Fish Physiol Biochem (2006) 32:209–220 DOI 10.1007/s10695-006-9002-z
ORIGINAL PAPER
Studies on digestibility and digestive enzyme activities in Labeo rohita (Hamilton) juveniles: effect of microbial a-amylase supplementation in non-gelatinized or gelatinized corn-based diet at two protein levels S. Kumar Æ N. P. Sahu Æ A. K. Pal Æ D. Choudhury Æ S. C. Mukherjee
Received: 22 April 2006 / Accepted: 4 July 2006 / Published online: 10 August 2006 Springer Science+Business Media B.V. 2006
Abstract A 60-days feeding trial was conducted to delineate the effect of both gelatinized and non-gelatinized corn with or without supplementation with exogenous a-amylase at two dietary protein levels (35% and 28%) on dry matter digestibility, digestive enzymes and tissue glycogen content of Labeo rohita juveniles. Three hundred and sixty juveniles (average weight 10±0.15 g] were randomly distributed into 12 treatment groups with each of two replicates. Twelve semi-purified diets containing either 35% or 28% crude protein were prepared by including gelatinized (G) or non-gelatinized (NG) corn as carbohydrate source with different level of microbial a-amylase (0, 50, 100 and 150 mg kg–1). The dry matter digestibility of G corn fed groups was significantly higher (P < 0.05) than that of the NG corn fed groups. Hepatosomatic index (HSI), liver glycogen and intestinal amylase activity of G
S. Kumar (&) Æ N. P. Sahu Æ A. K. Pal Æ D. Choudhury Department of Fish Nutrition and Biochemistry, Central Institute of Fisheries Education, Versova, Mumbai 400061, India e-mail:
[email protected] S. C. Mukherjee Department of Fish Pathology and Microbiology, Central Institute of Fisheries Education, Versova, Mumbai, India
starch fed groups were significantly higher (P < 0.05) than those of the NG corn fed groups. However, the reverse trend was found for gastrosomatic index (GSI), muscle glycogen and intestinal protease activity. Addition of 50 mg a-amylase kg–1 feed improved the dry matter digestibility of NG starch fed groups, which was similar to that of the G corn fed groups or NG corn supplemented with 100/150 mg a-amylase kg–1 feed. HSI, liver glycogen and intestinal amylase activity were significantly increased (P < 0.05) at minimum level of a-amylase in the feed (50 mg kg–1) and did not increase due to further inclusion of amylase in the diet. Supplementation with a-amylase at 50 mg kg–1 increased the intestinal amylase activity beyond which no significant changes were observed. Protease activity of liver and intestine was highest (P < 0.05) in higher crude protein (CP) fed groups, but protease activity of the intestine was significantly higher in the a-amylase supplemented groups. Hence, it was concluded that feed with 28% CP containing either G corn without a-amylase or NG corn with 50 mg a-amylase kg–1 may be used as the alternative carbohydrate source for L. rohita juveniles. Keywords Apparent digestibility Æ a-Amylase Æ Carbohydrate Æ Gelatinization Æ Glycogen Æ Labeo rohita Æ Protease
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Introduction Carbohydrate constitutes one of the three prime components of the fish feed that are used as energy source to support growth. Although carbohydrate is not indispensable in fish feed, it constitutes an inexpensive source of energy. In the absence of carbohydrate there is increased utilization of proteins and lipids as energy sources. In many species a dietary carbohydrate supply appears to be necessary, as it improves growth and, especially, protein utilization. It has been proposed that it is necessary to provide an appropriate level of this nutrient in the diet to ensure maximum utilization of other nutrients (Wilson 1994). Prather and Lovell (1973) reported that, if insufficient nonprotein energy is available, then protein is first used as an energy source rather than for growth purposes. Carbohydrate digestibility of carnivorous fish is poorer than that of the omnivorous and herbivorous fish. The carbohydrate digestibility in fish depends on the type of carbohydrate (simple/ complex), the dietary inclusion level and the processing treatments applied to it (raw/cooked/ extruded) as reported for carp, Cyprinus carpio (Chiou and Ogino 1975), for yellowtail, Seriola quinqueradiata (Shimeno et al. 1978), for rainbow trout, Salmo gairdneri (Bergot and Breque 1983; Ufodike and Matty 1984) and for cod, Gadus morhua (Hemre et al. 1989). Increased level of starch in the diet decreases its digestibility in carnivorous fish such as turbot (Inaba et al. 1963) and yellowtail (Shimeno et al. 1978). However, Mohapatra et al. (2002) reported a significant increase in the carbohydrate digestibility with increasing level of gelatinized carbohydrate (GC) in the diet of Labeo rohita fry. Fish fed higher levels (45% and 50%) of GC have higher carbohydrate digestibility (83%), indicating better utilization of carbohydrate by herbivores/omnivores such as carp. Cooking or gelatinization results in hydratization and shortening of starch chains and thus improves their digestibility. Processing of carbohydrate improves digestibility by destruction of amylase inhibitors and by shortening of
