Chapter 4. The crude and enzymatically processed guar seed meal as an alternative protein source for Labeo rohita fingerlings

Transcription

1 The crude and enzymatically processed guar seed meal as an alternative protein source for Labeo rohita fingerlings Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 108

2 4. 1. Introduction Nutritive value of fish diet depends on the quality of protein used in diet formulation (Glencross et al., 2007). Protein is an essential constituent in a balanced diet for (aquaculture) intensive fish culture. Fish meal is an important protein source in formulated diet for most cultivable fish species. In recent years, the scarcity of good quality fish meal and their increasing prices have generated renewed interest in the use of alternative protein sources for fishes. The need to replace fish meal in the supplementary feed of fishes has been recognized as a major research priority (Tacon, 1993). Plant protein sources, which are more consistently available at low cost, have been extensively used in combination with fish meal to formulate cost effective fish diets. The evaluation of various alternative plant protein sources as a partial or complete dietary replacement for fish meal has been carried out by numerous workers for a variety of fish species (Jackson et al., 1982; Ray and Das, 1995; Mukhopadhyay and Ray, 1996; Ramachandran and Ray, 2007). Inclusion of plant proteins in the diet of carp has shown that the partial replacement of fish meal is feasible. Most of the research was concentrated on the evaluation of legume seeds, oil seeds and their by-products like soybean, groundnut oil cake, canola, green gram, cowpea, sesbania and similar other feed for carps (Hossain et al., 2001; Garg et al., 2002; Khan et al., 2003). The presence of antinutritional factors and deficiency of certain essential amino acids have put limitations on the use of plant proteins as complete replacement of fish meal in the aqua feed (Tacon, 1997). Very few available raw materials have an amino acid balance suitable to serve as the sole protein source for fish and as a total substitute for fish meal (Gomes et al., 1995). Some of the plant sources, for instance, soybean (Wilson and Poe, 1985; Robaina et al., 1995), which is rich in protein and other nutrients, are successfully being used in fish diet in spite of the presence of ANFs and amino acid imbalance. Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 109

3 Guar, Cyamopsis tetragonoloba (L.) Taub, cluster bean, is a vigorous and drought-tolerant leguminous plant grown successfully in semiarid regions of many countries. It is native to India and Pakistan (Whistler and Hymowitz, 1979; Tayagi et al., 1982; Francois et al., 1990; Hassan et al., 2008). Guar meal has been widely used in animal feeds, particularly for ruminants, because of considerably high protein content (35-45%), which is also high in lysine and methionine (Couch et al., 1966; Thakur and Pradhan, 1975a, b; Kamran et al., 2002). Galactomannan is a major antinutritional factor present in guar meal. It is a polymer of D-mannose linked by α-1, 4 linkages with D- galactose attached to alternate mannose unit s β-1, 6. Guar gum is reported to contain 8-14% moisture, 75-85% galactomannan, 5-6% protein, 2-3% fiber and % ash (Maier et al., 1993). Feeding experiments using guar as a plant protein source in the diet of Tilapia have failed to achieve better growth performance of fishes (Al-Hafedh and Siddiqi, 1998). However, promising results were obtained on the growth of carp when guar was used in the diet after hydrothermical processing (Garg et al., 2002). The presence of gum (galactomanna) and other antinutritional factors in guar meal limit their use as an alternative plant protein source in fish diet. Enhancement of the nutritive value of guar meal by processing to increase the bioavailability of nutrients and to reduce or remove antinutritional factors by the inclusion of appropriate additive could result in plant material being incorporated at higher levels in fish feed (Wee, 1991). Exogenous enzymes are often used to increase the nutritive value of feed ingredients of plant origin in animal feed (Buchanan et al., 1997). There are several reports on enzyme supplementation in diets for fishes (Yan et al., 2002; Debnath et al., 2005; Drew et al., 2005; Lin et al., 2007). The viscosity of the soluble non starch polysaccharide (NSP) could be reduced by cleaving the polymers using appropriate enzymes (Choct, 1997; Simon, 1998). Endoglucanases and endoxylanases hydrolyse β-1, 4-bond in cellulose and xylan polymers respectively; and reduce the size of soluble non- Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 110

