Indian Journal of Experimental Biology Vol. 40, January 2002, pp. 83-88

Effect of formulated diet on digestive enzymes of Labeo rohita (Ham.)

T A Sethuramalingam'* & M A Haniffa2

'Centre for Aquafeed and Nutrition (CAFeN)

2Centre for Aquaculture Research and Extension (CARE)

Research Department of Zoology, St. Xavier's College (autonomous), Palayankottai 627 002, India.

Received 27 February 2001 ; revised 12 September 2001

Six sets of feeding experiments were carried out using formulated diets containing prawn head waste (PW), chicken in­testine waste (CW), banana flower (BF), cauliflower waste (CAU) Dolicos lab lab (DLL) and groundnut leaf (GNL) in four levels of inclusion (IS, 30,45 and 60%) to assess the pattern of distribution and activities of digestive enzymes like cellu­lase, amylase, maltase, invertase, protease and lipase in the digestive tracts of Labeo rohita fingerlings . A control group of fish was fed with diets containing antibiotics to destroy the digestive tract microflora which may induce digestive functions. In general, the activity of digestive enzymes depended on the amount and type of the ingredients present in the diets in­gested by the fish. Test animals showed both endogenous and bacterial cellulase activities which suggests the necessity for including cellulose (plant protein source) as dietary ingredient. Occurrence of higher amount of cellulase in the foregut and amylase in the fore and midgut influenced by DNL and GNL diets revealed the possibility of including less than 40% of the respective ingredients in the diet of rohu. Maltase and invertase were highly influenced by GNL, DLL and BF diets than PW and CW diets . More than 40% inclusion of PW and CW was found to increase protease and lipase secretion in the midgut and hindgut regions. The higher secretion of lipase in the midgut suggested the physiological versatility for lipid digestion in rohu fingerlings .

The ability of any fish to digest a given diet rests pre­dominantly on the appropriate enzymes. The digestion is not only associated with the type of nutrients but also by the feeding habits of animals. Studies on di­gestion and digestive enzymes in fish have been stud­ied ' .3. The organisation of intestinal tract was corre­lated with the production of enzymes4.5. The digestive processes aided by bacteria and fungi colonising the intestinal tracts were also studied6. Apart from these studies, food and feeding habits of fi sh related to di ­gestive enzymes are still scarce and scattered . Since carps especially Labeo rohita (Ham.) (order­Physostomi; family-Cyprinidae) show a clear dietary preference to certain plant and animal protein diets,7. )) the physiology of digestion seems to be more compli­cated and needs a detailed study. This communication reports on the effects of various formulated feeds pre­pared out of locally available selected plant and ani­mal ingredients on the pattern and activity o f di ges­tive enzymes in Labeo rohita fingerlings . Moreover, this study would help to identify alternative preferred feed items for inclusion in formulated diets of L. rohita.

Materials and Methods Rohu fingerl ings (5 .5 to.27g) recruited from poly­

culture pond of CARE (Centre for Aquaculture Re-

*Correspondent author

search and Extention) St. Xavier's College Palay­ankottai, India, were divided into 7 groups of five fish each and were reared In rectangular tanks (l20x80x80 cm) containing 40 I water. All the aquaria were connected through PVC pipes with a flowthrough system from a recirculating water tank having a water flow of 4 IIhour. During acclimatiza­tion and experimental periods the water samples were analysed for pH (7 to 7.6); DO (4.52 mg/I) ; tempera­ture (28° t 1°C) and alkalinity (62 t 5 mg/I) according to the standard methods '2. Six sets of pelleted diets were formulated using prawn head meal (PW), chicken intestine meal (CW), banana flower meal (BF), cauliflower meal (CAU) , Dolicos lab lab (field broad beans) meal (DLL) and groundnut leaf meal (GNL) as test diets and a control diet [rice bran (RB) (21 %), groundnut oil cake (GOC) (20%), fish meal (FM) (29%), beaf meal (BM) (20%), tapioca flour (TF) (7%)] was prepared for each test diet and incor­porated in the ratio of 85: 15; 70: 30; 55: 45 and 40: 60 consisting of control: test diets respectively (Table 1). Proximate composition of ingredients used in the formu lated feed is presented in Table 2. The experimental fish were fed with the respective formu­lated feed (produced in the form of dried pellets (size - 0.5 mm) with the help of a hand pelletizer with

84 INDIAN J EXP BIOL, JANUARY 2002

a 1.5 mm dye) for 41 days. For assessment of cellu­lose activity, the control diet with tetracycline (Pfizer & Co) at 1000 ppm was added along with the diet with a view to kill the mkroflora in the digestive tract which might induce digestive function. The feed was provided twice a day at 0800 and 1600 hrs. in equal proportions at 5% body weight on dry weight basis. Three replicates were used for each feeding scheme.

