Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 58 issue 3 (march 2024) : 433-439

​Comparative Evaluation of Dietary Raw and Solid-state Fermented Sesbania Leaf Meal in Labeo rohita (Hamilton, 1822)

Sudhanshu Raman1, Ashutosh Dharmendra Deo1,*, Md. Aklakur1, Narottam Prasad Sahu1, Manish Jayant1, Tincy Varghese1
1ICAR-Central Institute of Fisheries Education, Mumbai- 400 061, Maharashtra, India.
Cite article:- Raman Sudhanshu, Deo Dharmendra Ashutosh, Aklakur Md., Sahu Prasad Narottam, Jayant Manish, Varghese Tincy (2024). ​Comparative Evaluation of Dietary Raw and Solid-state Fermented Sesbania Leaf Meal in Labeo rohita (Hamilton, 1822) . Indian Journal of Animal Research. 58(3): 433-439. doi: 10.18805/IJAR.B-4889.
Background: Sesbania aculeata leaf are rich in nutrients which makes it a potential ingredient in fish feed preparation. Solid-state fermentation (SSF) process of leaf enhanced nutrient content and reduced antinutritional factors. This experiment was conducted to optimize inclusion of raw and fermented S. aculeata leaf meal (RSLM and FSLM) in the diet of Labeo rohita at fingerling stage at different level.

Methods: Nine isonitrogenous (30% CP) diet for rohu fingerlings were prepared using RSLM and FSLM. SSF of the leaf meals were carried out using Bacillus subtilis. Feeding trail was conducted for 60 days at RSLM inclusion level of 10, 20, 30 and 40% (T1, T2, T3, T4) and FSLM (T5, T6, T7, T8) in triplicate treatments with 0% inclusion as control (C).

Result: The fish fed with FSLM based diet had higher weight gain (%), SGR (%) and lower FCR when compared to RSLM and Control. S. aculeata can be incorporated in rohu fingerling’s diet upto 20% RSLM and 40% FSLM level without any adverse effect on the growth of the fish.
The growing human population has accelerated demands for not only food security and poverty alleviation but most importantly, nutritional security. Fish has been considered a cheap and nutritious food that plays significant role in nutritional security (Maulu et al., 2021). With increase in demand of fish, aquaculture has rapidly expanded, especially feed based vertical intensification, driving huge demand of fish feed. As feed attributed to about 60% to the total cost of production in commercial aquaculture (Silva et al., 1995), the market demand catalyzed escalation in prices of feed and its ingredients. Thus, Scientists are now exploring for cheap, easily available and nutritionally rich feed ingredients for use in fish feed industry. Dorothy et al., (2018) suggested various nutrient rich plants leaves that has cost-effective potential for preparation of fish feed. Sesbania aculeata is one of the nutrient rich and fast-growing green fodder crop commonly cultivated for cattle fodders and green manure (Rani and Kumar, 2020). The leaves are rich in protein and essential fatty acids (Anand et al., 2020). However, plant sourced ingredients often contain anti-nutritional factors (ANFs) and high fibers that requires certain processing procedures to reduce its negative impact on fish growth (Dorothy et al., 2018; Meshram et al., 2018). One of such processing method is SSF that is often used as an effective means to reduce ANFs and crude fibers and enhance protein contents (Anand et al., 2020). For experimenting the inclusion of SLM in the diet of fish at various levels in two different forms (RSLM and FSLM), Rohu (Labeo rohita) was selected as the experimental fish, as it is one of the most commonly cultured carp in the freshwater inland water bodies of India. The fish is a fast-grower with omnivorous feeding nature and have potential to utilize plant-based inputs.
Sixty days feeding trial was conducted at ICAR-Central Institute of Fisheries Education (CIFE), Mumbai, India with nine isonitrogenous (30% crude protein as per Renukaradhya and Varghese, 1986) experimental diets prepared (Table 1) with 10, 20, 30 and 40% inclusion level of RSLM (T1, T2, T3, T4), FSLM (T5, T6, T7, T8) and 0% inclusion of SLM as Control (C). The experiment, thus, comprises of 8 treatments (T1-T8) and a control(C), in triplicates, following a completely randomized design (CRD). Ten rohu fingerlings (4.43±0.12 g) were stocked in each FRP tank (300 L capacity) and were hand fed to apparent satiation with their respective experimental diets twice daily (6.00 h and 18.00 h). For sample analysis, two fish were randomly collected from each replicate of the experimental groups (n=6), anesthetized by immersing in 10 L water having clove oil (50 μl L-1) for 10 minutes prior to use (Anand et al., 2020).