4 starch polysaccharides (NSPs) within the digestive tract (Buchanan et al., 1997). Addition of multi-enzyme supplement to the grow-out diet of Atlantic salmon had a marked effect on the ability to use diet containing soybean meal (Carter et al., 1994). However, improvement in utilization of plant proteins using exogenous enzymes has not yet received much attention in aquaculture (Glencross et al., 2003). The aim of the present study is to evaluate the possibility of using feed enzymes, mainly multi-enzyme cocktail and protease to improve the nutritional quality of agro-based product, guar seed meal. The target of the study is to investigate the influence of enzyme treated guar meal as a protein source on the growth of carp Materials and methods Diet formulation and preparation The formulation and preparation of feed with guar and enzyme treatment to the feed was according to the procedure mentioned earlier (section 2. C. 2. 1). The treatment of guar with enzymes was carried out in glass Petri plates (24cmx36cmx6cm) by soaking guar with distilled water (1:1.5 ratio, by weight), containing 0.1%, 0.5% and 1% of commercial feed enzymes, protease and multi-enzymes cocktail. Since trypsin inhibitor (TI), tannin and phytic acid contents of the seed meal were minimum in groups processed for 3h, with protease and multi-enzymes at 1% level (data not shown); they have been used in the formulation of experimental diets along with raw guar (GC). Before diet formulation, the proximate composition of feed ingredients was determined (Table 1) and amino acid analysis of guar seed meal was carried out (Table 2). Seven iso-nitrogenous (37.5 % crude protein) and iso-energetic (15.95 KJ/g metabolic energy content) diets were formulated (Table 3). Each of raw, protease (7,500 IU/g) treated (1%) and multi-enzyme (7500 IU/g amylase; 4000 IU/g xylanase, 1100 IU/g phytase; 900 IU/g β-glucanase; 1000 IU/g pectinase; 7500 IU/g protease; IU/g cellulase) treated (1%) guar seed meal was Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 111

5 included in the experimental diets at 25% and 50 % replacement of fish meal and designated as D1 to D6 respectively. For the control diet, fish meal was used as a main protein source and designated as RD (Table 3) Experimental design Rohu fingerlings (mean weight 3.60 ± 0.12 g) were stocked at a density of 25 fishes per aquarium with three replicates for each treatment group. Acclimatization of fishes, periodic weight, faeces collection and experimental set-up were carried out as per the procedure given in Section 2. A The water quality parameters-like ph, dissolved oxygen and total organic carbon were monitored weekly following the methods of American Public Health Association (APHA, 1998) and are found to be , mg/l and mg/l respectively Chemical analysis Proximate analysis (moisture, crude protein, crude lipid and ash) of carcasses, feed ingredients and experimental diets and analysis of ANFs of test feeds have been carried out as per the methods described earlier (Section 2. A. 2. 3). The reducing sugar content of test feeds was estimated employing DNS method (Miller, 1979). All chemicals used for the analysis for above parameters were purchased from Qualigens, Qualigen Fine Chemicals, Mumbai, India Gut crude digestive enzyme extraction and estimation Gut digestive enzymes, protease, α-amylase and lipase were extracted and estimated as described in Section 2. A Nutrient digestibility The apparent nutrient digestibilities (AD) were calculated as per the method given in Section 2. A Calculations and statistical analysis Growth parameters were calculated according to formula given in Section 2. A ; and the statistical analysis was carried out using SPSS (section 2. A. 2. 6). Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 112

