Abstract: A 60 days feeding trial was conducted to delineate the effect of both gelatinized (G) and non-gelatinized (NG) corn supplemented with exogenous microbial α-amylase (0, 50, 100 and 150 mg kg-1) at either sub-optimal (28%) or optimal (35%) crude protein (CP) level on muscle protein, muscle protein/DNA ratio and DNA/muscle mass (wet wt.) ratio of Labeo rohita fingerlings. Three hundred sixty fingerlings (av. wt. 10±0.15 g) were randomly distributed in 12 treatment groups with each of two replicates. The muscle protein and muscle protein/DNA ratio of NG corn fed groups was significantly higher (p<0.05) than the G corn fed groups, whereas reverse trend was found for DNA/muscle mass (wet wt.) ratio. There was no significant effect on muscle protein, muscle protein/DNA ratio and DNA/muscle mass (wet wt.) ratio at both 28% and 35% CP. Supplementation of α-amylase at 50 mg kg -1 increased the muscle protein and muscle protein/DNA ratio beyond which no significant changes were observed but DNA/muscle mass (wet wt.) ratio was significantly higher in non α-amylase supplemented group. Hence it concludes that NG corn with 50 mg α-amylase kg -1 at 28% CP is optimum in the diet of L. rohita fingerling to improve muscle protein, muscle protein/ DNA ratio and DNA/ muscle mass (wet weight) ratio.
Introduction
Fish in general poorly utilize dietary carbohydrate. Furuichi and Yone (1980) observed depressed growth and feed efficiency in red sea bream, yellowtail and common carp fed high carbohydrate containing diets. A relative inability to metabolize carbohydrates had been reported in several studies in different species of fish (Furuichi and Yone, 1982b; Wilson and Poe, 1987; Gutierrez et al., 1991). This inability is reflected as persistent hyperglycemia (Palmer and Ryman, 1972; Furuichi and Yone, 1981; Wilson and Poe, 1987), a lower activity of liver hexokinase (Furuichi and Yone, 1982b), a lack of glucokinase (Nagayama et al., 1980; Cowey et al., 1977) and a lower number of muscle insulin receptors (Gutierrez et al., 1991).
Indian Major Carps (IMC) and exotic carps are considered to be the major aquaculture species in tropical countries, contributing about 97% of the total freshwater aquaculture production (FAO, 2001). Out of these, 90% of the aquaculture production is contributed only by IMC. Labeo rohita is the most preferred species, comprising about 35% of the total IMC production. Requirement of dietary carbohydrate has been reported to be 26% for L. rohita (Sen et al., 1978) but further study revealed that dietary level of 40% gelatinized carbohydrate improve the growth of the species (Mohapatra et al., 2002). Use of more carbohydrate in the fish diet is required as it significantly reduces the feed cost and hence production cost.
The relative use of dietary carbohydrates by fish varies and appears to be associated with the complexity of carbohydrate. The relative utilization of dietary glucose, dextrin and gelatinized starch has been compared in carp and sea bream. Growth and feed efficiency of carp were highest when fed the gelatinized starch diet, followed by the dextrin and glucose diets in decreasing order, whereas red sea bream did not show any significant difference in growth rates for the various carbohydrate sources (Furuichi and Yone, 1982a). Beside gelatinization, enzyme pretreatment of dietary plant ingredients with carbohydrases (α- amylase, β- glucanases and β- 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 the pig and poultry feed (Batterham, 1992; Farrell, 1992; Campbell and Bedford, 1992; Chesson, 1993; Bedford, 1996; Dudley-Cash, 1997). Stone et al. (2003) had also reported that starch digestibility was significantly affected by different level of Natustarch® (a commercial α- amylase supplement). They found that there was greater effect of Natustarch® on the diet containing raw wheat starch (starch apparent digestibility coefficient increased from 84% to 92%) than on the diets containing gelatinized wheat starch. Hilton et al. (1982) and Hilton and Slinger (1983) reported that excess of digestible carbohydrate causes reduction in growth of rainbow trout.Muscle growth in fish, including early myotome expansion, is a plastic process (Weatherley, 1990) that involves a combination of enlargement of the muscle fibres already existing (hypertrophy) and the recruitment of new fibres (hyperplasia) (Koumans and Akster, 1995; Rowlerson and Veggetti, 2001). The balance between these mechanisms determines the particular rate of growth and ultimate size of the species (Weatherley et al., 1988; Zimmerman and Lowery, 1999), but is dependent upon various internal and external factors. This is true for both `white' (fast contracting, glycolytic) and `red' (mainly slow contracting, oxidative) fibre types. DNA content is considered to be an index of cell number (Bulow, 1987), as all cells are assumed to contain the same amount of DNA (the nuclear and mitochondrial genomes). Foster et al. (1993) deals that protein/ DNA ratio is an index of cell size and DNA/ muscle mass ratio is an index of cell number. They concluded that muscle cell size increases whereas cell number decrease in growing fish. Relationship of dietary carbohydrate with cell size and cell number in fish is rarely reported.
