious tstes, therpeutic vlues nd vilility throughout the yer [10]. Among the four species (nmely Mystus gulio, Mystus leekeri, Mystus cvsius nd Mystus vitttus), Mystus gulio contins highest mount of protein % nd lipid % tht cn e conventionlly used to improve the protein qulity in our diet [10]. Considering the nutritionl significnce of protein content in this fish, the effect of fish protein concentrtion on growth performnce ws evluted y determining GBW, PER nd CER in the young lino rts fter feeding different mount of fish fillet with formulted cerels [12]. Moreover, to ssess the toxicologicl effect of this fish the hemtologicl nd histopthologicl studies were performed y determining the prmeters like hemogloin, red lood cells (RBC), erythrocyte sedimenttion rte (ESR), cholesterol, white lood cells (WBC) nd totl count of WBC [7,13]. For this purpose, the lino rts in oth control groups nd experimentl groups were ssulted y mintining the ll precution nd their lood, liver, hert, lung, spleen nd kidney were collected nd preserved in freeze for nlysis. These experiments were crried out y oserving ny chnges in the cellulr structures (degrdtion nd regenertion) of these orgns of the experimentl rts fter received fish fillet t different fish protein concentrtion levels for 32 dys with respect to control group. Although Mystus gulio is populr mong the people in our country, still now there is insufficient informtion ville on the nutritionl vlue nd toxicologicl effect of this species. Thus, the min ojective of this study ws to estlish the nutritionl significnce nd hygienic vlue of Mystus gulio s protein rich diet to the Bngldeshi people. MATERIALS AND METHODS Experimentl niml Twelve mle lino rts (Wistr Kyoto Outreed) hving ody weight g were used in this study. The experimentl nimls were kept in n niml house in Biochemistry nd Moleculr Biology, Rjshhi University, Bngldesh nd formulted cerel nd wter hd fed them for 32 dys. These nimls were distriuted rndomly into six groups (diet-a, diet-b, diet-c, diet-d, diet-e nd diet-f). Ech experimentl group contined two nimls nd they were lmost in similr size nd weight. Diet-A ws considered s controlled group wheres diet-b, diet-c, diet-d, diet-e nd diet-f were experimentl group. Diet formultion nd compositionl nlysis of fish protein Formulted cerel ingredients were collected from Interntionl Centre for Dirrhel Disese Reserch, Bngldesh (ICDDRB). The mjor ingredients of formulted cerel were corn, whet nd whet rn, grm, til oil cke, soy-44, slt, fish mel-60, rice polishing, nd soyen oil. The quntittive ingredients composition nd nutritive vlues of this formulted cerel hve een shown in Tle 1 nd Tle 2. Five experimentl diets (diet-b, diet-c, diet-d, diet-e nd diet-f) were prepred y supplementing fish protein concentrtion with formulted cerel. In these experimentl diets the level of fish protein concentrtion (FPC) nd ft content were 1.11 % to 5.55 % nd % to % (Tle 3). The percentge of totl protein nd ft in different diets were to nd 5.00 to respectively. 2