6 4. 3. Results Composition of experimental diets The chemical composition of the feed ingredients along with the levels of ANFs is shown in Table 1. Pre-treatment of guar meal with multi-enzyme resulted in significant (P<0.05) decrease in the levels of crude fibres, phytic acid and trypsin inhibitor activity; and also resulted in increase in the levels of protein. Protease treatment to the guar meal also resulted in significant (P<0.05) increase in protein and significant (P<0.05) decrease in TI activity. The amino acid content of fish meal and guar meal is presented in Table 2. Guar meal is not found to be deficient in any of the essential amino acids (EAAs) except lysine when compared to the requirement of carp (Ogino 1980). However, according to the requirement of rohu, as reported by Singh (1987), the guar is found to be deficient in sulphur amino acids and lysine. In comparison to javla fish meal, in guar meal aspartic acid, serine, glutamic acid, glycine, histidine and tryptophan levels were higher Fish growth No mortality was observed in any of the dietary groups during the present study. The percentage weight gain, specific growth rate (SGR), feed conversion ratio (FCR) and protein efficiency ratio (PER) as well as nutrient digestibility (APD and ALD) are presented in Table 4. 1) A significantly higher growth rate of the fishes was observed with fish meal based reference diet (RD) among the diet groups. 2) Inclusion of enzyme treated test feed in the diet, improved the growth of the fishes as compared to fishes fed with raw guar meal. 3) When multi-enzyme cocktail was used as feed additive to the guar meal, at both the inclusion level of guar meal, fishes exhibited significantly (P<0.05) higher growth rate in comparison to diets with crude guar meal (D1 and D2). Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 113

7 4) Protease treatment to the guar meal also improved the growth of fishes when incorporated in the diet; but not at the level of growth performance achieved with multi-enzyme treatment. 5) The digestibility values of protein (APD) and lipid (ALD) were found to be more or less similar with different diet groups (RD to D6); though, the nutrient digestibility values were some what higher with fish meal based reference diet in comparison to diets with test feeds (D1 to D6) Carcass composition and gut digestive enzyme activities The carcass composition and digestive enzyme activities before and after the experiment are shown in Table 5. The carcass quality and digestive enzyme activities are noted as followed: 1) The highest carcass protein and lipid as well as lowest carcass moisture and ash contents have been detected in the fishes fed with RD. 2) No significant (P>0.05) changes in carcass composition has been observed when fishes were fed with different test diets, processed with either protease or multi-enzyme cocktail, at each inclusion levels. The carcass moisture and ash levels have decreased with all the diet groups in comparison to the values at the start of experiment. 3) The values of carcass protein and lipid with diet D5 were found to be quite similar to that of RD; and these values were comparatively higher among experimental diet groups. 4) The gut digestive enzyme activities improved significantly with all the diet groups in comparison to values measured at the start of the experiment. 5) The α-amylase activity was quite similar among enzyme treated diet groups and control diet group; however, the activity was comparatively lower with crude guar meal group. 6) The protease and lipase activities were observed to be significantly lower with all the experimental diet groups in comparison to RD; however, the protease activity with D5; and lipase activity with diet D3 as well as with D5 were found to be quite similar to that of control group. Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 114

8 Table 1 Proximate composition of diet ingredients and antinutritional factors of treated and untreated Cyamopsis tetragonaloba (guar) meal (% dry matter unless otherwise stated). Nutrients Fish meal Corn gluten Rice bran Raw Guar meal Pre-treated Guar meal with Protease (1%) Dry matter Pre-treated Guar meal with Multienzymes (1%) Crude protein * Crude lipid Ash Crude fibre * NFE Gross energy (KJ/g) Antinutritional factors 2 Total phenols ± ± ±0.02 Tannins (ug/g) ± ± ±0.54 Phytic acids * (ug/g) Trypsin inhibitors(tiu/ g) ± ±0.92 ± * ±0.49 ± * ± Nitrogen free extract calculated as % (moisture-crude protein-crude lipid-ash-crude fibre). 2 Values are mean±s.e. * Values are significantly different (P <0.05) from Untreated guar meal. Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 115

9 Table 2 Amino acid of Guar meal and fish meal. Amino acid (g/16g N 2 ) Fish meal Guar Required for carp a Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenyl alanine Histidine Lysine Arginine Tryptophan a Data for EAAs requirement of carp from Ogino (1980). Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 116