The feed was analysed for proximate composition 13.

Moisture was determined by drying the sample to a constant weight in a hot air oven. Crude fibre content was found out by mild acid hydrolysis. Ash content was determined by burning the sample in a muffle furnace at 550°C for 8 hr. Gross energy value was determined by using semi-micro bomb calorimeter (Table 2) .

Preparation of enzyme source-After the experi­mental period (41 days) 3 test fish (8.1 ± 0.47 g) from each tank were removed and starved for 24 hrs and sacrificed. The whole alimentary tract was dissected out in ice cold fish ringer solution and thoroughly washed externally . The ti ssue was rinsed with cold distilled water and a portion each of alimentary canal, oesophagus, crop, liver and hepatopancreas and intes­tine was ligatured, split open and washed thoroughly. The tissues were homogeni sed separately with dis­tilled water using mechanical di spencer. The ho­mogenate was centrifuged at 40,000 rpm for 15 min at O°C using high speed refrigerated centrifuge (Remi­Model K- II) to prepare 1 to 10% of aquous extracts. The clear supernatant was used as the crude enzyme extract for subsequent assay.

Enzyme assay-Qualitative determination of car­bohydrases like cellulase, amylase, maltase and inver­tase was done l4 using Benedicts and Barford's re­agents. The protease was tested by using photographic film containing gelatin coating l5 . Lipase was tested l6

using milk as substrate.

Quantitative assay Cellulase activit/ 7-The reaction mixture con­

taining 100 mg carboxy methyl cellulose, 2 ml phos­phate buffer at pH 5.28 and 4 m! enzyme extract was added with 5 m! distilled water and incubated in a water bath at 36°C and after 5-7 min titrated against 0.1 N NaOH until it was exactly neutral to phenol­phthalein. Iodine (5 ml of 0.1 N) was added drop wise and then made acidic with 0 .1 N HCI until iodine was liberated with Na2S203. Unit of cellulase was meas­ured as the amount of enzyme required to hydrolyse glucose and to liberate iodine per min . Enzyme acti-

vity of cellulase was calculated by total hydrolysis of 100 mg of celluloselhr and specific activity expressed as Ilg glucose/mg of body proteinlhr.

Amylase and invertase activityl8-The reaction mixture containing 1 ml enzyme extract, 9.5 ml phos­phate buffer (PH 7.2) and 0.5 ml of 2% soluble starch solution (for invertase 2% sucrose was used at 6.2 pH) was incubated at 37°C for 3 min. The enzyme reaction was interrupted by adding 2 ml of 3, 5 dini­trosalicylic acid. After adding 20 ml of distilled water, the brown product was read at 580 nm. Maltose solu­tion (0.2 to 2 mg) was used as standard. The specific activity was expressed as Ilg maltose/mg of pro­tein/hr. Protein was determined by standard method 19.

Maltase activity2o- B.uffered (0.033 M) maltose solution (3 ml) was taken along with 10 Ilg/ml of no­tatin 0.02 M sodium azide in 0.1 M phosphate buffer (PH 6.8) in the main chamber of Warberg' s flask and 0.1 ml of enzyme added in side arm. After attaining steady state, the oxygen uptake in Ill/hr/m! of enzyme was measured equivalent to Ilg glucose formed/hr/m! of enzyme. Maltase unit is defined as the amount of enzyme, which would liberate Ilg of glucose/hr from maltose and specific activity is expressed as units/ mg of proteinlhr.