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starch chains (Silano et al. 1975; Hofer and Sturmbauer 1985). Digestibility of native starch is rather low (30–50%), whereas that of gelatinized starch is higher (50–90%). This has been shown in trout by Phillips et al. (1940), Pieper (1977), Bergot (1991) and Takeuchi et al. (1990), in carp by Chiou and Ogino (1975) and Mohapatra et al. (2002), in turbot by Jollivet et al. (1988), in red sea bream (Pagrua major) by Jeong et al. (1991) and in Penaeus vannamei by Davis and Arnold (1993). Pieper (1977) reported a higher digestibility of 95% for gelatinized corn starch in rainbow trout. Besides gelatinization, enzyme pretreatment of dietary plant material with carbohydrases (a-amylase, b-glucanases and b-xylanases) may enhance the energy digestibility in fish by releasing previously unavailable glucose, galactose, and xylose. Exogenous dietary enzyme supplements, isolated from plants and bacteria, have been used successfully in pig and poultry feeds (Batterham 1992; Farrell 1992; Campbell and Bedford 1992; Chesson 1993; Bedford 1996; Dudley-Cash 1997). The addition of exogenous carbohydrase enzymes to aquafeed has been reported to enhance the utilization of unavailable dietary carbohydrates by Atlantic salmon, Salmo salar, larval gilthead sea bream, Sparus aurata and tiger prawn, Penaeus monodon (Kolkovski et al. 1993; Carter et al. 1994; Buchanan et al. 1997). Stone et al. (2003b) has also reported that starch digestibility was significantly affected by different levels of Natustarch (a commercial a-amylase). They found that there was greater effect of Natustarch on a diet containing raw wheat starch [starch apparent digestibility coefficient (ADC) increased from 84% to 92%] than on diets containing gelatinized wheat starch. They also reported that the protein digestibility of the diet was not significantly affected by carbohydrate type but that the addition of Natustarch led to a slight increase in protein digestibility (P < 0.05). Hence, the aim of this study was to investigate the effects of the addition of exogenous amylase to diets containing either gelatinized or non-gelatinized corn at different protein levels on dry matter digestibility in the diet of L. rohita juveniles.
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Material and methods
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The degree of gelatinization was calculated as follows:
Fish and experimental conditions Gelatinization % ¼ A1 =A2 100 Labeo rohita (Hamilton) juveniles were brought from the Khopoli fish farm (Maharastra, India) to the Fish Nutrition Laboratory, Central Institute of Fisheries Education (Mumbai, India) and acclimatized for 24 days on the control diet (35% crude protein). At the beginning of the study the fish were weighed individually, and 15 fish (average weight 10 ± 0.15 g) were randomly transferred to each of 24 tubs (150 l). Aeration was provided to all the tubs, and manual water exchange was carried out every other day. Water quality parameters were checked every week, in accordance with the standard methods of the American Public Health Association (APHA) (1998). Gelatinization of corn The corn was ground to a fine powder and made into dough by the addition of the required amount of water, followed by cooking in an autoclave at 15 p.s.i. for 1 h to achieve maximum gelatinization. The cooked corn was then spread over a tray and dried in an oven at 60C. The dried mass was pulverized in a hammer mill with a 0.5 mm screen and stored in airtight containers until required. The degree of gelatinization of corn was determined in accordance with Guraya and Toledo (1993). A known amount (0.2 g) of corn powder was mixed with 15 ml of 0.2 N potassium hydroxide followed by intermittent stirring for 30 min. The pH of the mixture was adjusted to 5.5 with 2 N phosphoric acid, and the volume was made up to 100 ml with distilled water. Next, 100 ll of aliquot was transferred to a test tube and diluted to 5 ml with distilled water. Then, 50 ll of standard iodine solution (4% KI, 1% I2) was added, and the absorbance of the solution was taken at 600 nm (A1) against the reagent blank. Another aliquot was made by the same procedure by mixing 0.2 g of dried corn powder in 15 ml of 0.6 N potassium hydroxide, and the absorbance was taken at 600 nm (A2) as above.