10 Table 3 Ingredient and proximate composition of the experimental diets (on % dry matter basis) DIETS Cyamopsis tetragonaloba (Guar) seed meal Reference diet Raw GP1% GM1% RD D1 25% D2 50% Ingredient composition (% dry weight) D3 25% D4 50% D5 25% D6 50% Fish meal GC Corn gluten Rice bran Premix Oil pre mix Bentonite Cr 2 O Proximate composition (% dry matter) Moisture Crude protein Lipid Ash Crude fibre NFE Gross energy (KJ/g) 1 Corn gluten and Rice bran were purchased form Charotar Animal Feeds Pvt. Ltd., GIDC, V.V.Nagar, Gujarat, India. 2 Vitamin and mineral mixture (Vitaminetes Forte, Roche Products Ltd., Mumbai, India). 3 Oil Pre mix (2 Corn oil: 1 Cod liver oil) - Corn oil: Tirupati active, N.K.Proteins Co., Mehsana, Gujarat, India. - Cod liver oil: Seacod, Universal Medicare Pvt. Ltd., Mumbai, India. 4 Bentonite purchased from Gujarat Minechem, Bhavnagar, Gujarat, India. 5 Chromic Oxide (Cr2O3): Qualigens, Mumbai, India 6 Nitrogen-free extract Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 117

11 Table 4 Growth performance, feed utilization efficiencies and apparent digestibility in Labeo rohita fingerlings fed experimental diets for 60-days 1. Parame ters Initial wt. (g) Final wt. (g) Percent wt. gain (%) SGR RD D1 D2 D3 D4 D5 D a ± e ± e ± a ± ab ± ab ± a ± a ± a ± a ± bc ± cd ± a ± b ± bc ± a ± de ± de ± a ± cd ± cd ± e ± ab ± a ± cd ± bc ± de ± cde ±0.03 FCR 1.00 a ± c ± c ± b ± b ± ab ± b ±0.02 PER 2.77 e ± ab ± a ± cd ± bc ± de ± cd ±0.05 APD ALD Data are mean values ± SE. Values with the same superscript letters in the same row are not significantly different (P > 0.05) from each other. 2 Statistical analysis was not possible as determinations were performed on pooled samples. SGR: Specific growth rate (% day -1 ) = (log e W 2 log e W 1 )/t 100. FCR: feed conversion ration = feed intake (g) / live weight gain (g). PER: protein efficiency ration = weight gain (g) / crude protein intake (g) APD (%): Apparent Protein Digestibility ALD (%): Apparent Lipid Digestibility Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 118

12 Table 5 Proximate composition (%, wet weight) of the carcass and Digestive enzyme activity of the experimental fishes at the end of the 60-day feeding experiment 1. Parameters Initial RD D1 D2 D3 D4 D5 D6 Moisture Protein Lipid Ash a ± a ± a ± a ± b ± f ± d ± b ± bc ± bc ± bc ± ab ± c ± b ± b ± ab ± bc ± de ± bc ± ab ± bc ± bcd ± bc ± ab ± bc ± ef ± cd ± ab ± bc ± cde ± bc ± ab ±0.15 α- Amylase a ± c ± b ± b ± bc ± bc ± bc ± bc ±0.10 Protease a ± d ± b ± b ± bc ± b ± cd ± b ±0.01 Lipase a ± c ± b ± b ± c ± b ± bc ± b ± Data are mean values ± SE (n=5).values with the same superscript in the same row are not significantly different (P > 0.05) from each other. 2 α-amylase =mg maltose liberated h -1 mg -1 protein; Protease=mg tyrosine liberated h -1 mg -1 protein; Lipase=U/mg protein Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 119

13 4. 4. Discussion The guar meal, pre-treated with protease and multi-enzyme cocktail could be included in the diet of rohu up to 25% replacement of fish meal without any adverse effect on growth and feed utilization efficiency of fishes. The present experiment demonstrated that the pre-treatment of the plant based test feed with microbial enzymes resulted in improvement in nutritional quality as well as decrease in the antinutritional factors (ANFs). The processed guar meal in the diet also improved the growth and feed utilization efficiency in the fishes. It is well documented that the inclusion of plant protein sources in fish diet resulted in reduced growth; and this was attributed to the presence of ANFs and amino acid imbalance in plant based feeds (Hossain et al., 2001). Enhancement of the nutritive value of plant feed can be achieved by treating them with enzymes to increase the bioavailability of nutrients (Ng et al., 2002) as the feed enzymes, mainly from microbial source, as an additive in plant based feed are known to improve feed efficiency and growth in terrestrial animals (Bedford, 2000). During present investigation, addition of commercially available feed enzymes to the guar meal was found to be effective in reducing the anti-nutritional factors like total phenol, tannin, TI activity and fibres at significant level. Enzyme treatment also improved the nutritional quality of seed meal in terms of significant increase in protein. Our findings are in agreement with Ng et al. (2002) who reported an improvement in nutritional quality of palm kernel meal after enzyme treatment, mainly as increase in protein content and reducing sugar level with reduction in NSP level. The use of microbial enzymes to improve feed quality has been recently initiated. Enzyme application to plant based feed is reported to be specific to the composition of feed as well as developmental phase of livestock. Enzymes improve nutritional quality of plant based feed by removing antinutritional factors and toxins, increasing Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 120