Protease activity21-T he reaction mixture con­sisted of 1 ml of 1 % casein (as substrate), 0.5 ml of 0.1 M phosphate buffer (PH 7.6) and 1 ml of crude enzyme extract. The reaction was terminated after 30 min by adding 5 ml of 5% TCA solution. The mixture was centrifuged and tyrosine in an aliquot of sample mixture was determined by the colour given by Folin­Ciocalteu reagent in alkaline solution and the optical density was measured at 650 nm. The colour was compared against a standard tyrosine solution in I M HCI. Protein was estimated by standard method using BSA as standard and specific activi ~y was measured as Ilg of tyrosine/mg of protein/hr at 37°C.

Lipase activity-The lipase activity was deter­mined by titrimetric method22. The assay system con­sisted of 1.5 ml pure olive oil (99.9%) with 1.5 ml of 1 M Tris- HCI buffer (PH 8) to which 1 ml of crude enzyme extract was added and incubated for 6 hr at 37°C. The reaction was arrested by addition of 3 ml of 95% ethyl alcohol. This mixture was titrated against 0.01 N NaOH using 0.9% (w/v) thymolphthalein in ethyl alcohol as indicator. The specific activity was expressed as lipase/mg proteinlhr (l ml of 0.01 N NaOH is taken as corresponding 100 lipase units).

SETHURAMALINGAM & HANIFFA: EFFECT OF FORMULATED DIET ON DIGESTIVE ENZYMES OF LABED 85

The data were subjected to ANOY A for finding out the significant differences among the treatment groups and correlation coefficient was nsed to study the effect of inclusion levels of ingredients and en­zyme activity of rohu fed on different diets23.

Results and Discussion The results are presented in Tables 1-3 . The activity of the digestive enzymes, viz. cellu­

lase, amylase, maltase, invertase, protease and lipase in the fore, mid and hindgut regions of L. rohita de­pended on the type as well as the amount of ingredi­ents used it the diet. This relationship between the quantity and quality of enzyme secretions and that of ingredients used in the feed very well agrees with the adapti ve nature of the fish to the feed 16.

The cellulase activity was highly influenced by 30% GNL, 45% DLL and 45% CAU diets. A sharp decline in the cellulase activity was observed in the fish fed with diet containing antibiotics tetracycline which is supposed to kill intestinal microflora24 sug­gesting that cellulase activity in L. rohita is contrib­uted partially by microbes and also by endogenous secretion. The exogenous cellulase was secreted in the anterior digestive tract of carps fed with plant protein diet25 indicating the presence of microbial cellulase while the remaining cellulase activity takes place in the midgut region. The cellulase secretion was posi­tively correlated with the amount of plant detritus in the gut of carps26. 27.

The increased amylase activity in the foregut and midgut was influenced by the higher incorporation levels of DLL and GNL in the diets. The amylase was secreted more in the hepatopancreas than in the intes­tine of carps24.IO.I. The fish fed with PW and CW diets showed an elevated amylase secretion in the foregut but decreased gradually in the mjd and hindgut. In­corporation of animal protein sources in the diets of C. carpio and Ctenopharyngodon idella resulted in jncreased amylase secretion in the anterior part of the digestive traceS.29. The cyprinid (carp) larvae fed with natural diets (zooplankton) could digest starch and glycogen present in animal tissue even a few days after initial feedi ng3o.

The maltase activity was higher in the midgut than foregut and hindgut and the secretion was highly in­fluenced not only by the control diet but also by the incorporation level of PP diets up to 45% and AP diets up to 30%. This observation was corollary with the study on the secretion of maltase in the mjdgut of juvenile C. carpio fed with mixed diee ' and also in Dicentrarchus labrax4 larvae fed with algal diet. L. rohita was also found to digest mixed diets containing more animal proteinS.

Invertase activity was highly influenced by all PP diets than AP diets. The secretion was more in midgut than hindgut. Contrary to other enzymes secretion, the invertase in the foregut was found to decrease with increase in the incorporation levels of AP and PP in the diet. This may be an indication of "diet specific" secretion in L. rohita.

Table I-Percentage composition of ingredients used in the formulated diets of L rolli/a.