Diet preparation and feeding The composition of the experimental diet is given in (Table 1). Fat-free casein and gelatin were used as protein sources, whereas sunflower oil and cod liver oil were used as lipid sources and corn as carbohydrate source. Ingredients were finely ground and mixed thoroughly with water to make a dough. The dough was steam-cooked for 5 min in a pressure cooker. Vitamin–mineral premix was mixed after cooling, and the dough was passed through a hand pelletizer with a 2 mm die and then dried at 60C. The required amount of a-amylase (Aspergillus origin, HIMEDIA Laboratories Pvt. Limited, Mumbai, India) was dissolved in 50 ml of distilled water and sprayed over 1 kg of basal diet as described by Robinson et al. (2002). Thus, 12 experimental diets with 42.43% of non-gelatinized (NG) or gelatinized (G) corn, two levels of crude protein: 35% (optimum) or 28% (sub-optimum) and four levels of a-amylase: 0, 50, 100 and 150 mg kg–1 feed were prepared (Table 2). The feed was stored at 4C until required. Chromic oxide was added at 0.5% to all diets for digestibility studies during the last 20 days of the study. Each diet was fed twice daily (0800 and 1800 h) to satiation for 60 days. Sampling and analysis of samples Fish in each tub were weighed every 15 days to assess their growth. Faecal materials were collected daily, from the 41st to 60th day, for determination of dry matter digestibility. Faecal material was collected manually by being siphoned and strained through a fine-meshed net. Three hours after the first feeding (0800 h), uneaten feed, together with the faeces, was siphoned out and discarded. At 1400 h, faecal material was collected and dried to constant weight at 60C. The proximate composition of all the diets was determined by the standard methods of the Association of
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Table 1 Composition of the experimental diets [percentage dry matter (DM) basis] Ingredients
Inclusion (%)
Caseina Gelatinb Corn flour (NG or G)c Cellulosed Sunflower oil : cod liver oil (2 : 1) Vitamin + mineral mixturee Carboxymethyl cellulosef Vitamin Cg Vitamin B complexh Glycinei Butylated hydroxy-toluenej Chromic oxide a
28% CP
35% CP
26.57 4.00 42.43 15.00 8.00 2.60 1.00 0.10 0.10 0.20 0.02 0.05
30.57 8.00 42.43 7.00 8.00 2.60 1.00 0.10 0.10 0.20 0.02 0.05
Fat free: 75% CP (Himedia Ltd, India)
b
96% CP (HImedia Ltd)
c
Procured from Central poultry farm, Mumbai, India (NG - non gelatinized, G - gelatinized)
d
Sd Fine Chemicals (Mumbai, India)
e
Composition of vitamin–mineral mix (EMIX PLUS) (quantity/2.5 kg): vitamin A - 55,00,000 IU; vitamin D3 11,00,000 IU; vitamin B2 - 2,000 mg; vitamin E - 750 mg; vitamin K - 1,000 mg; vitamin B6 - 1,000 mg; vitamin B12 - 6 lg; calcium pantothenate - 2,500 mg; nicotinamide - 10 g; choline chloride - 150 g; Mn -27,000 mg; I - 1,000 mg; Fe - 7,500 mg; Zn - 5,000 mg; Cu - 2,000 mg; Co - 450 mg; Ca - 500 g; P - 300 g; L-lysine - 10 g; DL - methionine - 10 g; selenium - 50 ppm.