14 digestibility of nutrients and supplementing host endogenous enzymes (Campbell and Bedford, 1992; Classen, 1993; Wallace and Chesson, 1995; Bedford, 1996). The NSP degrading enzyme, glucanase is aimed at improving digestibility of barley and oats by reducing the viscosity in the gut lumen of broiler chicks and piglets. Xylanases are directed at degrading xylan polymers in wheat, rye and triticale (Jozefiak et al., 2007). Recent research suggests that mixture of xylanase, protease and amylase improve the digestion of corn and sorghum (Pack et al., 1998). Phytase is also being used on large scale to reduce the phosphorous content of feed and also to improve the digestibility of dry matter and protein (Jongbloed et al., 1997). Extensive work is being carried out in the areas of application of enzymes for the degradation of lectins and protease inhibitors, enzyme supplementation to augment the endogenous enzymes-protease, amylase and lipase and application to degrade fibers in the diet of monogastric animals and ruminants (Classen, 1993; McAllister et al., 1995). Presently, significant increase in the protein content of guar meal by protease treatment could be due to the release of bound proteins from the complexes. A significant decrease in trypsin inhibitor activity along with increase in protein content suggests that microbial protease in the feed is successful in degrading trypsin inhibitor, one of the frequently occurring antinutritional factors of legumes. Significant decrease in fibers, phytic acid and TI activity from guar meal after multi-enzyme treatment clearly indicates the roles of xylnase, cellulase, glucanase, phytase and protease present in multi-enzyme cocktail in reducing different ANFs. This also suggests the feasibility of multi-enzyme cocktail in improving the nutritional quality of legumes like guar meal to be incorporated as a plant protein source for carp diet. Guar meal is not found to be deficient in EAAs except lysine as compared to the requirement of carps (Ogino 1980; Singh 1987). Moreover, the level of certain EAAs like histidine and tryptophan are Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 121

15 found to be higher in guar meal as compared to the fish meal (Table 2). Thus guar meal appears to be a good protein source for carp although lacking in lysine content. The enzyme treated guar meal as an effective plant protein source was also observed in terms of improved percent weight gain, SGR, FCR and PER in rohu in comparison to untreated guar meal. However, the best growth performance of fishes has been observed with fish meal based RD (Table 4). The inclusion of multi-enzyme treated guar meal at 25% level (D5) was found to be more effective in improving the growth of fishes among experimental groups. This may be attributed to the improvement in the quality of protein and to the decrease in majority of ANFs at significant level. Al-Hafedh and Siddiqui (1998) also observed better growth performance of Nile tilapia when fed with guar meal at 25% replacement of fish meal. In the present experiment, untreated guar meal at graded level in the diet of rohu resulted in poor growth of fishes (Table 4). Mohanty et al. (1995) also reported poor growth rate in rohu fry fed diets containing graded levels of oil cakes and legumes. Guar beans are reported to be rich in guar gum galactomannans, the main storage carbohydrate (Azero and Andrade, 2006; Gallao et al. 2007). Guar gum galactomannan is reported to be responsible for reducing the availability of nutrients in the diet of salmonid and is found to be one of the factors responsible for hampering the growth of carp (Storebakken 1985; Garg et al. 2002). Deficiency of lysine and presence of guar gum (galactomannans, nonstarch polysaccharide) in the guar meal seem to be responsible for lower growth of fishes (D1 and D2). Several researchers have reported the effectiveness of enzyme application in poultry and livestock feed for their better growth and feed utilization (Campbell and Bedford 1992; Marquardt et al., 1994). The findings suggest that processed guar meal appear to be a better plant protein source for improving the growth of IMCs as compared to processed soybean, moong and several other processed plant protein sources for carp (Garg et al., 2002). Our results are in Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 122