Set I Set II Set III Set IV Ingredients (85: 15) (70:30) (55:45) (40:60)

Rice bran (RB) 20.89 18.71 15.22 11.40

Groundnut Oil Cake (GOC) 18.18 12.09 10.06 7.12

Fish meal (FM) 19.04 16.88 14.61 9.36 Beaf meal (BM) 18.89 14.96 9.11 7. 12

Tapioca flour (TF) 7.0 5.36 4.0 3.0

Vitamin and mineral mix* 3.0 3.0 3.0 3.0

Test diets

Prawn head meal (PW) 15 30 45 60

Chicken intestine meal (CW) 15 30 45 60 Banana flower meal (BF) 15 30 45 60 Cauliflower meal (CAU) 15 30 45 60 Dolicos lab lab meal (DLL) 15 30 45 60 Groundnut leaf meal (GNL) 15 30 45 60

*as 'vitaminets ' - ' Roche' pharmaceuticals, Mumbai , India.

86 INDIAN J EXP BIOL, JANUARY 2002

Table 2 - Proximate composition of formulated diets fed to L rohita fingerlings

Feed Sets Crude protein Crude fat Crude fibre Ash Energy type (%)

Control 38.76

PW 34.77

II 35.24

III 35.98

IV 36.82

CW I 33.63

II 34.11

III 34.87

IV 35.57

SF I 34.28

II 33.77

III 33.19

IV 32.69

CAU I 35.63

II 34.78

III 34.21

IV 33.57

DLL I 36.71

II 36.28

III 35.84

IV 35.26

GNL I 36.48

II 36.56

III 35.89

IV 35.31

PW and CW in all levels of incorporation signifi­cantly (P < 0.05) induced higher secretion of protease

in the midgut of L. rohita. Diet specific proteolytic enzyme activity in carps were also reported specifi­cally 1.2.7.32.33. Bovine trypsin in the diet was known to

increase the proteolytic activity in C. carpio34 and Dicentrarchus labrax larvae4• In Carasinus auratui ' , Chanos chanos35 , and C. idella24 proteolytic activity (trypsin) was observed in oesophagus and stomach fed with mixed diets.

Fish fed with diets containing AP strongly influ­enced lipase secretions in the midgut. This observa-. . h C . l 36 d' I 37 Th tlOn agrees WIt . mnga a an 111 te eosts. e

higher activity of lipase was also observed in the hepatopancreas of C. idella, C. carpio and in the gut of Hypophthalmichthys molytrix and Aristichthys no­bilislO. On the contrary a uniform lipase activity in the

gut and hepatopancreas of O. niloticus was ob­served lO, which suggests that food habits do not influ­ence lipase activity.

(%) (%) (%) U/mg)

7.28 5.26 5.61 21.73

5.72 4.82 6.78 20.59

5.89 4.97 6.32 20.88

6.12 5.27 6.11 2 1.l 7

6.53 5.34 5.96 21.33

5.41 4.72 6.71 20.38

5.78 4.97 6.43 20.67

5.93 5.14 6.19 20.88

6.18 5.22 5.71 21.03

5.55 9.17 12.58 17.62

5.01 10.59 11.27 16.74

4.73 12.06 10.86 16.65

4.32 13.33 10.03 16.09

6.37 8.14 10.66 17.45

6.01 8.79 10.71 17.63

5.83 9.57 10.83 17.89

5.28 10.33 11.76 17.91

6.81 8.22 10.73 17.51

6.54 9.59 12.31 17.83

6.08 9.86 12.88 17.98

5.89 10.09 13.26 17.34

6.54 8.13 12.37 17.39

6.04 8.94 13.27 17.93

5.47 9.56 13.92 18.08

5.12 10.87 14.98 17.12

The presence of endogenous cellulase reveals that L. rohita is a consumer of complex diets. The poten­

tial overall cellulase activity suggests that the fish could avail all the ingested dietary energy and also call for the necessity to provide more carbohydrate in the diets. Presence of higher secretion of amylase, protease and lipase suggests the potential for digestion of non-conventional plant and animal food sources like CW, PW (more than 40%), DLL, GNL (not more than 40%), CAU and BF (less than 30%) and hence

these could be incorported at desired levels in the diet of L. rohita.