Table 1: Feed formulation of the experimental diets (g kg-1 dry matter).

SSF of dried S.aculeata leaves were carried out using B. subtilis bacteria for 6 days with 100 g of leaf powder in 1000 ml conical flask. The total moisture content for fermentation mixture was maintained at 50% by adding 45 ml of distilled water. It was inoculated with 0.5 ml of spore suspension of B. subtilis (1 x 108 CFU), mixed and incubated at 37°C. The moisture and crude fiber content was determined by Aoac (1990), total nitrogen by automated nitrogen analyzer (KEL Plus-Classic DXVA, Pelican Equipment, India), ether extract by an automatic fat extraction system (SOCS PLUS-SCS 08 AS, Pelican Equipment, India). Total ash content was estimated in a muffle furnace (AI-7981, Expo Hi-Tech, Mumbai). The nitrogen-fee extract (NFE) content was calculated by subtracting the percentage of other nutrients content from 100. Phytic acid was estimated following Vaintraub and Lapteva (1988) while cyanid, tannin and saponin by Aoac (1990), mimosine by Megarrity (1978) and oxalate by Day and Underwood (1991).

Weight gain, specific growth rate (SGR), feed conversion ratio (FCR) and protein efficiency ratio of fish were evaluated at the end of feeding trial using the following equations;
The amino acid profiling was carried out at Aakar Biotechnologies Pvt. Ltd. Lucknow. Amylase activity was estimated by Rick and Stegbauer (1974) method while protease and lipase activity by Drapeau (1974) and Cherry and Crandall (1932) respectively, SOD assay by Misra and Fridovich (1972) method, Catalase activity by Takahara et al., (1960), lactate dehydrogenase (LDH) activity by Wroblewski and Ladue (1955) method, malate dehydrogenase MDH) activity by Ochoa (1955), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity by Wooten (1964). Amino acid profile of the prepared diet (Table 4) was within the recommended level for carps (Zehra and Khan 2013).

The statistical software package 231 SPSS (ver. 22) was used to analyse the experimental data in which data were subjected to one-way ANOVA and Duncan’s multiple range tests to determine the significant differences between the means.
Solid-state fermentation
The average crude protein content in the RSLM was 25.37% (Table 2), which is lower than the value (27.1%) reported by Ayssiwede et al., (2011) which may be attributed to the differences in the growth stage and geographical parameters (Anand et al., 2020). SSF with B. subtilis increased protein and ash content in the SLM which might be due to increase in bacterial biomass in the fermentation system which is in line with Shi et al., (2021) on increased crude protein in drumstick’s leaf meal after SSF with B. subtilis. The proximate composition of the experimental diet is given in Table 3 indicating SSF reduced NFE, fats and fiber content in SLM by 7.40%, 43.22% and 10.25% respectively suggesting that microbial fermentation consumed fat, digestible carbohydrate and fiber to synthesize essential amino acids and vitamins (Ramachandran et al., 2005). ANFs were decreased considerably after SSF (Table 2) which may be due to the secretion of tannase, phytase and other biological enzymes during fermentation process, resulting into breakdown of ANFs which is in line with the report of Meshram et al., (2018) that microorganisms reduce ANFs level during fermentation.

Table 2: Effect of SSF on proximate composition and ANFs of the leaf meals.

Table 3: Proximate composition of experimental diets (% dry matter basis).

Growth performance
The growth performance of fish was significantly higher in T7 (30% FSLM) with 150.53±3.88 % weight gain (Table 4,5). The least growth was recorded in T4 (40% RLSM) followed by T3 (30% RSLM). Thus, upto 20% RSLM and 40% FSLM level can be incorporated in diet of rohu fingerlings without compromising the growth performances of the fish. High inclusion of RLSM was found to negatively impact on the growth, FCR and PER which might be due to high fiber and saponin content in RSLM (Table 2) which might have reduced the protein digestibility either by inhibiting the activity of chymotrypsin or by forming saponin-protein complexes (Anand et al., 2020). Fish fed with FSLM based diet performed better due to the low saponin content after SSF processing.

Table 4: Amino acid compositions of experimental diets.

Table 5: Growth performance of rohu fingerling in the different treatment units.

