Aquaculture is becoming a major sector in the global food production cycle and contributing significantly to the increasing demand of animal-based protein worldwide (FAO, 2022). And it is considered one of the fastest-growing sectors in the world among all agriculture-based industries (Verdegem et al., 2023). Rohu (Labeo rohita Hamilton, 1822), a commercially important tropical cyprinid and one of the Indian major carps, dominates aquaculture production in the Indian subcontinent and is naturally distributed in the freshwater systems of South Asia (Jhingran and Pullin, 1985; Choudhary et al., 2024). Its culture practices have been expanded due to its high production potential, nutritional profile, good palatability and market acceptability (Shabir et al., 2025). Although intensive production practices boost yields but alters the culture environment as increased stocking densities and feed inputs lead to higher waste accumulation, greater disease risk and increased economic volatility (Ali et al., 2025). In such scenarios, high quality seeds and sustainable feeds become critical for rapid growth, efficient feed utilization and improving the tolerance capacity towards crowding conditions and variable water quality (Hamilton et al., 2022). Nutritionally balanced feeds have a significant impact in the productivity and health status of farmed fish (Coz-Rakovac et al., 2005). Plant based immunostimulant incorporated diet formulation has emerged as a promising avenue for positively modulating the growth, disease resistance and immune competence in L. rohita (Majumder and Saikia, 2020). Bioactive substances present in plants have antioxidant, anti-parasitic and antibacterial properties that stimulate the overall physiological responses and also produced healthy fish (Jahanjoo et al., 2018).
       
Moringa oleifera is well known for its rich nutritional profile, medicinal properties, pharmacological and immunological value and is increasingly explored as an important ingredient in aquafeeds (Ravani et al., 2017; Yadav and Ghimire, 2019; Trigo et al., 2020). The Moringa plant is abundant in calcium, potassium, vitamins, protein and essential amino acids (Gopalakrishnan et al., 2016; Kuswantoro and Rahayu, 2026). Karthivashan et al., (2015) reported that Moringa contains a wide range of bioactive compounds, including alkaloids, flavonoids, tannins, anthraquinones, carotenoids, anthocyanins and proanthocyanidins, along with specific phenolics such as kaempferol, zeatin, apigenin and quercetin. Due to its adaptability to diverse climatic conditions and drought tolerance, it is considered a dependable and sustainable resource for aquafeed formulation (Islam et al., 2021). Dietary incorporation of M. oleifera leaf has been shown to enhance growth performance, haematological parameters and digestive enzyme activity in L. rohita fingerlings (Dutta et al., 2026). Its antioxidant, anti-inflammatory and antimicrobial properties contribute to improved physiological stability and immune function in carps (Elabd et al., 2019). Traditionally, it has been valued for its therapeutic uses and all parts of this miracle plant are considered edible (Leone et al., 2015). Dietary supplementation with aqueous extracts of M. oleifera (MOAE) has been shown to promote growth performance and improve blood parameters in Nile tilapia (Emam et al., 2024). Similarly, Kaleo et al., (2019) demonstrated that incorporating 0.5% M. oleifera leaf extract improved growth efficiency, supported immune and physiological functions and reduced ammonia stress in freshwater shrimp (Macrobrachium rosenbergii). Despite several studies on various fish species, including Labeo rohita, dose-dependent, stage-specific growth studies remain limited. Therefore, the present investigation aimed to evaluate the effects of dietary supplementation with M. oleifera leaves at different inclusion levels on the growth performance of L. rohita fry, fingerlings and advanced fingerlings to demonstrate the comparative response to dietary dose across age groups.

Collection of plant materials
 
Leaves of Moringa oleifera have been collected from Dibrugarh University campus, Dibrugarh, Assam, India. The leaves were carefully cleaned with tap water to remove all impurities. The Moringa leaves were air dried for 7 days and processed into fine powder by passing through a sieve (200 µ) to create MOLP (M. oleifera leaf powder).
 