Rainbow trout fed a carbohydrate deficient diet showed a lower growth rate and reduced size of muscle cell (Peragon et al., 1999). But herbivorous fish may utilize the carbohydrate to a greater extent for growth, which can be measured in term of protein/ DNA ratio and DNA/ muscle mass ratio. However, these types of studies are rarely reported. In all fish, regulation of protein deposition is generally of great importance for control of whole body growth. In fish, other parameters related to tissue growth and protein deposition are content of nucleic acids, RNA and DNA (Buckley, 1984; Bastrop et al., 1992; Foster et al., 1993).
Excess protein in diet leads to utilization of protein for energy source (Phillips, 1972; Prather and Lovell, 1973). Protein sparing effect of carbohydrate is observed at low protein level in the feed (Shiau and Peng, 1993; Erfanullah and Jafri, 1995).
Thus, the aim of this study was to investigate effect of the gelatinized or non-gelatinised corn supplemented with exogenous amylase at sub-optimal (28%) or optimal (35%) protein level on muscle protein deposition, DNA concentration and their ratio in Labeo rohita fingerlings.
Materials and Methods
Fish and Experimental Conditions
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 with the control diet
(35% crude protein). At the beginning of the study, fish were weighed individually
and 15 fish (average wt. 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 following the standard methods of APHA (1998).
Gelatinization of Corn
The corn was ground to fine powder and made into dough by adding required
amount of water followed by cooking in as autoclave at 15 psi for 1 h so as
to get maximum gelatinization. The cooked corn was then spread over a tray and
dried in an oven at 600C. The dried mass was then pulverized in a
hammer mill with a 0.5 mm screen and stored in airtight containers until use.
The degree of gelatinization of corn was determined as 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 using 2N phosphoric acid and the volume was made upto 100
mL with distilled water. Next, 100 μL of aliquot was transferred to a test
tube and diluted to 5 mL with distilled water. Then 50 μL 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. The degree of gelatinization was calculated as follows:
Diet Preparation and Feeding
The composition of the experimental diet is given in Table
1. Fat free casein and gelatin were used as protein source, whereas sunflower
oil and cod liver oil were used as lipid source and G or NG corn as carbohydrate
source. Ingredients were finely ground and mixed thoroughly with water to make
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 600C. The required amount of α-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, twelve experimental diets
with 42.43% of non-gelatinized (NG) or gelatinized (G) corn, two level of crude
protein: 35% or 28% and four level of α-amylase: 0, 50, 100 and 150 mg
Kg-1 feed were prepared viz., T1 (NG, 35% CP, 0 mg kg-1
α-amylase), T2 (G, 35% CP, 0 mg kg-1 α-amylase),
T3 (NG, 28% CP, 50 mg kg-1 α-amylase), T4
(NG, 35% CP, 50 mg kg-1 α-amylase), T5 (G, 28% CP,
50 mg kg-1 α-amylase), T6 (G, 35% CP, 50 mg kg-1
α-amylase), T7 (NG, 28% CP, 100 mg kg-1 α-amylase),
T8 (NG, 35% CP, 100 mg kg-1 α-amylase), T9
(G, 28% CP, 100 mg kg-1 α-amylase), T10 (G, 35% CP,
100 mg kg-1 α-amylase), T11 (NG, 28% CP, 150 mg kg-1
α-amylase) and T12 (NG, 35% CP, 150 mg kg-1 α-amylase).
The feed was stored at 40C until use. Each diet was fed twice daily
(8.00 and 18.00 h) to satiation for 60 days.
