The rise in the price of MOC and its ANFs content limits its use in the aquafeed. In the present experiment, efforts have been made to replace the MOC with Moringa oleifera leaves in the raw and fermented form in Labeo rohita diet. The Moringa oleifera leaves are nutritionally rich and considered one of the potential alternative ingredients for fish feed. But the existence of some (ANFs), especially tannins, phytate, saponin, oxalate and alkaloid, limits its inclusion in fish feed. Therefore, it needs to be treated before using feed for optimum utilisation. Out of the different methods used to treat ANFs, solid-state fermentation has been proven to be one of the most effective techniques for reducing ANFs from plant-based ingredients and increasing their nutritional value (Shi et al. 2015, 2020). The decrease in ANFs levels in fermented MOLM may be due to the production of several enzymes such as tannase, phytase, etc. during the process to bring out this change (Hur et al. 2014; Shi et al. 2016). Ayuk et al. (2014) and Phulia et al. (2017) found reduced tannin content in A. niger mediated fermented Enterolobium cyclocarpum leaves and Jatropha curcas kernel meal, respectively. Zhang et al. (2020) found decreased phytic acid content in the A. niger mediated fermented leaf meals. Ayuk et al. (2014) found the reduced saponin content in A. niger mediated fermented E. cyclocarpum. The reduced oxalate content in A. niger mediated fermented J. curcas seeds (Oseni and Akindahunsi, 2011) and E. cyclocarpum leaf meal (Ayuk et al. 2014) could corroborate the present result. The increased total flavonoid content (by 31.77%) in the present study could be due to the complete breakdown of the plant cell wall matrix by the extracellular enzymes (cellulase, xylanase and pectinase) produced by the A. niger during fermentation. After the fermentation of MOLM, its crude protein and total lipid level have increased; however, the crude fibre level has decreased. The rise in crude protein level in FMOLM might be associated with the contribution of microbial protein (Ayuk et al. 2014) or from the breakdown of the protein-antinutrient complex by the microbial enzymes (Amankwah et al. 2009). In support of the present study, Zhang et al. (2020) reported the increased protein content in A. niger fermented M. oleifera leaves after 7 days of fermentation. The increased value of total ash content in FMOLM could be due to the microbes associated with the change in mineral composition (Ogodo et al. 2017). The decrease of crude fibre and nitrogen-free extract in FMOLM could be due to the degradation of soluble complex carbohydrates by the amylolytic enzymes (Roger et al. 2015) and the action of fibre degrading enzymes (Ojokoh and Bello 2014) of fermenting microbes. The fermentation process also releases bound phenolic compounds (Huynh et al. 2014).
The optimum water quality parameters should be maintained during aquaculture operation to obtain maximum growth, production, and profit. All the water quality parameters viz. DO, pH, temperature, ammonium-N, nitrite-N, nitrate-N, total alkalinity and total hardness were within the optimum ranges for carp culture (Bhatnagar and Devi 2013; Rajkumar et al. 2018).
After the experiment, the body protein of fish decreased on higher inclusion of raw MOLM, which could be attributed to the toxic effects of antinutritional factors present in MOLM that lowers the nutrient uptake capacity in fish by suppressing the function of intestinal enzymes (Shamna et al. 2017; Kumar et al. 2010). Similar observations were reported by Mehdi et al. (2016) and Ganzon-Naret (2014) in L. rohita and Lates calcarifer with 200 g/kg dietary MOLM. In contrast, dietary incorporation of 300 g/kg FMOLM showed significantly higher protein content in the body which might be due to the reduction of ANFs from the raw leaf meal by applying the SSF technology (Shamna et al. 2017). The body fat content in the FMOLM fed groups was significantly lower than the MOLM fed groups, which might be resulted from the more dietary lipid utilisation from the fermented diets at the cellular level of fish that leads to low deposition of fat in the adipose tissue of fish (Lee et al. 2015).
In the present study, growth suppression was shown at higher inclusion of raw MOLM might be due to a high level of saponin content which causes depletion of protein digestibility (Shimoyamada et al. 1998) and inhibition of nutrient transport (Francis et al. 2002). In corroboration with the present findings, Ganzon-Naret (2014) reported significantly lower growth performance in seabass fingerlings fed with 200 and 300 g/kg MOLM-based diets. However, the FMOLM group showed higher growth performance, which was attributed to the fermentation mediated decrease in the ANFs, resulting in an increased nutritional value of FMOLM. Thus, the resultant feed improves fish growth and nutrient utilisation (Ramachandran et al. 2005). Phulia et al. (2017) suggested that 30% fermented (with A. niger) Jatropha kernel meal in the diet of Labeo rohita fingerlings could show positive effects on growth performance. Saha and Ray (2011) found that Eichhornia crassipes leaf meal could substitute up to 40% of the fish meal. Similarly, better growth performances was observed in Labeo rohita fed with 30% fermented Lemna polyrhiza (Bairagi et al. 2002) or Ipomoea batatas leaf meal (Meshram et al. 2018). No mortality was observed in any treatment groups, which indicats that MOLM based diets did not have any acute toxic effects.