16 agreement with the findings of Ng et al. (2002) and Boonyaratpalin et al (2000) as they have too reported an improvement in growth and feed utilization in tilapia with enzyme treated palm kernel meal. The better FCR and PER in the fishes fed with multi-enzyme treated test feed at their lower inclusion level can be attributed to the availability of good quality protein to the fishes and the presence of ANFs in permissible limit in the test diets. The feed efficiency obtained with processed guar meal is found to be better in comparison to feed efficiency observed with several plant protein sources in carp as reported by Khan et al. (2003) and Garg et al. (2002). The APD and ALD values for diets prepared with raw and enzyme treated guar meal have been detected in the range of 85.95% to 89.13% and 86.17% to 88.67% respectively. The presently observed nutrient digestibility values are comparable with the values reported for mustard, linseed and sesame seed meal in carp (Hossain and Jauncey, 1989; Hasan et al., 1991). The presence of ANFs like tannins, NSPs, trypsin inhibitor and fibres are known to influence the digestibility of various nutrients in the diet (Lall, 1991). Tannin interferes with protein and dry matter digestibility either by inhibiting the protease and other enzymes or by forming indigestible complexes with dietary proteins (Klogdahl, 1989). Fibre content of plant ingredients could also be responsible for poor nutrient digestibility of plant feed (Jackson et al., 1982). Higher fibre level in the diet is known to retard the digestibility of feed in fishes (NRC-NAS, 1977; Edwards et al., 1985). Moreover, phytic acid is also known to influence protein digestibility by formation of phytic acid-protein complexes (Freancis et al., 2001). In the present investigation, better digestibility vales with enzyme treated test feed, especially treated with multi-enzyme cocktail clearly indicates the decreased content of different ANFs in the feed. During feeding trial, fishes fed with guar meal, pre-digested with multi-enzyme cocktail, exhibited comparatively higher deposition of protein and lipid as compared to other experimental diets; and found Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 123

17 to be almost at similar level as compared to RD. The major reasons that are responsible for improvement in the nutritional quality of carcass with processed guar meal could be 1) Improvement in the nutritional quality of guar during processing, 2) Improvement in the nutrient digestibility and 3) Improvement in the gut digestive enzyme activities. Crude guar meal as an alternative protein source resulted in lower level deposition of carcass protein and lipid at the end of the experiment. Presence of antinutritional factors, lower digestibility of test feed and probably amino acid imbalance seems to be responsible for lower nutritional quality of carcass. The improvement in gut digestive enzyme activities, α-amylase, protease and lipase in the fishes fed with enzyme treated experimental diets clearly indicates the role of multi-enzyme cocktail in improving the quality of feed, finally resulting in improvement in the gut digestive enzyme activities. The clear decrease in ANFs, mainly trypsin inhibitor, tannins and phytic acid and probably decrease in the level of NSPs by exogenous enzyme application appears to be responsible for the improvement in gut digestive enzyme activity levels. Tannins are known to inhibit protease and other enzymes by forming indigestible complexes with dietary protein (Klogdahl, 1989). Protease inhibitors present in plant feed are also reported to inhibit different digestive proteases in fishes (Francis et al., 2001). Moreover NSPs, mainly galactomannan, present in plant feed ingredients have been reported to increase viscosity of gut contents, finally preventing the action of enzymes on substrate Conclusion Present findings suggest that commercially available enzymes can be successfully used to improve nutritional quality of plant based feed like guar meal for carp. This approach can be successfully used for the formulation of cost effective diets for carp. Enzyme treated guar meal can effectively replace fish meal up to 25% level in practical diet Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 124

18 of rohu without any adverse effects on the feed utilization, growth and carcass quality of fishes. Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 125