| Table 1: | Composition of the experimental diets (%DM basis) |
| 1- | Casein fat free: 75%CP (HImedia ltd, India) |
| 2- | Gelatin: 96% CP (HImedia ltd, India) |
| 3- | Procured from Central poultry farm, Mumbai, India. |
| 4- | Sd Fine Chemicals Ltd., India. |
| 5- | 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 mcg; 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; Selenium 50 ppm; (Lactobacillus 120 million units and Yeast Culture 3000 crore units). |
| 6- | Sd Fine Chemicals Ltd., India. |
| 7- | Stay C (Hoffman La Roche, Nutley, NJ, USA) 15% ascorbic acid activity |
| 8- | Composition of vitamin B complex (quantity g-1) Thiamine mononitrate 20 mg; Riboflavin 20 mg; Pyridoxine hydrochloride 6 mg; Vitamin B12 30 mcg; Niaciamide 200 mg; Ca pantothenate 100 mg; Folic acid 3 mg; Biotin 200 mcg. |
| 9- | Himedia ltd, India. |
| 10- | Sd Fine Chemicals Ltd., India. |
| Table 2: | Proximate composition of the different experimental diets (DM basis %) |
| OM1 – Organic Matter, CP2 – Crude Protein, EE3 – Ether Extract, TC4 – Total Carbohydrate | |
Sampling and Analysis of Samples
The proximate composition of all the diets was determined following the
standard methods of AOAC (1995), Table 2. In brief, moisture
content was determined by drying at 1050C to a constant weight. Nitrogen
content was estimated by Kjeltec (2200 Kjeltec Auto distillation, Foss Tecator,
Sweden) and CP was estimated by multiplying nitrogen percentage by 6.25. Ether
extract (EE) was measured using a Soxtec system (1045 Soxtec extraction unit,
Tecator, Sweden) using diethyl ether (boiling point, 40-60°C) as a solvent
and ash content was determined by incinerating samples in a muffle furnace at
600°C for 6 h. Total carbohydrate was calculated by difference i.e., total
carbohydrate % = 100 - (CP% + EE% + ash %).
Sample Preparation
At the completion of experiment, fishes were anaesthetized with clove oil
at 50 Fl L-1 and killed by giving blow to the head and dissected
to collect the muscle for protein and DNA concentration estimation. The muscle
was taken from the caudal peduncle region after scraping off the scales. Immediately
a 5% homogenate in 250 mM sucrose was prepared for muscle tissues. The homogenate
was centrifuged at 5000 rpm for 20 min and the supernatant was collected in
a sample vial and kept at -20°C until use.
Protein Estimation
Quantification of protein in the muscle tissues was carried out using Lowry’s
method (Lowry et al., 1951). Tissue homogenate (0.1 mL) was taken and
precipitated using 1 mL of 10% TCA. The protein residue was obtained by discarding
the supernatant produced after centrifugation at 5000 rpm for 20 min. The residue
was dissolved in 0.5 mL of 0.1 N Na0H and 0.1 mL of the dissolved protein residue
was used for further analysis. Alkaline copper sulphate (5 mL) was added and
left for 10 min. To this 5 mL Folin’s reagent (1N) was added and incubated
for 30 min in the dark. Reading was taken at 660 nm against the blank. Bovine
serum albumin was used as standard.
Quantification of DNA
Quantitative determination of DNA in tissue was done by pentose analysis
(Schnieder, 1945) and was calculated as:
Statistical Analysis
The main effect was analyzed by using three-factor ANOVA with starch type
(gelatinized and non-gelatinized), 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 the Duncan’s Multiple Range
Test (DMRT). All value was compared at 5% level of significance. Statistical
evaluation of the data was carried out using the software SPSS version 11.0.
Results and Discussion
The muscle protein, muscle protein/ DNA ratio and DNA/ muscle mass (wet weight) ratio of L. rohita fingerlings fed different test diets are summarized in Table 3 and interaction effect of corn type x protein, protein x α- amylase and corn type x α- amylase are given in Table 4.
In general the muscle protein and muscle protein/ DNA ratio of NG corn fed groups were significantly (p<0.05) higher than their G counterpart irrespective of the protein and α- amylase level in the diet, whereas DNA/ muscle mass (wet weight) ratio was significantly (p<0.05) higher in G corn fed groups. Peragon et al. (1999) observed low growth rate of rainbow trout (Oncorhynchus mykiss) which was correlated with increased in relative DNA concentration and decreased total protein content in fish with reduced protein/ DNA ratio fed diet without carbohydrate. Feeding of G carbohydrate to L. rohita resulted increased DNA/ muscle mass, which indicate the increase in cell number and not cell size. This suggests that G carbohydrate at level of 42.43% seems to be higher causing metabolic stress as suggested by many authors (Hilton and Slinger, 1983; Kausik and de oliva-Teles, 1985).