Procurement and culture of fish specimens
 
The fish of different mean weight were procured from pike fish farm Dibrugarh, Assam and brought to the laboratory for acclimatization. Healthy Labeo rohita fry (mean total weight of 4.90±0.22 g and mean total length of 5.68±0.10 cm), fingerlings (mean total weight of 12.00±0.31 g and mean total length of 10.05±0.05 cm) and advance fingerling (mean total weight of 20.54±0.57 g and mean total length of 12.04±0.04 cm) were selected for the growth study.  For 15 days, they were kept in a concrete tank with a 5000 L capacity under controlled condition (Fig 1). Water quality parameters were also monitored regularly. Dissolved oxygen (mg/L) ranged between 6.5 to 7.5 mg/L (America Public Health Association, 2019), water temperature (°C) was measured by mercury thermometer (27.1 to 29.3°C) and pH (6.7 to 7.3) were measured pH meter (Systronics) respectively. A control diet (35% protein) was fed ad libitum during this period.



Experimental design
 
Three experiments were designed for different age groups with triplicate. Each experiment was conducted for a 60 day feeding trial (Goswami et al., 2020; Babitha et al., 2023; Dutta et al., 2026). Fish were grouped based on initial body weight following randomized design.  For each experiment a total of 120 fish with uniform initial body weight were selected and distributed in 12 (200 L capacity) aquarium with 10 fish each. Throughout the trial, fish were given 5% of their body weight every day at 9.30 and 16.30 hours.  The daily recovery of extra feed was oven dried for six hours at 60°C and weighed to estimate the feed conversion ratio (FCR). Following the first sample at 30 days, the daily ration was modified to adjust the new weight gained.
 
Experimental diet
 
Four isonitrogenous diets (three test diets, D1, D2, and D3 with 0.5%, 1.0% and 1.5%  MOLP respectively and C with no plant materials) were formulated using wheat flour (carbohydrate source), vitamin-mineral premix (Supradin), cod liver oil, dry fish powder and crude MOLP (Chakrabarti and Srivastava, 2012) (Table 1). All ingredients were properly mixed to achieve a homogenized mixture. A commercially available machine with different sieve sizes (0.5 mm-1 mm) was used to produce extruded strings, which were subjected to drying (2-3 days). The strings were crushed to get a 1 mm pellet size suitable for the experimental fish. Proximate composition of experimental and control diet has been analyzed utilized standard protocols described by AOAC (2012) (Table 1).


 
Sampling of fish
 
The initial weight and total length was measured at the start of the study and two sampling was carried out on the 30^th and 60^th day of the experimental trial with minimum stress.
 
Growth performance
 
The following techniques have been employed to assess fish’s growth performance (Bagenal and Tesch, 1978).
 
Absolute growth
 
Change in absolute body weight has been evaluated and documented to nearest gram using a digital balance (Aczet, CG302) by subtracting initial weight from the weight at time of harvest.
       
Similarly, body length gain of the fish has been calculated by subtracting initial length from final length to nearest centimetre using a dial vernier calliper. 
 
Survival rate
 
To calculate the survival rate, the difference in the number of fish between time of stocking and harvest has been calculated and reported as a percentage of the starting number of fish.

           
Specific growth rate (SGR)
 
Subsequent formula had been employed to get the SGR:

 
Feed conversion ratio (FCR)

 
Statistical analysis
 
The recorded data were analysed using one-way analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT) was applied to determine significant differences among treatments using SPSS (Version 20.0). The results (n=3) were shown as mean±SEM. A significance threshold of 0.05 has been used.

The exploration of phytogenic feed has increasingly attracted attention as a sustainable approach to improve fish health and productivity (Khan et al., 2021; Thakur et al., 2025). M. oleifera has drawn global attention due to its exceptional nutritional and pharmacological potential and considered a potential entrant for supplemented fish feed formulation (Panova et al., 2025). M. oleifera leaves contain essential amino acids and have a rich vitamin profile (Islam et al., 2021). The present investigation on the Moringa incorporated diet showed positive (p<0.05) effect on the growth parameters such as length gain (LG), final body weight (FBW), weight gain (WG), specific growth rate (SGR) and feed conversion ratio (FCR) of L. rohita at different life stages (fry, fingerling and advanced fingerling) in experimental diets D1, D2 and D3 (Table 2 and 3).  Due to its exceptional nutrient profile and wide availability, M. oleifera leaves have been extensively evaluated for their effects on growth performance in various fish species, including Oreochromis niloticus (Afuang et al., 2003), Nile tilapia (Richter et al., 2003) and Clarias gariepinus (Ncha et al., 2015). In this study growth response of L. rohita (Fry, fingerling and advanced fingerling stages) to dietary supplementation with Moringa leaf powder was evaluated and found that inclusion of 0.5-1.5% Moringa leave improved growth performance compared to the controlled group in all the life stages. Among the three experimental diet D2 containing 1.0% Moringa leave power showed optimum growth performance in all life stage. Moreover, it was observed that D2 diet has produce best result in the fry stage of L. rohita which was reflected in their highest weight gain, length gain, highest SGR and lowest FCR. A comparative growth performance of L. rohita fry at 30 and 60 days are shown in the Fig 2 and Fig 3 respectively, where it was observed that growth was better in the first phase as compared to the second phase.