As fish meal becomes scarce and costl ier, the above tested ingredients could be used as suitable pro­tein substitute for fish meal in diets of L. rohita. The study of digestive enzymes opened up a new phase to find out the suitable ingredients, which are physio­logically condusive for phenomenal growth of fish and to identify biologically valued nutrients from various non-conventional food sources. This also

Table 3-Specific activity (Ilglmg of body proteinlhr) of digestive enzymes in the digestive tracts of L rohita fed with vanous inclusion levels of animal and plant protein ingredients in formulated diets

Diets

Control

Set Control+Antibiotic (Ilg glucose/mg of

protcinlhr) F M H

PW I 25 27

II 49 41

IIT 61 47

IV 83 69

CW I 21 21

II 27 36

III 41 43

rv 58 70

BF J 120 89

II 243 231

III 287 244

IV 301 207

CAU I 57 42

II 184 164

III 192 149

IV 144 102

DLL 115 144

II 253 289

ill 276 384

IV 322 393

GNL I 143 129

II 269 284

III 289 361

IV 384 323

F = foregut; M = midgut; H = hind gut

Cellulase (Ilg glucose/mg of

proteinlhr) F M H

936 601

156 189

125 181

241 257

214 210

16& 181

187 247

183 263

223 314

1I35 887

1183 1157

1247 1189

1275 1048

995 942

1259 1083

1263 1139

1287 986

1227 1384

1372 1476

1526 1683

1451 1328

1366 1283

1425 1580

1512 1520

1478 1281

30

20

40

61

Amylase Maltase Invertase (Ilg maltose/mg of

proteinlhr) ().1g glucose/mg of

proteinlhr) (Ilg maltose/mg of

proteinlhr) F M H F M H F M H

824 436

626 480

794 505

860 542

862 566

585 412

819 460

893 468

940 510

625 505

825 550

891 576

1087 610

551 645

710 704

889 784

976 833

456 689

1143 781

1197 793

1246 844

673 724-

1097 807

1112 898 1296 980

209 348

355 341

286 638

336 534

339 508

310 310

261 521

213 527

174 485

180 515

163 525

141 814

124 627

196 436

173 559

143 653

121 547

136 563

121 547

119 786

110 572

106 654

99 714

106 769

198 623

510

548

497

436

417

537

435

421

408 665

640

476

396 491

463

418

389

787

724

709

683

843

810

767

728

142 186 344

235 285 234

184 256 246

216 224 278

211 208 216

216 294 258

1&9 271 268

159 267 276

143 232 224

III 537 385

107 369 435

97 307 467

83 241 460

107 415 391

92 403 427

83 327 498

62 298 422

73 475 627

63 449 751

52 407 818

44 247 746

82 427 547

76 415 663

63 409 784

53 383 718

116

115

183

285

316

163

186

214

254

195

214

234

246

207 224

263

268

234

268

294

317

230

253

231

256

Protease (Ilg tyrosine/mg of

protein/hr) F M H

280 899 482

187 639 485

207 779 605

226 955 678

249 1190 789

168 641 497

194 734 593

212 964 613

227 1146 738

121 535 436

169 626 482

179 683 497

192 696 533

179 595 422

194 667 438

218 696 489

227 750 447

185 580 463

209 687 4%

226 726 536

246 857 578

195 591 482

234 689 511

246 781 393

2!l1 963 417

Lipase ().1g Jipaselmg of pro­

tein/hr) F M H

694

626

649

698

727

638

683

706

764

365

407

427

459

314

339

371

384

334

368

394

443

347

389

410

456

316

249

279

363

410

226

240

249

394

175

183

165

235

82

99

131

167

136

156

183

199

141

163

144

188

en ~ ::c c: ~ 3:: );­r Z o );-3:: ~ ::c );-z ~ );-

tTl

~ o 'Tl

25 ~ c: r );-

til t:l t:l

~ o Z t:l Ci tTl en ...., <: tTl tTl Z N -< 3:: tTl en o 'Tl

~ Ct:l

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88 INDIAN J EXP BIOL, JANUARY 2002

helps in producing cost effective feeds for carps be­cause the utilization of these ingredients by the fish were found to be encouraging and they could be added in carp diets according to their digestive capacity.

Acknowledgement One of the authors (T AS) thanks Dr. K. Sampath,

Reader in Zoology, V. O. Chidambaram College, Tuticorin, for valuable suggestions and critical com­ments.

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