3 Tle 1. Compositions of formulted cerel. Ingredients % of ingredients Ingredients % of ingredients Corn Sortox 0.25 Whet Limestone 0.30 Whet rn Dichloride phosphte 0.25 Til oil cke 6.00 Choline chloride 0.10 Grm 8.00 Uitmine premix 0.30 Soy Slt 0.50 Skimoned milk powder 1.00 Fish mel Lysin 0.09 Soyen oil 1.00 Feedzine 0.05 Rice polishing 6.00 Sl kill 0.04 Tle 2. Nutritive vlues of formulted cerel. Proximte compositions of ingredients % in formulted cerel Protein Fier 5.15 Ash 5.90 Ft 5.00 Tle 3. Composition of experimentl diets. Nme of diets Composition of diets (g) % of protein(g) in diets % of ft (g) in diets Totl protein in diets (g) Totl ft in diets (g) Diet A 100% FC only Diet B 5% FF+ 95% FC FF =1.11, FC =19.95 FF =0.2625, FC = Diet C 10% FF+ 90% FC FF =2.22, FC =18.90 FF =0.5250, FC = Diet D 15% FF+ 85% FC FF =3.33, FC =17.85 FF =0.7875, FC = Diet E 20% FF+ 80% FC FF =4.44, FC =16.80 FF =1.050, FC = Diet F 25% FF+ 75% FC FF =5.55, FC =15.75 FF =1.3125, FC = Where, FF= Fish Fillet, FC= Formulted cerel Mintennce of nimls The nimls in controlled nd experimentl groups were housed individully in stinless steel cges with screen ottoms. The niml house ws well-ventilted, hygienic nd kept optimum room temperture with exposed light nd drk cycle of 12 h durtion ech. Both controlled nd experimentl nimls hd n ccess to 20 g of food in every 2-4 h intervl during 32 dys. The men weight of controlled nd experimentl nimls ws noted fter 4 dys intervl. Clcultion of PER nd CER Protein efficiency rtio (PER) ws defined s GBW per one grm of crude protein intke [12, 14]. Clorie efficiency rtio (CER) ws clculted from gin in ody weight (g) per 100 clories intke during the experiment [12]. PER = (1) CER = 100 (2) 3

4 Hemtologicl nd histopthologicl nlysis At the end of 32 dys, the lino rts were strved for overnight nd scrificed under mild ether nesthesi. Blood were collected from ech rt of either group y crdic puncture over EDTA for the mesurements of vrious hemtologicl prmeters [15]. Hemogloin (H) ws mesured ccording to the cid hemtin method of Shli [15]; peripherl count of RBCs nd totl count of white lood cells (WBCs) nd differentil count of WBCs were mesured ccording to mnul method with the help of hemtocytometer [7]. Erythrocyte sedimenttion rte (ESR) ws determined y Westergren Method. In this method, the lood ws mixed with suitle nticogulnt gent (3.1% trisodium citrte solution) nd ws mde to stnd verticlly [7]. Blood serum cholesterol ws determined y spectrophotometriclly ccording to Leiermnn-Burchrd rection method [7]. For histopthologicl investigtion, the liver, kidney, hert, lung nd spleen of ll rts were isolted, weighted nd recorded. The totl liver protein nd totl liver ft were estimted ccording to Bligh-Dyer method nd Microkjeldhl s method respectively [16]. A smll mount of these tissues were seprtely sliced, fixed in 10% formlin nd processed for prffin sections. After hemtoxylin-eosin stining, the section of the control group nd experimentl groups were crefully exmined under high power microscope nd were recorded y photogrphs. The histopthologicl nd hemtologicl exmintions were crried out t Rjshhi Medicl College, Rjshhi, Bngldesh. RESULTS AND DISCUSSION Fed intke nd ody weight gin Initil ody weights, totl food consumed, GBW for ech rt fter 32 dys nd weight of orgns hs een shown in Tle 4. The tle descries tht the ody weight of ll rts incresed grdully with incresing the fish protein concentrtion in spite of hving sme mount of food. Moreover, the given results lso confirmed tht the GBW of ech rt minly depended on supplied fish protein concentrtion, not on formulted cerel. Tle 4. Weight of different orgns of twelve lino rts fed on FPC (Mystus gulio) t different protein levels supplemented with formulted cerel. Diets Rt No. Initil ody weight (g) Food consumed (g) Finl ody weight (g) GBW (g) Weight of orgns (g) Liver Kidney Lung Spleen Hert Diet A Diet B Diet C Diet D Diet E Diet F Reference Vlues [17] During the experimentl period, the GBW for ech rt ws noted in every four dys (Tle 5). The men GBW of ll rts incresed with incresing dys nd reched t mximum vlues of 63.02g, 79.95g, g, g, g nd 118.9g respectively. 4