       
As aquaculture industry is fighting to cut the feed expenses in intensive farming systems (Fantatto et al., 2024), Moringa can became an alternative sustainable solution as it demonstrated effective FCR and high SGR value in L. rohita. In all the life stage a similar pattern was observed that the growth performance was better in the first 30 days of feeding as compared to the second half of the experiment. Growth performance of L. rohita fingerlings at 30 and 60 days are shown in the Fig 4 and Fig 5 respectively. Similarly, for advance fingerlings, shown in the Fig 6 and Fig 7 respectively, which exhibited better growth in the first phase as compared to the second phase. In all the three experiment the survival rate was 100% which can be due the optimum water quality and timely feeding and waste management scheme. Khalil and Korni (2017) observed improved   growth and immunity in Cyprinus carpio fed with Moringa-based diets. In our study, fish receiving 1% moringa supplementation showed the highest WG and SGR, supporting the hypothesis that Moringa rich nutritional profile and bioactive compounds contribute to enhanced feed utilization. Similarly, Ahmed et al., (2014) found improved growth efficiency in Oreochromis niloticus fed Moringa leaves up to 25% without adverse effect but Elabd et al., (2019) reported growth enhancement only at 1.5% inclusion level. Additionally, Choudhary et al., (2024) recorded optimum growth performance at 1.0% (10 g/kg) in L. rohita fingerlings. However, it is also reported that Moringa leaves have a variety of anti-nutritional elements, such as phytates that limit their use in fish diets because higher phytate levels restrict nutrient withholding, mineral and protein absorption (Soetan and Oyewole, 2009). Several studies have reported that high inclusion levels of M. oleifera (40-50%) can negatively affect the immune response in fish. Therefore, supplementation is generally recommended at levels not exceeding 15% of the diet (Sherif et al., 2014). Increase of M. oleifera in feed above 1.0% in L. rohita translated into slower growth performance possibly due to the adverse effects of certain anti-nutritional factors (Hlophe and Ngonidzashe, 2014). 

The findings of the present study clearly demonstrate that MOLP can be effectively utilized as an eco-friendly and sustainable feed additive to enhance the growth performance of Labeo rohita. Dietary supplementation with MOLP, particularly at an inclusion level of 1.0% (D2), resulted in significant improvements in WG, LG, SGR and FCR across all life stages. Among the different developmental stages, fry exhibited the highest responsiveness to the D2 (1% MOLP) diet, as evidenced by superior growth indices and lower FCR compared to fingerlings and advanced fingerlings. Consistent growth enhancement was observed throughout in the 30-day and 60-day feeding intervals, indicating the sustained efficacy of MOLP over time. However, diet with 1.5% inclusion level observed slightly lower growth performance compared to D2 diet. This observation highlights the need for further investigations aimed at identifying and mitigating such growth retarding factors through optimized feed formulation. The experimental diets (D1-D3) were well accepted by the fish and no deterioration in water quality was observed, underscoring the suitability of MOLP for sustainable aquaculture practices. Overall, the present study supports the inclusion of MOLP as a viable alternative to synthetic growth promoters, contributing to environmentally responsible and economically viable aquaculture development.

The Authors are grateful to the Department of Life Sciences, Dibrugarh University for providing necessary facility for carrying out the research work.
 
Ethical issues
 
None.

The authors declare no conflicts of interest.