The biometric parameters are the indicators of the general state of fish health and can be influenced by dietary composition (Singha et al. 2020b). Significantly higher HSI value of MOLM groups and control in the present study can indicate high lipid deposition in the liver (Kumar et al. 2012). It was reported that fermented foods have hypo-cholesterolemic effects (Şanlier et al., 2019), reducing lipid deposition around the liver. Therefore, significantly lower HSI values of the FMOLM fed groups in the present study could indicate the hypolipidemic actions of FMOLM.
In comparison to the HSI, the statistically similar ISI values among the different treatment groups revealed that fish fed FMOLM-based diets showed no significant improvement in the intestinal tract's physiological performances compared to the other groups (Fawole et al., 2016). A similar result was reported by Shamna et al. (2015) and Phulia et al. (2017).
Improvement in an animal's growth performance is directly related to the digestive enzymes' increased digestion of the dietary nutrients (Lemieux et al. 1999). Digestive enzymes directly reflect the dietary nutrient digestibility and utilisation in animals, including fish (Hlophe and Moyo 2014). Thus, in the present study, lower protease activity in the MOLM30 group could indicate the lowered dietary protein digestibility and utilisation leading to growth depression in fish. Similarly, Dyson and James (2019) reported significantly lower protease activity in Labeo rohita fed with 300 g/kg MOLM based diets. The antinutritional factor, phytic acid is reported to make a complex with dietary protein, thereby reducing the bio-availability of protein and growth of fish (Francis et al. 2001), which might have caused the reduction in protease activity. Higher protease and amylase activity in FMOLM groups might be due to the reduction of ANFs by SSF using A. niger.
Fermentation of ingredients may also enhance the soluble carbohydrate (NRC 2011; Anand et al. 2020). In support of the current work, Shamna et al. (2015) mentioned the highest protease activity in Labeo rohita fingerlings due to fermented Jatropha protein concentrate feeding. Bairagi et al. (2004) reported higher amylase activity in L. rohita of fermented Leucaena leaf meal fed group than its non-fermented counterpart. Lipase did not vary significantly among treatment groups due to iso-lipidic diets. Similar results were reported in Labeo rohita (Maiti et al. 2019; Garg et al. 2019) and Cyprinus carpio (Anand et al. 2020) when fed with leaf meal-based iso-lipidic diets.
The optimum energy utilisation, body protein synthesis and growth are directly influenced by a linkage between protein and carbohydrate metabolism provided by GOT and GPT (Singha et al. 2020b). Higher GOT and GPT in the liver and muscle of the FMOLM30 group might be due to the reduction of ANFs in the diet. This enhanced metabolism of amino acids improved body protein synthesis and growth performance of fish in this group. However, comparatively lower GOT and GPT found in the MOLM30 group might be due to the presence of more ANFs. Shamna et al. (2015) reported significantly higher GOT and GPT activities in Labeo rohita fingerlings fed Jatropha protein concentrate without fermentation
Fishes can scavenge excess oxygen-free radicals from the body during normal physiological conditions through their antioxidant defence mechanism. Antioxidant enzymes (e.g., SOD and catalase), being a crucial part of antioxidant defences play a critical role in protecting the fish from the harmful effects of free radicals and act as biomarkers for immunity and oxidative stress in fish (Shamna et al., 2017; Singha et al. 2020b). In the current study, higher oxidative stress enzyme activities noticed in the MOLM30 group which might be due to the presence of higher ANFs. Several reports described the positive relationship between dietary antinutritional factors and oxidative stress in fish (Olsvik et al., 2011). Accordingly, a high dietary level of gossypol-containing cottonseed meal in the diet of grass carp significantly increased the SOD and catalase activity (Zheng et al., 2012).
On the other hand, lower SOD and catalase recorded in the FMOLM30 group could probably be due to lower ANFs. Shamna et al. (2017) reported that Labeo rohita fed with fermented Jatropha protein concentrate had lower SOD activities than the non-fermented Jatropha protein concentrate group. Anand et al. (2020) also observed low SOD and catalase activities in common carp juveniles fed with fermented Sesbania aculeata leaf meal compared to the raw one. Thus, it is imprerative that the need of high antioxidant activities are alleviated by using fermented plant meals.