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

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).



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.




 
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.




 
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).


 
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.

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.