5 Tle 5. Men GBW t every four dys intervl of mle lino rts fter feeding FPC t different levels supplemented with formulted cerel. Dys Men GBW (g) t every four dys intervl Diet A Diet B Diet C Diet D Diet E Diet F According to Tle 6 it ws reveled tht the men GBW per one grm of food intke, PER nd CER incresed long with incresing protein concentrtion. The mximum vlue of these prmeters ws oserved 0.534g, 2.53 nd respectively in diet-d. Tle 6. The effect of feeding FPC t different levels supplemented with formulted cerel on GBW per one grm food, PER nd CER Diets Rt No. GBW (g) per one grm food Men Protein (g) intke y ech rt PER Men PER Clorie intke y ech rt CER % Men CER (%) A B C D E F When the levels of protein were further incresed through FPC, the GBW per one grm of food, PER nd CER were noticely decresed from diet-e (0.457g, 2.16 nd 11.15) nd diet-f (0.432g, 2.03 nd respectively). Rts under diet-a hd less effect on GBW (0.272g) per one grm of food intke, PER (1.30), CER (6.63) with compred to others. According to this dt, it ws found tht diet-d hd highest PER nd CER followed y diet E, C, F, B, nd A. It ws lso significnt tht PER vlues were directly proportionl to their respective CER vlues. From the vlues of PER, CER nd lso GBW per one grm of food in Tle 6, it ws inferred tht the effect of 15% FPC in diet-d ws remrkly high. Hemtologicl nd histopthologicl chnges According to Tle 7, it could e noticed tht hemtologicl prmeters of ll experimentl rts were lmost similr to tht of control group rt. The vlues of hemtologicl prmeters were within the norml rnge when compred to reference vlues provided y Mitruk nd Rmsley in Tle 8 [17]. 5

6 Tle 7. Hemtologicl profiles of lino rts for feeding FPC (Mystus gulio) t different protein levels supplemented with FC fter 32 dys Hemtologicl prmeters Rt No. Control rt with FC Percentge of fish filet of Mystus gulio in diet with formulted cerel Diet B Diet C Diet D Diet E Diet F Totl RBC count (10 6 / l) Hemogloin (g/dl) Men Men Cholesterol (mg/dl) Men Erythrocyte sedimenttion rte (ESR)(mm) Totl WBC Count (10 3 / l) Differentil count ) Neutrophil (%) ) Lymphocytes (%) c) Monocytes (%) d) Eosoniphils (%) Men Men Men Men Men Men Tle 8. Reference vlues for hemtologicl prmeters of the Sprgue Dwley rts [17]. RBC TWBC LYMPH NEUT MON EOS ( 10 6 / l) ( 10 3 / l) (%) (%) (%) (%) For ssessing histopthologicl prmeters, the weight of liver, kidney, hert, lung nd spleen of ll rts were compred to the reference vlues for Spgue Dwley rts nd oserved no normlities (Tle 4). During the whole experimentl period, locomotive ehvior, centrl nervous system, excittion, wekness, reflexes, slivtion, urintion, dirrhe etc. of ll lino rts were closely monitored nd found norml. Histopthologicl chnges in hert, liver, lung, kidney nd spleen of ll rts were oserved norml s stted in Tle 9. No morphologicl chnges were noticed in their microscopic view in displyed Figures

7 Tle 9. Histopthologicl exmintion of the section of Hert, Livers, Kidney, Lung nd Spleen of ll rts (Both control group nd experimentl groups) Oservtion Liver Kidney Hert Lung Spleen Control group No chnge Glomerulus s. No inflmmtory cell ws oserved Crdic muscle, no morphologicl chnge ws occurred nd no congestion ws found Alveoli, no congestion ws occurred No normlities were detected Experimentl Groups No normlities were detected No evidence of pthologicl chnge As sme s control group No chnge were occurred Sme s control group Figure 1. Hert tissues of control rt Figure 1. Hert tissues tretment with 10 % FPC Figure 1c. Hert tissues tretment with 20% FPC Figure 2. Kidney tissues of control rt Figure 2. Kidney tissues tretment with 10 % FPC Figure 2c. Kidney tissues tretment with 20% FPC Figure 3. Liver tissues of control rt Figure 3. Liver tissues tretment with 10 % FPC Figure 3c. Liver tissues tretment with 20% FPC Figure 4. Spleen tissues of control rt Figure 4. Spleen tissues tretment with 10 % FPC Figure 4c. Spleen tissues tretment with 20% FPC 7