f
Sd Fine Chemicals Ltd
g
Stay C (Hoffman La Roche, Nutley, N.J., USA) 15% ascorbic acid activity
h
Composition(quantity per gram): thiamine mononitrate - 20 mg; riboflavin - 20 mg; pyridoxine hydrochloride - 6 mg; vitamin B12 - 30 lg; niaciamide - 200 mg; Ca pantothenate 100 - mg; folic acid - 3 mg; biotin - 200 lg i
HImedia Ltd, India
j
Sd Fine Chemicals Ltd
Table 2 Details of experimental groups Experimental groups
Details
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12
NG, 35% CP, 0 mg a-amylase kg–1 G, 35% CP, 0 mg a-amylase kg–1 NG, 27% CP, 50 mg a-amylase kg–1 NG, 35% CP, 50 mg a-amylase kg–1 G, 27% CP, 50 mg a-amylase kg–1 G, 35% CP, 50 mg a-amylase kg–1 NG, 27% CP, 100 mg a-amylase kg–1 NG, 35% CP, 100 mg a-amylase kg–1 G, 27% CP, 100 mg a-amylase kg–1 G, 35% CP, 100 mg a-amylase kg–1 NG, 27% CP, 150 mg a-amylase kg–1 NG, 35% CP, 150 mg a-amylase kg–1
NG - non-gelatinized; G - gelatinized; CP - crude protein of feed
Official Analytical Chemists (AOAC) (1995), Table 3. In brief, moisture content was determined by drying at 105C to a constant weight; nitrogen content was estimated by Kjeltec (2200 Kjeltec
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Auto distillation, Foss Tecator, Sweden) and CP was estimated by multiplying nitrogen percentage by 6.25, ether extract (EE) was measured with a Soxtec system (1045 Soxtec extraction unit, Tecator, Sweden) using diethyl ether (boiling point 40–60C) as a solvent, and ash content was determined by incinerating samples in a muffle furnace at 600C for 6 h. Total carbohydrate was calculated by difference, i.e. total carbohydrate% = 100 – (CP% + EE% + ash%). Digestibility study Apparent digestibility coefficients (ADCs) for dry matter of different diets was measured by the indicator method using 0.5% chromium oxide as a marker (Hardy and Barrows 2002). Wet ashing of diets and faecal matters was carried out according to the AOAC (1995), and chromium content of feed and faecal matters was determined with a
Fish Physiol Biochem (2006) 32:209–220 Table 3 Proximate composition of the different experimental diets (percentage dry matter basis)
OM - organic matter; TC - total carbohydrate
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Treatment
OM
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12
96.47 96.77 96.81 96.55 96.67 96.69 96.59 96.72 96.73 96.71 96.72 96.61
CP ± ± ± ± ± ± ± ± ± ± ± ±
0.85 0.81 0.65 0.74 0.69 0.81 0.92 0.65 0.74 0.62 0.65 0.74
flame ionization atomic absorption spectrophotometer (AAS 4129, Electronics Corporation of India). The apparent dry matter digestibility was calculated by a standard formula: ADC ¼ 100 100 ½% marker in feed=% marker in faces
Sample preparation At the completion of the experiment, liver and intestine were taken from three fish from each tank for each treatment. The fish were anaesthetized with clove oil at 50ll l–1, killed with a blow to the head, and dissected to collect the liver and intestine for digestive enzyme (amylase and protease) estimation. Immediately, a 5% homogenate in 250 mM sucrose was prepared for liver and intestine tissues. The homogenate was centrifuged at 5,000 r.p.m. for 20 min and the supernatant was collected in a sample vial and kept at –20C until required. Whole intestine was used for amylase and protease assay. Before homogenization the intestinal contents were removed. Another three fish were anaesthetized with clove oil at 50ll l–1 and dissected to collect the liver and muscle for glycogen estimation. The muscle samples were taken from the caudal peduncle region after the scales had been scraped off. Liver and muscle glycogen, hepatosomatic index and gastrosomatic index Liver and muscle glycogen content was estimated colorimetrically by the method described