Proximate composition of fish carcass
Crude protein level was reduced in the carcasses of fish (Table 6) when RSLM is included in the diet which might be due to poor digestibility, absorption or bioavailability of proteins owing to the presence of ANFs such as trypsin inhibitors, tannin and saponin in the raw leafs (Francis et al., 2001). Increased lipid content in fish carcass in the treatment groups (both RSLM and FSLM) might be due to the toxic effect of saponin that interfered with the digestion of the fat. Ash content was higher in treatment groups compared to control which might be due to the phytic acid in plant leafs that reduces the absorption of minerals (Ranjan et al., 2018).

Table 6: Proximate composition of whole body of rohu fingerling (%wet weight basis).

Digestive and metabolic enzyme assay
Protease activity was significantly high in T7 group and lowest in T3 and T4 (Fig 1), as all experimental diets were isonitrogenous in nature, hence, improved protease activity might be due to the decrease in ANFs after SSF (Anand et al., 2020). Lipase activity was low in SLM diet which may attribute to the presence of saponin that binds with bile salt, forms insoluble complexes and creates large micelle (Forde-skjaervik et al., 2006). In the present study, T7 showed higher activity of AST and ALT in muscle and liver (Fig 2 and 3) indicating the synthesis of non-essential amino acids that might attribute to good growth performances in the fish. Similarly, lower activity of AST and ALT in T4 group reflects negative effect of ANFs. The elevated LDH activity of T3 and T4 groups may be due to metabolic stress caused by ANFs (Sahoo et al., 2020). Similarly, higher MDH activities in the RSLM fed group shows high energy demand leading to the activation of TCA cycle (Fig 4 and 5) which are in line with Anand et al., (2020) that elevated LDH and MDH pattern indicates metabolic stress. SOD and catalase activities were enhanced in RSLM fed groups (Fig 6 and 7) and reduced in FSLM groups indicating adverse effects on antioxidant status of rohu. Thus, fish fed with FSLM has improved antioxidant status.

Fig 1: Effect of SLM on digestive enzymes activities of rohu fingerlings.

Fig 2: AST in muscle and liver of rohu fingerlings fed with SLM-based diet.

Fig 3: GPT(ALT) in muscle and liver of rohu fingerlings fed with SLM-based diet.

Fig 4: LDH in muscle and liver of rohu fingerlings fed with SLM-based diet.

Fig 5: SOD in gill and liver of rohu fingerlings fed with SLM-based diet.

Fig 6: MDH in muscle and liver of rohu fingerlings fed with SLM-based diet.

Fig 7: Catalase in muscle and liver of rohu fingerlings fed with SLM-based diet.

SSF of SLM with B.subtilis for six days enhanced nutritional value by increasing crude protein (to 26.92%) and reduced ANFs and fibre. RLSM can be included in the diet of rohu fingerlings upto 20% and FSLM upto 40% level without compromising the growth performance of the fish. Fish fed with 30% FSLM based diet exhibited best growth and physio-metabolic responses.
All authors declared that they have no conflict of interest.

  1. Anand, G., Srivastava, P.P., Varghese, T., Sahu, N.P., Harikrishna, V., Xavier and Patro, D. (2020). Sesbania leaf meal as replacer of DORB in aquaculture feed: Growth, IGF 1 expression, metabolic and biochemical responses in Cyprinus carpio. Aquaculture Research. 51: 2483-2494.

  2. Aoac (1990). Official Methods of Analysis of the Association of Official Analytical Chemists (15th ed.). In [W. Horwitz (Ed.)], Arlington, VA: Association of Official Analytical Chemists. pp. 22201. 

  3. Ayssiwede, S.B., Zanmenou, J.C., Issa, Y., Hane, M.B., Dieng, A., Chrysostome and Missohou, A. (2011). Nutrient composition of some unconventional and local feed resources available in Senegal and recoverable in indigenous chickens or animal feeding. Pakistan Journal of Nutrition. 10: 707-717.

  4. Cherry, I.S. and Crandall Jr, L.A. (1932). The specificity of pancreatic lipase: Its appearance in the blood after pancreatic injury. American Journal of Physiology-Legacy Content. 100: 266-273.

  5. Day, R.A. and Underwood, A.L. (1991). Quantitative Analysis. NJ: Prentice Hall. (Vol. 27)

  6. Dorothy, M.S., Raman, S., Nautiyal, V., Singh, K., Yogananda, T. and Kamei, M. (2018). Use of potential plant leaves as ingredient in fish feed-A review. International. Journal of Current Microbiology and Applied Science. 7: 112-125.

  7. Drapeau, G.R. (1976). Protease from Staphyloccus aureus: In Methods in Enzymology. Academic Press. 38: 466-475.

  8. Forde-skjaervik, O., Refstie, S., Aslaksen, M.A. and Skrede, A. (2006). Digestibility of diets containing different soybean meals in Atlantic cod comparison of collection methods and mapping of digestibility in different sections of the gastrointestinal tract. Aquaculture. 261: 241-258.