8 Figure 5. Lung tissues of control rt Figure 5. Lung tissues tretment with 10 % FPC CONCLUSIONS Figure 5c. Lung tissues tretment with 20% FPC Hemtologicl studies indicted no ovious differences in ny one of the hemtologicl prmeters etween control group nd experimentl groups. Microscopic exmintion of treted tissues in ll group of rts hd no normlities nd no remrkle effect ws oserved. From this study it ws suggested tht the fish species Mystus gulio, the long whiskers ctfish could e considered s chep protein rich diet s well s hygienic to the Bngldeshi people without ny helth risk. ACKNOWLEDGMENTS The uthors thnk to Deprtment of Biochemistry & Moleculr Biology, Applied Chemistry & Chemicl Engineering, Rjshhi University, Rjshhi, Bngldesh for giving technicl nd finncil support. The uthors re lso grteful to Rjshhi Medicl College, Rjshhi, Bngldesh for giving io-technicl support. CONFLICT OF INTERESTS The uthors declre no conflict of interests. REFERENCES 1. Hrper A.E., Beneveng N.J., Wohlhueter R.M., Effects of ingestion of disproportionte mounts of mino cids. Physiol Rev. 50(3), Klhr S., Buerkert J., Purkerson M.L., Role of dietry fctors in the progression of chronic renl disese. Kidney Interntionl. 24(5), Devux C., Polzin D.J., Osorne C.A., Wht role does dietry protein restriction ply in the mngement of chronic renl filure in dogs? Veterinry Clinics of North Americ: Smll Animl Prctice. 26(6), Brzel U.S., Mssey L.K., Excess dietry protein cn dversely ffect one. J Nutr. 128(6), Reddy S.T., Wng C.Y., Skhee K., Brinkley L., Pk C.Y., Effect of low-crohydrte high-protein diets on cid-se lnce, stone-forming propensity, nd clcium metolism. Am J Kidney Dis. 40(2), Frntz N.Z., Ymk R., Friesen K.G., The effect of diet nd lysine: clorie rtio on ody composition nd kidney helth in geritric cts. J App Res in Veterinry Medicine. 5(1), Frid K.A., Textook of Pthology, Ludhok Prkshni: Dhk, Bngldesh. 8. Chukwu L.O., Histophysiologicl nd sl metolic responses of lino rt, rttus norvegicus (L.) exposed to queous pepper extrcts. Afri J Biochem. 5(13), Wikipedi. Histopthology. en.wikipedi. org/wiki/histopthology (Accessed Octoer 11, 2018). 10. Ar H., Jesmin M., Rhmn S.M., Krim K.M.R., Rhmn M.S., Proximte nd minerl composition of four tengr (Mystus S.P.) fishes of Bngldesh. KU. Studies. 7(2), Sfi M., Bngldesh Fisheries, Acdemic Press nd Pulishers Limited: Dhk, Bngldesh. 12. De H.N., Islm A., Mollh Y., Utiliztion of protein of fish flour or fish protein concentrte under different dietry levels in ody protein synthesis nd ft deposition in reltion to growth of immture nd dult rts. Scientific Res. 2(3), Kzi K.A., Prcticl Pthology, Kzi Tli Al- Mmun (Ashi): Dhk, Bngldesh. 14. Algoso S.O., Nwosu J.N., Njoku N.E., Okoye E.C., Eluchie C.N., Agunw I.M., Hemtology 8

9 nd growth study of lino rts fed vrying inclusions of cooked cnvli plgiosperm piper seed mel seddiets. J Food & Nutri Res. 5(9), Ro P., Sujth D., Rj K.R., Vishwnth S., Nrsimhmurthy K., Si P., Ro D.N., Divkr S., Sfety spects of residul ß cyclodextrin in egg treted for cholesterol removl. J Eur Food Res Technol. 211(6), Jyrmn J., Lortory Mnul in Biochemistry, Wiley Estern Ltd: New Delhi, Indi. 17. Mitruk B.M., Rmsley, H.M., Clinicl Biochemicl nd Hemtologicl Reference Vlues in Norml Experimentl Animls nd Norml Humns, 2nd ed., Yer Book Medicl Pulishers Inc: 35 Est Wcker drive, Chicgo. 9

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