34.73 34.78 26.68 35.05 26.68 33.75 27.01 34.82 27.01 34.82 26.68 34.78
EE ± ± ± ± ± ± ± ± ± ± ± ±
0.24 0.19 0.25 0.24 0.13 0.31 0.26 0.18 0.24 0.29 0.23 0.21
12.53 10.36 11.85 12.45 11.54 9.63 11.48 12.30 10.95 9.81 11.20 11.75
Ash ± ± ± ± ± ± ± ± ± ± ± ±
0.11 0.09 0.09 0.11 0.08 0.08 0.09 0.10 0.08 0.08 0.09 0.09
3.53 3.23 3.19 3.45 3.33 3.31 3.33 3.41 3.27 3.29 3.28 3.39
TC ± ± ± ± ± ± ± ± ± ± ± ±
0.02 0.01 0.02 0.02 0.02 0.00 0.01 0.02 0.02 0.02 0.01 0.01
49.21 51.63 58.28 49.05 58.45 53.31 58.18 49.47 58.77 52.08 58.84 50.08
± ± ± ± ± ± ± ± ± ± ± ±
0.32 0.41 0.36 0.38 0.41 0.32 0.25 0.25 0.35 0.38 0.35 0.28
by Hassid and Abraham (1957). The tissue was placed in a pre-weighed centrifuge tube containing 3 ml of 30% KOH. After the weight of the tissue had been recorded, the tubes were placed in a boiling water bath for 20 min. After cooling, 5 ml of 95% ethanol was added to precipitate the glycogen. The precipitate obtained was dissolved in 1 ml of distilled water and again precipitated with 95% ethanol and centrifuged. The glycogen precipitate was then dissolved in distilled water, and this solution was used to estimate the quantity of glycogen. To 0.1 ml of aliquot, 5 ml of anthrone reagent was added and mixed by swirling the tube. The tubes were covered with glass marble and heated for 10 min in boiling water, followed by cooling, and the absorbance was recorded at 590 nm. The reading was compared with that of standard glycogen. The hepatosomatic index (HSI) and gastrosomatic index (GSI) were determined using the following formulae: HSI ð%Þ ¼ ðwet weight of liver= whole wet body weightÞ
100
GSI ð%Þ ¼ ðwet weight of gastrointestinal tract= whole body weightÞ 100
Digestive enzymes Amylase activity of the juveniles was measured by estimating the reducing sugars produced by the action of gluco-amylase and a-amylase on carbohydrates, using di-nitro-salicylic acid (DNS) as described by Rick and Stegbauer
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(1974). The reaction mixture consisted of 1% (w/v) starch solution, phosphate buffer and the tissue homogenate. The reaction mixtures were incubated at 37C for 30 min, followed by addition of DNS, and kept in a boiling water bath for 5 min. After being cooled, the reaction mixture was diluted with distilled water and absorbance was measured at 540 nm. Maltose was used as the standard, and amylase activity was expressed as millimoles of maltose released from starch per minute at that temperature. Protease activity was determined as described by Drapeau (1974). The reaction mixture consisted of 1% casein in 0.05 M Tris-PO4 buffer (pH 7.8) and was incubated for 5 min at 37C. Then the tissue homogenate was added. Ten minutes later the reaction was stopped by the addition of 10% TCA, followed by filtration of the samples. The reagent blank was made by the addition of tissue homogenate just before stopping the reaction without incubation. One unit of enzyme activity was defined as the amount of enzyme needed to release acid soluble fragments equivalent to 0.001 A280 per minute at 37C and pH 7.8.