| Table 3: | Muscle protein, muscle protein/ DNA ratio and DNA/ muscle mass (wet wt.) ratio of L. rohita fingerlings fed with different experimental diets |
| Means with different superscript in a column differ significantly (p<0.05). Weight gain% (WG. %) = (final weight-initial weight)/initial weight x 100. NS- non- significant | |
| Table 4: | Muscle protein, muscle protein/ DNA ratio and DNA/ muscle mass (wet wt.) ratio of L. rohita fingerlings as influenced by starch type (G or NG), amylase, CP level and their interaction |
| Value with different superscript within a column differ significantly (p<0.05) | |
Muscle protein, muscle protein/ DNA ratio and DNA/ muscle mass (wet weight) ratio of fingerlings fed either 28 or 35% CP were similar, suggesting the protein sparing effect of carbohydrate at sub-optimum protein level (28%). Peragon et al. (1999) and Mohapatra et al. (2002) found that carbohydrate is used as energy source in fish by their protein sparing effect. Protein sparing effect of carbohydrate may be related to the fact that glucose is the preferred oxidative substrate for nervous tissue and blood cells and carbohydrate present in fish diets can depress gluconeogenic activity thus diverting amino acids away from oxidative pathways (Cowey et al., 1977; Sanchez-Muroz et al., 1996). The absence of carbohydrate in diet increased protein degradation and decrease the absolute protein synthesis rate in the white muscle of rainbow trout (Peragon et al., 1999).
Supplementation of α-amylase at different level in the diet significantly (p<0.05) varied the muscle protein, muscle protein/ DNA ratio and DNA/ muscle mass (wet weight) ratio of L. rohita fingerlings. Addition of α- amylase significantly improved the muscle protein and muscle protein/ DNA ratio irrespective of the starch type and protein level. However, DNA/ muscle mass (wet weight) ratio was significantly (p<0.05) decreased in the α-amylase supplemented groups. Muscle protein, muscle protein/ DNA ratio and DNA/ muscle mass (wet weight) ratio was similar at 50, 100 and 150 mg kg-1 α-amylase supplemented groups. 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 seabream, Sparus aurata and tiger prawn, Penaeus monodon (Kolkovvski et al., 1993; Carter et al., 1994; Buchanan et al., 1997). Thus, in the present study increase in muscle protein and muscle protein/ DNA ratio may be due to more utilization of NG carbohydrate by addition of dietary α-amylase in the diet as reported by Kumar et al. (2005).
There was no significant (p>0.05) interaction found between corn type x protein and protein x α-amylase on muscle protein, muscle protein/ DNA ratio and DNA/ muscle mass (wet weight) ratio. However, a significant (p<0.05) interaction was found between corn type x α-amylase on muscle protein, muscle protein/ DNA ratio and DNA/ muscle mass (wet weight) ratio. From the interaction effect it indicates that lowest muscle protein and muscle protein/ DNA ratio was observed with G corn fed groups without dietary α-amylase but reverse trend was found for DNA/ muscle mass (wet weight) ratio.
Protein/ DNA ratio is an index of cell size and DNA/ muscle mass ratio is an index of cell number (Foster et al., 1993). Thus, in the present study the increased muscle protein/ DNA ratio in NG corn fed groups supplemented exogenous α-amylase was due to increased cell size of the muscle (hypertrophy). Increased cell size was reflected as decrease in cell no. in the form of lower DNA/ muscle mass ratio. This can be correlated with our previous result (Kumar et al., 2005) that higher weight gain % and SGR was observed in NG corn fed groups supplemented with exogenous α-amylase. This is in agreement with Pelletier et al. (1995), who observed that protein/ DNA ratio increased with growth rate, whereas DNA/ muscle mass ratio decreased. Similarly, Stickland et al. (1988) in Atlantic salmon and Kiessling et al. (1991) in trout observed an increase in hypertrophy in muscle fibres of fish, where maximum growth was registered. Foster et al. (1993) also reported that muscle cell size increases whereas cell number decrease in growing fish.
From the present experiment, it concludes that supplementation of 50 mg α-amylase kg-1 is optimum in the diet of L. rohita juveniles containing 42.43% NG corn to improve muscle protein and muscle protein/ DNA ratio. Diet containing either 28 or 35% CP registered similar muscle protein, muscle protein/ DNA ratio and DNA/ muscle mass (wet weight) ratio. Thus a feed containing NG corn with 50 mg α-amylase kg-1 with 28 % CP, can be used to improve the muscle protein, muscle protein/ DNA ratio and DNA/ muscle mass (wet weight) ratio in the L. rohita fingerlings. This study also reveals that growth of L. rohita fingerling was due to increase muscular hypertrophy, not due to muscular hyperplasia. These data may be helpful to ascertain the effect of dietary carbohydrate on real growth response at short duration.
Acknowledgements
The authors are grateful to the Director, Central Institute of Fisheries Education, Mumbai, for providing facilities for carrying out the work. The first author is grateful to Central Institute of Fisheries Education for awarding the institutional fellowship.