  9. Francis, G., Makkar, H. and Becker, K. (2001). Antinutritional factors present in plant-derived alternate fish feed ingredients and their effects in fish. Aquaculture.199: 197-227.

  10. Maulu, S., Nawanzi, K., Abdel-Tawwab, M. and Khalil, H.S. (2021). Fish nutritional value as an approach to children’s nutrition.  Frontiers in Nutrition. 8: 1-10.

  11. Megarrity, R.G. (1978). An automated colorimetric method for mimosine in Leucaena leaves. Journal of the Science of Food and Agriculture. 29: 182-186.

  12. Meshram, S., Deo, A., Kumar, S., Aklakur, M. and Sahu, N.P. (2018). Replacement of de oiled rice bran by soaked and fermented sweet potato leaf meal: Effect on growth performance, body composition and expression of insulin like growth factor 1 in Labeo rohita, fingerlings. Aquaculture Research.  49: 2741-2750.

  13. Misra, H.P. and Fridovich, I. (1972). The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. Journal of Biological chemistry. 247: 3170-3175.

  14. Ochoa, S. (1955). Malic Dehydrogenase from Pig Heart. Methods in Enzymology. Academic Press. 735-739. 

  15. Ramachandran, S., Bairagi, A. and Ray, A.K. (2005). Improvement of nutritive value of grass pea seed meal in the formulated diets for rohu, Labeo rohita fingerlings after fermentation with a fish gut bacterium. Biosource Technology. 96: 1465-1472. 

  16. Rani, S. and Kumar, P. (2020). Proximate composition analysis of plant-based, Non-conventional feed ingredients having potential as ingredient in fish feed. Journal of Pharmacognosy and Phytochemistry. 9: 1853-1859.

  17. Ranjan, A., Sahu, N.P., Deo, A.D., Kumar, H.S., Kumar, S. and Jain, K.K. (2018). Comparative evaluation of fermented and non-fermented de-oiled rice bran with or without exogenous enzymes supplementation in the diet of Labeo rohita (Hamilton, 1822). Fish Physiology and Biochemistry. 44: 1037-1049.

  18. Renukaradhya, K.M. and Varghese, T.J. (1986). Protein requirement of the carps, Catla catla (Hamilton) and Labeo rohita (Hamilton). Proceedings: Animal Sciences. 95: 103-107.

  19. Rick, W. and Stegbauer, H P. (1974). α-Amylase Measurement of Reducing Groups. In: Methods of Enzymatic Analysis. Academic Press. pp: 885-890.

  20. Sahoo, S., Jain, K.K., Sahu, N.P., Deo, A.D., Shamna, N., Patro, D. and Maiti, M.K. (2020). Dietary optimisation of black gram leaf meal as substitute for deoiled rice bran in the diet of Labeo rohita. Indian Journal of Fisheries. 67: 71-79.

  21. Shi, H., Yang, E., Li, Y., Chen, X. and Zhang, J. (2021). Effect of solid-state fermentation on nutritional quality of leaf flour of the drumstick tree. Frontiers in Bioengineering and Biotechnology. 9: 267-269.

  22. Silva, S.D. Anderson, T.A. and Sargent, J.R. (1995). Fish nutrition in aquaculture. Reviews in Fish Biology and Fisheries. 5: 472-473.

  23. Takahara, S., Hamilton, H.B., Neel, J.V., Kobara, T.Y., Ogura, Y. and Nishimura, E.T. (1960). Hypocatalasemia: A new genetic carrier state. The Journal of Clinical Investigation. 39: 610-619.

  24. Vaintraub, I.A. and Lapteva, N.A. (1988). Colorimetric determination of phytate in unpurified extracts of seeds and the products of their processing. Analytical Biochemistry. 175: 227-230.

  25. Wooten, I.D. (1964). Microanalysis. Medical Biochemistry. 98: 101-107.

  26. Wroblewski, F. and Ladue, J.S. (1955). Lactic Dehydrogenase Activity in Blood. Proceedings of the Society for Experimental Biology and Medicine. 90: 210-213.

  27. Zehra, S. and Khan, M.A. (2013). Dietary lysine requirement of fingerling Catla catla (Hamilton) based on growth, protein deposition, lysine retention efficiency, RNA/DNA ratio and carcass composition. Fish Physiology and Biochemistry. 39: 503-512..

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