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type, a-amylase or protein content of diet and their interaction on apparent dry matter digestibility, are given in Table 4. Corn type and dietary a-amylase level had significant effect on apparent dry matter digestibility in the juveniles. A significant interaction (P < 0.05) was also recorded between corn type · amylase on apparent dry matter digestibility. The G corn fed groups registered significantly (P < 0.05) higher apparent dry matter digestibility than NG corn fed groups. Though addition of exogenous a-amylase in the diet significantly increased (P < 0.05) the apparent dry matter digestibility of dry matter, there was no significant difference (P> 0.05) among the amylase-treated groups. Apparent dry matter digestibility was not affected by a change in dietary protein level. Lowest apparent dry matter digestibility was registered in NG corn fed groups without supplementation with a-amylase. Supplementation with a-amylase at any level in the diet of G corn fed groups showed similar apparent dry matter digestibility (P < 0.05) to that in the non-amylase supplemented groups. Liver and muscle glycogen, HSI and GSI
Statistical analysis The main effect was analysed by three-factor analysis of variance (ANOVA) with starch type (gelatinized and non-gelatinized), two levels of protein (35% and 28%) and the amount of enzyme supplemented (0, 50, 100 and 150 mg kg–1) as three fixed factors. Where significant interactions were found between main effects, a one-factor ANOVA was used to compare the simple effects. When results were significant, comparison among the means were made using Duncan’s multiple range test (DMRT). All value were compared at the 5% level of probability. Statistical evaluation of the data was carried out with the software SPSS, version 11.0.
Results Apparent dry matter digestibility The apparent dry matter digestibility in juveniles fed different diets, and the main effect of corn
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A similar trend was observed for HSI as for ADC, but only corn type had a significant effect on GSI (Table 4). HSI of G corn fed groups was significantly higher (P < 0.05) than that of the NG corn fed groups. Supplementation with a-amylase significantly increased (P < 0.05) the HSI compared with that of the non-supplemented groups, but the values were similar (P > 0.05) among the a-amylase supplemented groups. A significant interaction was found between the corn type and a-amylase for HSI in the juveniles (Table 4). Unlike the HSI, the GSI in L. rohita fingerlings was significantly higher (P < 0.05) in NG corn fed groups than the G corn fed groups. As in ADC and HSI, a similar trend was also observed for liver glycogen content. However, muscle glycogen content was similar (P > 0.05) in all the groups (Table 4). G corn fed groups registered significantly higher (P < 0.05) liver glycogen content than the NG corn fed groups. Dietary CP level had no effect on liver glycogen content (Table 4). However, addition of a-amylase significantly (P < 0.05) enhanced the liver
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Table 4 Effect of dietary treatments on the apparent digestibility coefficient (ADC), hepatiosomatic index (HSI), gastrosomatic index (GSI) and liver and muscle glycogen concentration (mg glycogen g–1 wet tissue) of L. rohita juveniles Treatment
ADC (%)
HSI (%)
GSI (%)
Liver glycogen
Muscle glycogen
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12
56.38b ± 1.14 80.99a ± 1.05 80.16a ± 1.09 81.69a ± 1.14 82.23a ± 0.84 81.79a ± 0.83 80.19a ± 1.28 79.45a ± 0.89 81.76a ± 0.89 81.95a ± 0.97 80.74a ± 0.7 80.88a ± 0.52
0.73b ± 0.02 0.93a ± 0.04 0.87a ± 0.02 0.89a ± 0.05 0.93a ± 0.02 0.97a ± 0.03 0.89a ± 0.03 0.87a ± 0.02 0.91a ± 0.07 0.88a ± 0.05 0.90a ± 0.03 0.92a ± 0.05
2.41a ± 0.14 1.56b ± 0.12 1.81b ± 0.12 1.43b ± 0.13 1.55b ± 0.17 1.74b ± 0.09 1.59b ± 0.05 1.61b ± 0.05 1.42b ± 0.10 1.49b ± 0.15 1.73b ± 0.13 1.48b ± 0.05
97.13b ± 1.29 166.74a ± 3.25 171.36a ± 3.19 168.55a ± 5.50 172.91a ± 9.58 171.37a ± 7.73 172.09a ± 10.43 169.33a ± 6.86 175.31a ± 3.07 175.79a ± 11.19 176.99a ± 4.75 175.23a ± 7.95
13.10 14.18 11.56 14.10 14.44 12.85 11.70 13.68 14.52 10.33 13.94 12.36
ANOVA Corn type CP Amylase Corn type · protein Protein · amylase Corn type · amylase
